Lecture 11

Molecular Instrumentation
 Centrifuge
 Spectrophotometer
Spectrometer
 Mass Spectrometer
 HPLC
 NMR
 DNA Microarray
Lab on a Chip
1
Principles and Applications of
Ultracentrifugation
Introduction

 Centrifuges have been in use since mid-1800

 Low-speed (<10,000rpm)
- Purify and harvest intact animal and plant cells,
chemical precipitates, and micro-organisms.1
 High-speed (6000~20,000rpm)
- Purify and isolate mitochondria, viruses,
chloroplasts, and Golgi membranes etc.1
 Ultra-speed (30,000~120,000rpm)
- Purify and isolate endoplasmic reticulum,
ribosomes, plasmids, DNA, RNA, and proteins.1
 Theodor Svedberg – Nobel Prize in Chemistry in
1926
Introduction-cont’d
 Preparative & Analytical Ultracentrifuges
 Preparative Ultracentrifuge: Used to prepare and
purify a sample for subsequent experimental
usages.2
 Analytical Ultracentrifuge: Used to accurately
characterize sample properties such as
molecular weight, stoichiometry, conformation,
and shape.2
 This paper examines the theory and concept
behind centrifugation, discusses the important
rotor designs, and introduces various
applications of ultracentrifuges.
Theory - 1
• Centrifugal Force = Mω2r
where M is the molecular weight, ω is the
angular velocity (rad/sec), and r is the radius
of rotation
• Buoyant Force = Mω2rVρ
where V is the volume displaced by the
molecule and ρ is the density of the solvent
(g/ml)
4. Frictional Force = f(v) = f (dr/dt)
where f is the frictional coefficient and dr/dt is
the rate of sedimentation

for a spherical molecule, f=6πηrm

where η is the viscosity of the medium and rm is
the radius of the molecule. 1 3 4
Theory - 2
4. Centrifugal Force = Buoyant Force + Frictional Force
Mω2r = Mω2rVρ + f (dr/dt)
Mω2r - Mω2rVρ = f (dr/dt)
M (1-Vρ) ω2r = f (dr/dt)
M = [f/(1-Vρ)] × [(dr/dt)/ ω2r]

7. Sedimentation Coefficient (s)

s = v/ ω2r = (dr/dt)/ ω2r
M = (f)(s)/(1-Vρ) or s = M(1-Vρ)/f
since D=RT/f, where D is diffusion constant, R is
gas constant, and T is absolute temp,
f = RT/D  s = M(1-Vρ)D/RT
One unit of 10-13 seconds is defined to be one
Svedberg unit. 1 3 4
Theory - 3
1. Centrifugation
F = ω2r
F is expressed in g’s where g=980 cm/S2
Relative centrifugal force (RCF) = ω2r/980

However, rotor speed is commonly expressed in

revolutions per minute (rpm) where ω = π (rpm)/30

Therefore,
RCF = 11.17r (rpm/1000)2 where r is in cm
or RCF = 28.38r (rpm/1000)2 where r is in inches. 1 3 4
Design-1
Four main components to a Centrifuge:5
Driver and Speed Control
- negative feedback control
- prevent overspeeding

Temperature Control
- thermocouple temperature control

Vacuum System
- mechanical pump
- water-cooled diffusion pump

Rotors
- Fixed-angle rotor
- Swinging-bucket rotor
- Near vertical rotor
- Vertical rotor
- Zonal rotor
 Design-2
1. Fixed Angle Rotor:

Figure 1: Fixed Angle Rotor (angle set between 20 and 45 degrees)4

2. Swinging Bucket Rotor:

Start of centrifugation at 90 degrees

orientation
x

End of centrifugation with pellet at bottom

of tube
Supernatant Pellet

Figure 2: Swinging Bucket Rotor4

 Design-3
3. Near Vertical Rotor: 4. Vertical Rotor:

Near Vertical Rotor with θ = 7~100 16 Vertical Rotor16

5. Zonal Rotor:4
Application-1
Table 1: Summary of Ultracentrifuge Separation Methods 1
Preparative Ultracentrifugation Analytical Ultracentrifugation
Differential centrifugation - pelleting Sedimentation velocity – zonal
Density gradient centrifugation - rate zonal Sedimentation equilibrium - isopycnic
Density gradient centrifugation – isopycnic

Table 2: Summary of Classes

of Centrifuges and their
Applications4
 Application-2
I. Preparative Ultracentrifugation: b. Density Gradient Centrifugation: Rate
a. Differential Centrifugation – Zonal
Pelleting

Figure 6: Rate Zonal Centrifugation14

Figure 5: Differential centrifugation – pelleting15

c. Density Gradient Centrifugation:

Isopycnic

Figure 7: Isopycnic Centrifugation14

Application-3
Table 3: Different Rotors and Applications4
 Application-4
II. Analytical Ultracentrifugation:
Table 1: Sedimentation Velocity vs. Sedimentation Equilibrium12
Sedimentation Velocity Sedimentation Equilibrium
Shape and size Molecular Weight
Number of species in solution Stoichiometry
Diffusion constant Oligomerization

1. Sedimentation Velocity Experiment:

Concentration
Radius

Figure 8: Double-sector centerpiece. The sample

is placed in one sector and the reference is in the other.9 Figure 9: Concentration vs. radius9
 Application-5
II. Analytical Ultracentrifugation: cont’d
1. Sedimentation Equilibrium Experiment:

Figure 11: c1 (white circles, 40kD), c2

Figure 10: Schematic representation of sedimentation (triangles, 80kD), and c3(dark circles, c1+c2).9
equilibrium.9
 Newest Technology
1. Absorbance Optical System
(Scanning UV/Vis):
- scanning monochromator
- xenon flashlamp
- measure wavelength range
of 200 to 800nm
- most effective with macro-
molecules with strong
chromophores.11

2. Interference Optical System:

- Rayleigh interference optical
system
- Uses change in refractive index
to measure sample concentrations
- most effective with macro-
molecules that lack intense
chromophores.11

Figure 12: Absorbance Optical System10

Medical Instrumentation:
The Spectrophotometer

17
 Principles Behind the
Spectrophotometer
Light Source

Refracting Prism

Wavelength Selector

18
 Principles Behind the
Spectrophotometer
Sample

Photoelectric Tube

Digital Display

19
 Principles Behind the
Spectrophotometer
Beer-Lambert Law states:
– There exists a linear relationship between
absorbance and concentration of an
absorbing species

A = log10(I/I0 )

A=Kcl

20
 Principles Behind the
Spectrophotometer

Beer-Lambert Law

21
 Operation of a
Spectrophotometer
Setting the
Wavelength

Cuvettes

Sample Holder

Take Reading

22
 Application and Future of
the Spectrophotometer
UV/Visible Spectrophotometer
Fluorescence Imaging Micro-
Spectrophotometer
Dual Beam Spectrophotometer
Multi-channel Spectrophotometer

23
 Application and Future of
the Spectrophotometer
'Star Trek' technology for cancer screening
– Portable device using spectrophotometer principles
to help diagnose medical conditions in dermatology,
specific cancers, skin disorders, etc.

– See:
http://www.laboratorytalk.com/news/spa/spa100.html

24
 An Overview of Mass
Spectrometry Principles of
Operation

25
 Abstract
Mass spectrometry (MS) and its role in
cellular &molecular instrumentation

Concepts and principles of operation

Pertinent operating techniques

State of the art commercial product:

IonSpec Explorer Mass Spectrometer
26
 Introduction
Analytical tool
– Molecular and structural
identification of unknown
compounds
– Quantifying known materials
– Analysis of peptides,
oligonucleotides & proteins

27
Design/Methods – Operating
Principles
Operating Principle: molecular weight
evaluation
– Production of gas-phase ions
– Separation by mass to charge ratio (m/z)
– Detection in proportion of relative
abundance

28
 Design/Methods – Basic
Components
Method/device: introduce sample
Ionizing source: produce ions
Analyzer(s): separate product ions
Detectors: detect the various product
ions
Vacuum: prevent collision of ions
Computer: increase processing,
analytical power & speed
29
 Design/Methods:
Tandem MS
Block diagram of a typical tandem MS
Introduction of
sample

sample

Ionizing source

ions data system

control
Analyzer 1

parent ions

Analyzer 2

daughter ions
signal data
Detector Computer Mass Spectrum

modified from De Hoffmann, Charette, Stroobant, Mass Spectrometry Principles and Application, 1996, p. 8

30
 Design/Methods:
Ionizing Sources
Thermionic emission from heated filament
Electron beam strikes sample molecules (g)
Collision produces positively charged
molecules

Molecular ion: XY + e- → XY+ + 2e-

Fragment ions: XY + e- → X+ + Y + 2e-

31
 Design/Methods:
Ionizing Sources

Electrospray ionization (ESI)

– Ions from solution (sample & organic
solvent)
– Capillary tube: produce droplets
– Strong electric field applied: charge droplets
– “Coulombic explosion” => smaller droplets
– Allows sensitive analysis of large molecules
– Able to be coupled with liquid
chromatography (LC)
32
 Design/Methods:
Ionizing Sources

Typical layout of an ESI source

(from Cambridge University Mass Spectrometry Server, www-
methods.ch.cam.ac.uk/meth/ms/theory/history.html

33
 Design/Methods:
Ionizing Sources
Matrix Assisted Laser Desorption Ionization
(MALDI)
– Efficient direct energy transfer
– Sample co-crystallized with matrix compound
– Laser radiation: evaporate and form ions
– Excited cluster made up of matrix molecules and
single anayle molecule
– Matrix molecules evaporate => High yields of intact
analyte
– High accuracy and sub-picomolar sensitive
– Sample need not be in gaseous phase
34
 Design/Methods:
Ionizing Sources

Illustration of ion formation via MALDI

(from Scripps Center for Mass Spectrometry, http:\\masspec.scripss.edu)

35
Design/Methods – Analyzers

Separate ions according to m/z ratios

Relevant intensity recorded

2 common types of MS
– Field aka Sector

– Time of Flight (ToF)

36
 Design/Methods – Analyzers
Field (aka sector)
– Magnetic and electric fields applied
– Deviation of ion from path
– Ions have an initial kinetic energy from momentum
generated under an accelerating potential
– Under magnetic field B, magnetic force causes ion to
subscribe a circular trajectory of radius r
– Electric field passes ions further according to their kinetic
energy => improved resolution
– Ions of m/z sorted according to radius and kinetic energy

37
 Design/Methods – Analyzers
mv 2
Kinetic energy, Ek =zVs =
2

Equating magnetic force:

mv 2 m r 2B2
qvB=  =
r z 2Vs

Equating electric field force:

mv 2 m 2Ek
Diagram of a typical two-sector (electrostatic/magnetic) mass qE=  =
spectrometer
(from Cambridge University Mass Spectrometry Server, www-
r z qE
methods.ch.cam.ac.uk/meth/ms/theory/history.html

38
 Design/Methods – Analyzers
Time of Flight (ToF)
– Ions accelerated with same potential (same KE)
– Ion masses differ => time of flight differs
(Smaller or more highly charged ions reach detector
first)
mv 2
Kinetic energy, Ek =zVs =
2
d charge, q = ze
Total
Time of flight, t=
v
2
m  d2  m  t
t  
2
   2eVs  
z  2Vs e  z  d
39
Design/Methods – Detectors
Detect charged ion
– Intensity, position, and/or timing of arrival
Dynodes/Faraday Cup/Electron multiplier
– Ions strikes metal plate, change in charges
– Measurable current flow
– Electron multiplier: cascaded dynodes
Photomultiplier
– Scintillation: Production of photons as ions
strike phosphorescent surface
40
 Results/Data
Main performance characteristics
– Upper mass limit ~ highest m/z measurable
– Transmission ratio ~ ratio of number of ions
reaching the detector to that produced at
source
– Resolution ~ ability yield distinct signals
Upper mass limit improved
– by new ionization sources (ESA, MALDI)
– from 650 Da to >12,000 Da

41
 Results/Data
Improved resolution & sensitivity of mass
analyzers and through use of tandem
spectrometers

Resolution capabilities of normal mass spectrometers Resolution capabilities of MALDI Tof-Tof spectrometer
(from the British Mass Spectrometry Society, www.bmss.org.uk) (from the British Mass Spectrometry Society, www.bmss.org.uk)
42
 Results/Data
Overall performance improvements
(greater analytical capabilities) by tandem
mass spectrometry

A typical result from tandem mass spectrometry

(from University of Leeds MRes Informatics, www.bioinf.leeds.ac.uk/mres/home.html )
43
 Discussion: Fourier
Transform MS
IonSpec’s Explorer Mass Spectrometer
– State of the art commercially available product
– Utilizes both ESI & MALDI ionization sources
– Uses Fourier transform ion cyclotron resonance mass
spectrometry (FTMS) for mass analysis & detection
– Quick analysis, extremely high resolution (869,000
Da), high accuracy (0.9 ppm) and good sensitivity
(50:1 Sound-noise ratio)

44
 Discussion: Fourier
Transform MS
Theoretically
unlimited mass limit
– Use of ion trap
Speed of analysis,
resolution,
accuracy
– Measures whole
frequency spectrum
Picture of IonSpec’s Explorer Mass Spectrometer
– Frequency (from IonSpec, www.ionspec.com)

measurement most
accurate
45
 Conclusion
Mass spectrometry is a valuable tool
– Current resolution, sensitivity, accuracy & mass limit
capabilities are pretty good => enable much analysis
of peptides, oligonucleotides, proteins
Technology will improve further
– Current limitations: high cost associated with high
performance (tandem) mass spectrometers, slow
analytical times due to complex molecules
– Future: Further technological advances, more
widespread usage (economies of scale) and even
higher computer processing power

46
High Performance Liquid
Chromatography

47
 What is HPLC?

High Performance Liquid Chromatography

(HPLC) is a chromatographic method used to
separate molecules from an overall larger
mixture
– Molecule size range: large proteins and
polynucleotides to smaller inorganic molecules
– Soluble and insoluble

The term itself was coined to describe the

separation of molecules under high pressure
in a stainless steel column filled with a matrix
48
 HPLC History

 HPLC was first developed in the mid 1970’s as an

evolution of high pressure liquid chromatography
– Before this time, most scientists had to rely on methods such
as open-column, paper, and thin-layer chromatogrpahy
– These methods were inadequate for the quantification of
compounds and the resolution of similar compounds
 By the 1980’s, HPLC became the dominant assay
– New techniques improved separation, identification,
purification, and quantification far above previous methods
– Moreover, computer analysis tools and automation made these
HPLC systems even more convenient
 Currently, HPLC is used by a variety of fields including
biomedical research, the cosmetics, food, and energy
industries and numerous environmental groups
49
 Background
Liquid Chromatography (LC)
LC is a chemical separation process that uses a
mobile and stationary phase to purify mixed solutions
into individual compounds. The separation occurs
from the subtle chemical interactions of the mixture
with the two phases. This causes different
compounds to travel faster or slower than others
based on those chemical interactions, which is then
recorded by the detector.

High Performance Liquid Chromatography (HPLC) Figure 15

As instrumentation for LC became more advanced, it An example of an HPLC output. A plant

extract has been separated to detect
became necessary to use high pressure pumps to ecdysteroids.
force the flow of mixed solutions through the phases.
These advances allowed for better detection, higher
sensitivity, and faster analysis. This became known
as high pressure LC. Later on, when low pressure
techniques were also developed, the name was
changed to high performance LC.

50
 Basic HPLC Theory

HPLC works on the principle of absorption

followed by elution
Two main sections: Mobile Phase and
Stationary Phase
– Mobile Phase: Liquid carrier for the sample solution;
refers to the continuous application of the solvent with
sample to the column
– Stationary Phase: The solid support structure,
contained within the main column, that the mobile
phase continuously flows over; sample molecules
migrate through and get eluted from the stationary
phase
51
 Basic HPLC Theory

 While in contact with the mobile

phase, the stationary phase takes
away molecules of the sample in a
selective manner
 The sample molecules form
distinct bands in the column and
move through the stationary phase
according to their absorption
properties
 Later on in the process, the mobile
phase takes the absorbed sample
away from the stationary phase in
order to separate the various
components
 The stronger the interaction of the
sample with the stationary phase,
the longer it takes for the mobile
phase to pull it back out, and vice
versa
52
 Theory
Introduction Stationary Phase
Chromatographic separation is based on
chemical properties such as: Small porous solid particles are packed tightly
– Polarity inside the column. This packing is called the
– Size, Molecular Weight stationary phase. The size of the particles, size of
– Hydrophilicity the pores, and chemical properties on the surface
– Functional Groups of the particles affect how the analyte and mobile
The analyte (mixture to be separated) is phase will react to it. Particle sizes are usually
dissolved into the mobile phase (eluent) and between 3 to 5 micrometers3.
then pumped into a column containing the
stationary phase. As the analyte passes Separation
through the column, interactions between it
For example, if the stationary phase consists of
and the phases will separate the individual
components of the analyte. large, densely packed particles, then smaller
analyte components will travel faster through the
Mobile Phase column, and will be detected earlier. If the
The mobile phase is a solvent that is particles have pores in them, then small
continuously passing through the column’s compound will become trapped inside. Thus,
stationary phase. It allows the analyte to flow larger components would travel faster in this case.
through the column. The properties of the
mobile phase are defined by which type of
HPLC method is being used. Normally, the
chemical properties of the mobile phase are
very different from the stationary phase to
maximize separation.

53
 Test Results From Column

 The elution profile shows the

results from a BIOAdvantage
HL C18 4.6 x 150mm
column with Hodges Peptide
test mix, a challenging
peptide mixture that is used
to quantify HPLC peptide
column performance
 Shows very little noise and
good resolution, despite the
similarity of the compounds Fig 8: Hodges Peptide test mix with peak identities:
1. H2N-RGAGGLGLGK-amide
 Good reproducibility of 2. AC-RGGGGLGLGK-amide
results 3. AC-RGAGGLGLGK-amide
4. AC-RGVGGLGLGK-amide
5. AC-RGWGLGLGK-amide

54
 Stationary Phase Types
 Liquid-solid
– Based on polarity; polar
compounds bind more strongly
 Liquid-liquid
– Same as liquid-solid but better for
medium polarity entities
 Size-exclusion
– Based on molecular size; larger
molecules are excluded and elute
first
 Ion-exchange
– Selective exchange of ions in
sample with counterions in
phase; sample is retained by
replacing its ions with the
counterions
 Normal phase
– Stationary phase is polar,
mobile phase is non-polar;
non-polar (hydrophobic)
compounds elute faster
 Reverse phase
– Stationary phase is non-
polar (n-alkyl ligands),
mobile phase is polar; polar
(hydrophilic) compounds
elute faster
 Affinity
– Use immobilized
biochemicals that have a
specific affinity for the
compound of interest; other
compounds elute faster
55
 Mobile Phase Types
 Isocratic
– Compounds are eluted with a mobile phase of constant composition
– Advantages: Simple and inexpensive
– Disadvantages: Questionable resolution and extended retention of
certain compounds
 Gradient
– Uses increasingly stronger organic solvents in order to elute
different compounds; strength increased in a stepwise or linear
manner
– Same results as Isocratic but better bandwidth of elution profile
peaks
 Polytyptic
– Mixed-mode; uses many different techniques in the same column
– The columns contain rigid, macroporous hydrophobic resins
covalently bonded to a hydrophilic organic layer. By changing the
mobile phase, the mode of separation is thereby changed which
allows the user to achieve the desired selectivity in the separations

56
 Applications: Peptide/Protein HPLC using
Reverse Phase Chromatography (RPC)
 Five main categories of
HPLC applications:
– Purification
– Identification
– Preparation (combination of
purification and identification)
– Quantification
– Chemical Separation
 Myriads of molecules and
compounds that can be
studied by simply changing
mobile and stationary phase
conditions
 RPC: the most common form of HPLC
 A common and vital step in the
process of synthetic peptide
production and the purification of
natural peptide sequences
– Uses analytical columns that are more
suited towards identification and
quantification, but preparative in nature
due to the low number of active
proteins in tissue
– Biological activity is usually
maintained, as well as secondary and
tertiary structure of the protein
 Most commercially available RPC
columns use C8 (octyl chain) or C18
(octadecyl chain) phases to retain
hydrophobic peptide residues 57
 RPC HPLC of
Proteins/Peptides
 The BIOAdvantage column manufactured by Thomson Instruments
is one of the more reputable RPC columns on the market
 Specifications
– Silica based (ultra clean silica)
– 3 or 5 μm particle size
– 100Å or 300Å pore size
– C8 or C18 phases
– 0.17 to 4.6mm interior diameter
– 30 to 250mm in length
 High resolution with equal selectivity between all model variations:
allows user to maintain elution order throughout different
experiments => better reproducibility
 Modular design: can be used in almost any machine through the use
of a column-specific, adapter-like guard column

58
 Theory
Types of HPLC Detection
Adsorption The detection of compounds inside the column
are recorded with respect to time. A voltage
An adsorbent stationary phase is used to output is correlated to the detection peak. Fig 2
separate compounds with repeated shows a sample output2.
adsorption-desorption processes. There are
The capacity factor, k’, gives the peak of the
two types of adsorption HPLC:
eluting compounds relative to the unretained
– Normal Phase: a polar stationary phase with a solute.
nonpolar mobile phase t t
k' R 0
– Reverse Phase: a nonpolar stationary phase t0
with a polar mobile phase Resolution tells how close the spacing between
two peaks can be.
Ion-Exchange
t R 2  t R1
Ionically charged stationary phase is used to Rs 
0.5( w1  w2 )
attract oppositely charged particles in the
analyte.

Size Exclusion
Smaller molecules are separated from the
larger ones in the analyte by packing pores
of a specific size inside the column.

59
Fig. 2
 Instrumentation
HPLC System
Figure 3 shows the general setup for an
HPLC system. The sequence of events
are as follows:
1. The mobile phase is fed into
the pump, which pushes it
through the tightly packed
column.
2. As the mobile phase is
flowing through the column,
the analyte is injected via the
injection valve.
3. The loop shown in Figure 3 is
used to maintain consistency
in flow and pressure as the
analyte enters the column.
4. The detector’s output is then
sent to the data station.
5. The user controls all
configurations through the
interface, which can be Fig. 3 HPLC system setup1
manual or computer based.

60
 Instrumentation
Pump
The pump maintains the constant flow of eluent
and analyte into the column. Standard HPLC
pump requirements are1 :
– Flow rate range: from 0.01 to 10 ml/min
– Pressure range: from 1 to 5,000 psi
– Pressure pulsations: less than 1% for normal and
reversed phase mode less than 0.2% for size
exclusion mode.
• The most popular type is the reciprocating piston
pump (fig. 4). This pump allows for an unlimited
supply of eluent. However, because of its nature,
Fig. 4 Reciprocating piston pump
a pulse damper or dual reciprocating piston pump
must be used to counter the pulse effects (fig. 5).
Injector Column
The injector allows the analyte to enter the Columns are normally 5 to 25 cm in length with an
column without interrupting the mobile phase inner diameter of about 4 mm3. They are usually
flow, because the detector is sensitive to changes made of stainless steel because it is relatively inert
in flow rate and pressure. and can withstand high pressures. Columns typically
have an internal pressure of 24 to 100 bar2. The
stationary phase packing material is dependent on the
HPLC method and analyte composition. The most
common packing material is silica.

61
 HPLC Systems

 All HPLC systems have the same basic Protects column from
components; the only system innovation dirt/contaminants and
deals with the detector (RI, UV, MS, Near-IR, adapts it to other HPLC
NMR, LS) and the computer system systems
 Different molecules can be studied by
changing the specific combination of the
mobile phase, stationary phase, and detector

Delivers solvent at
Simple jar to hold solvent uniform rate; high
pressure b/c SP is Loads sample and injects it into mobile phase
small, tight particles
Solvent Injection Guard
Pump
Reservoir Port Column

Column

Waste
Detector Back Pressure Regulator Reservoir

Computer Simple jar to hold waste

Holds stationary Phase Modern HPLC System 62

 Instrumentation
Detector Photodiode Array (PDA)
There are many different types of detectors
that can be used with HPLC. The key The PDA is similar to the UV detector but it allows
characteristics of all are: for further qualitative analysis by being able to
– Low drift and noise detect absorption at specific wavelengths.
– High sensitivity Fluorescent
– Fast response
– Linearity A beam of xenon or deuterium radiation is emitted
– Low dead volume inside the flow cell to detect fluorescence of
– Insensitivity to changes in conditions conjugated systems of various compounds.
Detectors are usually attached to a separate Nuclear Magnetic Resonance (NMR), Mass
cell at the end of the column. The following
are the different types: Spectrometry (MS)
Refractive Index (RI) The latest advancements in HPLC deal with
combining the separating process of HPLC with
RI detector is the only universal HPLC analytical detection methods of NMR and MS.
detector. It simply measures the change in The combination gives faster identification of
refractive index of the solution flowing inside
the column. molecular species in one single test5.

UV Absorbance
The absorbance of UV light is detected based
on the Beer-Lambert law: A = ecb where A =
absorbance, b = length of cell, and e = molar
absorptivity2.

63
 Applications
Uses for HPLC
Chemical Detection in Food
HPLC is used in a wide variety of fields,
including pharmaceutical, petrochemical, Here, food dyes were analyzed (fig. 6) and then
and electronic industries. The following are detected in everyday foods, such as orange soda (fig
just a few examples of HPLC applications. 7)7.

Genetic Sampling
HPLC can be used to separate and analyze
various DNA components. Figure 5 shows
data from a mixture of purine bases,
nucleosides, and nucleotides using on-line
reverse phase (ODS) HPLC with anion
exchange HPLC6.
Fig. 6 Food dye detection

Fig. 5 Genetic material mixture Fig. 7 Food dye detection

Peaks: 1 – 8 = bases and nucleosides in orange soda 64
9 – 21 = nucleotides
 Conclusion
HPLC has proven to be the most powerful In addition to these benefits of using
of all chromatographic techniques. The HPLC, advancement in HPLC technology
benefits of HPLC are: are continuously being made. Future
– Separation and detection of focus will be on optimizing HPLC-NMR-
compounds that would be difficult or MS systems.
impossible to detect in other types of
separation methods
– Can separate a wide range of
compounds in a single run
– Versatility in choosing mobile and
stationary phase types
– Fast and highly efficient
– Affordable

References
1. Yuri Kazadevich, “Textbook on High Performance Liquid Chromatography”, http://hplc.chem.shu.edu/HPLC/index.html, last
modified January, 2002.
2. Sandie Lindsay, “High Performance Liquid Chromatography, Second Edition”, John Wiley & Sons, Inc, New York, 1992.
3. John G. Dorsey, "Liquid chromatography", in AccessScience@McGraw−Hill,
http://www.accessscience.com/server−java/Arknoid/science/AS/Encyclopedia/3/38/Est_386200_frameset.html, last
modified: June 3, 2002.
4. Richard A. Henry, “A Perspective on High-Performance Liquid Chromatography”, American Laboratory, June 2002, 25-31.
5. I. D. Wilson, "HPLC−combined spectroscopic techniques", in AccessScience@McGraw−Hill,
http://www.accessscience.com/server−java/Arknoid/science/AS/ResUpdates/2002/YB_020515_frameset.html, last
modified: March 25, 2002.
6. Richard L. Patience, and Elizabeth S. Penny, HPLC in Endocrinology, Advances in Chromatography 1987; 27: 38-69.
7. Harold T. McKone, “An Introduction to High Performance Liquid Chromatography: Separation of Some FD&C Dyes”, Journal of
65
Chemcial Education, 1980, 57:321-2.
Nuclear Magnetic Resonance

An Introduction to
Spectroscopy

66
 Introduction
Was discovered by Bloch and Purcell
in 1946
Is used as a cross-disciplinary tool
Relies on nuclear spins excited by
external magnetic fields
Is rich in measurable characteristics
(strength, frequency, decay) that
convey details of structure and
environmental interaction
67
Nuclear Magnetic Resonance

 Main magnetic field

– Align all spins with main
field
– Larmor frequency
ω0 = γB0 Fig. 1. Equilibrium condition due to
main magnetic field

 Radiofrequency pulse
– Excite all nuclei
– Tilts spins by 90°
– Spins precess back to Fig. 2. Magnetization after a 90°
equilibrium state RF pulse

D. Nishimura, Principles of Magnetic Resonance Imaging, MRSRL Press, 1996.

68
 Applications in NMR
Spectroscopy
 Elements can be identified
and their atomic and
molecular structure
elucidated by studying the
spectral lines

 Use magnetic fields to

excite material; then
measure change in Fig. 3. 31
P NMR Spectrum

magnetic moment

 Chemical shift
D. Nishimura, Principles of Magnetic Resonance Imaging, MRSRL Press, 1996.
69
 Spectroscopy
Instrumentation
Continuous wave

Fourier transform

Operation: Fig. 4. Free Induction Decay

– Select sample
– Pulse sample
– Measure FID
– Find Fourier
transform of FID
– Make prediction Fig. 5. Fourier transformed FID

J. Edwards, “Principles of NMR,” Process NMR Associates LLC, 1999.

70
Foxboro Process NMR
Analyzer
Seven components:
– Central computer
– Switching control unit
– Shimming control unit
– Power supply
– Magnet
– Probe
– Heater control unit Fig. 6. Foxboro Process NMR
Analyzer, Model NMRB, Style C

Foxboro Company, The, “I/A Series: Process NMR Analyzer, Model NMRB, Style C,”
Datasheet, The Foxboro Company (Foxboro, MA), 2000. 71
 Research Involving
Spectroscopy
 Chemistry Department:
– Molecular dynamics
– Inter-molecular recognition
– Protein organization

 Kennedy Krieger Institute

– fMRI and NMR spectroscopy
– Cellular function in brain
Fig. 7. Human spectroscopic data
– Metabolite levels and fluxes for several slices of the brain
– Relation to diseas
A. Horska, W.E. Kaufmann, L.J. Brant, S. Naidu, J.C. Harris, and P.B. Barker, “In vivo quantitative
proton MRIS study of brain development from childhood to adolescence,” Journal of Magnetic
Resonance Imaging, vol. 15(2), pp. 137 – 43, 2002. 72
 Current Issues in
Spectroscopy
 Technology
– Stronger signal
– Higher bandwidth
– Larger external
magnetic fields
– Hybridized magnets

 Research
– Improve signal quality,
acquisition speed and
SNR
Fig. 8. Department of Energy’s
– Hardware new 900 MHz magnet (largest in
the world)
– Procedures

S. Maloof, US Department of Energy, “World’s largest, most powerful NMR spectrometer,”

US Department of Energy Research News, Apr. 2002. 73
 Summary
Strong correlations between NMR
spectra and molecular organization
Reasonable predictions for structure
and function of unknown substances
Growing understanding of organ
function in medical studies
Developing technology spanning
many fields and applications
74
 References
 E. Atalar, course notes for 580.473: Magnetic
Resonance Imaging in Medicine, Johns Hopkins
University, Department of Biomedical Engineering,
Baltimore, MD, unpublished, 2003.
 F. Bloch, “Nuclear induction,” Physical Review, vol. 70,
pp. 460 – 473, 1946.
 P. Bottomley, “Instrumentation for MRI and MRS,”
course notes for 580.473: Magnetic Resonance
Imaging in Medicine, Johns Hopkins University,
Department of Biomedical Engineering, Baltimore, MD,
unpublished, 2003.
 P. Bottomley, “Introduction to magnetic resonance
spectroscopy in biomedicine,” course notes for
580.473: Magnetic Resonance Imaging in Medicine,
Johns Hopkins University, Department of Biomedical
Engineering, Baltimore, MD, unpublished, 2003.
 P. Bottomley, “Spatially localized magnetic resonance
spectroscopy,” course notes for 580.473: Magnetic
Resonance Imaging in Medicine, Johns Hopkins
University, Department of Biomedical Engineering,
Baltimore, MD, unpublished, 2003.
 Bruker-Biospin, NMR – Products – Systems,
http://www.bruker-
biospin.com/nmr/products/systems.html, 2003.
 Committee for High Field NMR, The, “A New Millenium
Resource,” report, unpublished, 2000.
 J. Edwards, “Principles of NMR,” Process NMR
Associates LLC, 1999.
 Foxboro Company, The, “I/A Series: Process NMR
Analyzer, Model NMRB, Style C,” Datasheet, The
Foxboro Company (Foxboro, MA), 2000.
 A. Garroway, P. Grannell, and P. Mansfield, “Image
formation in NMR by a selective irradiative pulse,”
Journal of Physics C: Solid State Physics, vol. 7, ppl.
L457 – L462, 1974.
 E. Haacke, R. Brown, M. Thompson, and R.
Venkatesan, Magnetic Resonance Imaging: Principles
and Sequence Design, Wiley, 1999.
 A. Horska, W.E. Kaufmann, L.J. Brant, S. Naidu, J.C.
Harris, and P.B. Barker, “In vivo quantitative proton
MRIS study of brain development from childhood to
adolescence,” Journal of Magnetic Resonance
Imaging, vol. 15(2), pp. 137 – 43, 2002.
 P. Lauterbur, “Image formation by induced local
interaction: Examples employing nuclear magnetic
resonance,” Nature, vol. 242, pp. 190 – 191, 1973.
 S. Maloof, US Department of Energy, “World’s
largest, most powerful NMR spectrometer,” US
Department of Energy Research News, Apr. 2002.
 D. Nishimura, Principles of Magnetic Resonance
Imaging, MRSRL Press, 1996.
 E. Purcell, H. Torrey, and R. Pound, “Resonance
absorption by nuclear magnetic moments in a solid,”
Physical Review, vol. 69, pp. 37 – 38, 1946.
 J.R. Tolman, H.M. Al-Hashimi, L.E. Kay, and J.H.
Prestegard, “Structural and dynamic analysis of
residual dipolar coupling data for proteins,” Journal of
the American Chemistry Society, no. 123, pp. 1416 –
1424, 2001.
 R. Weiss, “Two developers of MRI awarded Nobel
Prize,” Washington Post, Oct. 7, 2003, p. A02, 2003.
75
 Introduction
What is LC Technology?
 Lab-on-a-Chip (LC) is a new
technology developed from
microelectronics and
telecommunications that
incorporates microarray,
microfluidic and
microelectromechanical
systems

 LC has made possible rapid

miniaturization and low-cost
production of biological
laboratory analysis and
diagnostic testing

76
Introduction
LC Technology Applications
 Integrated into portable
devices for high
performance of multiple
applications and tens-of-
thousands of laboratory
tests

 Examples of applications
– Identification and
replication of specific
sequences of DNA
– Diagnosis of diseases
– Detection of biotoxins and
explosives 77
Introduction: Main
Processes/Characteristics
Two main processes performed on LC
microchips
– Chemical separation
– Chemical analysis

Main characteristics of LC microsystems:

– Reduced processing time
– Maximized sensitivity from minimal reagent input
– Low false positive rate
– Minimal power requirements
– Flexibility and reusability
78
 Basic Principles
Chemical Separation
Two major categories:
– Gas or Liquid Chromatograph – powerful
and widely-used separation techniques

– Electrokinetics – used for separation of

charged particles
• Electrophoresis
• Electroosmosis – less often used

79
 Basic Principles
Chromatography
Micro-distances on LC microsystem minimize
diffusion distances and maximize the use of gas and
liquid chromatography
Liquid chromatography
– Use open tubes etched in spiral
configuration
– Some use particle-packed columns
• Difficult to pack and may prevent
uniform particle densities
• Etched columns help resolve
problems with packing and particle
densities
80
 Basic Principles
Electrokinetics
Electroosmosis
– Uses continuous flow and an electrical potential to
separate charged particles
– Electrical potential minimizes back flow of particles
Electrophoresis
– Used most often in LC microsystems
– Separates larger molecules such as DNA or other
proteins
– Often used in conjunction with PCR
– Electric field separates particles based on size and
polarizability
81