Apexqe Series User Manual

apex-Qe series
generation II
User Manual
Bruker Daltonics
Version 2.0 (July 2008)
Introduction Bruker Daltonics Inc.
Chapter 1 Introduction
1.1 Table of Contents
Chapter 1 Introduction ....................................................................................................... 2

1.1 Table of Contents ................................................................................................................. 2
1.2 Copyright .............................................................................................................................. 7
1.3 Version History ..................................................................................................................... 7
1.4 Part number ......................................................................................................................... 7
1.5 Using This Manual ............................................................................................................... 8
1.6 Warranties ............................................................................................................................ 9
1.7 Warnings ............................................................................................................................ 10
1.8 Safety ................................................................................................................................. 11
1.9 Safety Labels ..................................................................................................................... 16
1.10 Bruker Daltonics Quality Policy .......................................................................................... 18
Chapter 2 Installation and Setup ..................................................................................... 19

2.1 Facility and Electrical Requirements .................................................................................. 19
2.2 Receiving............................................................................................................................ 19
2.3 Unpacking .......................................................................................................................... 19
2.4 Setup .................................................................................................................................. 19
2.5 How to Obtain Support....................................................................................................... 20
2.6 Remote Service Capability ................................................................................................. 21
Chapter 3 Instrument Overview ....................................................................................... 25

3.1 Introduction......................................................................................................................... 25
3.2 apex-Qe series system electronics .................................................................................... 28
3.3 Short electronics reference guide ...................................................................................... 31
3.4 System Control Unit ........................................................................................................... 36
3.4.1 System Power ................................................................................................................ 37
3.4.2 Bake-out......................................................................................................................... 37
3.4.3 Pump and Valve Control ................................................................................................ 40
2 of 256 apex-Qe Series User Manual, Version 2.0

Bruker Daltonics Inc. Introduction
Chapter 4 Theory of Operation ........................................................................................ 43

4.1 FT-ICR Mass Spectrometer ............................................................................................... 43
4.1.1 Ion Motion ...................................................................................................................... 43
4.1.2 ICR Cell .......................................................................................................................... 44
4.1.3 Trapping Plates .............................................................................................................. 45
4.1.4 Detection Plates ............................................................................................................. 45
4.1.5 Excitation Plates............................................................................................................. 46
4.2 The FTMS Experiment ....................................................................................................... 48
4.3 Signal Processing .............................................................................................................. 49
Chapter 5 Getting Started Guide ..................................................................................... 53

5.1 apexControl 3.0 Installation ................................................................................................ 54
5.2 Uninstalling apexControl 3.0 .............................................................................................. 64
5.3 Getting the first mass spectrum ......................................................................................... 67
5.3.1 Starting apexControl ...................................................................................................... 67
5.3.2 Closing apexControl....................................................................................................... 68
5.3.3 Getting ESI Signal .......................................................................................................... 69
5.3.4 Loading apexControl Methods ....................................................................................... 70
5.3.5 Acquiring Spectra and Basic Tuning.............................................................................. 74
5.3.6 Mass Calibration ............................................................................................................ 76
5.3.7 Defining Data Directories ............................................................................................... 78
5.3.8 MALDI signal .................................................................................................................. 79
Chapter 6 Calibration ........................................................................................................ 83

6.1 FT-ICR................................................................................................................................ 83
6.1.1 Mass Calibration ............................................................................................................ 84
6.2 Quadrupole ........................................................................................................................ 86
6.2.1 Principle of Quadrupole Calibration ............................................................................... 86
6.2.2 System Calibration ......................................................................................................... 89
apex-Qe Series User Manual, Version 2.0 3 of 256

Chapter 7 apexControl Reference Guide........................................................................ 97

7.1 Quick Reference Tables ..................................................................................................... 97
7.1.1 Toolbars ......................................................................................................................... 97
7.1.2 Shortcuts ........................................................................................................................ 99
7.2 apexControl Windows ........................................................................................................ 99
7.2.1 Display Windows............................................................................................................ 99
7.2.2 System Status Region ................................................................................................. 103
7.2.3 Instrument Tune Tabs .................................................................................................. 104
7.2.4 Readback and Peak List .............................................................................................. 105
7.2.5 Data Tree ..................................................................................................................... 106
7.2.6 Status Bar .................................................................................................................... 107
7.3 apexControl Tune Tabs.................................................................................................... 107
7.3.1 Standard Pages ........................................................................................................... 107
7.3.2 Expert Pages................................................................................................................ 121
7.4 apexControl Menus .......................................................................................................... 148
7.4.1 File................................................................................................................................ 148
7.4.2 Method ......................................................................................................................... 148
7.4.3 Acquire ......................................................................................................................... 149
7.4.4 Options ......................................................................................................................... 149
7.4.5 View ............................................................................................................................. 150
7.4.6 Expert........................................................................................................................... 150
7.4.7 Tools ............................................................................................................................ 151
7.4.8 Compass ...................................................................................................................... 152
7.4.9 Help.............................................................................................................................. 152
7.5 apexControl Context Menus (Right Click Menus) ............................................................ 153
7.5.1 Display Windows.......................................................................................................... 153
7.5.2 Data Tree ..................................................................................................................... 158
7.5.3 Peak List ...................................................................................................................... 158
7.5.4 Tune Diagram .............................................................................................................. 159
7.5.5 Quadrupole Calibration ................................................................................................ 159
7.6 apexControl Tool Windows .............................................................................................. 159
7.6.1 User Processing ........................................................................................................... 159
7.6.2 Quadrupole Calibration ................................................................................................ 162

7.6.3 ECD Regeneration ....................................................................................................... 164

7.6.4 Configure Instrument.................................................................................................... 169
7.6.5 View: Compiled Acquisition Event Sequence .............................................................. 171
7.6.6 View: Acquisition Event Property Files ........................................................................ 172
7.6.7 Excitation Control ......................................................................................................... 175
7.6.8 Peak Picking ................................................................................................................ 176
7.6.9 FID Advanced .............................................................................................................. 176
7.6.10 FFT Advanced.............................................................................................................. 179
7.7 apexControl Dialogs ......................................................................................................... 180
7.7.1 Hexapole RF Generator Switch ................................................................................... 180
7.7.2 MALDI Target Teaching ............................................................................................... 183
Chapter 8 Acquisition Workflows.................................................................................. 186

8.1 Fragmentation Methods ................................................................................................... 186
8.1.1 In-source Collision Activated Dissociation (IS-CAD) ................................................... 187
8.1.2 Collision Cell Activated Dissociation (C-CAD) ............................................................. 188
8.1.3 Sustained Off-Resonance Irradiation (SORI) .............................................................. 190
8.1.4 On-Resonance Collision Aided Dissociation ............................................................... 194
8.1.5 Infrared Multi-photon Dissociation (IRMPD) ................................................................ 195
8.1.6 Electron Capture Dissociation (ECD)........................................................................... 198
8.2 AutoMSMS ....................................................................................................................... 209
8.2.1 Overview of Decision Structure.................................................................................... 209
8.2.2 Charge State Determination ........................................................................................ 211
8.2.3 CAD Energy Correlation .............................................................................................. 211
8.2.4 Energy offset using the C_MsmsE parameter ............................................................. 212
8.2.5 Interfacing with Hystar.................................................................................................. 212
8.3 AutoRamp Mode .............................................................................................................. 216
8.4 MALDI Imaging ................................................................................................................ 220
8.4.1 General steps in performing an FTMS imaging run..................................................... 221
8.4.2 Setting up the apexControl method ............................................................................. 222
8.4.3 Hystar MALDI automation module ............................................................................... 223
8.4.4 Teaching ...................................................................................................................... 226

8.5 MALDI Automation ........................................................................................................... 227

8.5.1 Create apexControl Method ......................................................................................... 227
8.5.2 Create Sample Table ................................................................................................... 228
8.5.3 Define apexControl/Hystar workflow............................................................................ 229
Chapter 9 Instrument Maintenance ............................................................................... 232

9.1 Chemical Residues .......................................................................................................... 232
9.2 Biological Residues.......................................................................................................... 233
9.3 High Temperatures .......................................................................................................... 233
9.4 Hazardous Voltages ......................................................................................................... 234
9.5 Maintenance Schedule..................................................................................................... 234
9.6 Maintaining the apex-Qe SERIES .................................................................................... 235
9.6.1 Venting the Source or UHV chambers......................................................................... 235
9.6.2 Removing the Nebulizer............................................................................................... 236
9.6.3 Flushing the Nebulizer ................................................................................................. 236
9.6.4 Replacing the Nebulizer Needle .................................................................................. 237
9.6.5 Reinstalling the Nebulizer ............................................................................................ 238
9.6.6 Removing the Glass Capillary...................................................................................... 239
9.6.7 Cleaning the Spray Chamber....................................................................................... 240
9.6.8 Adjusting the Nebulizer Needle.................................................................................... 241
9.6.9 Abrading the Contamination ........................................................................................ 242
9.7 List of apex-Qe series Spare Parts .................................................................................. 243
Chapter 10 Further Documentation................................................................................. 244
Chapter 11 Appendix ........................................................................................................ 246

11.1 Abbreviations ................................................................................................................... 246
11.2 Bug Reporting and Requests for Enhancements in apexControl .................................... 248
Chapter 12 Index ............................................................................................................... 253

1.2 Copyright
July 2008
Bruker Daltonics, Inc.
Billerica, MA, USA
1.3 Version History
Version Date Changes Remarks

1.0 May 2007 Initial version apexControl 2.0
2.0 May 2008 Updated for apexControl 3.0
apex-Qe series
1.4 Part number
# 257038

1.5 Using This Manual
This manual applies to the apex-Qe series generation II FTMS

instruments from Bruker Daltonics. This series of instruments is
equipped with a new APEX COMMUNICATIONS HUB also referred to
as SHEDS (Smart Hub Ethernet Distribution to Serial). The latest
instrument in this series is the apex ultra.
Identification of an apex-Qe series generation II instrument:
The identification can be done by means of either a software tool or by

recognizing the physical device on the instrument:
Identification thru software tool:

1. open a windows internet browser
2. type in the address http://149.236.99.3:8080/sheds_serv2/
3. if the SHEDS hardware is connected the Status page of the SHEDS
Web server should be displayed
Identification on the instrument:

The communication hub can be found by opening both cover doors of
the two electronics bays on the vacuum cart of the instrument (see also
Chapter 3 Instrument Overview)
Apex communications hub - SHEDS

Note: From this point on the manual will use the shorter name apex-Qe series.
1.6 Warranties
The information contained in this document is subject to change without

notice.
Bruker Daltonics Inc. makes no warranty of any kind with regard to this
material, including, but not limited to the implied warrantees of
merchantability and fitness for a particular purpose.
Bruker Daltonics Inc. is not liable for errors contained herein or for
incidental or consequential damages in connection with the furnishing,
performance or use of this material.
Bruker Daltonics Inc. assumes no responsibility for the use or reliability

of its software on equipment that is not furnished by Bruker Daltonics
Inc.
Bruker Daltonics Inc. warrants the apex-Qe series to operate within

specifications for a period of one year following acceptance. This
warranty implies that the apex-Qe series and its electronics are not
modified by the user nor used for non-typical experiments.
Bruker Daltonics Inc. will repair or replace any component that is found
to be defective during the warranty period provided that malfunction is
not caused by abnormal operation on the part of the user.

1.7 Warnings
Connecting an instrument to a power source that is not equipped with a

protective earth contact creates a shock hazard for the operator and can
damage the instrument. Likewise, interrupting the protective conductor
inside or outside the instrument or disconnecting the protective earth
terminal creates a shock hazard for the operator and can damage the
instrument.
The instrument must be disconnected from its power source before any
cover, requiring the use of a tool, is removed or the system is opened.
All connections of the instrument must be used in correct way. The

instrument should only be used with the wires and cables delivered with
the system or otherwise provided by the manufacturer

1.8 Safety
For electrical safety, observe the following guidelines:
Service on electrical components should be performed only by a

qualified Bruker service engineer, service representative or similar
trained and authorized person.
Superconducting Magnet Safety Aspects
The superconducting magnet that is at the heart of the apex-Qe FTMS

system is of the latest, most advanced superconducting magnet
technology. However, there are safety concerns (though reduced for
these magnets), which need to be addressed. These include, stray
magnetic field, stored energy and cryogenic liquids. The siting of the
magnet should conform to the Food and Drug Administration (FDA)
safety guidelines.
Stray Magnetic Field
The superconducting magnet supplied with the apex-Qe FTMS system

is of the latest technology and employs state-of-the-art shielding.
However, even with this shielding technology, there exists a stray
magnetic field for a region around the superconducting magnet system.
Once the magnet is energized, the magnetic field is ALWAYS present,
even when all the power to the system has been turned off. The
magnetic field extends above, below and to the sides of the magnet. It
extends through doors, walls, ceilings, and floors. The specific extent of
this stray field is different for the various FTMS magnets.
The strength of the magnetic field increases dramatically as the distance

to the magnet is reduced. Objects and people at risk from the magnetic
field are considered safe outside the 5 gauss line.
Occasionally, fault conditions may cause magnets to loose their

superconducting properties (i.e. “quench” or “run down”), which in turn
will cause their magnetic field to collapse. However, for the actively
shielded magnets used in apex FTMS systems, before the magnetic
field collapses, it will momentarily “bloom” slightly beyond its normal
dimensions. This effect should be taken into account during siting of the
magnet.

Hazards Presented by the Magnetic Field
The strong magnetic field creates hazards because of its strong

attraction of ferromagnetic materials. Ferromagnetic metals generally
contain iron, nickel or cobalt and are used in most types of tools and
equipment and even in some surgical implants.
The strong magnetic field can cause the following hazards:

 Projectiles
 Displacement of surgical implants
 Stoppage of electrical and mechanical implants and devices
A loose metal object (such as a wrench or pen) becomes a projectile if it

gets too close to the magnet, with the force pulling the object towards
the center of the magnet. The metallic projectile can seriously injure
anyone standing between the object and the magnet. The effect is NOT
limited to light objects, but all ferromagnetic objects which may result in
a person being pinned between the object and the magnet (gas
cylinders, floor sweepers, etc).
Ferromagnetic materials are sometimes used in surgical implants and

prosthetic devises. The magnetic field can twist these items out of
place, which may result in tissue damage, pain, and may possibly be life
threatening.
Some cardiac pacemakers, bi-stimulators, and neuro-stimulators are

mechanically activated and may stop in the presence of the magnetic
field. This also could result in a life threatening situation.

FDA Requirements
The FDA requires that the hazards listed above are prevented by
establishing two secure and clearly marked zones:
Exclusion Zone
Security Zone
The exclusion zone comprises the area inside the magnet 5 gauss line.
Individuals with cardiac pacemakers or other mechanically active
implants must be prevented from entering the exclusion zone. The
exclusion zone must be enforced with a combination of warning signs
and physical barriers. Note that the exclusion zone may intrude into
floor immediately above and below the magnet.
The security zone is usually confined to the room that houses the
magnet and is established to prevent ferromagnetic objects from
becoming projectiles. Ferromagnetic objects should not be allowed
inside the security zone.
Emergency Discharge Switch
All of the magnets provided with the FTMS are equipped with an
emergency discharge switch. In the case of a life-threatening event
(e.g. a person pinned to the magnet by a ferromagnetic object), this
switch can be activated to rapidly and safely remove the field from the
magnet. This process will evolve a great deal of gaseous helium, which
if not vented from the room via a quench duct, may create an
asphyxiation hazard (see below).
Hazard in Case of Fire
The exclusion zones described above MUST be conveyed to fire safety

personnel. It is important for them to know that there are extreme
hazards in the zones besides fire and smoke. It is important to review
these hazards on a regular basis with fire-fighting personnel so that
ferromagnetic material is not brought into the security zone during the
fighting of a fire. These personnel should be made aware of the location
and operation of the emergency discharge switch.

Cryogenic Liquids
The superconducting magnets that are supplied contain cryogenic

liquids. Conventional, actively shielded magnets contain liquid helium
and liquid nitrogen. The R series of UltraShield Plus magnets contain
liquid helium only.
Helium is a naturally occurring inert gas that becomes liquid at about 4 K

(-269 C). It is odorless, colorless, non-flammable, and non-toxic. The
liquid helium is used to keep the superconducting magnet at its required
operating temperature.
Nitrogen is also a naturally occurring gas (about 70% of air) that

becomes liquid at approximately 77 K (-196 C). It is also odorless,
colorless, non-flammable, and non-toxic. It is used in the non-
refrigerated series magnets in addition to the helium. A reservoir filled
with liquid nitrogen serves as a cool shield surrounding the helium
reservoir. In the refrigerated (R) series magnets, this function is
replaced by the cryo-refrigerator.
Asphyxiation Hazard: A large quantity of gas released in an area that

is not well ventilated can cause asphyxiation due to displacement of
oxygen.
Cryogenic Burns: Cryogenic liquids or cold gasses can cause
cryogenic burns.
Fire Hazard: The filling procedure produces an oxygen rich liquid,
which is a fire hazard if it drops on combustible material.
The large quantity of nitrogen or helium that can be released into a room
during filling or a magnet quench can create an asphyxiation hazard by
displacing the oxygen in the room. The expansion ratio of liquid helium
at 4.2 K to gas at room temperature is about 700:1. Correspondingly,
the expansion ratio of liquid nitrogen at 77K to room temperature is
about 680:1. Hence a volume of 100 liters of liquid, if released as gas,
would displace 70 cubic meters of air! Hence, care should be taken to
insure that the site is well ventilated. Bruker Daltonics recommends that
the site have an oxygen monitor that would alert personnel in the case
of local depletion of oxygen.

Also, it is recommended that the magnet’s helium vent be connected to

a “quench duct” that would direct the outflow of helium outside the room
to a safe location. Details of these measures are given in the site
planning guide.
The extremely low temperatures of the cryogenic liquids, materials in

contact with cryogenic liquids, and the gases evolved from them can
cause severe cryogenic burns and frostbite. This is mainly a danger
during the filling process or the cold-head servicing on R series
magnets. To prevent cryogenic burns, protective gloves and face
shields should be worn and long sleeved clothing, long trousers, and
closed shoes are recommended.
During the filling process, atmospheric air condenses on the fill lines and
pipes. This condensate is an oxygen rich liquid (or even liquid oxygen)
which could start combustion if allowed to drip on flammable material.
Hence the secure zone should exclude all sources of ignition. All tools
and equipment used for the transfer of cryogenic liquids should be kept
clean and free of grease or oil. The magnet room should be designated
as non-smoking.
Stored Energy
The energized magnets store a considerable amount of energy; in

general several MJ in the magnetic field. This energy is equivalent to
lifting the cryomagnet systems, weighing tons, to a height of the order of
100 m.
In the event of a “quench”, this energy is release in the form of heat,

which in turn evaporates the cryogenic liquid surrounding the magnet
and also heats the magnet. This causes most of the helium in the
dewar to be evolved as gas. (Please see the above section on
asphyxiation hazards). This gas should be directed out of the room via
a “quench duct”. The magnet is protected from accidental damage
during a quench by an internal electrical protection network.

1.9 Safety Labels
These labels are placed on the instrument to indicate the potential dangers.
NOTE
This symbol is placed on the product where it is
necessary for you to refer to the manual in order to
understand a hazard.
WARNING CAUTION
This symbol is placed on the product within the area
where hazardous voltage is present or shock hazard can
occur. Only trained service persons should perform work
in this area.
WARNING CAUTION
where hot parts and surfaces are present. Allow the
product to cool before performing work in this area.

WARNING
where biohazards are present. Handle these areas with
the respective care.
WARNING CAUTION
The source chamber may not be opened until the sample
flow has stopped.
Health Risk!
Fire danger!
Contamination of the Environment and Air!
CAUTION
Laser radiation
Indicates that laser light may be present.
The instrument with the closed safety cover is a Class I
Laser product.
With safety cover opened it turns to a Class IV Laser
product.

1.10 Bruker Daltonics Quality Policy
Quality Policy
It is the policy of Bruker to provide customers with products and
services of the highest quality. Bruker is focused on continually
improving our products and our internal processes so that our
customers are satisfied with the products we deliver, and have
lasting confidence in what we offer.
Because the success of our quality management system is essential for our
competitiveness, it is vital that the employees of Bruker understand and
adhere to our Quality Policy.
Bruker's top management is committed to ensure that the quality policy:
- Is appropriate to the purpose of our organization.
- Communicates our commitment to meeting requirements and to
continually improving the effectiveness of our quality management
system.
- Provides a focus for continual improvement through establishing,
tracking, reviewing and maintaining Bruker’s quality objectives.
- Is continuously reinforced by management to ensure understanding
and commitment throughout our organization.
- Is reviewed for continuing suitability during the Management Review
Process.

Bruker Daltonics Inc. Installation and Setup
Chapter 2 Installation and Setup
2.1 Facility and Electrical Requirements
For detailed facility and electrical requirements for the apex-Qe Series FTMS
instruments please refer to the apexTM ultra Q MASS SPECTROMETER SITE GUIDE
AND PRE-INSTALLATION INSTRUCTIONS, which has been provided to you prior to
delivery of the instrument
2.2 Receiving
After receiving packages and instrument crates please report any damages to the
outside of the packaging caused during shipment immediately to Bruker Daltonics, Inc.
2.3 Unpacking
Do not unpack any package or crate yourself. Unpacking on your own might
cause damage to the equipment and will void Warranty. !
2.4 Setup
The installation of the apex-Qe series FTMS instrument is performed by a trained

Bruker service engineer who will also perform the onsite acceptance measurements
and initial training in basic operation and maintenance.

Installation and Setup Bruker Daltonics Inc.
2.5 How to Obtain Support
If you encounter problems with your system please contact a Bruker representative in
your area, or:
Company headquarters in US:

Bruker Daltonics, Inc.
40 Manning Rd
Billerica, MA 01821
U.S.A.
Call: +1 978 663 3660; Ext. 1445
Fax: +1 978 667 5993
Email: service-support@bdal.com
Internet: http://www.bdal.com/
Main branch in Europe:

Bruker Daltonik GmbH
Fahrenheitstr. 4
28359 Bremen
Germany
Call: +49 421 2205 248
Fax: +49 421 2205 107
Email: ftms_support@bdal.de
Internet: http://www.bdal.de/

2.6 Remote Service Capability
http://bdal.webex.com
http://bruker.webex.com
Remote
Service
Customer
Side Daltonics
Service Board
Figure 2-1 Operating principle of the remote service
To obtain a maximum operating time the apex-Qe series FTMS is equipped with a
remote service capability (Figure 2-1). This feature allows for remote troubleshooting
via the internet. Thus problems can often be solved efficiently with the customer PC
being fully controlled by an off-site Bruker Service engineer. Both software and
firmware updates are possible in this manner.
Moreover, the service process can be sped up, as the service engineer can arrive on
site with the appropriate spare part, after remote diagnosis.
Prerequisite:
The customer must have Internet access on the Control PC of the apex-Qe SERIES
FTMS.
Select http://bdal.webex.com (Europe) or https://bruker.webex.com (Americas/Asia).
Figure 2-2 appears.

Figure 2-2 Webpage of Bruker Enterprise

Select Join on the web page.
Note: The “http”-connection changes to a “https”-connection, coded with 128-bit to

provide the highest possible level of security!
Figure 2-3 appears.
Please, enter the required information. A call to Bruker Service on +49 421 2205 248
(Germany) or +1 978 663 3660, Ext. 1445 (USA), will provide you with the Support
Session Number. Press the “Submit” button and follow the instructions to successfully
connect the remote service to your apex-Qe series.

Figure 2-3 Enter the required information

Bruker Daltonics Inc. Instrument Overview
Chapter 3 Instrument Overview
3.1 Introduction
Sample Introduction
ESI spray chamber
Superconducting Vacuum cart

magnet
Figure 3-1 View of the apex-Qe series FTMS from the user side
Figure 3-1 shows the apex-Qe series instrument with its two major components, the
vacuum cart and the superconducting magnet. The sample for electrospray ionization
is introduced at the ESI spray chamber interface on the right hand side.

Instrument Overview Bruker Daltonics Inc.
Sprayer
Superconducting Sample
Inlet
Magnet
UHV pulsed
gas inlet Nebulizer Gas N2
Collision Gas Ar
Spray (pressure controlled)
UHV (flow controlled)
Chamber Drying Gas N 2(flow and
Vacuum Beam temp. controlled)
Stage 6 Valve
ECD Vacuum Vacuum Desolvation
Vacuum Vacuum Stage 2
He ater
Cathode Stage 5 Stage 4 Stage 3 assembly
IR laser
beam <10 -9mbar ~8x10 -6mbar ~5x10 -4mbar ~0.1mbar
(IRMPD
optional) ~4mbar
TP4 TP3
Vacuum Stage 1 MALDI plate
TP2 TP1
UHV Source
Rough pump Rough pump
Exhaust
Analyzer
(Infinity Cell) Collision Source Dual stage Ion funnel
Ion Transfer optics cell Mass selective Hexapole
Quadrupole
Detector Ion Transfer Optics Qh Interface Ion Source
Figure 3-2 Schematic overview of the ion optics and

pumping system of the apex-Qe series FTMS
The instrument can be divided into 4 major sections, the atmospheric pressure
ionization source (API), the Qh-Interface, the ion transfer optics and the detector. Six
differential pumping stages maintained by four turbo-molecular pumps (TP1 – TP4) and
two mechanical roughing pumps allow the introduction of ions at atmospheric pressure
while providing ultra high vacuum of <10-9 mbar necessary to operate the analyzer.
Ions are separated from the solvent with the assistance of nebulizer and counter-drying
gas in the atmospheric pressure spray chamber and then pass through a glass
capillary into the first vacuum stage at ~4 mbar. Here they enter orthogonally into the
first funnel stage and are focused into a defined ion beam with the help of DC and RF
voltages. Alternatively, if optionally equipped with the Dual source (ESI & MALDI), ions
can be generated using a focused laser beam on the MALDI plate positioned in front of
the ion funnel.

The ions then enter the second funnel in the vacuum stage 2 at ~0.1 mbar. Here they
are decelerated before they experience further cooling in the vacuum stage 2 of the
source hexapole. Ions can be accumulated in this region for a defined time or simply
passed through, before they enter the Qh-interface.
 For a detailed description of the ion sources please refer to the manual Apollo II
Dual ESI/MALDI Source (Part number 600392).
Ions are subsequently focused into the mass selective quadrupole which allows the
selection of a desired mass window (1 amu – 6000 amu) around a given m/z to pass
through into the collision cell where they can be stored for a selected time.
The collision cell is at an elevated pressure of ~1x10 -3 mbar of typically Argon (Ar). If
desired the conditions can be changed to induce fragmentation of the mass selected
precursor ions. Fragment ions are then pulsed out of the collision cell and are
accelerated into the ion transfer optics towards the analyzer.
 For a detailed description of the mass selective quadrupole and the collision cell
please refer to the manual Qh-Interface User and Service Manual (Part number
600393).
The ions are initially accelerated up to ~3kV to focus them through the flow restriction
between the differential pumping stages into the ultra high vacuum (UHV) of <1x10-9
mbar. In this phase ions can be steered using two sets of horizontal and vertical beam
deflectors to correct for alignment of the instrument. After passing through the gap of
the beam valve that separates the Qh-Interface from the UHV region ions are
decelerated and focused through the inhomogeneous fringe field of the
superconducting magnet before they reach the analyzer, the Infinity CellTM .
The analyzer is a cylindrical ion trap. Ions are confined radial by the high magnetic field
(7-15T) and axially by two trapping electrodes at either end of the trap. Ions are
accelerated to higher radii by means of rf excitation across two opposing electrodes
(excite plates). At the appropriate time the rf field is turned off and the now coherent
package of ions is detected by another set of two opposing electrodes of the trap. The
ions which revolve around the axis of the homogeneous magnetic field with their
individual cyclotron frequencies induce mirror current which is amplified by a
preamplifier (see Figure 3-7) mounted on the back flange of the vacuum system (for
more detail on the analyzer see Chapter 4).

3.2 apex-Qe series system electronics
Spray
Chamber
UHV inlet
system
Vacuum
Display
Electronics Electronics
Bay 2 Bay 1
UHV Vacuum Source

gauge Vacuum gauge
SCU –
System
Control Unit QFSCU –
Quadrupole
Frequency System
SHEDS – Smart Hub Control Unit
for Ethernet
Distribution to Serial
QTIC –
Quadrupole
Transfer Ion
optics Control
TOPPS – Transfer
Optics Power
Figure 3-3 Main components of cart electronics accessible to the user

Electronics Electronics
Bay 2 Bay 1
UHV Vacuum Source Vacuum

gauge gauge
SCU – System
Control Unit
QFSCU –
Quadrupole
Frequency System
SHEDS – Smart Control Unit
Hub for Ethernet
Distribution to
Serial
QTIC –
Quadrupole
TOPPS – Transfer Ion optics
Transfer Optics
Power Supply
Figure 3-4 Alternate Layout (later generation apex-ultra)  Main components of cart
electronics accessible to the user

Acquisition
Console
(MicroBay)
PDU – Power
Distribution
door
Figure 3-5 Acquisition electronics (microBay console) and power distribution panel
on a apex-Qe
Figure 3-6 Acquisition electronics (nanoBay console) on an apex-ultra

3.3 Short electronics reference guide
Figure 3-3, Figure 3-4 and Figure 3-5 depict the major electronic components that are
important for the operation of the apex-Qe series instrument. The following list will
introduce you to the individual components and their function. Figure 3-7 shows a
simplified block diagram on how these components relate to each other.
 Host PC (not shown): runs the apexControl software, which is the main GUI
for the user to interact with the instrument and acquire data. The host PC
communicates with the acquisition console to set up acquisitions on the
detector and to receive the data. It also communicates with SHEDS (Smart Hub
for Ethernet Distribution to Serial, see Figure 3-7), which controls all the devices
in the vacuum cart.
 SHEDS: Smart Hub for Ethernet distribution to Serial; This unit manages the
device control between the host PC, the vacuum cart and the detector (Infinity
Cell).
 SCU: System Control Unit; controls the vacuum system, houses the main
power switch and allows the user to monitor the status of the turbo molecular
pumps and valves. It also allows individually venting the source region (first 4
vacuum stages) or the UHV system (see Figure 3-2) after closing the beam
valve that separates the two sections. It connects to the source and UHV
vacuum controllers for pressure monitoring.
 QTIC: Quadrupole Transfer Ion Controller; supplies all necessary DC and RF

voltages for the atmospheric pressure ionization interface (electrospray (ESI),
atmospheric pressure chemical ionization (APCI) and Nano-electrospray (Nano-
ESI), the dual stage funnel and the source hexapole. It also provides all the DC
voltages to the quadrupole interface (Qh-interface)
 QFSCU: Quadrupole Frequency System Control Unit; supplies RF voltages to

the mass selective quadrupole and the collision cell hexapole. It also supplies
the mass selective DC level and the DC bias to the main rod of the mass
selective quadrupole. The calibration of the mass selective quadrupole is stored
on this unit in a calibration grid.
 please refer to the Qh-Interface – User and Service Manual (600393.pdf)
for further reading on the theory of operation and a detailed description of
quadrupole calibration.

 TOPPS: Transfer ion OPtics Power Supply; supplies all the voltages to the ion
transfer optics that guide the ions from the collision cell through the fringe field
of the superconducting magnet into the analyzer (Infinity Cell).
 ICC2: Infinity Cell Controller ; supplies the power to the preamplifier and
controls all the DC voltages on the analyzer. It also controls the ECD (Electron
Capture Dissociation) electron gun. The ICC2 unit consists of two parts, the
ICC2 Controller and the ICC2 power supply and is located inside the acquisition
console.
 Acquisition Electronics: contains all electronics to support the experimental

timing, pulse control and ion detection. The major components are the console
rack, the Infinity Cell Controller, the power amplifier (for ion excitation) and an
Ethernet hub.
The apex-Qe series generation II has two variants of the acquisition electronics
– microBay and nanoBay console, the latter one being part of the apex-ultra
instruments.
Specifics of microBay console (see also Figure 3-7):

The AQS console rack of the microBay can be divided into two main parts,
receiving part and signal generation. The receiving part consists of an analog
receiver and is connected to the preamplifier which is mounted on the vacuum
flange of the analyzer. The signal is passed from the receiver to an ADC and
after digitization the data is stored on the RCU (Receiver Control Unit) board
before the data is transferred to the host PC and displayed in apexControl. The
signal generation part mainly consists of the TCU (Timing Control Unit), the
FCU (Frequency Control Unit) and the SGU (Signal Generating Unit). Using the
clock from the reference board the TCU maintains the timing of the experiment
sequence embodied in the acquisition event sequence (also called pulse
program) that is defined in apexControl. The TCU also generates all the
necessary TTL pulses to devices in the vacuum cart and to the ICC2. The FCU
is uploaded with the appropriate frequency list and the SGU generates the
corresponding waveform for excitation, which is amplified by the power
amplifier. The power amplifier passes the excitation through a power splitter
(0,180 phase splitting) to the excitation plates on the analyzer.
 please refer to the AQS Avance User Manual (600876.pdf) for further
reading on operation, system component description and wiring.

Specifics of nanoBay console (see also Figure 3-8):

The AQS/2 console rack of the nanoBay can be divided into two main parts,
receiving part and signal generation. The receiving part consists of an analog
receiver and is connected to the preamplifier which is mounted on the vacuum
flange of the analyzer. The signal is passed from the receiver to the DRU
(Digital Receiver Board) board for digitization, accumulation and storage. The
data is transferred directly from the DRU board to the host PC and displayed in
apexControl. The signal generation part mainly consists of the TCU (Timing
Control Unit), the FCU (Frequency Control Unit) and the SGU (Signal
Generating Unit). Using the clock from the reference board the TCU maintains
the timing of the experiment sequence embodied in the acquisition event
sequence (also called pulse program) that is defined in apexControl. The TCU
also generates all the necessary TTL pulses to devices in the vacuum cart and
to the ICC2. The FCU is uploaded with the appropriate frequency list and the
SGU generates the corresponding waveform for excitation, which is amplified
by the power amplifier. The power amplifier passes the excitation through a
power splitter (0,180 phase splitting) to the excitation plates on the analyzer.
 please refer to the NANOBAY FTMS User Manual (601799.pdf) for further
reading on operation, system component description and wiring.

Ion Transfer Optics Qh-Interface Ion Source

Ar
Collision
Gas Control
Shutter
(QTIC)
Magnet
VS6 VS5
Preamp VS4 VS3 VS2
Analyzer
(ICR-Cell)
VS1
MicroBay AQS
Console Rack QFHRFG QFQRFG
CCU Rx
RS232
ICC2 Controller
QHRF
System
ICC2 PS
Interlock
J101 QFSCU QTIC
J102 J424
Power Amp J410
Ultra HUB J26 TOPPS
J21 RS232
RS485
RS232
SHEDS
Ethernet
Vac
PC Ready SCU
Interlock
Vacuum Sense
Vacuum
V 3501-UHV VGC 3581 - SRC
V Cart
Electronics
DC voltages & control RF connections Serial Connection Ethernet (TCP/IP) Signal/Data flow Interlock
VS1 Vacuum Stage 1
Figure 3-7 Block diagram of apex-Qe series instrument with microBay console

Figure 3-8 Block diagram of apex-Qe series instrument with nanoBay console

3.4 System Control Unit
The main power, the bake-out of the UHV and the vacuum system are controlled by the
System Control Unit (SCU). The SCU monitors the speed of all turbo pumps, the
position of vacuum valves and the set-points on the vacuum gauge controllers for the
UHV and the Source, respectively. It also controls a hardware interlock to the system’s
electronics.
Pump and
Power Bake-out Valve
switches control Control
Figure 3-9 System Control Unit (SCU) and its 3 sections, power switches,
bake-out control and pumping and valve control
From left to right the SCU divides into three sections. The first section controls the
power to the whole vacuum cart (“Main”) and to the turbo pumps (“Turbo”). The
second section is the Bake Out control for the UHV system. See section (b) below for
a description of the complete bake-out procedure. The third section controls the UHV
and Source turbo pumps along with the corresponding rough pumps. This section also
allows for the control of roughing valves and the Beam Value, which separates the
source and UHV chambers. Indicator lights provide status on pump speeds, valve
positions and mode settings.

Figure 3-10 Power and Bake-out control sections of the System Control Unit (SCU)
3.4.1 System Power

Power for the entire vacuum cart is enabled with the MAIN switch shown in Figure
3-10. An additional switch, labelled TURBO, enables the power for the system’s turbo
pumps.
3.4.2 Bake-out
A timed bake out function is provided as part of the SCU to improve the ultimate
vacuum in the UHV chamber. For this procedure, the cart first must be rolled out of the
magnet and a bake-out jacket attached (see Figure 3-11) to the flight tube. To do so
first disconnect the RF leads from the power splitter box (see Figure 3-12). All other
cables (cell voltages, preamp power, signal cable CH1 & CH2) should be long enough
– nevertheless make sure these have enough slack to be pulled through the whole
length of the magnet. Pull out the vacuum cart until the flight tube is fully visible (see
Figure 3-13).
Also move out the LASER tubing of the IRMPD unit if mounted (see IRMPD manual for
details). The bake out timer (see Figure 3-10) can be set to run up to a maximum of ten
hours in 1 hour increments, after which it automatically shuts off. This allows the
system time to return to room temperature before it is rolled back into the magnet

(typically a bake-out is preformed overnight). The bake out time is set, and started, by
moving the toggle switch to the “START” position. The longer the switch is held in the
start position, the longer the bake-out will be (up to 10 hrs). The ON time is indicated
by the LED above the control switch, where one LED is equal to one hour of bake-out.
When the desired time is reached the switch is returned to the middle position and the
bake out begins. To either reset the time or turn the bake out off, the control switch
should be moved to the “RESET” position.
Figure 3-11 Bake-out jacket
Figure 3-12 Power Splitter. Disconnect both RF-power leads.

Make sure to stop any acquisitions first.

Figure 3-13 Instrument rolled out. Wrap bake-out jacket around the flight tube (cable
facing vacuum cart) securing it with the built-in Velcro strip. Connect the
bake-out jacket to mating connector on vacuum cart (see insert)

3.4.3 Pump and Valve Control

The SCU provides control of the system pumps, roughing valves and gate valves
(Figure 3-14). An overview of the system’s pumping configuration is shown below in
Figure 3-15. The vacuum system is divided into two parts, UHV and SOURCE that are
separated by a BEAM VALVE.
Figure 3-14 Vacuum control section of the System Control Unit (SCU)
To turn the vacuum system ON, the square black button in each pumping section is
pressed. The Green LED in the button indicates that the pumps have been turned on.
Once started, there is a 1 minute roughing delay before the Turbo pumps are activated.
Below these On/Off buttons are the Turbo pump speed indicators for the various
vacuum stages . An LED is lit for every 10% increase in rotor speed. So when the last
green LED is on, the Turbo pump is at 100%. There is an internal timer that will shut
down the pumps if 100% is not reached within 10 minutes of turning on the pumps.
When turning the turbo pumps OFF, the roughing pumps remain ON until the turbo
reach 50% (3 red bars and two orange) at which point the vent valve opens and the
pumps are turned off.

N2
QTIC N2 Flow
regulator
V5 Ar
Ar
R1 G5 Shutter V2
V6 Pulsed Valve 1x10 -3mbar
G4 G2
TP4 TP3 TP2 TP1
V1
V3
G1
V4 RP1
RP2
Turbo Pump
Rough Pump
Vacuum Gauge VS6 VS5 VS4 VS3 VS2 VS1
<10 -9 2x10-8 5x10-6 5x10-4 0.1mbar ~3mbar
Regulated Valve
Shutoff Valve Vacuum stages
Figure 3-15 Overview of the pumping system. The beam valve separates vacuum
stages 4 & 5.

When the turbo pumps are at full speed, the vacuum gauges need to be turned on
(Figure 3-16). If the pressure is below a programmed set point, the BEAM VALVE can
be opened. A SAFTEY switch is provided that when in position ON uses vacuum set
points to prevent valves from being opened when there is no vacuum. In special
cases, this logic can be bypassed by switching SAFETY mode to OFF.
Figure 3-16 Vacuum gauge controller
When the BEAM VALVE is OPEN, it provides a hardware interlock signal that allows
the cart electronics to be enabled. This is indicated by the HVI (High Voltage Interlock)
status LED becoming green (located directly below the beam valve switch). The key is
that the interlock is daisy-chained starting at the SCU. TOPPS then has to be enabled
when it receives the signal from the SCU to in turn provide an enable signal.
Additionally for QTIC to be enabled, the QFSCU has to be on and has to have received
a positive interlock signal from TOPPS. By connecting the interlock system like this, all
of the system electronics will be shut down when the BEAM VALVE is closed. This
means that all of the system electronics can remain powered on, but they will be
automatically disabled when, for example, the source is vented.
The SCU also provides control of both the SOURCE and UHV roughing valves (V1/V2
and V3/V4, respectively). The INLET valves are located on the UHV rough pump.
When ON, they close roughing to the turbo pumps and provide roughing for the
vacuum inlet system (see Figure 3-15). The status of the valves is indicated within the
STATUS section of the SCU. With the inlet switch OFF (roughing the turbo pumps),
the FLV (Fore line Valve) LED is green and the INV (Inlet valve) LED is red (Fig. 3-8).
If the INLET switch is changed to ON, first the FLV LED becomes red allowing the port
to the turbo pumps to close. Then the INV LED goes green, providing pumping for the
inlet system. The extra step of closing both valves ensures that the UHV turbo pumps
do not see a possible high gas load coming from the vacuum inlet system.
The Source INLET toggle (Fig. 3-8) provides switching between EI/MAL and ESI
modes. With either the Apollo II source or the Apollo II Dual ESI/MALDI source, the
operational position should be in ESI. This provides roughing to the first 4 vacuum
stages in Figure 3-15. The EI/MAL position is required for systems that do not have a
quadrupole interface.

Bruker Daltonics Inc. Theory of Operation
Chapter 4 Theory of Operation
4.1 FT-ICR Mass Spectrometer
4.1.1 Ion Motion

Ion cyclotron resonance mass spectrometry is fundamentally based on the tendency of
charged particles in a magnetic field to gyrate with a frequency that is proportional to
their charge and mass. The classical dynamics behind this process are illustrated in
Figure 4-1.
An ion that is originally traveling along a linear path with initial velocity V 0 (orange solid
line) interacts with the magnetic field (B o ), shown directed into the plane of the paper, to
produce a resultant circular trajectory. The inwardly directed force vector shown in the
diagram (green arrow) is the cross product of the magnetic field strength, number of
charges on the molecule, and velocity in the plane perpendicular to the magnetic field.
It is useful to mention here that there is no work function involved with velocity
components of charged particles traveling along (parallel to) magnetic field lines. The
magnet only provides radial containment.
Figure 4-1 Classical representation of force for ions trapped in a magnetic field

Theory of Operation Bruker Daltonics Inc.
The term cyclotron motion is used to represent this type of ion movement in the
magnetic field. From the relationship of the linear and angular force vectors described
above, one can derive a simple equation that elegantly expresses cyclotron motion in
terms of mass spectrometry:
qBo
c 
m
Since the angular rate of cyclotron motion, cyclotron frequency (ωc) is proportional
(inversely) to the mass-to-charge ration of an ion, we measure this property for mass
spectrometry. Notably absent from this relationship is velocity, meaning that the
measurement of m/z is ideally completely independent of the initial kinetic energy from
ion formation or transfer.
While an exhaustive discussion of more complex modes of ion motion and electrostatic
interaction is beyond the scope of this simple introduction, the reader is directed to
several useful reviews and texts that cover the subject in far greater detail. (Alan G.
Marshall and Francis R. Verdun, Fourier Transforms in NMR, Optical, and Mass
Spectrometry, Elsevier, New York, 1990.)
4.1.2 ICR Cell

In the apex-Ultra line of instruments, detection and measurement of ions occurs in the
ICR cell, located in the center of the superconducting magnetic field. Although there
are historically many types of geometric arrangements, most ICR cells share
commonality to the closed cylindrical geometry shown in Figure 4-2.
In this diagram, the magnetic field lines follow the red arrow along the central axis of
the right cylinder, so the cyclotron motion of the ions will be perpendicular to this axis,
or in the cylindrical plane.
In general, there are 3 sets of electrically isolated conductive cell plates that make up
the ICR cell; excitation plates, detection plates, and trapping plates.

Figure 4-2 Typical ICR cell geometry
4.1.3 Trapping Plates

As mentioned in the previous discussion, ions can move freely along magnetic field
lines. In the ICR cell shown in Figure 4-2, this predicts that while ions are contained
radially within the cylinder of the cell volume by the magnetic field, they could easily
escape along the axis. To provide axial containment of the ions, a small voltage (0.5-
1.5V) of the same polarity as the trapped ions is applied to the trapping plates to
provide a potential barrier to retain ions within the cell volume and establishing the
trapping condition. Ions trapped in this manner could be theoretically confined
indefinitely as long as the electric and magnetic fields remained intact.
4.1.4 Detection Plates

Ion cyclotron resonance does not use impact devices such as channeltron multipliers
or faraday cups for ion detection. Instead, the interaction of the electric field generated
by the ions gyrating between the orthogonally situated detection plates of Figure 4-2 is
used to detect ions in a non-destructive manner. The signals from these antennae are
differentially amplified and converted into a single RF voltage signal as shown in Figure
4-3 depicting a cross-sectional view of the ICR cell. This process results in a
sinusoidal response containing both frequency and magnitude information that
describes the rotating ions in the time domain. This response function is generally
referred to as a transient, with an amplitude that decays over time due to various
broadening mechanisms including collisions with background gas.

Figure 4-3 Cross sectional view of the ICR cell
4.1.5 Excitation Plates

In the ideal detection scheme illustrated in Figure 4-3, the single ion is assumed to
have enough cyclotron energy to be able to move out near the cell detection
electrodes. In reality, trapped ion populations consists of many m/z values with only
near thermal energies that result in cyclotron radii of ≤1 mm. For efficient detection,
the cyclotron radius should be much higher so that the electric fields of the ions are
more proximal to the detection plates.
The tendency of ions to randomly spread out among the cyclotron orbit is another
limitation to detection. In the worst case, two ions of the same m/z are 180 o out of
phase with each other, resulting in equivalent voltages being differentially amplified
resulting in a zero (net) signal observed at the output stage of the amplifier shown in
Figure 4-3.
To place ions in a larger cyclotron orbit and provide phase coherence for optimal
detection conditions, ions are treated with an externally amplified RF signal of a
frequency matching the cyclotron frequency presented in equation 1. As shown in
Figure 4-4, ions starting in the middle of the cell absorb energy from the amplifier on
the condition that the frequency resonates with the cyclotron frequency of the trapped
ions. After some time period, the externally applied signal is switched off. The final
radius achieved by the ions is a function of the excitation amplitude and radiation time.

Ideally, detection occurs just after excitation, although a short (~ 1-10 ms) time is
usually required for the excitation amplifier to become quiet enough to start recording a
transient signal. For most experiments, the excitation waveform is a frequency sweep,
or chirp, that scans through a range of frequencies that correspond to the m/z ratios of
the trapped ions. Although excitation is primarily used to prepare ions for detection, it
may also be used as a method of ejecting ions from the ICR cell by resonant excitation
to the radial cell boundaries. Since discreet frequencies or frequency ranges can be
selected in this manner, very high resolution isolations can be performed in the ICR
cell.
Figure 4-4 Excitation of ions in ICR cell

4.2 The FTMS Experiment
The FTMS experiment consists of a list of pulses or events that are carried out over the
duration of the experiment, ending in excitation and detection of the ions. For the basic
experiment, such as Q-CAD, the number of events is minimal.
Figure 4-5 graphically illustrates this sequence of events for the simple experiment. As
mentioned in the previous section, the FT-ICR experiment uses non-destructive
detection of the trapped ion population, so ions from the previous experiment are
usually present in the cell, requiring a purge or quench event before the cell can be
loaded with a new set of ions. This event will most likely initiate the experimental pulse
sequence, and is performed by simultaneously raising one trap plate to a high positive
voltage, while lowering the other trap plate to a large negative voltage. This asymmetry
destabilizes axial trapping to the point where the trapping condition is no longer met,
and ions leave the cell in a short period (< 1 ms) of time.
Figure 4-5 Sequence of events in FTMS experiment

The next event in the basic sequence is the ion injection, or ionization event. In the
contemporary arrangement for FT-ICR instrumentation, such as the apex-ULTRA,
places the ionization source outside of fringing magnetic field up to 1m away from the
ICR cell. The ion injection event transfers ions from the external source or trap to the
ICR cell and establishes the trapping condition for the ions. At this point, the ions can
be manipulated or dissociated with a more complex pulse sequence.
The excitation and detection events end the basic pulse sequence. The excitation
event usually lasts less than 5 ms and is followed by a short (1-5 ms) delay. During
detection, no RF fields are introduced into the cell as they might be picked up by the
extremely sensitive preamp and complicate (or obliterate) the mass spectrum. The
length of the detection event is determined by the user, and can last from several
milliseconds up to several minutes depending on the requirements of the experiment.
4.3 Signal Processing
As shown in Figure 4-3, the signal produced by ion movement in a circular path
between differentially amplified plates is a sinusoid with a periodicity related to the m/z
of the ion or ions trapped in the cell as given in equation 1. In the simplest case, this
waveform would look like a simple sine wave, and the frequency could be easily
determined by examining the distance between the peaks. Due to the naturally
occurring stabile isotopes common to most samples of chemical interest, the single m/z
case is not likely to occur.
Since many ions of discreet m/z are detected simultaneously (Fellgett advantage), the
waveform becomes more complex as sample heterogeneity increases. This
complexity is apparent in both the waveform periodicity and amplitude as frequencies
constructively or destructively interact (superposition). To convert this complex time
domain waveform into mass spectral data, the fast fourier transform (FFT) is applied to
the time domain transient to produce frequency indexes. From these frequencies, a
calibration function is used to transform the data into the m/z domain and a traditional
mass spectrum results. For a typical ESI experiment (400-1500 m/z) on a 7T system,
the frequency range would cover from 270 kHz for m/z 400 to about 70 kHz for the high
m/z end of the spectrum, illustrating the inverse nature of the frequency and mass
scales.

In Figure 4-6 below, a transient signal is displayed for two closely spaced small
molecules in the top frame. The bottom frame contains the mass spectrum resulting
from FFT processing and frequency to mass conversion.
FFT
Figure 4-6 Conversion of transient signal (Top) into a mass spectrum (Bottom)
One important experimental feature of transient collection is deciding what length of

transient is desired for a given experiment. The length of the transient in data points is
restricted to a multiple of 2n points to meet input requirements imposed by the FFT
algorithm. This means that experimentally adjusting the length of the transient must
incrementally double or halve the collection time. Since the relationship b etween time
and mass resolving power is linear, halving the transient time will halve the resolving
power. A simple calculation to predict the mass resolving power of a particular
transient for a given m/z value is shown in equation 2, where f is the observed
cyclotron frequency and t is the transient length in seconds. This relationship assumes
that the transient lasts the entire length of the acquisition period.
ft
RPFWHH 
2

a) t detect = .26 ms
RP FWHH = 64
b) tdetect = 260 ms
RPFWHH = 64,000
Figure 4-7 Resolving power for the same compound with 0.26 ms transient (a) and
with 260 ms transient (b)
Above in Figure 4-7a, a transient signal of .26 ms results in a mass resolving power of
64 after FFT processing. The same compounds acquired with a 260 ms transient as
shown in Figure 4-7b provided a mass resolving power of about 64,000, clearly
resolving the two closely spaced components in the mixture. One of the primary
strengths of FT-ICR is the ability to allow the user to acquire any desired resolution by
simply changing the parameters of the experiment.

The only ‘costs’ associated with achieving this higher resolution is a longer experiment
time and more space on the storage device associated with the acquisition computer.
Notice in Figure 4-7a that the sinusoidal nature of the signal is clearly visible.

Bruker Daltonics Inc. Getting Started Guide
Chapter 5 Getting Started Guide
Figure 5-1 apexControl splash screen
apexControl 3.0 is a program designed to configure and operate the apex-Qe series
mass spectrometer and to record and collect mass spectra. Permanently saved to the
hard disk, data can be reloaded at a later time for post processing with the Bruker
DataAnalysis software.
apexControl 3.0 is installed together with Microsoft® Windows XP ® (SP1 or higher) on

personal computers.
apexControl employs standard IBM-PC and Microsoft Windows conventions for using
windows, m enus, dialog boxes, and the mouse.

Getting Started Guide Bruker Daltonics Inc.
5.1 apexControl 3.0 Installation
The instrument will always come with the apexControl software already installed. In
the case of installing apexControl for the first time, re-installing the program or
upgrading to the latest version, detailed installation instructions are provided.
Important Note: For apexControl 3.0 to function properly, the instrument electronics
must have the correct version of Firmware installed. Detailed instructions on how to tell
if the Firmware is up-to-date, and if necessary, how to update the Firmware are given
on the Compass 1.4 Installation DVD.
Important Note: Before the installation of apexControl 3.0, any previous versions of
the software must be uninstalled. Detailed instructions on how to remove older versions
of apexControl can be found in the next section.
Once ready, the following steps outline the installation process:
Place the Compass 1.4 Installation DVD into the DVD drive (e.g., E) and the following
the Menu appears:
Figure 5-2 Compass 1.4 for apex main menu

Select “Install Software” and the following screen appears:
Figure 5-3 Compass 1.4 software installation menu
Detailed installation instructions are also provided on the DVD that outline the
installations of Hystar, DataAnalysis, apexControl and flexImaging. In the case of a
completely new installation, it is recommended to first install Hystar, then DataAnalysis
and finally apexControl. Additionally, for MALDI instruments the installation of
flexImaging is necessary to perform MALDI Imaging experiments and should be
installed last.
Once Hystar and DataAnalysis have been successfully installed, click on the “Install
apexControl” link to proceed with the installation of apexControl. The following screen
will then appear:

Figure 5-4 Removal of installation file
Choose Next
Figure 5-5 Initial apexControl version screen

Choose Next
Figure 5-6 Welcome screen
Choose Next

Figure 5-7 License agreement

To accept the terms of the license agreement choose Yes
Figure 5-8 Legacy data path

Change or accept the current recommended directory for storage of acquired data and
choose Next.

Figure 5-9 SHEDS option
If the instrument is equipped with a SHEDS controller (in most cases it will) select
SHEDS controller button, then choose Next

Figure 5-10 Installation destination path
Choose the destination location for installation of apexControl, then choose Next.
Figure 5-11 Installation Setup type
Select the Acquisition radio button in the type of Setup, then choose Next.

Figure 5-12 Installation status window
Figure 5-13 Completed installation window
Select Yes and click Finish to restart the computer.

Figure 5-14 Completing the installation process upon restart of computer
The installation process will complete when the computer is restarted.
Start apexControl by clicking on the apexControl Expert icon below:
Figure 5-15 The RunExpert icons launches apexControl in expert mode
If this is the first time apexControl 3.0 is launched, the FFTW configuration window will
appear.
Figure 5-16 FFTW Configuration setup
The configuration process optimizes the FFT algorithm for the quickest performance
depending upon the computer’s specific hardware configuration and the dataset sized
used. This process typically takes 1 hour to complete and the window will disappear
when the setup is done.

apexControl then needs to be configured for the correct FTMS Instrument hardware.
In the tools menu select Configure Instrument to access the FTMS Instrument
Configuration window.
Figure 5-17 FTMS Instrument Configuration dialog
First make sure that the system information is correct, and if not, update the necessary
fields (Console, Magnet and Model). Then select the correct hardware present on the
instrument. If unsure, contact a Bruker service representative for help. With the correct
information in place, click Update Changes. This will update all of the hardware
configuration files. If the Console Type was changed, it is necessary to run a full
system configuration. Before this is done, make sure that the console is on and running
and then click Run Full Configuration. This may take several seconds to complete. A
configuration window will appear indicating the results of the configuration process,
which should be reviewed before closing. Then “Close” the instrument configuration
window. If a full configuration was performed or if any FTMS Hardware settings were
changed, it is necessary to restart apexControl.

5.2 Uninstalling apexControl 3.0
The following steps will uninstall apexControl:
From the windows start menu, open the Control Panel and select Add Remove
Programs. From the program list, select “Bruker Compass apexControl 3.0.0”
Figure 5-18 Add/Remove program entry
Clicking on the Change/Remove button will open the following apexControl dialog:
Figure 5-19 Initial splash screen
Select Next

Figure 5-20 Modify, repair or remove menu
Select the Remove option and click Next
Figure 5-21 Confirm the uninstall of apexControl
Confirm the uninstall process by clicking OK

Figure 5-22 Removal status
The progress of uninstalling apexControl will be indicated in the status window. When
complete, the window will shut automatically.

5.3 Getting the first mass spectrum
5.3.1 Starting apexControl

apexControl has two operational user levels: standard and expert. The user level is set
at the start up of apexControl and can not be changed once the program is started.
Standard mode is for users who intend to run predefined Methods only. The user
interface has been simplified to ease the use of operation. Expert mode is intended for
users that will perform Method development and are given full access to all of the
instrument parameters and additional control functions.
To launch apexControl in Standard mode, click on the window start button and select
all programs. Go to the Bruker Daltonics folder and select apexControl.
A second way to launch apexControl is by double clicking the corresponding icon on

the Windows desktop.
Launches apexControl in Standard mode and
Launches apexControl in Expert mode.

Figure 5-23 apexControl Standard mode user interface
5.3.2 Closing apexControl

To exit apexControl, from the File Menu select Exit,
or press Alt + F4. Answer the confirmation request(s) that may appear.

5.3.3 Getting ESI Signal

In order to obtain initial ESI signal, it is recommended to use HP Tuning Mix (1:10
dilution in acetonitrile) or a 1uM solution of reserpine in MeCN/H 2O (50:50) 0.1%
Formic Acid as an ESI standard. Later on, other standards can be used that are
tailored towards a specific application. Once the standard is prepared, load it into the
100 uL or 250 uL syringe. Set the syringe pump flow to 120 uL/h, connect the syringe
to the injection port and turn on the flow on the pump. Fast forward the sample through
the tubing to remove any air in the system.
Syringe
Syringe
Coupling
Syringe
Pump
Figure 5-24 Syringe pump – to fast forward press right arrow and run/stop buttons
simultaneously
Make sure that the instrument BEAM VALVE is open; after that the instrument should
be ready for operation.
The easiest way to obtain ESI signal is to load a previously created ESI Method,
described in detail in the next section. A second option is to load a previously acquired
dataset file from the “data tree” window of apexControl. The dataset is loaded into
apexControl by double-clicking it

5.3.4 Loading apexControl Methods
Figure 5-25 Method Menu

To load the apexControl method select the “Open apexControl Part” in the Method
pull-down menu. The method contains ALL of the acquisition parameter settings
needed in order to get an ESI signal. A method only differs from a dataset in that it
contains NO data.
Figure 5-26 Load Method window in apexControl

Select an available method. For new systems, the user will find several Methods that
were created during the installation process. In general, ESI_Pos.m
A data file may also be opened that was created by a Bruker engineer in the Install
directory. Once opened in apexControl, the data file will load a set of instrumental
parameters associated with this file. To do so just click the file of interest in the tree
view menu of apexControl
Figure 5-27 Data tree window

The instrument parameters are automatically downloaded to the hardware once the
method is successfully loaded. Before attempting to acquire data, please make sure
that there is capillary current present. In the “Readback” tab please check if the
Capillary Current reads 15-20mA. The “Readback” tab is located in the lower right
corner of apexControl GUI and might be hidden if the “Peak List” tab is activated.
Figure 5-28 Readback window
If there is no current, please check if the “Nebulizer Gas Flow” and the “Drying Gas
Flow” are turned ON in the API Source Tab. Also check if the “ESI High Voltage” is
turned on (System Status Region). The “Drying Gas Heater” must be turned on as
well, and the “Dry Temp” set to ~200 oC. In case there is still no current please check if
the sprayer is clogged.
Figure 5-29 API Source Tab

The sprayer can be checked by forcing the liquid through the sprayer using the syringe
and checking whether it goes through.
WARNING: When checking the sprayer, use protective gloves and safety
glasses. Avoid contacting the fluid.
To do so open the spray chamber and put a dry clean cloth inside. Now carefully push
the syringe being connected to the sprayer forward (Fig. 5-2) to see whether liquid
emerges from the sprayer tip or not. In case the sprayer is clogged, see the section for
maintenance information.
Figure 5-30 The sprayer should emit a fine spray when liquid is run through it

5.3.5 Acquiring Spectra and Basic Tuning

In the tuning mode of operation, apexControl acquires and displays the real time data
but does not write the data to disk. This acquisition mode is initiated by pressing the
Tune/Operate button.
Figure 5-31 Operational run mode buttons

This mode allows the operator to optimize signal, resolution, and MS/MS fragmentation
efficiency in real time while observing the effects of parameter changes on the signal.
The Acquisition mode of operation is different from the tuning mode in that the signal
is averaged and saved into a designated file name and directory (see below).
The detection mode is also selected on the mode page. Broadband detection covers a
wide m/z rage while narrowband detects, with greater mass resolution, over a small
m/z range. For Broadband detection, the mass range of interest is edited by setting the
“Low Mass” and High “Mass” fields in the “Mode” tab.
Figure 5-32 Mode tab parameters

To set the number of scans to average for a typical infusion acquisition, set the
“Average Spectra” field accordingly (typical values are 10-20 scans). It should be noted
that it is not possible to change any of the instrumental parameters during acquisition.
In order to acquire a data file, it has to be given a name and placed in an appropriate
subdirectory. To do so edit the “Prefix” field and the “Subdirectory” field accordingly
within the “Sample Info” tab.
Figure 5-33 Sample Info tab, showing Data File Column
The “Sample Name” and “Sample Comment” fields are also available if there is a need
to record any additional information about the sample.
Figure 5-34 Sample Info tab, showing Sample Parameter Column

After the above steps have been completed, the system is ready to acquire a spectrum.
The acquisition of the spectrum is initiated by the operator in the apexControl by
pressing the Acquisition button.
Once the instrument averages the set number of scans the acquisition will stop and is
ready to start another acquisition. The acquisition may be stopped at any time during
the run by clicking the Stop button.
5.3.6 Mass Calibration

Once a spectrum has been acquired, the FT-ICR must be calibrated in order for the
m/z scale to be accurate. Calibration of the quadrupole should be done subsequently
and is described in detail within the calibration chapter. In apexControl, the calibration
window is accessed using the Calibration tab (see Figure 5-37).
Figure 5-35 Selection of ICR accurate mass list
Internal calibration of the acquired mass spectrum is done through comparison to

reference accurate mass lists. As set of mass lists are provided with apexControl and
are stored in the following directory:
C:\Bruker\apexControl\CurVer\resource\cal_ICR\

Figure 5-36 ICR reference mass lists
The Browse button will display the set of reference mass list (Figure 5-36). Select the
desired mass list for the acquired mass spectrum and click Open. The reference
masses will now be displayed within the calibration tab. Next select the calibration
mode: Cal2 (linear fit) or Cal3 (quadratic fit).
Figure 5-37 FT-ICR Calibration window

The process begins by selecting a peak in the current spectrum to be calibrated. This
is done by clicking in the “Current Mass” column in the row of the corresponding
“Reference Mass”. A useful tool at this point is the zoom feature. When enabled,
apexControl zooms in on the selected peak. This allows the user to verify that the
correct peak was picked, as indicated by the red line. For Cal2, a minimum of 3 peaks
are required, while Cal3 needs at least four peaks to perform the calibration. Certain
peaks may not initially line within the calibration and can be removed using the Clear
button at the bottom of the window. All of the selected peaks are removed with the
Clear All button. Once an acceptable set of calibration points has been selected, the
Accept button updates the FT-ICR calibration constants. To see how well the
calibration work, select the Automatic button. All of the peaks that fall within a given
error range will be selected. Again, using the zoom feature, select the various masses
to ensure that the correct peak is being picked for the calibration and accept. Repeat
this process until the entire mass range is calibrated.
5.3.7 Defining Data Directories

It is useful to have various data directories for separate users, experiments etc.
However, the data root directory used in apexControl was defined and fixed during the
installation process. Separate directories can be created using the Subdirectory field of
the Sample Info Tab, shown below.
Figure 5-38 Definition of data subdirectories on the Sample Info tab
The subdirectories are created by simply entering a name into the field. Once created,
a pull down menu (indicated with the downwards arrow) allows the user to choose from
the various Sub-data directories.

5.3.8 MALDI signal

Once an ESI Method is created, instruments equipped with an Apollo II Dual ESI
MALDI source can easily switch to MALDI mode. Typically a MALDI method is present
and the user can simply load it, setting all of the parameters. Manually, MALDI mode is
enabled via a checkbox within the System Status Region. When switching to MALDI
only mode, it is recommended to disable the ESI High Voltage, as shown below in
Figure 5-39.
Figure 5-39 Enabling MALDI mode in the System Status Region
When MALDI is enabled, the MALDI tune tab becomes active. When tuning for initial
MALDI signal, it is recommended to use the apex prepared standard target (P/N
253860). To load the target into the source, click on the Load Target button. This will
eject the target tray from the source. Once the target is in place, click Load Target
and the target will be loaded into the vacuum housing. If a target is already in the
source, the Load Target button will read Unload Target and clicking on this will eject
the target plate.
Typical values for the MALDI source are given in Figure 5-40. All other instrument
parameters should be left as set in the ESI method.

Figure 5-40 Typical parameter setting in the MALDI tune tab
For improved sensitivity, set the Capillary Exit value to zero. This is done within the
Source Transfer Optics tab.
Figure 5-41 Setting the Capillary Exit voltage to zero improves MALDI performance
With all of the instrument parameters set, make sure the correct target geometry file is
selected. Specific spots on the target are selected using the displayed target geometry.
Used spots are indicated by a filled red circle, and the Active spot is highlighted in
green. Guide lines are provided to ensure the correct position is selected.

Figure 5-42 Selection of a spot position on target plate
Once the spot have been selected, fine tuning the laser position is done on the camera
image of the spot. Additionally, the video image quality can be adjusted via the Set
Levels button.
Figure 5-43 Fine tuning the target position is done directly on the image display.

The laser spot can be manually positioned throughout an acquisition using this window.
However, it is possible to automate moving the spot position during the acquisition by
selecting the Random Walk option. The Random Walk option will randomly move the
laser position from scan-to-scan. The spot is divided into an 8x8 grid. The Scans Per
Grid parameter defines the limit for the number of movements within one grid section
before moving on to the next random position. The spot Diameter defines the range of
the random walk in µm. The default is defined by the geometry file, but the user can
reduce this value, to additionally constrain the movement.
Once a MALDI dataset has been acquired, the user should save a new MALDI Method.

Bruker Daltonics Inc. Calibration
Chapter 6 Calibration
6.1 FT-ICR
Mass spectrometers apply numerical calibration functions to convert measured raw

signals to m/z values based on a relationship between the measured property and
known calibrants. This raw value can be a variety of physical indices such as time,
applied voltage, magnetic field strength, or observed cyclotron frequency as in the case
of the FTMS analyzer.
For analyses where high mass accuracy is crucial, properly performed calibration of the
FTMS experiment as described in this chapter is an essential step. In general, the
accuracy stability of the FTMS is directly related to the magnet stability, better than
1ppm/day, meaning that for experiments performed in the same fashion can be
calibrated several hours apart without a significant impact on the maximum measured
mass accuracy. Additionally, any experiments that require a change in the ICR
analyzer cell conditions (trap voltages, excitation RF, etc.) from those used for the
calibration measurement necessitate that the calibration be repeated.
This chapter introduces the user to the methods used for mass calibration performed
using the apexControl acquisition software. It should be noted that since the
exceptional mass stability of the FTMS allows calibration to be performed before or
after acquisition, mass calibration can also be performed post-acquisition in the
DataAnalysis processing package. While the calibration is fundamentally identical in
both cases, DataAnalysis offers additional options such as lockmass correction that
can only be performed after the measurements are complete. For more information on
calibration in DataAnalysis, the user is directed to the online help section of DA.

Calibration Bruker Daltonics Inc.
6.1.1 Mass Calibration

Once a spectrum has been acquired, the FT -ICR must be calibrated in order for the
m/z scale to be accurate. Calibration of the quadrupole should be done subsequently
and is described in detail in Section 6.2. In apexControl, the calibration window is
accessed using the Calibration tab (see Figure 5-37). Internal calibration of the
acquired mass spectrum is done through comparison to reference accurate mass lists.
As set of mass lists are provided with apexControl and are stored in the following
directory:
C:\Bruker\apexControl\CurVer\resource\cal_ICR\
The Browse button will display the set of reference mass lists (Figure 6-1). Select the
correct mass list for the acquired mass spectrum and click Open. The reference
masses will now be displayed within the calibration tab. Next select the calibration
mode (Cal2 or Cal3).
Figure 6-1 Reference mass lists

Figure 6-2 FT-ICR Calibration window
The process begins by selecting a peak in the current spectrum to be calibrated. This
is done by clicking in the “Current Mass” column in the row of the corresponding
reference mass. A useful tool at this point is the zoom feature. When enabled,
apexControl zooms in on the selected peak. This allows the user to verify that the
correct peak was picked, as indicated by the red line. To apply a linear calibration,
Cal2, a minimum of 3 peaks must be selected. To use a quadratic calibration, cal3,
needs at least four peaks to perform the calibration. Certain peaks may not initially line
within the calibration and can be removed using the Clear button at the bottom of the
window. All of the selected peaks are removed with the Clear All button. Once an
acceptable set of calibration points has been selected, the Accept button updates the
FT-ICR calibration constants. To see how well the calibration works, select the
Automatic button. All of the peaks that fall within a given error range will be selected.
Again, using the zoom feature, select the various masses to ensure that the correct
peak is being picked for the calibration and accept. Repeat this process until the entire
mass range is calibrated.

6.2 Quadrupole
6.2.1 Principle of Quadrupole Calibration

The mapping of the exact rf amplitude and DC voltage to a desired mass and window
width is called the calibration. Ideally this can be written as two equations with four
parameters:
VRF = mass * k1 * M2RF - width * k4 * R2RF

U DC = VRF * k2 * RFDC - width * k3 * R2DC
k 1 to k 4 are defined by the geometry and resonant frequency of the quadrupole itself.
M2RF, R2RF, RFDC, and R2DC are correction values stored in the nonvolatile RAM of
the quadrupole generator. These reflect the individual differences of the quadrupoles
and the RF generators among one another. However, this is not an ideal situation. So
the linear equations above provide only a first-order functionality of the quadrupole
mass filter. To make the calibration more robust, more non-linear corrections have to
be made at several points of the calculation. This problem is solved using a multipoint
calibration with linear interpolations between the measured reference points.
6.2.1.1 The Raw Calibration Data

The easiest way to explain this process is to look at an example. Using a standard
calibration mixture, ions of three different m/z values with different window widths are
chosen for the calibration, e.g.:
m/z = 622 width = 10

m/z = 922 width = 5
m/z = 1522 width = 17
After scanning over the peaks, the following results were obtained:
required required measured measured

mass width center width
mass
622 10 618 15
922 5 910 8
1522 17 1505 23

W e know that the values in the column "measured center mass" is not really a mass
value but a scaling value mapped to the RF amplitude set at that point. So when this
table is sent to the instrument, the device knows which RF amplitude and DC voltage
corresponds to the values in the columns "measured center mass" and "measured
width" and adds these voltages to the table:
required required measured measured RF DC

mass width center width amplitude voltage
mass
622 10 618 15 1107 89
922 5 910 8 1640 133
1522 17 1505 23 2708 220
This is called a "raw calibration table" and is stored in the nonvolatile memory of the rf -
generator. The columns "required mass" and "required width" are used for sorting of
the table only.
6.2.1.2 The Raw Calibration Grid

There is a two-dimensional dependency for each rf amplitude and dc voltage to the
desired mass and window width. To get the voltage values for any combination of mass
and width different from those in the raw calibration table, a linear interpolation in each
of the two three-dimensional spaces must be preformed:
RF amplitude DC voltage
width width
mass mass
Figure 6-3: Linear interpolation of the rf and dc dependencies

The linear interpolation is done using plain areas that are defined by three points.
These three points have to be linearly independent, which means the points are not on
a straight line. Fortunately this is the case for the example above.
Unfortunately, this linear dependency covers only a small range of masses and widths.
In real systems, calibration data is needed at several points. When using such a
calibrated system, the device has to decide which three points should be used for the
interpolation for any desired mass / width combination:
width
w4
w3
wx
w2
w1
mass
m1 m2 mx m3 m4
Figure 6-4: An example of the Raw calibration grid

In another example, assume there are raw calibration data points at (m 1 , w2 ), (m1, w4 ),
(m 2, w 1), (m 3 , w3), (m 4 , w1 ), (m4 , w4 ). To determine the necessary RF amplitude and DC
voltage for a new point, (mx, w x), it is not trivial to decide the three raw calibration data
points that should be used for the interpolation. To make this decision easier a
complete calibration grid is generated from the raw calibration data that holds the
interpolation coefficients at each grid element. This "active calibration grid" is
generated at every boot of the instrument or upon special request.

6.2.1.3 The Active Calibration Grid

Linear interpolation in the three-dimensional space is done with a plain area. This area
is mathematically described by:
z = a·x + b·y + c
In our application z is the needed voltage, x is the mass, y is the width and a, b, c are
the interpolation coefficients. We need three points in the mass / width area to define
the coefficients. There are four different sets of coefficients for each point reflecting the
four quadrants around that point.
voltage width
w3
Q2 Q3
w2
Q0 Q1
w1
mass
m1 m2 m3
Figure 6-5: An example of the Active calibration grid
The numbering of the quadrants is not what is typically used in mathematics, but it is
simpler to get these numbers into program code.
6.2.2 System Calibration

Q1 must be calibrated in order for the Q1Mass and Q1Res settings to be accurate.
The calibration has to occur upon initial set up of a Qh-Interface, and then every one to
three months of operation depending on the reproducibility that is desired as well as
several other experimental conditions.

The general idea is to operate the instrument such that the ICR is used as a “detector”
for the quadrupole mass filter. The quadruple is operated as a mass spectrometer,
scanning across some mass range, and the intensity of a peak in the ICR spectrum is
used to recreate a peak shape within the quadrupole mass spectrum. The position of
the recreated peak is then used to calibrate the mass range of Q1 and the width of the
peak is used to calibrate the resolution scale. Due to the overall low repetition rate of
the FTMS instrument, it can take close to a minute to acquire a single quadrupole peak
shape. The entire process is automated by apexControl, so that once the instrument is
running it can be left by the user until the calibration procedure is finished.
As noted above, calibrations should not need to be performed more than once a
month. Please see the trouble-shooting section if it is found that the mass calibration is
drifting more than one m/z unit in less than one month. As long as the calibrations
have not drifted too far from acceptable values, a single run of the resolution calibration
and then the mass scale calibration should be adequate to fully recalibrate Q1.
The recommended sample to use for calibration is HP Mix (Bruker PN 552781

“Calibration Kit”), diluted 10:1 in Acetonitrile. HP Mix is a good choice because it
provides good peaks across a large mass range and is also relatively easy to obtain
intense signal from. Other samples such as peptide mixtures and polymers would also
be acceptable and the calibration procedure provides the functionality to use mass
values different from HP Mix. The only sample requirement is that it creates fairly
intense peaks across the mass range of interest. Peak intensity is important because
calibration has to occur at very narrow isolation widths resulting in loss of a large
fraction of the initial ion intensity.
6.2.2.1 User Interface

To access the calibration window from apexControl, click on the Tools menu and select
Quadrupole Calibration. This will produce the calibration window shown in Figure 6-6.
The window is divided into four sections. The upper left section is used to input the
calibration scan data, while the lower left displays the results of the scans. Also in this
section are the button used for manipulating the calibration data. The right portion of
the window displays the actual peak shape observed and used for the calibration. The
upper right window displays the active scan data, while the lower right window shows
all of the results for a given mass. The window is initialized with values in several
fields, but these are all editable by the user. Pre-defined scans (mass pairs and scan
parameters) can be chosen from the top pull-down menu. These are also editable by
the user.

Figure 6-6: Quadrupole calibration window
Various controls for the calibration procedure are described below:
Add Row Opens a window that allows the user to enter a calibration mass
along with the resolution value for that mass.
Delete Row Removes the above entry from the calibration window.
Open File Open previous scan data (both input and output data)
Scan Width Factor Allows increasing the range for the scan. The range is
determined by the current resolution times the scan width factor.
Scan Offset Offsets the scan range by the amount in the entry, e.g. “–1” will
shift the scan range to lower mass by 1 amu for both calibration
modi. This feature should only be used initially when the
calibration on the Q is off by more then ~1 amu.
NS The number of scans used in apexControl for each point in the
calibration

TD The dataset size used in apexControl

Update/Overwite Used to update (or refine) the active calibration table. It is also
used to overwrite the existing table, provided the DAC values are
present
Save Permanently Permanently save the calibration data to the QFSCU, the host
computer (as a file) and adds the data into the active
apexControl method
Read Back Reads back the active calibration table on the QFSCU
Clear Tables Clears the active calibration table on the QFSCU
6.2.2.2 Calibration Procedure

The General procedure for calibration of the Q is as follows:
1. Tune the instrument with the desired sample in isolation mode

2. Stop the acquisition and open the Quadrupole calibration window
3. Either select a pre-defined calibration run (i.e. Final Calibration) or enter the
desired mass/width pairs along with the scan parameters.
4. Start the calibration – the results of the calibration can be monitored in real-time
in the calibration window.
5. When the calibration is finished, confirm all of the peaks have a “good” shape
and the results seem reasonable.
6. Only results that have the Accept column check will be used in the calibration
7. Update the active calibration grid with “Update”
8. If the calibration is complete, proceed to the next step; if not, repeat steps 3-7
9. Save the calibration permanently.
10. Close the Quadrupole calibration window.
6.2.2.3 Initial System Calibration

If both the mass and resolution calibrations are far from acceptable (i.e. when a new
Qh-Interface is being tested) then the mass and resolution calibration usually has to be
performed. Following the general calibration procedure outlined above, step 1& 2
should be taken before performing the actual Q-calibration, which is described in detail
below.
Step 1: Clear Previous Calibration Data
NOTE: This step is only performed when the system calibration is far from acceptable.
The procedure to refine the current/active calibration table is given in Section 6.2.2.2.

The first step is to check if there is any calibration data present on the QFSCU. This is
done by clicking on the Read Back button. A window will pop-up displaying the
calibration data (if the window is blank then there is no calibration data present). The
results window can be closed. To remove this data, click Clear Tables. Double-check
this worked by again clicking the Read Back button. An empty results window should
appear. The results window can be closed.
Step 2: Input Scan Data
For the initial calibration, a set of default calibrations input files are provided. It is
recommended to start with Initial_Cal_1 and work sequentially to Final_Cal. To
account for the difference between instruments, all of the control fields are editable.
Custom scans can also be run. Use the Add Row button to enter in a specific m/z and
the corresponding widths to calibrate. Continue to add entries with the Add Calibrant
Row button. When done, click on the Done/Close button.
Figure 6-7: Predefined calibration menu
Figure 6-8: Manual mass and resolution input window

Step 3: Start Calibration
Once the all of the scan data has been entered, the START button becomes active and
the calibration run can be started. Only the rows that have the process box checked
will be run. During a calibration run, the START button changes to ABORT/PAUSE. A
left mouse click on this button will abort the scan while a right mouse click will pause
the scan (a right mouse click will resume the scan). This feature is useful for refilling
the syringe during a very long calibration.
Figure 6-9: Typical calibration input values

Step 4: Select and Update Calibration Data
By default, the “Accept” column is checked when the calibrated peak’s FWHM is within
10% of the requested resolution. The row can be added to the calibration list, if the
data looks like it falls in line with the general trend, by checking the accept box.
Once all of the data has been reviewed, the table is sent to the QFSCU using the
Update Button. This updates the calibration table in the QFSCU’s active memory only.
It does NOT save this data anywhere. Once the data has been updated, it is removed
from the calibration window.
Figure 6-10: Quadrupole calibration data window

Step 5: Save Calibration Data
To permanently save the calibration data that is in active memory on the QFSCU,
select Save Permanently. This will also bring up a prompt to save the file onto the host
computer. The file needs to have the extension .QDC in the filename. The QDC file
stores the mass and resolution pairs along with the calibrated DAC (Digital to Analog
Converter) voltage values in hardware. This file can be used to restore a certain
calibration, if something goes wrong in future scans. Additionally, the calibration data
is added into the active apexControl method.
Figure 6-11: Buttons for manipulating calibration data
Step 6: Repeat Steps 2 – 5
The above steps are preformed for a given input file, i.e. Input_Cal_1. It is common to
observe a very narrow mass range when first calibrating. A key feature of the
calibration procedure is that the calibration table can be refined at any time. This can
include adding higher m/z entries or including more resolution entries for a given mass.
By sequentially running the Input_Cal files, a very robust calibration is obtained after
performing Final_Cal. When running a custom calibration, repeat the above steps until
all of the calibration entries have the accept column checked.

Bruker Daltonics Inc. apexControl Reference Guide
Chapter 7 apexControl Reference Guide
7.1 Quick Reference Tables
The following topics provide quick reference to toolbars and shortcuts available in
apexControl 3.0:
7.1.1 Toolbars
apexControl 3.0 provides the following buttons in its toolbar. All tool buttons offered are
referenced below with the corresponding menu command and description. Shortcuts, if
available, are given in the following section.
Figure 7-1 apexControl 3.0 standard toolbar
Button Menu – Command Used to…
Tune Start Tune Mode
Acquisition Start Acquisition Mode
Stop Stop current acquisition
Display FID Show FID in display window
Display Spectrum Show Spectrum in display window
Arrange Windows Arrange window layout as shown

apexControl Reference Guide Bruker Daltonics Inc.
Reset Horizontal Axis Reset horizontal axis to full data scale
Reset Vertical Axis Reset vertical axis to full data scale
Reset Both Axes Rest both axes to full data scale
Zoom Horizontal Zoom in on horizontal axis
Zoom Vertical Zoom in on vertical axis
Zoom In Manual zoom in
Zoom Out Manual zoom out
Undo Zooming Undo last zoom command
Redo Zooming Redo last zoom command
Zoom Box Change curser to zoom box
Export View Export view to file system
Print View Print selected view
Copy Graph Copy current graph to clipboard
DA Open Data Analysis

7.1.2 Shortcuts
apexControl 3.0 provides the following shortcuts, which are also included in the menus.
Shortcut Menu – Command Used to …
Alt+X File – Exit Exit apexControl

Ctrl+T Acquire – Tune Start tune mode
Ctrl+A Acquire – Acquisition Start acquisition mode
Alt+S Acquire – Stop Stop current acquisition
Ctrl+Alt+S Acquire – Abort Immediate Abort current acquisition
Ctrl+F View – FID mode Change display to FID mode
Ctrl+M View – Spectrum mode Change display to spectrum mode
F1 Help – Help Contents Launch apexControl online help
7.2 apexControl Windows
The main GUI window is used to view data. Depending on the run mode, the main
window will display any one of 5 data types: Mass Spectrum, FID, Frequency
Spectrum, TIC and Optimize. Additionally, from the tool bar, the main window can be
partitioned from one maximized display in to four separate displays, each being able to
show a different mode.
7.2.1 Display Windows

Spectrum Mode

Spectrum mode displays the total mass spectrum, which has been calculated from the
acquired raw data (FID). This window usually displays the entire spectrum, but can
zoom in on regions/peaks of interest. Left-clicking will select a peak and highlight it in
the peak table. Left-clicking while simultaneously holding down the control (ctrl) key will
bring up the peak tuner. The peak tuner displays information on that peak (S/N,
resolution, etc), shows the calculated noise level as a dashed line and also remains
visible during tuning.
Spectrum mode window showing Peak tuner box. Peak information is available both
while in tune mode and in acquisition mode. Arrows are provided to indicate the peak
center and FWHM.

FID Mode
FID mode displays the acquired raw data (FID). While In tune mode, only a partial FID
is displayed.
Frequency Display
Frequency mode displays the total peak spectrum as a function of frequency, which
has been calculated from the acquired raw data (FID). This window usually displays the
entire spectrum, but can zoom in on regions/peaks of interest. Right-clicking in the
window brings up a context menu. Left-clicking will select a peak and highlight it in the
peak table. Left-clicking while simultaneously holding down the control (ctrl) key will

bring up the peak tuner. The peak tuner displays information on that peak (S/N,
resolution, etc), shows the calculated noise level as a dashed line and also remains
visible during tuning.
TIC Display
TIC mode displays in real time the Total Ion Current for each acquisition in a serial 2D
run as a function of time.
Optimize Display
The Optimize window displays the Extracted Ion Chromatograms (EIC) of the selected
peaks that are used in the parameter optimization process.

7.2.2 System Status Region

The System Status Region is located on the left portion of the GUI. Here the user is
able to adjust the acquisition state of the instrument. Additionally, various global
parameters are available. apexControl always reflects the current instrument status
and will report any errors or warnings to the user.
Operational States
The user has the choice of two main operation states: Tune and Acquisition. When the
Tune button is selected, the instrument is operated in online mode and data is not
recorded. The operational mode is stopped by clicking on the Stop button (it is not
possible to switch directly between operational states). When the user turns on
Acquisition mode the online data is averaged and recorded into a dataset.
Instrument Status
There are several Global instrument parameters that control and reflect the operational
status of the ion sources (Polarity, MALDI Enable, Source Quench Enable), ECD and
Chromatography Mode.

Events and Errors

Any instrument related errors are reported to the user in the bottom portion of the
instrument status region. If an error occurs, a warning window appears that describes
the errors. A log of the reported warnings and errors is viewed by clicking on the View
Event Table button.
7.2.3 Instrument Tune Tabs

The bottom half of apexControl contains the Instrument Tune Tabs. The region is
outlined using notebook style tabs. Each tab contains a page with a variety of
parameters used to adjust the mass spectrometer. Depending upon the operational
mode (Basic or Expert) the number of tabs and the contents of the pages will change
accordingly. Expert mode is used to define new Methods whereas Basic mode is used
to run predefined Methods. A more detailed description of the various pages for both
modes is given in the “apexControl Tune Tabs” sections (see 7.3).

7.2.4 Readback and Peak List

The lower right hand side of apexControl contains the Readback and Peak List Region.
This region contains a tab structure, allowing the user to select either the Readback
panel or the Peak list panel.
Peak List Panel Readback Panel

7.2.5 Data Tree

The upper left region of the GUI contains the Data Tree. This window displays the data
root directory, allowing the user to navigate through data in the various subdirectories.
Data files can be loaded into both apexControl and DataAnalysis or permanently
deleted.

7.2.6 Status Bar

The Status Bar is permanently displayed at the bottom of the GUI. It provides a real
time display for the current method, acquisition state, MS mode, NS, ECD current and
peak position. Additionally, when any parameter is changed, it is displayed in the left
most section.
7.3 apexControl Tune Tabs
The Tune Tabs in apexControl provide the user access to instrument and acquisition
parameters. Two operational modes are available and configured when apexControl is
launched. Standard mode gives very limited access to instrument parameters and is
intended to operate using pre-defined Methods. Expert mode gives access to a huge
number of instrument parameters. This mode is used to create new Methods.
7.3.1 Standard Pages
7.3.1.1 Mode
The Mode page is used to specify Acquisition and Accumulation parameters.
In Detection Mode Broadband

In Detection Mode Narrowband
Acquisition Mass Control

Detection Mode Switches between Broadband mode (typically several
thousand m/z range) and Narrowband (typically zoomed
around a center mass with an m/z range of 1-50
Acquisition Size Number of data points – defines the file size and the
length of the transient (i.e. detection time)
Low Mass Low mass end of Broadband acquisition
High Mass High mass end of Broadband acquisition
Center Mass Defines center mass for mass window in Narrowband
mode
Mass Window m/z width around the center mass in Narrowband mode
Max. Resolution Estimated resolution to be expected roughly based on
low mass and acquisition size
Transient length Length of transient = detection time of ions in analyzer
Accumulation
Average Spectra Number of scans accumulated for one spectrum
Source Accumulation Accumulation time of ions before ejecting them into the
collision cell
Ion Accumulation Time Accumulation time of ions from the source into the
collision cell
TOF Time in seconds for the ions to travel from the collision
cell to the Analyzer

7.3.1.2 API Source

The API Source page is used to adjust parameters for the API-interface (Atmospheric
Pressure Ionization). Depending on the source attached and selected on the System
Status Region, one or more parameters may be grayed (deactivated), as they are not
required to run this specific ion source.
API Source Control

Source Type Pull down allowing the selection of different API source
types – ESI, APCI, APPI, Nano-ESI_Offline and Nano-
ESI_Online
APCI High Voltage Enables the high voltage provided to the Corona Needle
Nebulizer Gas Flow Enables/Disables the sheath gas flow
Drying Gas flow Enables/Disables the counter drying gas flow
Drying Gas Heater Enables/Disables Drying gas heater – linked to Drying
Gas flow  when gas flow is off the heater is
automatically disabled
Nebulizer Gas Heater Enables/Disables Nebulizer gas heater
Spray Chamber
Capillary Absolute voltage on the front of the glass capillary (the
voltage is negative for positive ions and positive for
negative ions, respectively
Spray Shield Absolute voltage of the spray shield (the voltage is
negative for positive ions and positive for negative ions,
respectively
Corona Needle Set current in [nA] for the corona discharge in APCI
Neb Gas Flow Set value of Nebulizer gas flow in l/min
Dry Gas Flow Set value of Drying Gas flow in l/min
Dry Temp Set value of Drying gas temperature in [C]
APCI TEMP Set value of Nebulizer Gas temperature in [C]

7.3.1.3 MALDI
The MALDI page is used to adjust parameters for the MADLI source (Matrix Assisted
Laser Desorption Ionization). The MALDI tab is activated when the check box is
selected on the System Status Region.
Geometry File Lets the user select from a library of targets

Unload Target Unloads the target from the source
Load Target Loads the target into the source
Teach Target opens the MALDI Target Teaching Dialog
Clear Target Clears all spot markers (green and red) from the target
geometry GUI
Laser Shots Defined the number of laser shots per scan – effects the
accumulation time in MALDI  (Accumulation time = D28
x Laser Shots) with D28 delay in pulse sequence defining
the repetition rate of the laser
Laser Power Laser power in % - as it has been calibrated
MALDI Plate Voltage applied to the target plate
Enable Random Walk Enables random walk with change of target location
between scans
Spot Diameter defines the range for the random walk in μm – default is
defined in the geometry file
Scan Per Grid The random walk pattern is organized in a 8x8 grid over
the target spot, it defines the number of movements
constraint within one grid section before moving on to the
next random grid position

Laser Focus Only available if configured with SmartBeam component;

pull down to define laser focus as Standard (~100-300
μm) or as Imaging size (~30-100 μ m)
Video Control
Stop/Start Video Stops and starts the video feed in apexControl

Set Levels Allows setting of Brightness, Contrast, Hue and
Saturation
Lamp Power Sets the power level of the target illumination in [%]
7.3.1.4 MS/MS
The MS/MS page is used to adjust parameters for manual MS/MS operation. This
includes In-source Fragmentation, Quadrupole MS/MS and various types of Internal
MS/MS.
In-source Fragmentation
Skimmer 1 allows fragmentation of ions between Funnel 1 and 2 by raising

Skimmer 1 voltage for positive ions
Quadrupole MS/MS
Isolate Toggles on/off isolation of the defined Q1 mass using the
mass selective quadrupole
Isolation window defines the m/z isolation window width for isolation in the
mass selective quadrupole

Collision Voltage Induces collision activated dissociation in the collision cell

after passing through the mass selective quadrupole by
lowering the voltage (i.e. making it more negative for
positive ions)
Internal MS/MS
Isolation
Isolation Mode Pull down selection of Correlated Sweep, Correlated

Shots, Combination of Correlated Sweep and Shots and
Multi-CHEF excitation for ion isolation in the detector
(ICR-cell)
Parent Ion Sets the m/z value of the precursor ion to be isolated
Isolation Power Power level in [%] for the correlated sweep excitation
Low Mass low mass m/z value for the correlated sweep
High Mass high mass m/z value for the correlated sweep
Safety Belt defines width of notch around the precursor m/z not
covered by the correlated sweep excitation
Cleanup Shots File loads the text file – also called the m1 list – in
C:\Bruker\apexControl\goApex\exp\lists\m1 that has to
contain the m/z values to be eliminated
Pulse time excitation pulse length in [s] per m/z value in the Cleanup
Shots file
Cleanup Shot Power Power level in [%] of cleanup excitations

Multi-CHEF mass list
If Multi-CHEF is selected as the Isolation Mode a separate entry column to enter

masses to be isolated simultaneously becomes available. The entry allows entering 8
different m/z values to be isolated at the same time.
Correlated Sweep excitation

A correlated sweep is a frequency ‘chirp’. The individual frequencies making up the
‘chirp’ are “correlated” with the ion m/z (frequency) to be isolated. This correlation
minimizes off-resonance excitation of the ion. This is often an important consideration
when ion energy can be an influencing factor, such as in kinetics or fragmentation
threshold studies.
Correlated Shot excitation

Correlated shots are single frequency pulses used to eject individual ions within the
mass spectrum. T he actual ejection frequency is shifted slightly from the value
corresponding to the mass given in the m1-list. This shift is calculated such that the
broadcast frequency “correlates” (contributes minimal off-resonance excitation) with the
frequency corresponding to the parent ion m/z value. Because the frequency shift is
small, usually on the order of a few millihertz, a strong overlap with the m/z to be
ejected is maintained.

Correlated Combination excitation

This mode implements the uses of correlated shots immediately after correlated sweep
excitation to remove any remaining peaks that are not efficiently isolated.
PULSED VALVE
Check box to define whether pulsed valve #1 should be activated when necessary
during the experiment.
SORI-CAD
Parent Ion Precursor m/z value of interest (usually should match the
Parent Ion mass in the isolation setup
SORI Power Power level for SORI (Sustained Off Resonance
Irradiation) excitation of a precursor ion
Pulse Length Pulse length of SORI excitation of precursor ion
Frequency Offset Frequency offset in [Hz] of the excitation for
fragmentation from the mass of the parent ion.

IRMPD
IR Pulse Length Pulse length of IR laser in [s]

IRMPD Power Power level of IR laser in [%]
ECD
ECD Pulse Length Pulse length of electron beam irradiation of multiply

charged ions
ECD Bias Bias voltage on the surface of the electron cold cathode
emitter – defines electron energy
ECD Lens Lens element between cold cathode emitter and detector
ECD Heater Heater current of cold cathode emitter in [A]

7.3.1.5 AutoMS
The AutoMS page is used to define parameters for automatic MS/MS operations.
Precursor Selection
Include
Ions within the defined mass range(s) are included for MSMS analysis. This function is
complementary to the Exclude function. Only ions that fall within the included mass
range will be selected for MSMS. Ions defined within the Prefer list will be selected
whether or not they fall within the Include list. Mass ranges defined within the Exclude
list will override any overlapping mass ranges defined in the Include list, causing these
ions to not be selected for MSMS.
Exclude
Ions within the defined mass range(s) are excluded for MSMS analysis. This function is
complementary to the Include function. Mass ranges defined within the Exclude list
will override any overlapping mass ranges defined in the Include list, causing these
ions to not be selected for MSMS. Ions listed in the Prefer list will always be selected
for MSMS even if these ions fall within excluded mass ranges.
Prefer
Ions defined within this list are analyzed by MSMS first if present, regardless of these
ions fall within mass ranges defined in the Exclude list. If these ions are not observed
in the MS spectra, other ions will be analyzed.
Number of Precursor Ions

Determines how many precursor ions should be analyzed.

Selection Mode
Allows the user to select the range in which peaks are chosen for MSMS analysis:
Above Minimum, Below Maximum and within range.
Min. Threshold
Signal below this level is disregarded in peak picking
Max. Threshold
Signal above this level is disregarded in peak picking
Active Exclusion
Temporarily excludes the number of greatest peaks. The “Excluded after” parameter
sets the number of spectra after which the most intense peak(s) are excluded. The
“Release after” parameters sets the time period after which the excluded peaks are
released again for analysis.
Dynamic Exclusion
When enabled, all of the precursor ions in the previous AutoMS scan are excluded
from the current AutoMS scan. This feature is only available when running under
apexControl and not Hystar.
Source Quench Enable

When this checkbox is selected, the source quench event is included in the active
pulse program; conversely when un-checked, the source quench event is removed
from the pulse program.
Base Peak
When selected, only the most intense peak (within the set limits) is chosen for analysis.
This limits the number of available precursor ions to 1. The base peak option excludes
the Prefer Multiply Charged option.
Prefer Multiply Charged

When checked, sets the rules for peak picking to preferentially choose multiply charged
ions for analysis. This option excludes the Base Peak option.
Mass Accuracy
Set s the accuracy for the peak picking algorithm.

Fragmentation Types
CAD Set the method of MSMS fragmentation to CAD
ECD Set the method of MSMS fragmentation to ECD
IRMPD Set the method of MSMS fragmentation to IRMPD
CAD+ECD This mode of fragmentation performs a single MS scan and then

on the chosen peak performs a CAD MSMS and an ECD MSMS scan. The
accumulation time for the MSMS scan can be set to a longer time than the MS scan
using the “CAD+ECD Pulse Length” entry.
Energy Calculation
MS/MS Isolation
Enable MS/MS isolation
Fixed Collision Voltage

For CAD fragmentation, set the collision energy to a fixed value for all ions
Energy File Editor

Opens the file that contains slope and intercept values specific to each charge state
that are used to calculate the energy required to effectively dissociate a given ion.
AutoMS Modes
Boost
The mode sets separate accumulation times for MS and MSMS analysis. This allows
for a shorter MS scan and a longer MSMS accumulation scan. The time for the MSMS
scan is set with the “MS/MS Duration” entry field. Using the boost functionality forces
the acquisition into an alternating mode: each MS acquisition is used to select only one
precursor ion for subsequent MS/MS analysis before reverting back to an MS
acquisition.
FIA
Flow Injection Analysis (FIA), when enabled, sets the mass spectrometer to
continuously run in a data dependant mode. All of the peaks (within the set limits) in
the MS spectrum are analyzed via MSMS.

7.3.1.6 Sample Info

The Sample Info page is used to specify the name and the path where data files are
stored. Additionally the user may add sample information and specific comments that
are saved along with the spectra.
ESI
Prefix/Counter or Manual toggles between either automatically advancing

the counter (Prefix/Counter) or a manual mode
where the user has to define the Prefix for each
spectrum
Prefix Prefix of the filename
Counter Counter for the filename
Subdirectory Subdirectory within Data Root
Data Root Data Root directory
Sample Name Allows entry of the name of the sample
Sample Comment Allows entry of comments regarding the analysis
MALDI
In MALDI the filename is the combined Prefix - usually signifies the type of analysis
(could be the sample name)-, the Spot number and the counter. The counter separates
different analysis on one target spot.

7.3.1.7 Calibration
The Calibration page is used to recalibrate the ICR section (Infinity Cell) of the mass
spectrometer. A very detailed description on how to calibrate the ICR is given in the
Calibration Chapter (see Chapter 6).
Reference List Browse and select from a repository of reference mass lists for
calibration
Zooming defines the level of zooming in the spectrum display when
highlighting a row in the mass calibration list
Name Name (e.g. molecular formula) of reference mass entry
Reference Mass m/z value of reference mass
Current Mass m/z value of assigned mass peak in spectrum
Resulting Mass corrected mass in spectrum after applying calibration
Error(ppm) resulting mass error based on the reference mass and resulting
mass
Resolution reported mass resolution for an included mass peak
Clear clears a highlights entry from the current reference mass table

Clear All clears all entries from the current reference mass table displayed
in the tab
Add Allows the addition of a reference mass table in the current mass
table on the tab (note: this will not add a reference mass to the
original file)
Fix
Mode allows switching between cal2 and cal3 calibration modes applying
either linear (two constants) or quadratic (three constants)
correction
ML1, ML2, ML3 calibration constant that will be applied after accepting the
calibration results
Automatic automatically tries to find and match reference masses with the
current spectrum
Accept accepts the calibration fit and applies it to the method
Current
CalStatus Displays the calibration status of a data set displaying calibration
mode, calibration constants and reference masses used for the fit
7.3.2 Expert Pages
7.3.2.1 Mode
The Mode page is used to specify Acquisition and Accumulation parameters.
In Detection Mode Broadband

In Detection Mode Narrowband
Acquisition Mass Control

Detection Mode Switches between Broadband mode (typically several
thousand m/z range) and Narrowband (typically zoomed
around a center mass with an m/z range of 1-50
Acquisition Size Number of data points – defines the file size and the
length of the transient (i.e. detection time)
Low Mass Low mass end of Broadband acquisition
High Mass High mass end of Broadband acquisition
Center Mass Defines center mass for mass window in Narrowband
mode
Mass Window m/z width around the center mass in Narrowband mode
Max. Resolution Estimated resolution to be expected roughly based on
low mass and acquisition size
Transient length Length of transient = detection time of ions in analyzer
Accumulation
Average Spectra Number of scans accumulated for one spectrum
Source Accumulation Accumulation time of ions before ejecting them into the
collision cell
Ion Accumulation Time Accumulation time of ions from the source into the
collision cell
TOF Time in seconds for the ions to travel from the collision
cell to the Analyzer

7.3.2.2 MALDI
The MALDI page is used to adjust parameters for the MADLI source (Matrix Assisted
Laser Desorption Ionization). The MALDI tab is activated when the check box is
selected on the System Status Region.
Geometry File Lets the user select from a library of targets

Unload Target Unloads the target from the source
Load Target Loads the target into the source
Teach Target opens the MALDI Target Teaching Dialog
Clear Target Clears all spot markers (green and red) from the target
geometry GUI
Laser Shots Defined the number of laser shots per scan – effects the
accumulation time in MALDI  (Accumulation time = D28
x Laser Shots) with D28 delay in pulse sequence defining
the rep rate of the laser
Laser Power Laser power in % - as it has been calibrated
MALDI Plate Voltage applied to the target plate
Enable Random Walk Enables random walk with change of target location
between scans
Spot Diameter defines the range for the random walk in μm – default is
defined in the geometry file
Scan Per Grid The random walk pattern is organized in a 8x8 grid over
the target spot, it defines the number of movements
constraint within one grid section before moving on to the
next random grid position
Laser Focus Only available if configured with SmartBeam component;
pull down to define laser focus as Standard (~100-300
μm) or as Imaging size (~30-100 μ m)

Video Control
Stop/Start Video Stops and starts the video feed in apexControl

Set Levels Allows setting of Brightness, Contrast, Hue and
Saturation
Lamp Power Sets the power level of the target illumination in [%]

7.3.2.3 API Source

The API Source page is used to adjust parameters for the API-interface (Atmospheric
Pressure Ionization). Depending on the source attached and selected on the System
Status Region, one or more parameters may be grayed (deactivated), as they are not
required to run this specific ion source.
API Source Control

Source Type Pull down allowing the selection of different API source
types – ESI, APCI, APPI, Nano-ESI_Offline and Nano-
ESI_Online
Source type selector select after right mouse click on tune diagram
background

APCI High Voltage Enables the high voltage provided to the Corona Needle
Nebulizer Gas Flow Enables/Disables the sheath gas flow
Drying Gas flow Enables/Disables the counter drying gas flow
Drying Gas Heater Enables/Disables Drying gas heater – linked to Drying
Gas flow  when gas flow is off the heater is
automatically disabled
Nebulizer Gas Heater Enables/Disables Nebulizer gas heater
Spray Chamber
Capillary Absolute voltage on the front of the glass capillary ( the
voltage is negative for positive ions and positive for
negative ions, respectively
Spray Shield Absolute voltage of the spray shield ( the voltage is
negative for positive ions and positive for negative ions,
respectively
Corona Needle Set current in [nA] for the corona discharge in APCI
Neb Gas Flow Set value of Nebulizer gas flow in l/min
Dry Gas Flow Set value of Drying Gas flow in l/min
Dry Temp Set value of Drying gas temperature in [C]
APCI TEMP Set value of Nebulizer Gas temperature in [C]

7.3.2.4 Source Transfer Optics

Capillary Exit Voltage on the vacuum side of the atmospheric pressure

glass capillary
Deflector Plate Determines voltage deflecting ions from the end of the
glass capillary into the ion funnel (Note: in case of a dual
source the voltage is applied to the MALDI plate and the
actual deflector plate is resistively connected to the
MALDI plate)
Funnel 1 Voltage at the entrance of the RF ion funnel 1
Skimmer 1 Voltage at the exit of the RF ion funnel 1
Funnel 2 Voltage at the entrance of RF ion funnel 2
Skimmer 2 Voltage at the exit of RF ion funnel 2
Hex DC DC Offset voltage of the source RF hexapole
Trap Voltage on exit lens of source RF hexapole during source
accumulation time (D1)
Extract Voltage on exit lens of source RF hexapole during
extraction time of ions into the collision cell (D10)
Hexapole RF Amplitude Storage hexapole RF amplitude
Hexapole Frequency Storage hexapole RF frequency (either 5 (standard) or
optional 2.5 MHz (note: requires physical change of
separate RF driver box)
Source Accumulation Accumulation time (D1) in source RF hexapole

7.3.2.5 Qh

Qh Optics
Focus Lens Focus lens to guide the ions into the mass selective Quadrupole
Entrance Lens Entrance lens to the mass selective Quadrupole
Pre-Filter DC Bias RF only pre filter DC bias
DC Bias DC bias on all four rods of the mass selective Quadrupole
Post-Filter DC Bias RF only post filer DC bias
Q1 Mass sets the centre mass to be isolated
Isolation Window sets the mass width of the isolation window around the Q1 mass
Entrance Lens Trap Voltage of entrance lens to the collision cell during
injection of ions from the source into the collision cell
Entrance Lens Extract Voltage on entrance lens of the collision cell during
ejection of ions from the collision cell into the ICR cell
Collision Voltage DC bias on collision cell hexapole during injection and
storage of ions in the collision cell (the more negative the
voltage is set the higher the collisional energy – the
collisional energy is defined by the difference between
the source hexapole DC offset and the collision voltage)

DC Extract Bias Bias voltage of the collision cell hexapole during ion
extraction from the collision cell into the analyzer (ICR
cell)
Exit Lens Trap Voltage of exit lens of the collision cell during ion injection
from the source into the collision cell
Exit Lens Extract Voltage of exit lens of the collision cell during ion
extraction from the collision cell into the analyzer (ICR
cell)
Gas Control
Collision Gas Flow Collision gas flow in l/s
Collision Gas Flush Time Sets flush cycle time
Collision Gas Enable Enables the collision gas flow when checked
Collision Gas Flush Enable flush cycle (on, off, on, etc…) to atmosphere
Collision Gas Bypass Continually flushes gas line to atmosphere
7.3.2.6 ICR Transfer Optics

Accelerator 1 first element of accelerating einzel lens after the collision

cell
Accelerator 2 second element of accelerating einzel lens – split lens
Vertical Beam Steer 1 Differential voltage between the two segments of
accelerator 2 to allow vertical beam steering
Accelerator 3 third element of accelerating einzel lens – split lens

Horizontal Beam Steer 1 Differential voltage between the two segments of
accelerator 2 to allow horizontal beam steering
Vertical Beam Steer 2 2nd vertical beam steer in HV section

Horizontal Beam Steer 2 2nd horizontal beam steer in HV section
Focusing Lens 1 Decelerating focus lens part 1


7.3.2.7 Analyzer
Sidekick Differential voltage between the two halves of the split

entrance lens
Sidekick Offset split entrance lens to provide the SideKick – offset
voltage

Excitation Amp Excitation Attenuation in dB

Front Trap Plate front trapping plate to confine the ions in z-direction of the
magnetic field
Back Trap Plate back trapping plate to confine the ions in z-direction of the
magnetic field
Analyzer Entrance Entrance element that ions have to pass coming from the
transfer optics
7.3.2.8 MS/MS
The MS/MS page is used to adjust parameters for manual MS/MS operation. This
includes In-source Fragmentation, Quadrupole MS/MS and various types of Internal
MS/MS.
In-source Fragmentation
Skimmer 1 allows fragmentation of ions between Funnel 1 and 2 by raising

Skimmer 1 voltage for positive ions
Quadrupole MS/MS
Isolate Toggles on/off isolation of the defined Q1 mass using the mass
selective quadrupole
Isolation window defines the m/z isolation window width for isolation in the mass
selective quadrupole
Collision Voltage Induces collision activated dissociation in the collision cell after
passing through the mass selective quadrupole by lowering the
voltage (i.e. making it more negative for positive ions)

Internal MS/MS
Isolation
Isolation Mode Pull down selection of Correlated Sweep,

Correlated Shots, Combination of Correlated
Sweep and Shots and Multi-CHEF excitation for
ion isolation in the detector (ICR-cell)
Parent Ion Sets the m/z value of the precursor ion to be
isolated
Isolation Power Power level in [%] for the correlated sweep
excitation
Low Mass low mass m/z value for the correlated sweep
High Mass high mass m/z value for the correlated sweep
Safety Belt defines width of notch around the precursor m/z
not covered by the correlated sweep excitation
Cleanup Shots File loads the text file in
C:\Bruker\apexControl\goApex\exp\lists\m1 that
has to contain the m/z values to be eliminated
Pulse time excitation pulse length in [s] per m/z value in the
Cleanup Shots file
Cleanup Shot Power Power level in [%] of cleanup excitations

Multi-CHEF mass list

If MULTI-CHEF is selected as the Isolation Mode a separate entry column to enter
masses to be isolated simultaneously becomes available. The entry allows entering 8
different m/z values to be isolated at the same time.
Correlated Sweep excitation

A correlated sweep is a frequency ‘chirp’. The individual frequencies making up the
‘chirp’ are “correlated” with the ion m/z (frequency) to be isolated. This correlation
minimizes off-resonance excitation of the ion. This is often an important consideration
when ion energy can be an influencing factor, such as in kinetics or fragmentation
threshold studies.
Correlated Shot excitation

Correlated shots are single frequency pulses used to eject individual ions within the
mass spectrum. T he actual ejection frequency is shifted slightly from the value
corresponding to the mass given in the m1-list. This shift is calculated such that the
broadcast frequency “correlates” (contributes minimal off-resonance excitation) with the
frequency corresponding to the parent ion m/z value. Because the frequency shift is
small, usually on the order of a few millihertz, a strong overlap with the m/z to be
ejected is maintained.

Correlated Combination excitation

This mode implements the uses of correlated shots immediately after correlated sweep
excitation to remove any remaining peaks that are not efficiently isolated.
PULSED VALVE
Check box to define whether pulsed valve #1 should be activated when necessary
during the experiment.
SORI-CAD
Parent Ion Precursor m/z value of interest (usually should match the
Parent Ion mass in the isolation setup
SORI Power Power level for SORI (Sustained Off Resonance
Irradiation) excitation of a precursor ion
Pulse Length Pulse length of SORI excitation of precursor ion
Frequency Offset Frequency offset in [Hz] of the excitation for
fragmentation from the mass of the parent ion.

IRMPD
IR Pulse Length Pulse length of IR laser in [s]

IRMPD Power Power level of IR laser in [%]
ECD
ECD Pulse Length Pulse length of electron beam irradiation of multiply

charged ions
ECD Bias Bias voltage on the surface of the electron cold cathode
emitter – defines electron energy
ECD Lens Lens element between cold cathode emitter and detector
ECD Heater Heater current of cold cathode emitter in [A]

7.3.2.9 AutoMS
The AutoMS page is used to define parameters for automatic MS/MS operations.
Precursor Selection
Include
Ions within the defined mass range(s) are included for MSMS analysis. This function is
complementary to the Exclude function. Only ions that fall within the included mass
range will be selected for MSMS. Ions defined within the Prefer list will be selected
whether or not they fall within the Include list. Mass ranges defined within the Exclude
list will override any overlapping mass ranges defined in the Include list, causing these
ions to not be selected for MSMS.
Exclude
Ions within the defined mass range(s) are excluded for MSMS analysis. This function is
complementary to the Include function. Mass ranges defined within the Exclude list
will override any overlapping mass ranges defined in the Include list, causing these
ions to not be selected for MSMS. Ions listed in the Prefer list will always be selected
for MSMS even if these ions fall within excluded mass ranges.
Prefer
Ions defined within this list are analyzed by MSMS first if present, regardless of these
ions fall within mass ranges defined in the Exclude list. If these ions are not observed
in the MS spectra, other ions will be analyzed.
Number of Precursor Ions
Determines how many precursor ions should be analyzed.
Selection Mode

Allows the user to select the range in which peaks are chosen for MSMS analysis:
Above Minimum, Below Maximum and within range.
Min. Threshold
Signal below this level is disregarded in peak picking
Max. Threshold
Signal above this level is disregarded in peak picking
Active Exclusion
Temporarily excludes the number of greatest peaks. The “Excluded after” parameter
sets the number of spectra after which the most intense peak(s) are excluded. The
“Release after” parameters sets the time period after which the excluded peaks are
released again for analysis.
Dynamic Exclusion
When enabled, all of the precursor ions in the previous AutoMS scan are excluded
from the current AutoMS scan. This feature is only available when running under
apexControl and not Hystar.
Source Quench Enable
When this checkbox is selected, the source quench event is included in the active
pulse program; conversely when un-checked, the source quench event is removed
from the pulse program.
Base Peak
When selected, only the most intense peak (within the set limits) is chosen for analysis.
This limits the number of available precursor ions to 1. The base peak option excludes
the Prefer Multiply Charged option.
Prefer Multiply Charged
When checked, sets the rules for peak picking to preferentially choose multiply charged
ions for analysis. This option excludes the Base Peak option.
Mass Accuracy
Set s the accuracy for the peak picking algorithm.
Fragmentation Types
CAD
Set the method of MSMS fragmentation to CAD
ECD
Set the method of MSMS fragmentation to ECD
IRMPD
Set the method of MSMS fragmentation to IRMPD
CAD+ECD

This mode of fragmentation performs a single MS scan and then on the chosen peak
performs a CAD MSMS and an ECD MSMS scan. The accumulation time for the
MSMS scan can be set to a longer time than the MS scan using the “CAD+ECD Pulse
Length” entry.
Energy Calculation
MS/MS Isolation
Enable MS/MS isolation
Fixed Collision Voltage
For CAD f ragmentation, set the collision energy to a fixed value for all ions
Energy File Editor
Opens the file that contains slope and intercept values specific to each charge state
that are used to calculate the energy required to effectively dissociate a given ion.
AutoMS Modes
Boost
The mode sets separate accumulation times for MS and MSMS analysis. This allows
for a shorter MS scan and a longer MSMS accumulation scan. The time for the MSMS
scan is set with the “MS/MS Duration” entry field. Using the boost functionality forces
the acquisition into an alternating mode: each MS acquisition is used to select only one
precursor ion for subsequent MS/MS analysis before reverting back to an MS
acquisition.
FIA
Flow Injection Analysis (FIA), when enabled, sets the mass spectrometer to
continuously run in a data dependant mode. All of the peaks (within the set limits) in
the MS spectrum are analyzed via MSMS.
7.3.2.10 Sample Info

The Sample Info page is used to specify the name and the path where data files are
stored. Additionally the user may add sample information and specific comments that
are saved along with the spectra.

ESI
Prefix/Counter or Manual toggles between either automatically advancing

the counter (Prefix/Counter) or a manual mode
where the user has to define the Prefix for each
spectrum
Prefix Prefix of the filename
Counter Counter for the filename
Subdirectory Subdirectory within Data Root
Data Root Data Root directory
Sample Name Allows entry of the name of the sample
Sample Comment Allows entry of comments regarding the analysis
MALDI
In MALDI the filename is the combined Prefix - usually signifies the type of analysis
(could be the sample name)-, the Spot number and the counter. The counter separates
different analysis on one target spot.

7.3.2.11 Calibration
The Calibration page is used to recalibrate the ICR section (Infinity Cell) of the mass
spectrometer. A very detailed description on how to calibrate the ICR is given in the
Calibration Chapter (see Chapter 6.1).
Reference List Browse and select from a repository of reference mass lists for
calibration
Zooming defines the level of zooming in the spectrum display when
highlighting a row in the mass calibration list
Name Name (e.g. molecular formula) of reference mass entry
Reference Mass m/z value of reference mass
Current Mass m/z value of assigned mass peak in spectrum
Resulting Mass corrected mass in spectrum after applying calibration
Error(ppm) resulting mass error based on the reference mass and resulting
mass
Resolution reported mass resolution for an included mass peak
Clear clears a highlights entry from the current reference mass table
Clear All clears all entries from the current reference mass table displayed
in the tab
Add Allows the addition of a reference mass table in the current mass
table on the tab (note: this will not add a reference mass to the
original file)
Fix
Mode allows switching between cal2 and cal3 calibration modes applying
either linear (two constants) or quadratic (three constants)
correction
ML1, ML2, ML3 calibration constant that will be applied after accepting the
calibration results

Automatic automatically tries to find and match reference masses with the
current spectrum
Accept accepts the calibration fit and applies it to the method
Current
CalStatus Displays the calibration status of a data set displaying calibration
mode, calibration constants and reference masses used for the fit
7.3.2.12 Optimize
Allows the user to automatically increment one parameter at a time between a min and
max value after taking consecutive mass spectra. Pre-configured tables for the different
sections of the instrument are provided. The EIC (Extracted ion chromatogram) for
defined masses are plotted in one of the spectrum displays. It provides the option to
save the data into a serial file which can be analyzed further in Data Analysis (called
AutoRamp mode).
Columns of Optimize/AutoRamp input table
Parameter Name of method parameter

Common Name Name of parameter in the GUI with units in […]
Min. Minimum value of parameter at start of Optimize/AutoRamp run
Max. Maximum value of parameter at end of Optimize/AutoRamp run
Increment Step size for the Optimize/AutoRamp run between Min. and Max.
value
Optimized optimized value (parameter value of highest signal intensity) as
defined automatically after the run or later by selection on graph
by the user
m/z peaks entry to add m/z values to be monitored and plotted during a run

Ins Inserts an m/z value from the entry to the mass list
Del Deletes a highlighted m/z value in the mass list
Clr Clears the Optimize/AutoRamp m/z list
Save Data When checked the data is saved into a serial run like in
chromatography mode (AutoRamp)
Optimize Button starting the Optimize or AutoRamp run respectively
Add Add a parameter to optimize or AutoRamp in the parameter
name column
Remove Remove a highlighted parameter line from the current loaded
table (note: will not remove it from the predefined setup files
Accept Accept the optimized value
File Pull down to select pre-configured parameter tables for runs
Average Spectra Number of scans per spectrum and parameter value
accumulated
TD (FID Size) Data set size for the Optimize/AutoRamp run
Optimize Display
Left mouse clicking in the Optimize Display window displays a vertical bar for visual
selecting of the value to use. To actually submit this value requires a right mouse click
to bring up the context menu which has the 'Submit Parameter <param name value>'
enabled. Once this Submit Parameter popup menu action is selected, then the
displayed parameter value is accepted.

7.3.2.13 Tune Diagram
The tune diagram is an interactive user interface that allows the user to set up a
customized tuning interface using mouse button actions as described below. The
configuration is stored and will be restored after restart of apexControl.
Left mouse click on element pop up of entry widget of associated parameters,

entries widgets can be pinned else they close
when opening another entry widget
Right mouse click on element allows select of parameter and jumps to tuning tab
associated with the parameter and highlights the
entry field
Right click on background opens context menu to reset the size of the
diagram, select source type and configure read-
back features on the tuning diagram
Right click and drag moves tuning diagram on background of tab
Scroll with mouse wheel zooms in and out on the tune diagram
Hovering mouse over element Display of parameter and values

7.3.2.14 Parameter Control

CAUTION: only for very experienced user – errors could break a method:
Allows the change of all method parameters using their device commands (CORBA
names).
7.3.2.15 Console
Displays logging information – Note logging is also written to log files in directory
C:\Bruker\apexControl\Data\datahome\logs

7.4 apexControl Menus
apexControl provides the following menus in its menu bar:
7.4.1 File
The File menu offers the following commands:
Command Used To…

Exit Close apexControl
7.4.2 Method
The Method menu offers the following commands:
Command Used To…

Open apexControl Part Loads apexControl parameters from method
Save apexControl Part Save apexControl parameters to current method
Save apexControl Part As… Save apexControl parameters to new method
Save Entire Method As… Save all parameters to new method
Add DataAnalysis Part… Add DataAnalysis parameters to current method

7.4.3 Acquire
The Acquire menu offers the following commands:
Command Used To…

Tune/Operate Start tune mode
Acquisition/Single Spec. Start acquisition mode
Stop Stop run mode
Abort Immediate Abort run mode
7.4.4 Options
The Options menu offers the following commands:
Command Used To…

Rest Tab order Re-order Tabs to default settings
Console Pops ups console window commands
Pointing to Console offers the following commands
Command Used To…

In Tune Tab Activates console tab
Log Console window Logs console window output
Hide Console Removes Console tab from Tab windows

7.4.5 View
The View menu offers the following commands:
Command Used To…

FID Mode Displays FID in active window
Spectrum Mode Displays spectrum in active windows
7.4.6 Expert
The Edit menu offers the following commands:
Command Used To…

RunMode Pops up Run Mode commands
ControlCommands Pops up Control Commands
Parameter Controller Launch parameter controller window
Pointing to RunMode offers the following commands:
Command Used To…

Abort Immediate Aborts current run mode
Stop Stops current run mode
Time Calculation Calculates time of currently defined acquisition

Tune / Operate Starts tune / operate mode

Acquisition / Single Spec. Starts acquisition mode
Pointing to ControlCommand offers the following commands:
Command Used To…

FT Off Turns online FT off
FT On Turns online FT on
Online Display Off Turns off online display
Online Display On Turns on online display
Resume in TCU Sends resume command to TCU
7.4.7 Tools
The Tools menu offers the following commands:

Command Used To…

Open In DataAnalysis Loads last acquired data set into DA
User Processing Opens User Processing window
Quadrupole Calibration Opens Quadrupole calibration window
ECD Regeneration Opens ECD Regeneration window
Configure Instrument Opens Instrument configuration window
View: Compiled Acq… Displays compiled pulse program
View: Acquisition Event… Displays acquisition event properties files
Excitation Control Advanced control of excitation for detection, allows to set
the excite mass range separately as well as the direction
of the excitation sweep
Peak Picking Opens peak picking window
FID Advanced Opens FID advanced window
FFT Advanced Opens FFT advanced window
7.4.8 Compass
The Compass menu offers the following commands:
Command Used To…

Operator… Set current operator
Compass Desktop Launch Compass Desktop
DataAnalysis Launch DataAnalysis
Hystar Launch Hystar
7.4.9 Help
The Help menu offers the following commands:

Command Used To…

Help Contents Open apexControl online help
Report bug or Request Feature Opens Bug/Feature report window
About apexControl Displays apexControl 3.0 Information
7.5 apexControl Context Menus (Right Click Menus)
apexControl offers the following context menus, available by right clicking in the
following GUI regions.
7.5.1 Display Windows

Spectrum Mode Menus
Command Used To…

Spectrum Mode Changes window to Mass Spectrum Display
FID Mode Changes window to FID Display
Frequency Display Changes window to Frequency Display
TIC Display Changes window to TIC Display (2D runs only)
Optimize Display Changes window to Optimize Display
Select peak for MS/MS Sets selected peak to Q1 mass
Peak Resolution Determines peak resolution and S/N
Mass Differential Determines peak spacing in amu

Zoom Pops-up zoom commands

Properties Pops-up Properties commands
Copy-to-Clipboard Copy graph to clipboard
Print View Print select view
Pointing to Zoom offers the following commands:
Command Used To…

Zoom In Sets scaling arrow mode to zoom in
Zoom Out Sets scaling arrow mode to zoom out
Zoom X Scales x-axis according to above mode
Zoom X Scales y-axis according to above mode
Rest All Rests x- and y-axis to full display range
Rest X Rests x-axis to full display range
Rest Y Rests y-axis to full display range
Zoom Box/Stretch Sets manual axis zooming mode to either box or stretch
mode
Pointing to Properties offers the following commands:
Command Used To…

Show Grid Displays a grid within the display window

FID Mode Menus
Command Used To…


Frequency Display Menus
Command Used To…


TIC Display Menus
Command Used To…

Close TIC Display Changes TIC display to last display mode
Insert new segment Creates a new segment
Edit segment limits Edits selected segments limits (in times)
Remove selected segment Removes selected segment from TIC display
Delete all segments Delete all segments from TIC display
Optimize Display Menus
Command Used To…

Close Optimize Display Changes Optimize window to last display mode

7.5.2 Data Tree
Command Used To…

Load Analysis Loads selected analysis into apexControl
Open In DataAnalysis Opens selected analysis into DataAnalysis
Remove Analysis Deletes a single analysis
Remove Subdirectory Deletes entire subdirectory
Remove Analyses Deletes multiple analysis
Refresh Updates the data tree
Combine MS/MS2 Combines MS and MSMS analysis for DA
7.5.3 Peak List
Command Used To…

Peak Resolution Determines peak resolution and S/N
Mass Differential Determines peak spacing in amu
Add Frequency Column Adds/removes peak Frequency list column
Print Peak Table Prints peak table

7.5.4 Tune Diagram
Command Used To…

Reset Image Resets tune diagram images to default
Source Selector Selects the API source type (ESI, APCI…)
ParameterReadback Selector Opens parameter readback selection window
7.5.5 Quadrupole Calibration
Command Used To…

Add Row Adds a new mass/resolution calibration entry
Delete Row Removes a mass/resolution calibration entry
7.6 apexControl Tool Windows
The Tools Menu provides access to many advanced features in apexControl and is
only available for the EXPERT level user.
7.6.1 User Processing

The User Processing window is available from the Tools Menu. It allows the user to
reprocess acquired data. All reprocessed data must be saved to a new file name so it
can be loaded into a program like DataAnalysis. Either single spectra (also called 1d
or single fid spectra) or chromatography runs (also referred to as 2d or serial spectra)
can be reprocessed.

7.6.1.1 Buttons
The following buttons are available:
Button Used To…

Reprocess data Reprocesses the data set
Reset Resets all post processing calculations

7.6.1.2 Processing Options
The following processing options are available:
Advanced Parameters
See FID Advanced Section 7.6.9 below
Baseline Correction
Window Correction
FFTW
SI Dataset processing size (Real Transform Size)
Phase Correction
See FFT Advanced Section 7.6.10 below
Peak Pick
See Peak Picking Section 7.6.8 below
Serial (appears only if a 2D data set (e.g. chromatography run) was loaded)
Number of Experiments Displays the total number of experiments in a serial run
Total Time (minutes) Displays the total run time of the serial run
Experiment Number Allows the selection of an experiment number to be
processed
Time (minutes) Allows the selection of a time, the processing tool will find
the closest experiment number matching the time in
minutes
Save
SAVE 1D DATASET Saves the reprocessed 1d data set or reprocessed
experiment of a 2d (serial/chromatography) run into a
new 1d data set with a new name that has to be
specified.
SAVE 2D DATASET Saves the reprocessed 2d data set to a 2d data set with a
new name. Processing will be applied to all experiments
within the chromatography run.

7.6.1.3 Menus
File
The File menu offers the following commands:
Command Used To…

Open File Loads a data set into the User Processing Window
Save 1D Data Saves the reprocessed 1D data set to a new name
Save 2D Data Saves the reprocessed 2D data set to a new name
Exit Closes User Processing Window
Options
The Options menu offers the following commands:
Command Used To…

Reprocess data Reprocesses the data set
Reset Resets all post processing calculations
7.6.2 Quadrupole Calibration

The Quadrupole Calibration window is available from the Tools Menu. It allows the user
to calibrate the quadrupole so that the Q1 Mass and Isolation Window settings are
accurate. A very detailed description on how to calibrate the quadrupole is given in the
Calibration Chapter (see Chapter 6.2).

User Input File: Loads predefined input parameters for calibration

Start/Abort/Pause: Starts (also Aborts and Pause) the calibration run
Add Row: Opens a window that allows the user to enter a calibration mass along with
the resolution value for that mass
Delete Row : Removes the above entry from the calibration window
Open File: Open previous scan data (both input and output data)
Scan Width Factor: Allows increasing the range for the scan. The range is determined
by the current resolution times the scan width factor.
Scan Offset : Offsets the scan range by the amount in the entry, e.g. “–1” will shift the
scan range to lower mass by 1 amu. This feature should only be used initially when the
calibration on the Q is off by more then ~1 amu.
NS: The number of scans used for each point in the calibration
TD: The dataset size used for the calibration

Use Factory: Loads factory calibration data into memory

Update/Overwrite: Used to update (or refine) the active calibration table. It is also
used to overwrite the existing table, provided the DAC values are present.
Readback: Reads back the active calibration table on the QFSCU
Save Permanently: Adds Calibration data to the method and save a copy of the
calibration results along with the corresponding DAC values.
Load Previous: Loads the last user saved calibration table.
Clear Tables: Clears the active calibration table on the QFSCU
7.6.3 ECD Regeneration
7.6.3.1 NanoBay
The ECD Regeneration window is available from the Tools Menu. It allows the user to
condition (regenerate) the ECD cathode.
Selecting ECD Regeneration pops-up the follow ECD Regeneration window:

The first step is to set the maximum operating current for the ECD cathode. The
regeneration process will condition the cathode by slowly increasing the cathode
current.
Clicking the Start Regeneration button will initiate the regeneration procedure –
indicated by the Regeneration Status entry changing to “Regeneration Active. Set

Parameter entries will then by greyed out and the Start Regeneration button will
change to Stop Regeneration.
When the regeneration process is complete, the Regeneration Status will respond with
Cathode ON. The user can now exit the window by clicking on the Close button,
leaving the Cathode ON at the set regeneration current. The cathode can also be
turned OFF by clicking the Cathode Off button.

When the cathode is turned off, it takes 90 seconds to ramp the current down to 0. The
“Cooling Down” process is indicated in the Regeneration Status entry. During this time
the Close button is greyed out.

Once cool, the Regeneration Status turns to “Cathode OFF and the Close button
becomes active. The user can now exit the ECD Regeneration window.
Upon closing the window, the user is asked for confirmation.
7.6.3.2 AQS
For an AQS console, the regeneration process has to be preformed manually. For this
load a non-ECD method then:
1. Activate the ECD Heater On/Off switch
2. Check ECD in the Internal MS/MS section of the MS/MS

3. Slowly increase the ECD Heater current in 0.1A steps and at the same time
watch the pressure in the UHV; if the pressure starts to rise wait until it starts
dropping again before you increase it by the next 0.1A
4. When you reached 1.5A leave this setting and allow the cathode to outgas for
the next 12 hours (typically done overnight).
5. After this extended period of time the pressure in the UHV should drop back
into the lower 10-9 or upper 10-10 mbar range.
7.6.4 Configure Instrument

The FTMS Instrument Configuration window is available from the Tools Menu. It allows
the user to configure the FT mass spectrometer.

7.6.4.1 FTMS System Info

The FT mass spectrometer is configured using the following fields:
Console Type
Selects the instrument console type: AQR, AQS and NanoBay
Magnetic Field
Selects the Magnet’s Field Strength: 3, 4.7, 7.0, 9.4, 12.0 and 15.0 T
FTMS Model
Selects the model of the instrument
7.6.4.2 FTMS Hardware
SHEDS
Selects if the SHEDS device is present, and if so whether it is enabled or disabled.
Note that in the disabled state no communication with SHEDS is attempted by
apexControl and all instrument conditions controlled by SHEDS are effectively
disabled.
Primary Source
Selects the primary Ion Source
Secondary Source
Selects if a secondary ion source is present

Accessories
Selects any instrument accessories
Q Controller
Selects the type of Quadrupole control electronics present
7.6.4.3 Action buttons

Run Full Configuration
Performs a full configuration of the console acquisition hardware. Depending upon the
hardware the system has, this step may take 1-2 minutes. It is recommended that the
“Update Changes” is clicked (if active) before performing a full configuration.
View Console Config

Displays the current console configuration report in a pop-up window.
Update Changes
Updates the currently selection options to the “hardware.properties” file
Close
Closes the FTMS Instrument Configuration window.
7.6.5 View: Compiled Acquisition Event Sequence

The Acquisition Event Sequence window is available from the Tools Menu. It allows the
user to view the “compiled” version of BASIC (or any PULPROG in uses) for the active
acquisition. The “compiled” event sequence is the full pulse program that is used by
the acquisition hardware to control the experiment.
This file is generated (compiled) for every acquisition using the combination of:
1. the acquisition event sequence file defined by PULPROG (e.g. BASIC)
2. the individual event definitions themselves created from the “key-name” file
(e.g. EventKeyNameDefs_Bruker.properties) and hardware specific event
definition file (e.g., AQS_Bruker.properties).

7.6.6 View: Acquisition Event Property Files

The Acquisition Event Property Files window is available from the Tools Menu. It allows
the user to view specific acquisition event related files.

Configuration Specific Files

Default event definition files that are compatible with the currently configured
acquisition hardware
Bruker Default Files

All available event definition files provided by Bruker, to support the various types of
acquisition hardware
User Files
User editable property event definitions. These include master.properties and any
property files created with the “Save File” button.
7.6.6.1 Example Edit

Warning: Any edits to BASIC (Bruker Acquisition event Sequence for Instrument
Control) WILL NOT be supported within the GUI. The specific events and their
ordering in BASIC are dynamically linked to the user interface to provide the
user with maximum functionality and ease of use. The user assumes all risks
involved when proceeding. The following section is intended for highly advanced
users only and is not recommended for the majority of users.
One way BASIC can be customized is through the use of USER_EVENTs. There are 5
available USER_EVENT_KEYS defined within the acquisition event property files. The
following example shows how to properly include a USER_EVENT into BASIC for a
Nanobay console:

Part I – Creating user editable property files:
1. In apexControl, from the Tools Menu, open the Acquisition Event Property Files
window, select Configuration Specific Files, highlight Nano_Bruker.properties,
and click “Save File”. The “Save File” command automatically removes “Bruker”
from the filename and replaces it with the current user name (e.g.
Administrator).
2. Repeat the same steps to re-save the EventKeyNameDefs_Bruker.properties

file.
3. The files are saved into the following folder:

C:\Bruker\apexControl\CurVer\config\pulseprogram
Part II – Selecting user editable property files to be used by apexControl:
4. Within the pulseprogram folder, open the master.properties file with a text editor
(WordPad).
5. Modify the console specific event definition line. It is recommended that a copy
of the original “Bruker” entry is kept as a reference. The original entry should
be copied and then commented out with a # symbol. In this example, Nano is
set equal to the newly saved Nano_Administrator file:
#Nano = Nano_Bruker
Nano = Nano_Administrator
6. In the same manor, modify the Event Key line:
#EventKeyNameFile = EventKeyNameDefs_Bruker
EventKeyNameFile = EventKeyNameDefs_Administrator
*** Remember that the .properties extension is added automatically and should not be
added to the filename.
Part III – Adding user specific event definitions:
7. Open the Nano_Administrator.properties file with a text editor
8. Modify the USER_EVENT_1_KEY entry. For this example it is shown how to

include a “wait for trigger” input:

# USER_EVENT_1_KEY:
USER_EVENT_1.lines = 2
USER_EVENT_1.1 = " trigne4 ; wait for trigger (trig3)"
USER_EVENT_1.2 = " d14 ; delay"
Part IV – Updating the BASIC event sequence:
9. In the C:\Bruker\apexControl\goApex\exp\lists\pp.ApexControl directory, copy

the file BASIC and rename the copied file to something else: BASIC_TRIG
10. Open BASIC_TRIG with a text editor
11. Add USER_EVENT_1 into the desired position. Please note that
“USER_EVENT_1” and not “USER_EVENT_1_KEY” is used:
;--------------------------------------
;IRMPD Block
;--------------------------------------
USER_EVENT_1
IRMPD
12. In apexControl, from the Param Control tab, type in PULPROG and enter in the
new name, BASIC_TRIG
13. Run an Acquisition and save a new method.
7.6.7 Excitation Control

The Excitation Control window is available from the Tools Menu. It allows the user to
set the excitation mass range separately as well as the direction of the excitation
sweep.
Frequency Sweep Direction Sets direction of frequency sweep

Excitation Low Mass Sets low mass for excitation sweep
Excitation High Mass Sets high mass for excitation sweep

7.6.8 Peak Picking

The Peak Picking window is available from the Tools Menu. It allows the user to
change the method used for peak picking.
Peak Picking Method

Apex: uses a 3 point interpolation at maximum
Maximum: simply takes the maximum point
Maximum Number of Peaks

The total number of picked peaks (GUI limit 10,000)
Maximum Intensity Intensity which above no peaks will be picked
Minimum Intensity Minimum required intensity for a peak to be picked
Peak Sensitivity
Gives the minimum necessary intensity for a peak – the lower the sensitivity the more
peaks will be found
7.6.9 FID Advanced

The FID Advanced window is available from the Tools Menu. It allows the user to
change how the raw data (FID) is processed.

FID
TD: Time domain data set size
TDeff: Number of points of the FID used for processing, if 0 process with TD
TDoff: Start of the display offset
Baseline Correction
None: No correction to FID
Single: Adds a constant to the current FID in order to eliminate any dc-offset present
in the signal. The constant is determined by calculating the average value of the last
quarter of the FID. In the case of two-channel data ('quad'), the correction is carried
out for each channel separately. Baseline correction is automatically performed before
the execution of exponential multiplication, Gaussian multiplication, or Fourier
Transform; providing that Baseline Correction is set to Single. The correct acquisition
mode ('quad') is automatically used, depending on the type of acquisition data.
Windowing
Exponential: multiplies the data point i by the factor:

i1 
LB

  
 2SW  

e
The processing parameter Line Broadening (LB in the above equation, in Hertz)
determines the resultant line broadening. Line Broadening is normally greater than
zero and improves the sensitivity, but with the disadvantage of line broadening. In the
above equation, SW, is the sweep width of the spectrum (in Hertz). Exponential
multiplication is automatically preceded by Baseline Correction
Gaussian: multiplies the data by the function:

e a
t b
t2 
where 'a' is negative and 'b' is positive. The FID originally has the exponential envelope
of the form:
t 
 
e T 2 
therefore a Gaussian line shape results in:

 
e b
t2
when a 
1
T2
Since this line shape has less extensive wings than a normal line, the resolution of
overlapping resonances is improved greatly. A suitable choice of parameters can lead
to further reduction in the half-line width. 'a' and 'b' have the form
a
a LB and b
2 GB AQ 
where Line Broadening (LB) and Gaussian Broadening (GB) are processing
parameters and the acquisition parameter (AQ) is the detection time of an FID (without
including ion accumulation times). The function has a maximum value of
 AQ 

LB 
e  2 
when T max GB 
AQ
Tmax and, therefore, GB may be determined (once the data has been weighted by a
suitable LB exponential and transformed), by taking the reciprocal of the half line width
of the line required to be 'sharpened'. To execute Gaussian multiplication, Line
Broadening should be entered as a negative value. Valid values for Gaussian
Broadening lie between zero and one. If GB=0.33 the Gaussian function has a
maximum after 1/3 of the detection time. Gaussian multiplication is automatically
preceded by Baseline Correction, unless Baseline Correction is set to none..
Sine-Bell: multiplies the data by a sine function of the form:
 t   

sin 

PHI 

 
PHI ,0<t<AQ, where PHI 

 AQ   SSB
The processing parameter Sine Bell Shift Factor (SSB) may take on the values 0, 1,
2, 3... A pure sine wave is obtained when SSB=0 or 1; a pure cosine wave when
SSB=2. PHI approaches zero for greater values of SSB.
Square Sine Bell Multiplication: multiplies the data by the square of this function.

7.6.10 FFT Advanced

The FFT Advanced window is available from the Tools Menu. It allows the user to
change how the time domain data (FID) is transformed into spectral data.
FFTW
SI: the spectra display data set size
Phase Correction
Phase Correction:
The data, consisting of real and complex points (R(i), I(i)), is phase corrected using the
formulae:

Ri R i
 cos aiI 
isin 
 ai

i I 
I icos aiR i
sin 
ai

where R' and I' represent the corrected values and
a
iPHC0 
i 1
PHC1
, where i>0
PHCO and PHC1 are the zero and first-order phase parameters, respectively, and may
be set by the user (in degrees) as processing parameters. Phase correction is mainly
used to phase a number of similar spectra with the same phase parameters.
Phase correction always operates on the processed data if available (e.g. after
Exponential Multiplication or Fourier Transform), otherwise the acquisition data (FID)
are used. In order to force phase correction to use the acquisition data even though
processed data exist, the processed data must be deleted.

Magnitude Calculation: replaces the real part of the spectrum by its absolute value
according to the formula:
i  R(i) 2 
I (i) 2
where R is the real part and I is the imaginary part of the spectrum. Magnitude
calculation can also operate on the FID like phase correction.
Power Calculation: The real part of the spectrum is replaced by the square of the
absolute value:
2
i
Power calculation can also operate on the FID like phase correction.
7.7 apexControl Dialogs
apexControl has several dialog windows that provide the user step-by-step instructions
for completing certain tasks.
7.7.1 Hexapole RF Generator Switch

For high mass MALDI (or ESI) operation, it may be beneficial to change from the
standard 5 MHz source hexapole rf generator to an optional 2.5 MHz generator.
Lowering the rf operating frequency will increase the transmission of higher m/z ions
(and conversely hinder the transmission of lower m/z ions). The user has to physically
change the rf generator on the instrument. apexControl provides a dialog to guide the
user through the process.
In the Source Transfer Optics page change the Hexapole Frequency from 5 MHz to 2.5
MHz. This will cause the dialog window to open.

The window will display the current generator status and resonant frequency. Once the
Generator Status is OFF, the rf generator can be physically changed. At this point, the
Next button is clicked.
Once the generator status changes to ON, the re-resonate button becomes active and
should be selected.

When the re-resonating process begins the status will reflect this and the frequency will
be set 0.
After the process has run to completion, the Finish button will become active and is
used to close the dialog window.

7.7.2 MALDI Target Teaching

The procedure for teaching the MALDI target assures a proper correspondence
between the position of the target movements and the camera image in apexControl.
The target teaching dialog is available from the MALDI Tune tab. First make sure the
correct target geometry is chosen from the Geometry File pull-down menu. Then click
on the Teach Target button.
The Teach Positions dialog window will pop up as shown below. Note that at the same
time the target will already roughly move to the first of three teaching positions (A1 in
this example).
Fine tune the position with the spot control in apexControl until the target spot is in the
center of the video image. Then click on Reached. This will initiate the software to
move the target to the next teaching spot (A24 in this example). At the same time you
can see the relative motor control values for the first spot in the “Relative Coordinates:”
column.

Again fine tune the position and acknowledge with the Reached button.
After the third teaching position is reached repeat the steps and again acknowledge.

Now the Save, Save As and Save As Default buttons become available. Use anyone
of them to save and update the default teaching file. The Save and Save As buttons
will open the following save window:
In general the default.teach file is always used; however and “.teach” file can be saved.
Then make sure you exit the Teach Positions dialog window with the OK button to use
the teaching with the current target.
NOTE:
If the range of movement does not allow you to get the target spot all the way into the
center of the video display, try to get it as close as possible, save the teaching as the
default, close the window with OK and repeat the teaching – now you will start with the
full range of movement around the already closer position of the target spots.

Acquisition Workflows Bruker Daltonics Inc.
Chapter 8 Acquisition Workflows

Detailed information on the various workflows in apexControl can be found in this
chapter:
8.1 Fragmentation Methods
The apex ULTRA FTMS offers a variety of ion dissociation methods for MS/MS
experiments. In the following section we introduce the basic principle of operation of:
 In source Collision Activated Dissociation (IS-CAD)
 Collision Cell Activated Dissociation (C-CAD)
 Sustained Off-Resonance Irradiation Collision Activated Dissociation (SORI-CAD)
 On-resonance CAD in the ICR cell
 Infrared Multi-Photon Dissociation (IRMPD)
 Electron Capture Dissociation (ECD)
SORI-CAD
IRMPD
ECD IS-CAD
C-CAD
Collision gas
for SORI-CAD
UHV
Collision gas
for C -CAD
ECD
Heate r
Cathode
IR laser
beam
(IRMPD
optional)
Analyzer
(Infinity Cell) Collision
Ion Transfer optics Source Dual stage Ion funnel
cell Mass selective Hexapole
Quadrupole
Figure 8-1 Overview of dissociation methods on the apex ULTRA FTMS

Bruker Daltonics Inc. Acquisition Workflows
Figure 8-1 shows the respective location of these different fragmentation methods and
this chapter will introduce the methods in logical order from the source side to the
analyzer.
8.1.1 In-source Collision Activated Dissociation (IS-CAD)

The Apollo II ESI only and Dual sources are designed to gently transmit ions from the
source region into the mass spectrometer. Changing the way ions are accelerated
through the source optics can increase the number of collisions with background gas
molecules; resulting in fragmentation of the ions. In-source fragmentation of volatile
analyte ions (e.g. Angiotensin I) is generally achieved by raising the Skimmer 1
potential. To fragment a more strongly bound species (e.g. Reserpine), ions have to be
accelerated through both funnels as shown in the voltage profile of Figure 8-4 as dotted
red line by raising Skimmer 1, Funnel 2 and Skimmer 2 voltage.
Figure 8-2 Typical In-source Fragmentation tuning (e.g. Angiotensin) on the MS/MS
tab

8.1.2 Collision Cell Activated Dissociation (C-CAD)

The design of the apex ULTRA allows for single parameter MS/MS operation. This is
enabled simply by adjusting the collision cell pole bias voltage during trapping in the
Collision Cell (the Collision Voltage parameter shown in Figure 8-3). The resulting
difference between ion funnel 2 and the collision cell pole bias voltages is termed the
collis ion energy. The collision energy will cause the ions to undergo energetic
collisions with the gas as they are accelerated into the cell. If enough collisions occur
and if they are energetic enough, then it is possible that the ion will gain enough energy
to dissociate and form products. The product ions are then cooled and trapped by
further collisions with the gas. Again, after trapping the pole bias voltage is pulsed to
the extraction value so that the ions can be efficiently transferred into the Analyzer cell.
Figure 8-4 shows a voltage profile for the source ion optics when ions are accelerated
into the collision cell and dissociated.
Figure 8-3 MS/MS tuning tab

Post-Filter DC Bias
Trap/Extract
Focus Lens Funnel 1
Entrance Lens Skimmer 1
Trap/Extract Entrance Lens Funnel 2
Exit Lens
Trap/Extract Pre-Filter DC Bias Skimmer 2
H
ea
ter
DC Bias Hex DC
Collision Voltage /
DC Extract Bias
Deflector
Plate
300
250
200
150
IS-CAD
100
50
-50
C-CAD
-100
-150
Figure 8-4 Lens voltage profile for IS- and C-CAD operation

8.1.3 Sustained Off-Resonance Irradiation (SORI)
The SORI-CAD experiment employs a low energy, off-resonance rf pulse during a high
pressure burst of an inert gas (e.g. Argon) inside the ICR cell. The effect is that
energetic collisions occur between the precursor ions and the collision gas. If enough
collisions occur, the ions will gain enough energy to dissociate and form products.
Step 1: Optimizing the collision gas
Before executing the MSMS experiment the appropriate pressure in the collision gas
reservoir has to be set. Open the cover of the UHV inlet system (see Instrument
Overview Chapter). For a system with one pulsed valve you will find a set of two
valves, the roughing vacuum valve (green) and the sample inlet valve (red) (see Figure
8-5).
Roughing
vacuum
valve
Sample
Inlet valve
Gas inlet for ¼” tubing

or glass vials
Figure 8-5 UHV gas inlet system for the pulsed valve – valves shown in closed
position (to open turn anti-clock wise)
The next step is to rough out the reservoir (inside the cart not shown) and the gas lines
inside the vacuum cart from the sample inlet valve to the pulsed valve. To activate the
rough pump switch the INLET switch on the System Control Unit (SCU) to the ON
position (see Figure 8-6).

Figure 8-6 Switching the UHV roughing pump to INLET ON position
Now open the green roughing valve on the inlet system and pump on the inlet system
until you reach a pressure in the ~1x10 -2 mbar range (see Instrument Overview Chapter
– set the aux selector to Pulsed Valve 1 and observe 3rd display on top of the
instrument).
Connect flushed gas line of Argon (ultra high purity) to the gas inlet port (see Figure
8-5) with slightly higher pressure than atmosphere, close the green rough valve and
open the red sample inlet valve to fill the reservoir. If the inlet system has not been in
operation for a while then repeat the roughing and filling procedure several times.
After the actual fill of the reservoir, set a pressure of 6-8mbar by operating the green
rough valve (at this point the red sample inlet valve has to be closed). After the right
pressure in the reservoir is established switch the INLET back to the OFF position on
the SCU.
Now activate the pulsed valve by selecting the Isolation, SORI and the Pulsed Valve
On/Off check boxes on the MS/MS tab in apexControl. Set SORI power to 0. To test
the appropriate amount being pulsed into the cell, run a tune experiment and observe
the UHV pressure. It should rise shortly to ~1x10 -8 mbar within each scan. You will also
notice that the experiment (scan rate) becomes slower. This is due to the 3s pump
down delay, which is automatically added when activating the pulsed valve.
Step 2: Isolation of parent ion mass in the ICR cell
Before the SORI-CAD experiment is performed the isolation of the parent ion needs to
be set up. To isolate an ion you want to fragment make sure the Isolation check box on
the MS/MS tab in apexControl is still checked (see Step 1). This will expand several
entries you need to set. The isolation is a correlated sweep excitation over a broad
mass range with a notch at the mass to be isolated. Enter the parent ion mass

accurately (not just nominal mass but with several digits after the decimal point).
Specify the low and high mass value of the correlated sweep. Here you want to cover
the whole range of unwanted ions to be eliminated. Then enter a Safety Belt around
the parent ion mass. This will allow you to expand the m/z notch so that it contains the
complete isotopic envelope of your parent ion. This is in particular important if you
fragment multiply charged ions and you want to retain charge state information in the
product ions.
While tuning, carefully raise the Excitation Power [%] within the column of entries for
Isolation starting with 0. When you approach the right excitation power you should see
unwanted ions disappear while the parent ion remains. If too much power is used, even
the wanted parent ion will disappear. Typical parameters are shown in Figure 8-7.
Step 3: Fragmentation in the ICR cell using SORI-CAD
When the SORI-CAD box within the MS/MS tab of apexControl is checked during Step
1 the SORI-CAD parameters are activated to the right of the Isolation parameters. The
parent ion mass will be of course the same as in the isolation step. Carefully start
increasing the SORI Power from value 0 in fractions of percent until you start to see
fragments.
SORI experiments are longer and use lower amplitudes than on-resonance CAD (see
next chapter). Typical values for power (amplitude), pulse length and frequency offset
for SORI are given in Figure 8-7.
Figure 8-7 MS/MS tuning tab showing typical Isolation and SORI-CAD parameters

Figure 8-8 Schematic of the SORI process

8.1.4 On-Resonance Collision Aided Dissociation

To perform on-resonance CAD simply use SORI-CAD on the Internal MS/MS tab and
set the Frequency Offset to “0” (zero). On-resonance CAD experiments are shorter and
use higher amplitudes than SORI. Typical values for power (amplitude), pulse length
and frequency offset for on-resonance CAD are given in Figure 8-9.
Figure 8-9 MS/MS tuning tab showing typical Isolation and On-Resonance CAD
parameters

8.1.5 Infrared Multi-photon Dissociation (IRMPD)
8.1.5.1 Introduction and Principals

Infrared multi-photon dissociation (IRMPD) is the technique of fragmenting parent ions
by irradiating them with an infrared (IR) laser inside the ICR cell. Typically a CO 2 laser
at 10.6 m is used. For peptides the ions generated are b and y sequence fragment
ions.
Figure 8-10 IRMPD fragmentation spectrum of a peptide
8.1.5.2 Operating the IRMPD Hardware

To run the IRMPD experiment, first turn on the IR Laser by pressing the red on/off
button shown below.

Figure 8-11 IRMPD Equipment
Then press the laser on/off button on the laser power controller. This step is not
required for the apex ULTRA instruments, as it is controlled directly via apexControl.
Figure 8-12 Laser power controller
At this point the laser is ready for IRMPD operation.
8.1.5.3 Controlling IRMPD in apexControl

In order to begin the IRMPD experiment make sure you enable the IRMPD checkbox
within the MS/MS tab (Figure 8-13). IRMPD can not be turned on or off during tuning.

In order for the change to take place tuning mode has to be stopped and resumed after
the IRMPD radio button is checked.
Figure 8-13 Software interface for IRMPD Operation
The only tunable parameters for IRMPD operation are the IR Pulse Length and the
laser power. Typically the pulse length is set to 0.01-0.5s (see Figure 8-13) and the
laser power is 30-70%. In apexControl 3.0, the laser power can be set directly from the
MS/MS tab. In earlier version of the software, the laser power is adjusted via the knob
on the Controller (Figure 8-12).
The idea behind tuning is to maximize the overlap between the ions in the ICR cell and
the IR beam. Since the laser fires into the center of the ICR cell Sidekick should be
minimized for successful IRMPD operation; which is typically set around +-2.0 volts
(Figure 8-14).
Figure 8-14 Analyzer Parameters

8.1.6 Electron Capture Dissociation (ECD)
8.1.6.1 Introduction and Principals

In the process of ECD (Electron Capture Dissociation), the ESI-produced multiply
protonated peptides and proteins, [M+nH] n+, capture a low-energy (<0.2eV) electron to
produce the odd-electron ion [M+nH](n-1)+•, which dissociates via an energetic H•
transfer to the backbone carbonyl group to form c and z sequence ions. ECD is a mild
fragmentation technique and is often used for analysis of post-translational
modifications; it is also preferentially used for top-down analysis of proteins, no larger
then 35 kDa. The principal of operation is shown in Figure 8-15 below. The electrons
produced by the hollow cathode are pulsed into the ICR cell, which causes
fragmentation of the ions which are already trapped in the ICR cell.
Figure 8-15 Electron injection system with an ICR cell

8.1.6.2 Conditioning and Operating the ECD cathode
Warning
Never operate the dispenser cathode without high vacuum (> 10 -8 mbar) in the
analyzer part of the instrument. Damage to the cathode may result.
Warning
The heating of the cathode to the final temperature should be done slowly
because of possible damage of the heater and the dispenser cathode. Please
follow the instructions carefully.
When using the ECD cathode, there are two possible states the cathode could be in:
conditioned and unconditioned. The cathode is considered to be unconditioned if this
is the first time the cathode has been turned on after a vent of the UHV system. In this
case, the ECD cathode has to be carefully regenerated. This should be ideally set up at
the end of a day as the full regeneration takes up to 12h. A conditioned cathode is
one that has been successfully regenerated. Even with a conditioned cathode, care
should be taken when bringing the cathode up to its operating current.
The regeneration process is different depending on the type of console: AQS or

Nanobay. For an AQS console, the regeneration process has to be performed
manually, whereas this process has been automated for Nanobay consoles. The
following sections outline both processes:
8.1.6.2.1 AQS - Cathode regeneration

For this load a non-ECD method then:

1. Activate the ECD Heater On/Off switch
2. Activate ECD in the Internal MS/MS section of the MS/MS tab
3. Slowly increase the ECD Heater current in 0.1A steps and at the same time
watch the pressure in the UHV; if the pressure starts to rise wait until it starts
dropping again before you increase it by the next 0.1A
4. When you reached 1.5A leave this setting and allow the cathode to outgas for
the next 12 hours (typically done overnight).
5. After this extended period of time the pressure in the UHV should drop back
into the lower 10-9 or upper 10-10 mbar range.
8.1.6.2.2 Nanobay - Cathode regeneration

The ECD regeneration window is available from the Tools Menu in apexControl.
Selecting ECD Regeneration pops-up the follow ECD Regeneration window:

The first step is to set the maximum operating current for the ECD cathode. The
regeneration process will condition the cathode by slowly increasing the cathode
current.
Clicking the Start Regeneration button will initiate the regeneration procedure –
indicated by the Regeneration Status changing to Active. The set parameters will then
by greyed out and the Start Regeneration button will change to Stop Regeneration.

When the regeneration process is complete, the Status will automatically respond with
Cathode ON and Stop Regeneration will change to Cathode Off. The user can now
exit the window by clicking on the Close button, leaving the Cathode ON at the set
regeneration current. The cathode can also be turned OFF by clicking the Cathode Off
button.

When the cathode is turned off, it takes 90 seconds to ramp the current down to 0. The
“Cooling Down” process is indicated in the Regeneration Status window. During this
time the Close button is greyed out.
Once the cathode is cooled down, the Status turns to OFF and the Close button
becomes active. The user can now exit the ECD Regeneration window.
Upon closing the window, the user is asked for confirmation.
8.1.6.2.3 Cathode Operation
If the cathode has already been regenerated the user should nevertheless carefully
prepare the instrument for an ECD experiment, especially if the ECD cathode has not
been in operation for some time (e.g. several days).

Operating the cathode with a nanobay system only requires setting the ecd heater to
the desired value (but below 1.8A) in the Internal MS/MS section of the MS/MS tab.
For an AQS system there is no automatic ramping of the heater current as is in

nanobay systems. Therefore, after checking the ECD box in the Internal MS/MS
section of the MS/MS tab slowly increase the ECD Heater current. The time table
(Table 8-1) for slowly heating the cathode to the typical final value of the filament
current of 1.7 A without damaging the dispenser cathode and the cathode heater is
shown below. Be careful to increase the current of the dispenser cathode heater
slowly! While heating the cathode always monitor the pressure in the analyzer region, it
should not exceed 10 -8 mbar!
Table 8-1 Time table for heating the ECD Cathode (Note: these values are
recommended for warm-up after a proper cathode regeneration, see
8.1.6.2.1)
Time ECD Heater
parameter
0 min 0.5 A
2 min 1.0 A
3 min 1.3 A
4 min 1.5 A
5 min 1.7 A
7 min 1.5 A
This operation takes about 7 minutes. The short increase of the heating current to a
higher value than the setting during operation is needed for surface activation of the
dispenser cathode. Also after finishing the heating process, the activation of the
surface can take several more minutes.
During this operation the acquisition of mass spectra for accurate mass measurements
should not be carried out. This is due to the hot dispenser cathode resulting in an
increase of the pressure in the analyzer cell, which will effect signal intensity, signal
shape and mass accuracy of the measurement.

8.1.6.3 ECD Measurement
Before starting with ECD measurements, the dispenser cathode has to be heated
stepwise to the right temperature for electron emission as describe in the previous
section. The ECD heater is turned on and off with the Global checkbox in the System
Status Region.
Note: Whenever the Global switch is On and a method is loaded with ECD checked in
the Internal MS/MS section of the MS/MS tab, the cathode current will be changed to
the value of the method. However if the Global switch is turned Off, loading an ECD
method will not turn on the cathode. In addition when apexControl is started, the
software will check the last instrument state – if the cathode was turned on in the
previous session of apexControl the Global switch will be set accordingly to the
checked status.
The ECD measurement is performed in the same manner as other MS/MS

measurements with and without peak isolation, however based on the electron capture
process for dissociation only multiply charged ions can be fragmented.

Select the ECD checkbox in the Internal MS/MS column to active the pulsed ECD
event within the acquisition – the Global switch does not active the ECD event within
apexControl. For all systems, the ECD event can not be turned on/off during tuning. In
order for the change to take place, tuning has to be stopped and resumed after the
ECD on/off buttons are checked.
The standard values for ECD operation are shown in Table 8-2. The typical values for
the ECD Pulse Length of the electron beam is can vary from 0.2s to 0.001s with ECD
bias (ionization energy of electrons) of ~1.0-3.0V. However, the ECD Bias value for
ECD operation correlates with the rear trapping potential in the ICR cell (Analyzer tab).
Therefore, with high rear trapping potentials higher ECD Bias values are needed for the
ECD measurements. ECD lens parameter allows focusing of the electron beam and
has a standard setting of ~15.0V. Sidekick value (Analyzer tab) should be in the range
of +-6V.
The ECD Heater parameter should be to the standard value of 1.6-1.8A as done
previously during the procedure of stepwise heating the dispenser cathode to the final
temperature for operation.
In the following table the typical parameter setting for ECD measurements with the
hollow dispenser cathode for the fragmentation of positive multiple charged ions are
summarized:
Table 8-2 Typical ECD parameters
Parameter Value
ECD Bias 0.5 – 1.5 V
ECD Pulse Length about 0.2s -0.001s
ECD Lens about 10.0-15.0 V
ECD Heater 1.6-1.8 A
Sidekick high value (e.g. +-6 V)
The correct setup of the parameters of the dispenser cathode for ECD can easily be
done with the peptide substance P. The most intensive ECD fragment ion of substance
P is the c5 + fragment with m/z 624 (Figure 8-16). The values of ECD Pulse Length and
ECD Bias should be adjusted for the highest intensity of this c 5+ fragment. However,
too long of an electron beam length and a too high cathode potentials lead only to a
decrease of the parent ion intensity and to the same intensity of the c 5 + fragment ion.
The general rule for tuning the ECD for other molecules is the larger the molecule the
shorter the ECD Pulse Length and the Higher the ECD Bias values.

[M+2H]2+
c 5+ +
c7
+ c 6+
c4
c10 +
c 9+
c 8+
c 2+ [M+H]+
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z
Figure 8-16 ECD spectrum of substance P
8.1.6.4 Calibration of ECD Spectra
The calibration of an ECD spectrum can be done externally using the ECD spectrum of
substance P. The calibration file “SubstanceP_ECD.ref” for calibration of the ECD
spectrum of substance P can be found in the calibration file directory. In the following
table the exact masses for this calibration are listed:
Table 8-3 Calibration masses
ECD Fragment Exact Mass [m/z]

c1 + 174.13494
+
c2 271.18770
+
c4 496.33543
c5 + 624.39400
c6 + 752.45258

c7 + 899.52099
c8 + 1046.58941
c9 + 1103.61087
+
c10 1216.69494
[M+H]+ 1347.73542
An example of a typical calibration result for the calibration of substance P is shown in

the following table using nearly all c-fragments of this peptide.
Table 8-4 Typical results for the calibration of substance P
Ref. Mass [m/z] Exp. Mass [m/z] Difference [ppm]
271.187700 271.187704 0.0149
496.335430 496.335474 0.0883
624.394000 624.394008 0.0134
752.452580 752.452384 0.2610
899.521000 899.520407 0.6588
1046.589410 1046.589103 0.2934
1103.610870 1103.610883 0.0115
1216.694940 1216.697276 1.9203
1347.735420 1347.734542 0.6513

8.2 AutoMSMS
The apex-Qe and ULTRA series instruments have been specifically designed for data
dependent MS/MS analysis. The idea behind a data dependent experiment is for the
data contained in an MS scan to be used to decide which peaks should be investigated
via MS/MS analysis in the subsequent scan(s). This is particularly useful when
interfacing the instrument to a liquid chromatography system and/or when the user
does not have prior knowledge of the composition of the analyte of interest, i.e. a
protein digest.
Data dependent operation within apexControl is turned on and off using the Auto
MS/MS checkbox within the AutoMS Tab. If Auto MS/MS is checked then apexControl
will control the quadrupole (Qh-Interface) in a data dependent fashion as described
below. Data dependent acquisitions are run as 2-D datasets in serial mode. Common
settings for data dependent runs are given in Figure 8-17. Typically Hystar, not
apexControl, is used to control data dependent acquisitions, as most experiments are
LC-MS/MS. In these cases, apexControl is initially used to create a method that Hystar
accesses during the LC run.
Figure 8-17: Typical data dependent conditions set on the AutoMS tab
8.2.1 Overview of Decision Structure

A block diagram of the complex decision structure that is used to decide which peak in
the MS spectrum is selected for MS/MS analysis is shown in Figure 8-18 (single
precursor ion selection example).

Choose Species for MS/MS
Peak Pick
Calculate Energy
to Use
Determine Charge States
Acquire MS Inclusion List Update QTIC
Valid Intensity?
Pulse Program Resume Pulse
Suspends Valid Charge State? Program
Exclusion List
(dynamic and static) Acquire MS/MS
Figure 8-18 Decision structure for data dependent operation
After each acquisition the pulse program is suspended using the “autosuspend”
command. At this point the acquisition computer (apexControl) analyzes the mass
spectrum and decides which peak to pick for MS/MS investigation. The choice is
based on a large number of rules regarding the ions presence in various inclusion or
exclusion mass lists, the peak intensity and the charge state of the ion (see Section
8.2.2). First the inclusion list is used. If an ion on the inclusion mass list is found in the
spectrum it is preferentially chosen for MS/MS analysis. If no inclusion masses are
found then the masses that are found are evaluated based on their intensity, charge
state and the exclusion mass list. In most cases the most intense peak is chosen.
However, it is possible to use additional rules such as the preferential selection of
multiply charged ions, as well as the selection of ions whose peak intensity fall below a
maximum threshold. All of the possible options for this peak selection can be setup by
the user. The multiple rules and order of their use have been carefully implemented so
as to produce the maximum amount of information from the sample that is presented to
the instrument.
Once the peak of interest has been chosen, the MS/MS energy is calculated based on
the charge state of the ion and its m/z value (see Section 8.2.3). The QTIC is then
updated with the parameters for the next experiment. Once a positive response is
received back from the QTIC, a resume command is sent to the console to terminate
the suspend state and the next acquisition is performed. This mass spectrum is then
stored and the process starts again.

8.2.2 Charge State Determination

The charge state of the various isotopic clusters observed in the mass spectrum is
determined by apexControl during the data dependent decision making phase. Charge
states are determined based on the nominal mass spacing of adjacent isotope peaks.
Only if the spacing and the absolute peak intensities match what would be expected for
that m/z value is a charge state assigned. If no charge state is assigned to a particular
peak then this peak is assumed to be due to noise and is not considered for MS/MS
analysis. The charge state determination algorithm is able to handle up to charge state
6+ provided that the SI value used for dataset processing gives enough resolution to
identify individual isotope peaks.
8.2.3 CAD Energy Correlation

As shown in Figure 8-18, the MS/MS energy used to dissociate a given ion is
calculated by apexControl so that the proper voltages can be set on the QTIC. The
energy is calculated as a function of the ion’s m/z value and its assigned charge state.
In general the energy required to dissociate an ion increases with m/z and decreases
with charge state. The relationship that apexControl uses for this calculation is stored
in a collision energy file (.cef). This file can be viewed through apexControl, as shown
in Figure 8-19, by clicking on the Energy File Editor button (Figure 8-17). The Energy
File Editor button becomes active only when the Fixed Collision Voltage box is un-
checked.
Figure 8-19 Editable collision energy window showing the peptides.cef file
The file contains slope and intercept values specific to each charge state that are used
to calculate the energy (volts) required to effectively dissociate a given ion. New energy
correlation files can be produced by the user by modifying the default.cef file and
saving it under a new name. Files are generally store in the path:

C:\Bruker\apexControl\CurVer\config\automs
These files should be edited with caution. To obtain the necessary slope and intercept
values the user needs to calibrate the collision energy for the analyte family of interest
(the particular collision gas pressure and type will also affect the optimum collision
energy). There is a file provided with the instrument (peptides.cef) that is suitable for
peptides, i.e. protein digest fragments, but other classes of chemicals will likely need a
different calibration. Performing the calibration involves measuring the optimum
collision energy with respect to m/z and charge state. Note that the collision energy
is defined in the software as the voltage difference between the source hexapole
dc bias and the collision cell dc bias. Once several m/z values have been
measured for each charge state (at least 3 per charge state), a plot is made of collision
energy vs. m/z. A linear fit is then applied to this plot and the slope and intercept is
!
used in the .cef file.
8.2.4 Energy offset using the C_MsmsE parameter

The energy values calculated from the .cef energy file can be offset by a fixed value
using the C_MsmsE parameter. This might be helpful to get a little more overall
fragmentation without having to recalibrate the energy file.
This parameter is directly accessible in the Param Control Tab when running in Expert
Mode. The parameter is edited using the command line by first entering in the
parameter name. This will display the current value that can be set accordingly.
e.g.: an entry of –10 will result in 10eV increase of all the calibrated energy values (for
positive ions). An entry of +10 will result in a decrease by 10eV for all the calibrated
energy values (for positive ions).
8.2.5 Interfacing with Hystar

For coupling of High Performance Liquid Chromatography (HPLC) systems to
instruments of the apex series it is easiest to use Bruker’s HyStar software.
When Compass is opened it will display a menu on the right side:

Upon right-clicking on an entry either the respective program will open with the
corresponding page activated (e.g. Hardware Setup will open HyStar with the
Hardware selection window being active) or a pop-up menu with further choices will be
displayed.
For any LCMS experiment the logical workflow is to follow the items from the above
menu from top to bottom. Here a brief description of the workflow will follow, for details
of HyStar operation please refer to the HyStar User’s manual.
Before you want to start an LC coupling make sure that apexControl is running and a
method has been optimized and saved for your application (use “save apexControl part
as” from the method menu in apexControl).

First go to hardware set-up and specify (or define a new) LC-system and mass
spectrometer (apex series) you will use. Note: Hardware setups can be saved and
reloaded, if you always run the same hardware you can skip that menu item.
For LC method development open the Method item. For a detailed description please
refer to the HyStar User’s manual.
If you want to use standard methods (e.g. for a Peptide mass Fingerprint) which have
been set up earlier you can go directly to the Sample Table item.
TIP: A very convenient way of working is opening an old Sample Table and save it
under a different name. Using the Sample Table menu item will open a browsing
dialogue where one can select a sample table. Then you can edit the sample table and
save it again. Once you selected a row you can change to the acquisition window.

Within the acquisition window the status of the different modules is color coded (green
= ready, yellow = not ready, blue 0 under control by HyStar, red = error).
If all items are green one can start the acquisition by clicking “Start”
If more than one line has been checked in the Sample Table there are two operation
modes: either one runs a single LC analysis or a sequence of which.

During an LCMS or LCMSMS acquisition the devices actually controlled by HyStar are
depicted in blue in the acquisition module. If one opens the Sample Table while a
sequence is running the status of individual acquisitions can be reviewed (to go back to
the acquisition window simply close the Sample Table).
8.3 AutoRamp Mode
In this optimization strategy, selected ion peak intensities are monitored while a single
instrument parameter is ramped. Based upon the Optimize mode, AutoRamp mode
differs in that the Acquisition data is saved. AutoRamp mode is enabled within the
Optimize page by selecting the Save Data checkbox.
The following outlines the steps using AutoRamp to find the optimum voltage for In -
source dissociation of Angiotensin.
1. From the file menu, select the parameter file for the Tab that contains the
parameter to be ramped. In this case Skimmer 1 will be ramped, so the
“Source Transfer Optics” file is selected.

2. Once the parameter file is selected, the input window will be populated with all
of the parameters on the “Source Transfer Optics” tab. The Min, Max and
Increment fields have default values, but can be adjusted for the desired
experiment. It should be noted that the parameter which is highlighted will be
ramped.
3. Several peaks can be monitored during the AutoRamp. Enter the desired peak
into the top field on the “m/z peaks” sections. To add the peak to the monitor
list, click the “Ins” button. The “Del” button will delete a selected peak from the
list and the “Clr” button will completely clear the list.
4. If desired, the number of Average Spectra (per data point) and dataset size can
be adjusted at the bottom of the Optimize Tab.

5. To Save the acquisition data, enable the Save Data checkbox.
6. Click on the Optimize button to start the ramping procedure
7. Ramping results are displayed in the Optimize Window. If this window is not
shown, it can be displayed by right clicking on any display window and selecting
“Optimize Display”.
8. Intensities for the selected peaks are displayed as a function of the ramped
parameter. A legend at the bottom right corner of the Optimize window identifies
the peak associated with each trace. The current parameter setting is displayed
in the upper left corner of the window.

9. When completed, the traces will be shown in the Optimize window and an
Optimized value will appear in the optimized column of the input table. The
optimized value is NOT automatically up-dated in the method. This is done by
clicking on the accept button.
10. Results are saved into a single 2D dataset that can be brought into DA for
further post processing.
AutoRamp will not automatically proceed to the next parameter. To ramp another
parameter, it must first be highlighted in the parameter input table and then needs to be
started by clicking on the “Optimize” button again.

8.4 MALDI Imaging
The FTMS imaging experiment is performed using flexImaging, Hystar and

apexControl. Similar to TOF instruments, the FTMS imaging experiment is setup as a
series of measurements at defined x-y-positions of the sample. FlexImaging is used to
create a new sequence and to define the measurement area. The imaging workflow
(autoXecute sequence) is then sent to Hystar where the method is fine tuned. Hystar
then triggers apexControl to measure the spectra.
FTMS workflow in an automatic acquisition of a MALDI image

In flexImaging, the automatic sequence is started by clicking Start Automatic Run,

which then triggers the following series of events:
 flexImaging creates a geometry for the sample. This geometry contains the
information on which points of the sample the spectra will be acquired.
 flexImaging creates an autoXecute run (Hystar sample table). This run
contains the geometry and defines the order of the measurements. It also
contains the autoXecute method.
 The autoXecute method is then sent to Hystar, where it is combined with the
apexControl method. Additionally, method parameters can be fine tuned, such
as the laser power, the number of laser shots and the number of scans
summed per pixel .
 Hystar then triggers apexControl to measure the spectra according to the
autoXecute method.
 After the automatic measurement is finished flexImaging automatically reads
the acquired data according to the settings in the sequence properties set in
fleximaging.
For FTMS data it is recommended not to use the baseline subtraction option in the
flexImaging method and to read in the raw spectra ( see the flexImanging manual for
more information). This will save time when loading spectra into flexImaging.
8.4.1 General steps in performing an FTMS imaging run

1. Setup data acquisition method in apexControl
2. Open the Hystar Acquisition/MALDI Automation module
3. Select the MALDI Imaging workflow
4. Create a new sequence in flexImaging
5. Teach new sample image to motor positions using apexControl (via
Hystar/flexImaging)
6. Define measurement regions in flexImaging
7. Send autoXecute method to Hystar
8. Add apexControl method to autoXecute method
9. Start automatic run from Hystar

8.4.2 Setting up the apexControl method

The general apexControl MALDI method needs be fine tuned for the specific tissue
sample to be imaged (see Sample Preparation section of the flexImaging manual). To
do so, first load the sample into the source. Using the displayed geometry file, e.g.
MTP 384 ground steel, move the sample carrier a spot on the tissue (again see FI
manual for more details). In Tune mode, adjust the laser power and number of laser
shots to optimize signal intensity for several spots on the tissue. Additional parameters
such as mass range, Q1 mass and data set size are adjusted now. Depending on the
desired spatial resolution one should also choose a wider or smaller laser focus now.
This is accomplished by either setting it to imaging (ca. 40 µm) or standard (ca. 170
µm).
With these optimized settings it is recommended to move to a prepared spot for

calibration (e.g on a peptide standard) and perform a calibration prior to saving the
acquisition method.

8.4.3 Hystar MALDI automation module

Communication between flexImaging and apexControl is controlled via Hystar. For
imaging experiments, the “MALDI Automation” Acquisition module in Hystar is required.
The MALDI Automation module is necessary for all communication between

flexImaging and apexControl. Specifically, it is responsible for communications during
Teaching (see FI manual for more information) of the sample image and for running the
autoXecute sequence. Hystar does not have an interactive role during Teaching, but is
required to be open when doing so.
MALDI Automation has two types of workflows to choose from: Automation and
Imaging. The workflow defines how Hystar and apexControl will communicate during
the experiment. To select the type of workflow, in the Checklist click on “Choose
Apex/MALDI Workflow.
This will give the following menu. For all Imaging experiments, choose the MALDI
Imaging work flow.

The MALDI Automation module also combines the autoXecute sequence with the
apexControl method. The autoXecute sequence is loaded automatically from
flexImaging when the automatic sequence is started in flexImaging. To include the
apexControl method, use the browse button in the Method Name column to select the
correct method.
Typically if a good method is used it is not necessary, but, if required, several of the
MALDI method parameters are editable within Hystar. By default, the parameter
columns are labeled with a “d” to indicate that the value defined within the method will
be used. If the user chooses, new values can be added that will overwrite the values
defined in the method.

Once the apexControl method is loaded, it is necessary to “click” out of the field. The
Start button is then active. The start button will begin a multi-fid acquisition in
apexControl. With in a single LC-type run, each position (sample) defined in the
autoXectue sequence is run and written to an individual file. In Hystar all of the
positions, or Pixels, are given as individual entries in the Pixel tab.

8.4.4 Teaching
For the FTMS experiments, teaching is done following the same procedure as the TOF
experiment. The only difference being that apexControl (with Hystar in the background)
is used rather than flexControl. For more detail on teaching the image, please refer to
the flexImaging manual.

8.5 MALDI Automation
The automated MALDI experiment is performed using both apexControl and Hystar.
For a given run, a single apexControl method is defined for every selected spot on the
MALDI target plate. Experiments ranging from complex LC-MALDI to the analysis of
several separately spotted peptides are easily acquired. Automation workflows can be
defined by Warp-LC or manually created from a template. The workflow is then loaded
into Hystar where the method parameters can be fine tuned. The run is then started
via Hystar, which triggers apexControl to measure the spectra.
8.5.1 Create apexControl Method

The MALDI Method to be used for automation is first setup in apexControl. Parameters
such as random walk, Q1 mass and data set size should be optimized for the specific
sample to be analyzed. If needed, several of the MALDI specific parameters (LP, LS,
NS) are adjustable in Hystar if required. Another important aspect of the apexControl
method is the target teaching. A poorly taught target can lead to samples not being
analyzed. Once the acquisition parameters are optimized, the entire data acquisition
method is saved.

8.5.2 Create Sample Table

If an LC-MALDI run is set-up via Bruker’s WARP-LC software, you only have to import
the XML file which is generated by the AutoeXecute Run Editor (for details please refer
to WARP-LC user’s manual).
However, most of the parameters for acquisition are stored in the apexControl method.

8.5.3 Define apexControl/Hystar workflow

The following steps outline how to setup and run a MALDI Automation experiment:
1. Choose type of workflow in Hystar
From the main Hystar menu, select Acquisition and then choose MALDI Automation.
MALDI Automation has two types of workflows to choose from: Target Automation
and Imaging. The workflow defines how Hystar and apexControl will communicate
during the experiment. To select the type of workflow, in the Checklist click on
“Choose Apex/MALDI Workflow.
This will give the following menu. For all automation experiments, choose the MALDI
Target work flow.

2. Load/add Sample
From the Checklist, select Load/add Sample and browse to the input sample file.
3. Include and refine Method
Once the sample table has been loaded, the apexControl method needs to be
included. Mouse over the method name column and the following browse icon
appears.
Navigate to and select the required apexControl method.
Once the apexControl method is loaded, it is necessary to “click” out of the field for
Hystar to accept it. Typically if a good method is used it is not necessary, but, if
required, several of the MALDI method parameters are editable within Hystar. By
default, the parameter columns are labeled with a “d” to indicate that the value defined
within the method will be used. If the user chooses, new values can be added that will
overwrite the values defined in the method.

4. Start the run
Before the run can begin, apexControl must be open. This is indicated in Hystar by the
apexControl status box showing ready and apexControl will be highlighted in green.
When all of the Hystar Method fields have valid entries, and apexControl is ready,
Hystar will indicate that it is now ready to start the automation run.
Once all the lights are green, the start button becomes active and the run can begin.
Once started, the status boxes will indicate the current run mode.
Hystar will now trigger apexControl to make measurements for all of the entries in the
sample table.
It should be noted that the data generated via the MALDI Automation workflow can not
be re-loaded into apexControl due to the required folder convention. The data can be
directly loaded into DataAnalysis for further post processing.

Instrument Maintenance Bruker Daltonics Inc.
Chapter 9 Instrument Maintenance
Note: During maintenance operators may be exposed to:
 Chemical Residues (section 9.1)

 Biological Residues (section 9.2)
 High Temperatures (section 9.3)
 Hazardous Voltages (section 9.4)
9.1 Chemical Residues
Not all of the sample and solvent are ionized. The majority of sample and solvent
passes through the interface without being ionized. The vacuum pumps of the apex-
Qe series pump away the unionized sample and solvent. The exhaust from these
pumps can contain traces of your samples and solvents. Vent all pump exhaust
outside or into a fume hood. Comply with your local laws and regulations.
WARNING: The exhaust fumes from the vacuum system and spray chamber will
contain trace amounts of chemicals from sample analysis. Health hazards include
chemical toxicity of solvents, samples, buffers, and pump fluid vapor, as well as
potentially biohazardous aerosols of biological samples. Vent all exhausts outside the
building where it cannot be re-circulated by the environmental control systems. Do not
vent the exhaust into the laboratory. See the warning labels on the instrument.
WARNING: When replacing pump oil, use protective gloves and safety glasses. Avoid
contacting the fluid.
WARNING: Fluid drained from the spray chamber is composed of solvent and sample
from the analyses. The oil in the mechanical pumps collects traces of the samples and
solvents. In addition, non-nebulized solvent and sample accumulate at the bottom of
the spray chamber. Connect the drain at the bottom of the spray chamber to a closed
container. Handle and dispose all fluid with care appropriate to its chemical and/or
biological content. Handle all used pump fluid as hazardous waste. Dispose used
pump fluid as specified by your local laws and regulations.

Bruker Daltonics Inc. Instrument Maintenance
9.2 Biological Residues
Not all of the sample and solvent are ionized. The majority of sample and solvent
passes through the interface without being ionized. Any sample and solvent passes
through the interface without being ionized. The vacuum pumps of the apex-Qe series
pump away the unionized sample and solvent. The exhaust from these pumps can
contain traces of your samples and solvents. Vent all pump exhaust outside or into a
fume hood. Comply with your local regulations and laws.
WARNING: Fluid drained from the spray chamber is composed of solvent and sample
from your analyses. The fluid in the mechanical and diffusion pumps collects traces of
the samples and solvents. In addition, non-nebulized solvent and sample accumulate
at the bottom of the spray chamber. Connect the drain at the bottom of the spray
chamber to a closed container. Handle and dispose all fluid with care appropriate to its
biohazardous and biological content. Handle all used pump fluid as hazardous waste.
Dispose of used pump fluid as specified by your local laws and regulations.
WARNING: The needle in the NanoSpray source is extremely thin. Avoid touching and
getting hurt especially on measuring dangerous and toxic substances.
9.3 High Temperatures
Many parts of the apex-Qe Series operate at temperatures that can cause serious
burns. These are for example:
 Mechanical pumps
 Drying gas heater
 Drying gas
 APCI heater (vaporizer)
 Capillary and capillary cap
 Spray shield
 Any other parts that come in contact with the drying gas (the entire spray
chamber) capillary, capillary cap and lamp
Most of these parts are normally covered or shielded. Therefore, also the covers
become hot. Avoid touching these parts!
WARNING: Many of these parts remain hot for a lot of time after the apex-QE series
has been shut down or switched off. Pay attention when working on a recently shut
down instrument!

9.4 Hazardous Voltages
When the apex-Qe series is connected to the mains, hazardous voltages are applied
to assemblies, such as:
 Mechanical pumps
 Transformers and power supplies in the vacuum cart and console
 High voltage electrodes (capillary and end plate) in the spray chamber
 Drying gas heater
 APCI heater
 APCI corona needle
 Wiring and cables between components
 rf generators
 Lens voltage cables
9.5 Maintenance Schedule
General maintenance tasks are listed in the table below. Performing these tasks on
schedule avoids problems, prolongs system life, and reduces overall operating costs.
Keep a record of all performed system performance characteristics and maintenance
operations. This facilitates detecting deviations from normal operation.
Table 9-1 Maintenance Schedule
Task Daily Bi-weekly As needed
Flush ESI sample path X

Clean spray chamber, X
spray shield, capillary
cap, contacts and the tip
of the corona needle
Check rough pump fluid X
level
Replace rough pump fluid X
Replace nebulizer needle X
Clean or replace entire X
capillary
Magnet Refer to the magnet
manual for the system

9.6 Maintaining the apex-Qe SERIES
9.6.1 Venting the Source or UHV chambers

 Close the Beam valve
 Turn of the vacuum gauge for the section you will be venting.
 Press the source or UHV button on the SCU (see Figure 9-1)
 The green led on the switch will turn off after approx 1 second and the venting
procedure will begin.
 The SCU will maintain the power to the source roughing pumps until the source
turbo pumps are all below 50% speed.
 The turbo pump speed is indicated by the LED bar graph below the switch.
 When the pumps are below 50% speed the source roughing pump will shut off
and the source vent valve will open.
Figure 9-1 SCU control panel

9.6.2 Removing the Nebulizer

Remove the nebulizer for visual inspection.
Required tools:
Gloves, latex.
WARNING: The tip of the nebulizer may be very hot. Let it to cool down.
Procedure:
 Shut off the flow of LC solvent.
 Shut off the flow of nebulizing gas.
 Push back the nebulizer’s plastic cover.
 Disconnect the LC tubing and nebulizing gas tubing from the nebulizer.
 Turn the nebulizer counterclockwise and disengage it from the retaining screws.
 Carefully lift the nebulizer out of the spray chamber.
9.6.3 Flushing the Nebulizer

It is recommended to flush the nebulizer tubing and valves after a measurement series
with:
Isopropyl alcohol, reagent grade or better, and
Water, reagent grade or better.
Procedure:
 Remove the nebulizer.
 Mix a solution of 50 % isopropyl alcohol and 50 % water.
 Use a syringe and a hose to pump this mixture through the nebulizer several
times.
 Clean the tip of the Nebulizer in an ultrasonic bath.
Note: Apply this to both Electrospray and APCI nebulizer.

9.6.4 Replacing the Nebulizer Needle

A clean and undamaged needle is essential to achieve good electrospray
conditions. Flush the needle before and after each analysis. Flushing helps to keep
the needle clean and reduces the frequency of needle replacements.
Replacing the Electrospray nebulizer needle when:
 The needle is visibly bent or damaged.

 The spray is inclined to the direction of the tip of the needle assembly.
 The needle is blocked. Common symptoms are increased LC backpressure or
off-axis spraying or dripping from the nebulizer.
 When data shows excessive noise or the current signal is unstable the needle
must be replaced.
Required tools:
Adjustment fixture.
Gloves, latex.
Wrench 3-mm, open-end.
Wrench 8mm.
Required spare parts:

Nebulizer needle (ES-shipping kit)
Procedure:
 Remove the nebulizer from the spray chamber.
 CAUTION: Be very careful when inserting the needle. The tapered end of the
needle must pass through restrictions in the nebulizer shaft. The end of the
needle can be damaged if it is forced through.
 Install the nebulizer in the adjustment fixture.
 Loosen the locked nut next to the zero-dead-volume (ZDV) union.
 Remove the union from the nebulizer.
 Pull the needle and ferrule out of the nebulizer.
 Push a new ferrule, large end first, onto the blunt end of a new needle. The
tapered end of the ferrule should be even with the blunt end of the needle.
 Very carefully push the tapered end of the needle into the nebulizer until it
appears at the tip of the nebulizer.
 Reinstall the union.
 Tighten the locked nut against the union.
 Adjust the Electrospray needle position before reinstalling the nebulizer in the
spray chamber.

Figure 9-2 Mounting the Nebulizer needle
9.6.5 Reinstalling the Nebulizer

Make sure the nebulizer needle is correctly adjusted. Make sure the nebulizer cover is
open.
CAUTION: Be careful not to bump the tip of the needle on inserting the nebulizer. The
tip of the needle is easily damaged.
Procedure:
 CAUTION: Do not tighten the LC fitting too much. This can crush the tubing, or
creating a restriction.
 Slowly and carefully insert the nebulizer into the spray chamber.
 Reconnect the nebulizing gas tubing to the nebulizer.
 Finish inserting the nebulizer into the spray chamber.
 Turn the nebulizer clockwise to lock it into place.
 Reconnect the LC tubing to the zero-dead-volume union.
 Close the nebulizer cover.

9.6.6 Removing the Glass Capillary

Required tools:
Gloves, latex.
WARNING: The spray chamber operates at very high temperatures. Let it cool down
before proceeding.
CAUTION: Pull the capillary straight out while rotating it clockwise along its long axis.
The capillary is made of glass and can break it during handling!
Procedure:
 Vent the Source Chamber
 Open the spray chamber.
 Remove the spray shield.
 Remove the capillary cap from the end of the capillary.
 Carefully pull the capillary straight out of the desolvation assembly while
rotating it clockwise.

9.6.7 Cleaning the Spray Chamber

Required material:
Cloths, clean, lint free.
Gloves, latex.
Isopropyl alcohol 99,5 %, reagent grade or better.
Mix a solution of 50 % isopropyl alcohol and 50 % water.
WARNING: The spray chamber operates at high temperatures. Let it cool down to
ambient temperature before continue working.
Note: If contaminations or discolorations of the spray shield and capillary cap cannot
be removed by polishing abrading may help (section 9.6.9).
Procedure:
 Remove the Nebulizer (section 9.6.2).
 Dampen a clean cloth with the mixture of isopropyl alcohol and water.
 Remove spray shield and capillary cap.
 Put both parts into a solvent and clean it with an ultrasonic cleaner.
 Reinstall the spray shield.
 Wipe all other accessible surfaces. Pay special attention to the bottom of the
spray chamber near the drain hose and to areas that are discolored.
 Close the spray chamber.
 Reinstall the Electrospray nebulizer.

9.6.8 Adjusting the Nebulizer Needle

Adjusting the needle becomes necessary after replacement, or if symptoms indicate
that the needle is not correctly adjusted.
Required tools:
Adjustment fixture.
Gloves, latex.
Magnifier.
Wrench 3-mm, open-end.
Wrench 8mm.
Wrench 1/2-inch x 9/16-inch, open-end.
Remove the nebulizer from the spray chamber. If necessary, replace the Electrospray
nebulizer needle.
CAUTION: Be careful and avoid knocking the tip of the nebulizer against anything.
Any damage will have a large, negative effect on system performance.
Note: The resulting position will work very well for a wide range of LC flows. If you
intend to work exclusively with flows above 0.5 ml/min, you can achieve even better
performance by adjusting the needle so that it is even with the tip of the nebulizer.
Procedure
 Place the nebulizer in the adjustment fixture.
 Loosen the needle holder locked nut.
 Position the magnifier so you can view the tip of the nebulizer.
 Adjust the needle holder until the needle extends just slightly less than 1/2 its
own diameter beyond the tip of the nebulizer.
 Tighten the locknut. Make sure this does not change the position of the needle.
 Remove the nebulizer from the adjustment fixture.
 Reinstall the nebulizer in the spray chamber.

9.6.9 Abrading the Contamination

Abrading the spray shield or capillary cap is necessary, if significant discoloration or
deposits cannot be removed by polishing.
Required tools:
Sand paper, 8000 grit,
Cloths, clean, lint free,
Gloves, latex.
Isopropyl alcohol 99,5 %, reagent grade or better,
Use a solution of 50 % isopropyl alcohol and 50 % water for cleaning.
CAUTION: Because the spray shield and capillary cap are made of stainless steel,
they can safely be abraded. However, they are the only parts that should be cleaned in
this way. Many other metal parts, such as the spray chamber, may look similar to
stainless steel, but are made of much softer metals or are plated by materials that will
be damaged by abrading.
WARNING: The spray chamber operates at high temperatures. Let it cool down before
proceeding.
Procedure:
 Remove the spray shield.
 Remove the capillary cap.
 Clean the spray shield: Only the large flat surface needs to be cleaned unless
there are obvious deposits elsewhere on the shield. The inner rim of the main
hole in the shield may also occasionally need cleaning. Using the Cotton-
Tipped Applicators and mixture of isopropyl alcohol and water can do it.
 Clean the capillary cap with the sand paper. Only the end surface of the cap
needs to be sandpaper cleaned unless there are obvious deposits elsewhere
on the shield. The inner rim of the hole in the cap may occasionally need
cleaning.
 Put the capillary cap into a solvent and clean it with an ultrasonic cleaner.
 Reinstall the capillary cap.
 Reinstall the spray shield.
 Close the spray chamber.

9.7 List of apex-Qe series Spare Parts
Description Part Number
Sprayshield 556638
Capillary Cap 556639
Contact spring (Gold plated) 554046
Glass Capillary 600um/150mm (AP 2 ESI) 556463
600um/300mm (AP 2 MTP) 557031
Nebulizer 554744
Nebulizer Needle (ES shipping kit) 554183
APCI / APPI Source

Nebulizer APCI / APPI assembly 555037
Nebulizer spray needle kit APCI / APPI 554435
APCI Corona needle 554345
APCI / APPI Heater 601699
APPI Krypton Lamp 243897
Rough Pump
Oil Qe Series grade 19 for Rough Pump 553754
Exhaust Filter Alcatel OME 25 555534
Exhaust filter Edwards EMF20 210370
Syringe 500µl 554438

Further Documentation Bruker Daltonics Inc.
Chapter 10 Further Documentation

These manuals are provided on the Compass 1.4 for apex-Qe Series installation DVD.
No Manual Description Document #

1 Apollo II Dual User manual for Apollo II sources 600392.pdf
ESI/MALDI (ESI only and Dual ESI/MALDI)
2 Qh Interface Advanced user and service manual 600393.pdf

for the Qh Interface; incldg.
Theoretical background on Q
operation, tuning and calibration,
wiring diagrams
3 Mechanical Installation Instructions, wiring, 600880.pdf
Shutter spare parts
4 TOPPS Operation, Troubleshooting, wiring 600879.pdf

Transfer Optics
Power supply
5 Infinity cell Operational manual covering wiring 600878.pdf

and preamplifier
6 Infinity cell Installation, wiring, ECD cathode 600877.pdf

controller regeneration

Bruker Daltonics Inc. Further Documentation
No Manual Description Document #

7 MicroBay Operation, system component 600876.pdf
Console description, wiring
8 NanoBay Operation, system component 601799.pdf

Console description, wiring

Appendix Bruker Daltonics Inc.
Chapter 11 Appendix
11.1 Abbreviations
The following abbreviations are used throughout this manual:
API Atmospheric Pressure Interface

APCI Atmospheric Pressure Chemical Ionization
APPI Atmospheric Pressure Photo Ionization
BASIC Bruker Acquisition Sequence for Instrument Control
CAD Collision Activated Dissociation
C-CAD Collision Cell Activated Dissociation
ECD Electron Capture Dissociation
EIC Extracted Ion Chromatogram
ESI Electrospray Ionization
FIA Flow Injection Analysis
FID Free Induction Decay
FWHM Full Width at Half Maximum
FFT Fast Fourier Transform
FFTW Fastest Fourier Transform in the West
FT Fourier Transform
GUI Graphical User Interface
Hz Hertz
ICC2 Infinity Cell Controller
ICR Ion Cyclotron Resonance
IRMPD Infrared multi-photon dissociation
IS-CAD In-Source Collision Activated Dissociation

Bruker Daltonics Inc. Appendix
M megaword (= megapoint) & (4 points = byte)

MALDI Matrix Assisted Laser Desorption Ionization
MS Mass Spectrometer & Mass Spectrometry
Multi-CHEF Multi-Correlated Harmonic Excitation Frequencies
m/z Mass-to-Charge Ratio
ppm Parts Per Million
QTIC Quadrupole Transfer Ion Controller
QFSCU Quadrupole Frequency System Control Unit
RAM Random Access Memory
ROI Region Of Interest
SCU System Control Unit
SHEDS Smart Hub for Ethernet Distribution to Serial
S/N Signal-to-Noise Ratio
SORI Sustained Off Resonance Irradiation
TP Turbo-molecular Pump
TFA Trifluoroacetic acid
TIC Total Ion Chromatogram
TOPPS Transfer Ion Optics Power Supply
UHV Ultra High Vacuum
XML Extensible Markup Language

11.2 Bug Reporting and Requests for Enhancements in

apexControl
A bug/request management interface in apexControl allows the user to report bugs

and request new features in the software.
Note: This bug reporting process is web based and is secured using SSL encryption in
passing all information between the user and Bruker Daltonics. Prerequisite to use this
feature is that the computer running apexControl has internet connection.
To start the bug/request management interface in apexControl select “Report bug or

!
request feature” in the “Help” pull-down menu (see Figure 11-1).
Figure 11-1 Starting the bug/request management interface in apexControl
The apexControl customer feedback login window will pop-up. First time users click on
the create new account button as shown in Figure 11-2, if the user forgot the password,
the user can click on the “Forgot password” item, and then fill out the user’s e-mail
address and the password will be e-mailed to the user right away.

Figure 11-2 apexControl customer feedback login screen
New users are required to fill out the create account form as shown in Figure 11-3.
Once the form is submitted, the user will quickly receive e-mail with a password and
account information.
Figure 11-3 Create new account window

Once the user has an account, they then use their e-mail and password to login on the
“apexControl customer feedback login screen” (see Figure 11-2).
After successful login the main feedback portal of the bug/request management
interface will show (see Figure 11-4). The main portal allows the user to submit new
bug reports or change requests, and also allows monitoring of the status of submitted
reports and even the addition of new information.
Figure 11-4 apexControl feedback portal
To submit a new report select the appropriate item (e.g. “bug”) from the pull down
menu and click on the submit button as shown in Figure 11-4.
Figure 11-5 shows the form for creating a new bug report (change request is also
similar to this). Items marked with an “*” asterisk are required, however, the user
should try to provide as much information as possible. By default, the apexControl log
file is added to the report automatically unless unselected by the user (in most cases
the log file should be attached). Other files/images can be attached if necessary to
clarify the problem or request.

Figure 11-5 Bug report window (note request form is similar)

Once the form is filled out, click the “submit” button at the bottom of the form (not
shown in Figure 11-5 – scroll down).
A detail report of interest can be viewed, as shown below in Figure 11-6, by clicking on
the id hyperlink of an already submitted (see Figure 11-4). The detail view will show
the current status plus all comments added by others. New comments can also be
added that will help to provided more information.
Figure 11-6 Bug report detail after selecting ID link of submitted report on the
apexControl feedback portal window (see Figure 9-4)

Bruker Daltonics Inc. Index
Chapter 12 Index
A D
Abbreviations 246 Data Directories 78
Abrading off Contamination 242 Detection Plates 44, 45, 46
Acquire Menu 149
Acquisition Event Property Files window172 E
Acquisition Event Sequence window 171 ECD Regeneration window 164
Acquisition Mode 74, 103 Electron Capture Dissociation
Adjusting the Nebulizer Needle 241 ECD 32, 186, 198, 246
Analyzer Page 133 ESI Initial Signal 69
apexControl 3.0 Installation 54 Excitation Control window 175
API Source 72, 109, 125 Excitation Plates 46
AutoMS Page 116, 139 Exclusion Zone 13
AutoMSMS Workflow 209 Expert Menu 150
AutoRamp Mode 216 Expert Mode 67, 104, 107
B F
Bake out 37 FFT Advanced window 179
Beam Valve 42 FID Mode 101, 150, 153, 155, 156
Biological Residues 233 File Menu 148
Broadband Detection74, 107, 108, 121, 122 flexImaging 220
Bug Reporting 248 Flushing the Nebulizer 236
Frequency Display 101, 153, 155, 156
C
FT-ICR Calibration 83, 84
Calibration page 120, 143 FTMS experiment 48
Chemical Residues 232 FTMS Instrument Configuration window 169
Cleaning the Spray Chamber 240
Collision Cell Activated Dissociation G
C-CAD 186, 188, 246 Graphical User Interface 97
Compass Menu 152
Console Page 147 H
Context Menus 153 Hazardous Voltages 234
Correlated Combination excitation 114, 137 Help Menu 152
Correlated shots 113, 136 Hexapole RF Generator Switch 180
Correlated Sweep 112, 113, 114, 135, 136, Hystar 209, 220, 223, 227
137, 191 HyStar 212
Cryogenic Liquids 14
Cyclotron Motion 44 I
ICR Transfer Optics Page 131
Infinity Cell Controller

Index Bruker Daltonics Inc.
ICC 2 32, 246 Q

Infrared Multi-photon Dissociation
IRMPD 195 Qh Page 129
Initial MALDI signal 79 Quadrupole Calibration 86
Quadrupole Calibration Procedure 92
In-source Collision Activated Dissociation
IS-CAD 187 Quadrupole Calibration window 162
Ion Motion 43 Quadrupole Frequency System Control Unit
QFSCU 31, 247
M Quadrupole Transfer Ion Controller
QTIC 31, 247
Magnet Safety Concerns 11
Quality Policy 18
Maintenance 232
Maintenance Schedule 234 R
MALDI Automation 227
Readback Panel 105
MALDI Imaging 55, 220, 221, 223
MALDI mode 79 Reference Mass Lists 77, 84, 120, 143
MALDI page 110, 123 Reinstalling the Nebulizer 238
MALDI Target Teaching 183 Remote service capability 21
Mass Calibration 76 Removing the Glass Capillary 239
Method Menu 148 Removing the Nebulizer 236
Microbay Console 30, 32, 34 Replacing the Nebulizer Needle 237
Mode page 107, 121 Resolving Power 50
MS/MS page 111, 134 S
Multi-CHEF 112, 113, 135, 136, 247
Safety Labels 16
N Sample Info page 119, 141
Nanobay console 30, 32, 33, 35 Security Zone 13
Nano-ESI_Offline 109, 125 Shortcuts 97, 99
Nano-ESI_Online 109, 125 Shut down options 235
Narrowband Detection 74, 108, 122 Signal Processing 49
Smart Hub for Ethernet distribution to Serial
O SHEDS 31
Source Roughing Valve 42
On-Resonance Collision Aided Dissociation
Source Transfer Optics Page 80, 127, 180,
194
216, 217
Optimize Display 102, 145, 153, 155, 156,
Spare Parts 243
157, 218
Spectrum Mode 99, 150, 153, 155, 156
Optimize Page 144
Standard Mode 67, 68, 107
Options Menu 149
Status Bar 107
P Support 20
Sustained Off-Resonance Irradiation
Parameter Control Page 147 SORI 186, 190
Peak List Panel 105 System Power 37
Peak Picking window 176 System Status Region 72, 79, 103, 109,
Peak Tuner 100, 102 110, 123, 125, 205
Precursor Selection 116, 139
Pump Control 40 T
TIC Display 102, 153, 155, 156, 157
Tool buttons 97

Bruker Daltonics Inc. Index
Tools menu 90, 151 Uninstalling apexControl 64

Transfer ion OPtics Power Supply User Levels 67
TOPPS 32 User Processing window 159
Trapping Plates 45
Tune Diagram Page 146
V
Tuning Mode 74, 197 Venting the Instrument 235
View Menu 150
U
UHV roughing valves 42

Index Bruker Daltonics Inc.

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apex-Qe series

Chapter 1 Introduction ....................................................................................................... 2

Chapter 2 Installation and Setup ..................................................................................... 19

Chapter 3 Instrument Overview ....................................................................................... 25

2 of 256 apex-Qe Series User Manual, Version 2.0

Chapter 4 Theory of Operation ........................................................................................ 43

Chapter 5 Getting Started Guide ..................................................................................... 53

Chapter 6 Calibration ........................................................................................................ 83

apex-Qe Series User Manual, Version 2.0 3 of 256

Chapter 7 apexControl Reference Guide........................................................................ 97

4 of 256 apex-Qe Series User Manual, Version 2.0

7.6.3 ECD Regeneration ....................................................................................................... 164

Chapter 8 Acquisition Workflows.................................................................................. 186

apex-Qe Series User Manual, Version 2.0 5 of 256

8.5 MALDI Automation ........................................................................................................... 227

Chapter 9 Instrument Maintenance ............................................................................... 232

Chapter 10 Further Documentation................................................................................. 244

Chapter 11 Appendix ........................................................................................................ 246

Chapter 12 Index ............................................................................................................... 253

6 of 256 apex-Qe Series User Manual, Version 2.0

1.3 Version History

Version Date Changes Remarks

1.4 Part number

apex-Qe Series User Manual, Version 2.0 7 of 256

1.5 Using This Manual

This manual applies to the apex-Qe series generation II FTMS

Identification of an apex-Qe series generation II instrument:

The identification can be done by means of either a software tool or by

Identification thru software tool:

Identification on the instrument:

Apex communications hub - SHEDS

8 of 256 apex-Qe Series User Manual, Version 2.0

The information contained in this document is subject to change without

Bruker Daltonics Inc. assumes no responsibility for the use or reliability

Bruker Daltonics Inc. warrants the apex-Qe series to operate within

apex-Qe Series User Manual, Version 2.0 9 of 256

Connecting an instrument to a power source that is not equipped with a

All connections of the instrument must be used in correct way. The

10 of 256 apex-Qe Series User Manual, Version 2.0

For electrical safety, observe the following guidelines:

Service on electrical components should be performed only by a

Superconducting Magnet Safety Aspects

The superconducting magnet that is at the heart of the apex-Qe FTMS

Stray Magnetic Field

The superconducting magnet supplied with the apex-Qe FTMS system

The strength of the magnetic field increases dramatically as the distance

Occasionally, fault conditions may cause magnets to loose their

apex-Qe Series User Manual, Version 2.0 11 of 256

Hazards Presented by the Magnetic Field

The strong magnetic field creates hazards because of its strong

The strong magnetic field can cause the following hazards:

A loose metal object (such as a wrench or pen) becomes a projectile if it

Ferromagnetic materials are sometimes used in surgical implants and

Some cardiac pacemakers, bi-stimulators, and neuro-stimulators are

12 of 256 apex-Qe Series User Manual, Version 2.0

Emergency Discharge Switch

Hazard in Case of Fire

The exclusion zones described above MUST be conveyed to fire safety

apex-Qe Series User Manual, Version 2.0 13 of 256

The superconducting magnets that are supplied contain cryogenic

Helium is a naturally occurring inert gas that becomes liquid at about 4 K

Nitrogen is also a naturally occurring gas (about 70% of air) that

Asphyxiation Hazard: A large quantity of gas released in an area that