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Tech Tip

The Role of Isotope Peak Intensities

Obtained Using MS in Determining an
Elemental Composition, Part 1

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Analysis of beer using time-of-flight technology for gas
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Issue 5: September 2010

The Role of Isotope Peak Intensities Obtained

Using Mass Spectrometry in Determining an
Elemental Composition, Part 1
O. David Sparkman

The atomic mass of an element is the weighted average of the masses of the naturally
occurring isotopes of that element. These dierent isotopes of an element have
dierent masses that are almost an integer in value because of dierent numbers of
neutrons in their nuclei; e.g., carbon has two primary naturally occurring isotopes, 12C
and 13C. These two isotopes have respective integer masses of 12 and 13. Atoms of 12C
have one less neutron in their nuclei than do atoms of 13C. This means that a mass
spectrum of an ion containing carbon will be represented by peaks that are one m/z unit
apart. The lowest m/z value peak represents an ion where all the carbon atoms are 12C.
The peak one integer m/z value higher represents an ion where one of the carbon atoms
is 13C. The peak one m/z unit higher than that represents the ion where two of the
carbon atoms are 13C and so on.
The lowest m/z value peak expressed as
an integer is referred to as the nominal
mass1 peak or the nominal m/z value peak.
The peaks representing ions that are one
mass unit greater than the nominal mass
peak are isotope peaks. The intensity of the
isotope peaks relative to the intensity of
the nominal mass peak can often be used
in the determination of an ions elemental
composition. This is possible because the
abundances of the various isotopes of an
element are constant. The intensities of
the isotope peaks relative to the nominal
m/z value peak are because of the number

of atoms of the element present and the

probability of finding an ion containing one
of these high-mass isotopes based on their
natural abundances.
The elements and their isotopes
The common elements encountered in organic
mass spectrometry are carbon, hydrogen,
nitrogen, oxygen, silicon, sulfur, phosphorus,
and the halogens (F, Cl, Br, and I). Potassium
and sodium need to be added to this list
because of their ubiquitous presence in
ions produced by electrospray, which has
become an important part of todays mass

1 The nominal mass of an element is the integer mass of the most abundant naturally occurring isotope of the element. The nominal mass of an elemental composition is determined from the number of each
element in that composition and the nominal mass of those elements.
2 Many authors use the symbols A, A+1, and A+2 because A is the IUPAC symbol for an elements mass number. The peak representing the various isotopic compositions of an ion can have any m/z value;
therefore, it is felt that X is a more appropriate symbol.

spectrometry. Table 1 shows these elements

and how they are categorized. These elements
are separated into three primary types: X, X+1
and X+2.2
The X elements are those that have ONLY
ONE naturally occurring isotope (P, F, I, and Na).
The X+1 elements are those that have ONLY
TWO naturally occurring isotopes, and they
are ONLY ONE MASS UNIT different (C and N).
The X+2 elements are those that have isotopes
that DIFFER BY TWO MASS UNITS. It does not
matter how many isotopes an X+2 element
has, only that it has isotopes that are two mass
units apart (O, Si, S, Cl, K, and Br). Hydrogen
has two naturally occurring isotopes that are
one mass unit apart; however, the amount
of deuterium (2H) relative to the amount
of protium (1H) is so small that hydrogen is
considered to be an X element for all CHcontaining ions under 1,000 Da (daltons).
If an ion contains a single atom of carbon,
such as the molecular ion (M+) of methane
(CH4), the intensity of the X+1 isotope peak
will be the relative percent abundance of 13C
(abundance of 13C / abundance of 12C 100%
= 1.1% / 98.91% 100% = 1.1%). The number
1.1 is the X+1 factor for carbon. The X+1 factor
times the number of atoms of carbon presence
in an ion will equal the intensity of the X+1
peak relative to the nominal m/z value peak.
If there is only one atom of carbon in the ion,
there is a 1.1% probability that there will be
an ion with an atom of 13C; however, if the ion
has two atoms of carbon, there is twice the
probability that there will be an ion with a
single atom of 13C. If the ion has ten atoms of
carbon, there is ten times the probability that
an ion will be found with one atom of 13C than
there is when the ion has only a single carbon
atom. These increased probabilities result in
increased relative intensities of the X+1 isotope
peak. Setting the M+ peak of benzene (m/z 78)
to 100% means that the intensity of the X+1
peak will be 6.6% (the carbon X+1 factor times

Table1

Type

Element

Symbol

Integer
Mass1

Exact
Mass2

Percent
Abundance

Hydrogen

1.0078

99.99

D or

2.0141

0.01

12

12.0000

98.91

13

13.0034

1.1

14

14.0031

99.6

15

15.0001

0.4

16

15.9949

99.76

17

16.9991

0.04

18

17.9992

0.20

19

18.9984

Si

28

27.9769

92.2

X+1
Factor 3

X+2
Factor 4

1.1nC

0.0060nC2

X+1

Carbon

12

13

X+1

Nitrogen

14

15

X+2

Oxygen

16

17
18

Fluorine

X+2

Silicon

28

Si

29

28.9765

4.7

Si

30

29.9738

3.1

Sodium

Na

23

22.9898

100

Phosphorus

31

30.9738

100

X+2

Sulfur

32

32

31.9721

95.02

33

32.9715

0.76

34

33.9679

4.22

Cl

35

34.9689

75.77

Cl

37

36.9659

24.23

39

38.9637

93.26

40

39.9640

0.013

41

40.9618

6.74

Br

79

78.9183

50.5

Br

81

80.9163

49.5

127

126.9045

33

34

X+2

Chlorine

35

37

X+2

Potassium

39

40
41

X+2

Bromine

79

81

Iodine

0.04nO
0.20nO

100

30

29

0.37nN

5.1nSi
3.4nSi

0.8nS
4.4nS
32.5nCl
0.012nK
7.22nK
98.0nBr

100

Table 1: List of the common elements encountered in mass spectrometry along with their types and isotope abundance factors.
The integer mass of the most abundant* naturally occurring stable isotope of an element is the elements nominal mass. The nominal mass of an ion is the sum of the nominal masses of the elements in its
elemental composition (e.g., C3H6O+ has a nominal mass of 58).
The exact mass of the most abundant* naturally occurring stable isotope of an element is the elements monoisotopic mass. The monoisotopic mass of an ion is the sum of the monoisotopic masses of the elements in its elemental composition (e.g., C3H6O+ has a monoisotopic mass of 58.0417).
3
Assume X = 100%; X represents the relative intensity of the first peak in a cluster of peaks corresponding to isotopic variants of a given ion.
4
The factor is multiplied by the number (n) of atoms of the element present to determine the magnitude of the intensity contribution for a given isotope. For example, the contribution at X+1 because of 15N for
an ion containing three nitrogens would be 0.37 3 = 1.11 relative to 100 at X.
* This may not always be the lowest mass naturally occurring stable isotope of the element, as is the case with the elements in this table. The lowest mass isotope of Hg is 196 and the nominal mass isotope is
202, seventh from the lowest mass isotope.
1

the number of atoms of carbon in benzene (6).

As is seen in Table 1, carbon has an X+2
factor. This is not because of the presence of
14
C. If 14C was a factor, carbon would be an
X+2 element rather than an X+1 element.
The abundance of 14C is so small, it is of no
consequence. The X+2 factor of carbon relates
to the probability of the ion having two atoms
of 13C. This probability is much smaller than
the probability that an ion will have a single
atom of 13C. Consider the binomial expression
(a + b)n where a is the percent abundance
of the most abundant isotope, b is the
abundance of the second isotope of an X+1
element, and n is the number of atoms of the
X+1 element. Solving this expansion results
in an X+2 factor for the probability of having
two atoms of carbon being 0.0060 times the
square of the number of carbon atoms (0.006
nC2). Carbon is usually the most ubiquitous
element in ions encountered in organic
mass spectrometry; therefore, based on the
large numbers of carbon atoms that may be
present and the abundance of 13C, the X+2
factor for carbon can be significant as seen in
Table 2.
The contribution to the relative intensity
of each element present can be considered
additive. The relative intensity of the X+1 peak
can be considered to be the intensity of the
contribution resulting from the probability of
an ion with an atom of 13C (nC 1.1) plus the
contribution from the ion having an atom of
15
N (nN 0.37) plus the contribution of the
ion having an atom of 33S (nS 0.8) and so
on. The same is true for the relative intensities
of the X+2 peaks; i.e., (nC2 0.0060) + (nCl
32.5) + (nO 0.2) + (and the number of atoms
of the other X+2 elements times their X+2
factors).

Table 2

C Number

X+1

X+2

C Number

X+1

X+2

C1

1.1

0.01

C21

23.1

2.6

C2

2.2

0.02

C22

24.2

2.9

C3

3.3

0.05

C23

25.3

3.2

C4

4.4

0.10

C24

26.4

3.4

C5

5.5

0.15

C25

27.5

3.7

C6

6.6

0.22

C26

28.6

4.1

C7

7.7

0.29

C27

29.7

4.4

C8

8.8

0.38

C28

30.8

4.7

C9

9.9

0.49

C29

31.9

5.0

C10

11.0

0.60

C30

33.0

5.4

C11

12.1

0.73

C31

34.1

5.8

C12

13.2

0.86

C32

35.2

6.1

C13

14.3

1.0

C33

36.3

6.5

C14

15.4

1.2

C34

37.4

6.9

C15

16.5

1.3

C35

38.5

7.3

C16

17.6

1.5

C36

39.6

7.8

C17

18.7

1.7

C37

40.7

8.2

C18

19.8

1.9

C38

41.8

8.7

C19

20.9

2.2

C39

42.9

9.1

C20

22.0

2.4

C40

44.0

9.6

Table 2: X+1 and X+2 relative intensities for ions containing atoms of carbon and hydrogen.
The isotopic contributions from other elements are additive in experimental data (e.g., the X+1 contributions from N, O, S, etc. are combined with the above-listed X+1 contribution from carbon; similarly,
the X+2 contributions from elements that have X+2 isotopes are combined with the X+2 contribution from carbon).

Elemental composition based on isotope

peak intensities
Just as the intensity of the isotope peaks
relative to the nominal m/z peak can be
predicted, the intensity of the isotope
peaks in the mass spectrum of an unknown
relative to the intensity of the nominal
m/z value peak can be used to propose an
elemental composition. Having an elemental

composition is an important step in the

identification of an ion.
Most mass spectrometers used in organic
chemistry produce spectra where the
isotope peak intensities are no better than
10% of the theoretical values. Better values
can be obtained using isotope ratio mass
spectrometers, but these instruments will not
be considered in this discussion. The accuracy
and precision of isotope peak intensities

is highly dependent on signal strength. The

precision, from spectrum to spectrum, is much
better when the signal strength is high. The
accuracy will fall much closer to the theoretical
value within that 10% window when the
signal strength is high; however, the about
10% limit should be used as a guideline
when determining an elemental composition
using the relative intensity of isotope peaks.
Once a contribution has been reconciled to
within this 10% limit, move to the next step
in the procedure. Failure to do so may result
in over-interpretation of the data. It is always
possible to return to a previous step and make
adjustments.
The assignment of the various elements
must follow a specific order. The X+2 elements
should be assigned first. If the number of
carbon atoms are assigned before the X+2
elements, a serious error can result because
silicon and sulfur (as well as potassium) have
significant contributions at X+1. When the
number of X+2 elements are assigned, do
not consider oxygen. The number of atoms
of oxygen should not be assigned until the
number of atoms of carbon has been assigned.
The contribution at X+2 for a single atom of
oxygen is 2% (relative). The X+2 contributing
for an ion with five atoms of carbon is 0.22%.
Before considering the number of oxygen
atoms present, the contribution at X+2
because of the possibility of two atoms of
carbon 13 should be taken into account.
After assigning the number of X+2 elements
(with the exception of oxygen3), assign the
number of X+1 elements. If nothing is known
about the elemental composition of the ion,
start with carbon and then assign the number
of atoms of nitrogen. If the ion is known to be

a M+ and the m/z value is odd or it is known

to be a protonated molecule (MH+), hydride
abstracted molecule ([M H]), an electrospray
adduct ([M Na]+ or [M K]+), a deprotonated
molecule ([M H]+), or some negative ion
adduct such as ([M Cl]+) and has an even m/z
value, then the ion will have an odd number of
nitrogen atoms4. In this case, the reconciliation
should begin with a single atom of nitrogen. If
a reasonable solution cannot be reached with
a single atom of nitrogen, try three nitrogen
atoms. Remember, the contribution from three
atoms of nitrogen at X+1 is almost exactly the
same as the contribution due to one atom of
carbon.
After the number of X+1 elements have
been assigned, assign the number of atoms
of oxygen. The number of oxygen atoms can
be in error by 1. Do not exceed the mass
balance at this point regardless of what the
renaming unassigned isotope peak intensity
data indicates. The mass balance must always
reconcile to zero.
Once the number of atoms of X+2 and X+1
elements and the number of oxygen atoms
have been assigned, assign the number of X
elements (H, F, Na, P, and I). Sodium is only a
consideration when the data are acquired by
electrospray. If peaks representing sodium
adducts are present, there are usually peaks
representing the corresponding protonated
molecule 22 m/z units lower (unless there are
multiple-charge ions). It is always important to
keep in mind that the mass must reconcile to
zero.
After an elemental composition is proposed,
a good way to determine if it is unreasonable is
through a rings-plus-double-bonds calculation
(R+dB).

R+dB =

#C
Si

#H + #N
F
P
Cl
Br
I
Na
K

+1

Elements with the same valence are treated

the same. The number of atoms of oxygen
and sulfur are not used in the calculation;
however, the double bonds associated with
them are determined by the calculation
(provided these two elements are present with
a valence of 2). It is important to remember
that the R+dB calculation is ONLY VALID when
ALL the elements are at their lowest valence
state. That is to say, double bonds associated
with sulfur at a valence of 4 or a valence of
6 and phosphorous at a valence of 5 are not
determined by this calculation. A negative
value resulting from a R+dB calculation is
not valid. Too large of a value should be
questioned. A phenyl group results in a R+dB
count of 4. When a large number results from
a R+dB calculation, phenyl rings (or other
aromatic structures) should be considered. A
triple bond will result in 2 rings plus double
bonds.
If the R+dB calculation ends in a whole
number, the ion is an odd electron ion (OE).
If the calculation ends in , the ion is an
even-electron ion (EE)5. Whether the R+dB
calculation ends in an integer or can be
used as a validation of the proposed elemental

3 Only consider potassium when the data are generated by electrospray ionization.
4 The Nitrogen Rule is an important concept in mass spectrometry. The Nitrogen Rule states that any molecule that contains an odd number of nitrogen atoms will have an odd nominal mass; therefore, any molecular ion (M+) that has an odd m/z value will contain an odd number of nitrogen atoms. When that ion
fragments through the neutral loss of a radical and if the resulting ion retains an odd number of nitrogen atoms, the resulting even-electron ion will have an even mass. A corollary to the Nitrogen Rule is that all molecules that do not contain an odd number of Nitrogen atoms will have an even m/z value. Another
corollary to the Nitrogen Rule is that all even-electron single-charge ions like MH+ produced in ESI and chemical ionization that contain an odd number of nitrogen atoms will have an even m/z value.
5 Mass spectrometers measure the abundance of ions according to their mass-to-charge ratios (m/z). All ions have either a positive or a negative charge. All molecules have an even number of electrons and are neutral; therefore, they cannot be detected or separated according to their mass by a mass spectrometer.
The molecules are converted into ions. Electron ionization (EI) mass spectrometry results in an ion having the same elemental composition as the molecule but one less electron. This ion is called a molecular ion (M+). It is also a positive-charge odd-electron ion (OE+). Ions representing intact molecules formed by
electron capture ionization are also odd-electron ions (OE-). Ions formed by adduction of a proton or another cation (MH+ or MNa+), hydride abstraction ([M H]+), or proton abstraction ([M H]) are even-electron ions (EE). Fragment ions formed from molecular ions by the neutral loss of a radical are also evenelectron ions.

composition based on whether or not the ion

is presumed to be an odd-electron or evenelectron ion.
Another confirmation of an elemental
composition determined using isotope
peak ratios is to obtain accurate mass data
for the ion. When the mass of the ion can
be determined to the nearest 0.1 mmu
(millimass unit), it is usually possible to
propose a unique elemental composition
for ions that have masses under 500 Da.
Accurate mass information is much easier to
obtain than previously. Time-of-flight (TOF)
instruments will produce these types of data.
The TOF mass spectrometer does not always
produce good isotope peak intensity data,
but improvements are being made. There is
software (MassWorks from Cerno Bioscience,
http://www.cernobioscience.com/) that can
be used with data acquired in the profile
mode on standard transmission quadrupole
mass spectrometers such as those available
from Thermo Fisher, Agilent, Perkin-Elmer,
Shimadzu, Bruker, and others to produce
accurate mass information. These instruments
normally produce integer m/z data when
used in their native centroid mode, but can
yield information to provide unambiguous
elemental composition using MassWorks
when the data are acquired in the profile
mode. It should be noted that elemental
compositions from isotope peak data can
also be used for confirmation of elemental
compositions obtained from accurate mass
data.
Once the elemental composition has been
established, it can be searched against
various databases to determine possible
compound identification. Such databases
are the NIST08 Mass Spectral Database, the
Chemical Abstracts Service registry number
database, the Merck Index, etc. The Formula
Search in the NIST MS Search Program of the
NIST MS Database is very useful especially

for data that result from LC/MS applications.

These data usually have peaks representing
ions that have a mass one less or one more
than the mass of the analyte. This difference
needs to be taken into account by the
addition or subtraction of a H from the
formula that resulted from the elemental
composition determination. The returned
spectra from such a search also contain what
other databases the compounds are in and
synonyms that often include common/trade
names. Such information can be used to
restrict the number of possible candidates for
identification. Another point about isotope
peak intensities in LC/MS data is that the ion
abundances for the ions representing the
intact molecule are usually high and therefore
the intensities of the isotope peaks will be
more accurate than when the ion current is
low or dispersed over a number of m/z values.
In part 2 of this article, David Sparkman
provides an illustrative example of the use of
isotope peak ratios to determine an elemental
composition.
Suggested Additional Reading
J. Throck Watson and O. David Sparkman
Introduction to Mass Spectrometry:
Applications, Instrumentation and
Strategies for Data Interpretation; Wiley:
Chichester, UK, 2007.
R. Martin Smith, Understanding Mass
Spectra: A Basic Approach; Wiley: 2004.
Jrgen H. Gross Mass Spectrometry: A
Textbook; Springer: Berlin, 2004.
O. David Sparkman is currently an
Adjunct Professor of Chemistry at the
University of the Pacific in Stockton,
California; Consultant to the National
Institute of Standards and Technology

Mass Spectrometry Data Center; President

of ChemUserWorld.com; and a former
American Chemical Society Instructor and
American Society for Mass Spectrometry
Member-at-large for Education.
At the University of the Pacific he
teaches courses in mass spectrometry
and analytical chemistry and manages
the mass spectrometry facility. Over the
past 28 years, he has developed and
taught five dierent ACS courses in mass
spectrometry. He is the author of Mass
Spectrometry Desk Reference.
He also provides general consulting
services in mass spectrometry for a
number of instrument manufacturers,
manufacturing companies, and
government agencies.

Featured Applications
Application Note: ANBT10

Accurate and reliable analysis of beer using time-of-flight

technology for gas chromatography
Introduction
Any product that is derived primarily
from natural ingredients will inherently
comprise a tremendous range of
chemical components. Beer, usually
made from water, malt, hops and yeast, is
no exception. Variations in ingredients
give different beers their distinct
flavours. Typical compounds responsible
for flavour can have extremely low (ppt)
olfactory thresholds, meaning significant
compounds may be present at trace
levels. As such, component
concentrations within the beer may vary
by several orders of magnitude. Accurate
chemical profiling has previously proved
challenging when trying to
accommodate a sample with such
chemical complexity and dynamic range.

of sample matrix with physically large

sorbents, such as PDMS, is the key to the
sensitivity advantage over the
aforementioned techniques.
Sorptive extraction is complemented by
thermal desorption (TD), which
concentrates the sample, making ppt
detection possible, and provides efficient
injection directly into the GC.

at conventional SIM detection levels

whilst still producing classical spectra
that are compatible with both
proprietary mass spectral libraries and
commercial databases (e.g. NIST).

A reliable analysis of beer using time-of-flight technology for gas chromatography

Company: ALMSCO International

The high sensitivity BenchTOF-dx

(figure 2) may be used in combination
with GC for routine screening of the
chemical profile of a sample. In this
application, four commercially available
beers were selected and their chemical
profiles analysed to test the ability of the
TOF-MS to determine both high and low
level compounds.

Products such as beer, derived primarily from natural ingredients will inherently comprise
a tremendous range of chemical components. Typical compounds responsible for flavour
can have extremely low (ppt) olfactory thresholds, meaning significant compounds may
be present at trace levels. This application note from ALMSCO describes how SPEthermal
desorption offers a simple but very sensitive means of extracting volatile components from highly
complex aqueous products such as beer. In addition, the introduction of a novel time-of-flight MS
provided a detector which can fully characterise a sample of wide dynarange, providing sensitivity
even for very low level compounds.
Volatile extraction

It is essential to extract the widest

possible range of compounds from a
sample in order to comprehensively
characterise its chemical profile. Solid
phase micro-extraction (SPME) and
conventional equilibrium (static)
headspace are techniques which have
previously been used to extract volatile
organic compounds (VOCs) from liquid
samples; however, neither method
provides the sensitivity to allow full
characterisation of the VOC profile.

Sorptive extraction using SPE-tD

cartridges (figure 1) is a simple yet highly
sensitive technique used for extracting
organic compounds from aqueous
matrices. The enrichment factor possible
from thorough mixing of large volumes

Figure 1. Long, hollow, PDMS-coated SPE-tD

cartridges

MS capability

Recording full spectral information using

GC quadrupole MS (or MS-MS) in scan
mode leads to the identification of a
large range of compounds in a sample,
however it does not provide the required
sensitivity for the identification of tracelevel compounds. Therefore, any
important flavour compounds are
unlikely to be detected. Conversely,
selected (or single) ion monitoring (SIM)
conditions provide the sensitivity for
trace-level identification but preclude
the gathering of spectral information.
Advances in time-of-flight (TOF)
technology have resulted in the
development of a TOF-MS with the
ability to record full spectral information

Figure 2. BenchTOF-dx mass spectrometer

High/low analysis

To ensure trace-level components in a

particularly complex matrix are precisely
measured (i.e. for quantification), a highly
concentrated sample needs to be
introduced to the GC/MS; however this
may exceed the capacity of a highly
resolving column and result in detector
overload. By contrast, desorbing a
smaller sample to the column will allow
correct characterization & measurement
of the major components, but then the
low-level compounds will be lost.

WWW.ALMSCO.COM

Gwaun Elai Medi Science Campus | Llantrisant | RCT | CF72 8XL | United Kingdom
T: +44 (0)1443 233920 | F: +44 (0)1443 231531 | E: enquiries@ALMSCO.com

Download

Application Note: ANBT11

Comprehensive analysis of crude oil by two-dimensional GC

(GCxGC) and time-of-ight (TOF) MS
Introduction
Crude oil is the generic term for the
unrened ammable liquid that is mined
from the ground. It contains vast
amounts of organic compounds ranging
from light hydrocarbons to complex
biomolecules, derived from the remains
of ancient marine organisms and
bacteria.

One-dimensional gas
chromatography
Within the complexity of crude oil, the
compounds of most interest to the
petroleum industry are relatively volatile
(boiling points generally below 400C)
and non-polar, therefore separations are
predominantly performed by GC with a

non-polar column. The resulting

chromatograms are highly convoluted
and usually characterised by a matrix of
unresolved material that appears as a
signicant background hump beneath
the partially resolved non-polar
compound peaks (Figure 1).
A background subtraction/library search
method is able to identify the non-polar
compound peaks, but for those lost in
the matrix, there is little hope for a
positive identication. Incomplete
characterisation limits the understanding
of the oil samples geochemistry and
provides no insight into issues that might
arise during extraction, transport, or
rening. In addition, as the more easily
extracted light crude oils become
depleted, oil companies are moving to

reservoirs containing heavier crudes,

bituminous shales and tar sands. These
contain higher proportions of involatile,
polar compounds, exacerbating the
problem of incomplete analysis1 and
such constituents are thought to
contribute to the process of
solidication of oil in transmission
pipes. Others might have relevance to
the environmental impact of oil, a factor
that will gain further attention with the
introduction of programs such as
European REACH (Registration Evaluation
Authorisation and Restriction of
Chemicals), which focuses on the
identication of chemicals in order to
assess their toxicological impact.

Comprehensive analysis of crude oil by two-dimensional GC (GCxGC) and

Time-of-Flight (TOF) MS
Company: ALMSCO International

Comprehensive (2D) gas

chromatography

This application note demonstrates the capable of producing classical spectra that are
comparable to commercial spectral libraries. The high sampling frequency of BenchTOFdx enables the spectral quality and sensitivity to be realised in this GCxGC application
and, equally, any high-speed GC analysis. This capability would benefit laboratories without the
time, budget or ability to develop custom spectral libraries, or those possessing proprietary spectral
libraries previously developed on quadrupole systems. The power of this abilityillustrated here in the
separation and identification of hopanes in a sinanalysis.
Figure 1. GC/MS total ion chromatogram of a sample of lubricating oil (a distillate fraction of a crude
oil)

Comprehensive two-dimensional
chromatography (GCxGC) is a technique
which has benetted the petrochemical
industry signicantly due to its ability to
separate very complex mixtures2,3. This
technique requires a conventional nonpolar column to be connected to a short
length of a polar column with a GCxGC
modulator. The modulator collects time
slices of efuent from the rst column
(typically 5 seconds wide) and re-injects
them onto the short polar column. The
result is a separation that details both
polar and non-polar elements along two
planes. With two levels of separation, the
complexity of the matrix is pulled apart,
increasing the resolution and allowing
the identication of far more
components.

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Application Note: ANTV12

Application of TargetView software in the fragrance industry:

Accurate identication of allergens with fast GC
Introduction
The need to screen consumer products
for specic organic compounds is
increasing across many industry sectors,
both for product quality assurance and
as a consequence of regulatory
requirements. An example is the
monitoring of allergenic compounds in
fragranced products per EU Directive
76/768/EEC. Twenty six fragrance
compounds which have the potential to
cause contact allergy, as assigned by the
Scientic Committee on Consumer
Products (SCCP), are incorporated into
this Directive. Of these 26, 24 are volatile
and can be analyzed by GC(MS).
The International Fragrance Association
(IFRA) species a GC/MS method which
complies with EU Directive 76/768/EEC,
allowing the analysis of the 24
compounds of interest1. To complement
this method and simplify in-house data
interpretation, TargetView software has
been designed to automatically identify
predetermined (target) compounds
within GC(MS) proles. A user can either
create their own library of target
compounds or import established ones;
this library may extend from an
individual entry up to several hundred if
more comprehensive sample screening is
required.

algorithm is applied to distinguish

between peak spectra. This is then
followed by principle component
analysis (PCA) to locate specic
compounds of interest.
To demonstrate the datamining
prociency of TargetView, a fragrance
sample was analysed for the presence of
the 24 SCCP allergens. The sample was
run under both slow and fast GC/MS
chromatographic conditions, the latter
signicantly compressing the
chromatography and, as a consequence,
increasing compound co-elution. The
results from each analysis were then
compared to see if the increased
complexity (i.e. co-elution) of data
acquired under fast chromatographic
conditions compromised compound
identication by TargetView.

Method

Oven (fast):

80C (1 min),
35C/min, 260C
(4 min) Run time
10.4 min

Injection volume: 0.2 L split 100:1

MS
Instrument:

BenchTOF-dx
(TOF-MS)

Mass range:

15350 amu

Acquisition rate:

5 Hz (10 kHz
continuous), 2000
pulses/cycle

Application of TargetView software in the fragrance industry: Accurate identification of

allergens with fast GC
Company: ALMSCO International

N.B. BenchTOF-dx data les are automatically

converted post-run into le formats compatible with
existing, commercially available software platforms.

TargetView
A library of the 24 allergenic compounds
listed by the SCCP was created in
TargetView using their NIST library
entries. Table 1 lists these compounds
with their International Nomenclature of
Cosmetic Ingredients (INCI) name and
CAS number.

TargetView software has been designed to automatically identify predetermined (target)

compounds within GC(MS) profiles. A user can either create their own library of target
compounds orimport established ones; this library may extend from an individual entry
up to several hundred if more comprehensive sample screening is required.
TargetView incorporates a sophisticated
dynamic background compensation
(DBC) algorithm to minimise baseline
noise from the total ion chromatogram
(TIC). The software then adopts a
chemometric approach to data analysis;
initially, a spectral deconvolution

Liquid samples were directly injected

into the GC/MS system under the
conditions listed below.
Analytical conditions
GC

Column:

30 m, 0.25 mm i.d.,
0.25 m HP INNOWax

Column ow:

1.5 mL/min constant

ow

Sample volume:

0.2 l, split 100:1

Oven (slow):

80C (1 min), 5C/min,

250C (2 min) Run
time 37.0 min

The resulting GC/MS data les were

processed by TargetView with the
Allergens library.

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Featured Applications
Application Note: ANBT10

Accurate and reliable analysis of beer using time-of-flight

technology for gas chromatography
Introduction
Any product that is derived primarily
from natural ingredients will inherently
comprise a tremendous range of
chemical components. Beer, usually
made from water, malt, hops and yeast, is
no exception. Variations in ingredients
give different beers their distinct
flavours. Typical compounds responsible
for flavour can have extremely low (ppt)
olfactory thresholds, meaning significant
compounds may be present at trace
levels. As such, component
concentrations within the beer may vary
by several orders of magnitude. Accurate
chemical profiling has previously proved
challenging when trying to
accommodate a sample with such
chemical complexity and dynamic range.
Volatile extraction
It is essential to extract the widest
possible range of compounds from a
sample in order to comprehensively
characterise its chemical profile. Solid
phase micro-extraction (SPME) and
conventional equilibrium (static)
headspace are techniques which have
previously been used to extract volatile
organic compounds (VOCs) from liquid
samples; however, neither method
provides the sensitivity to allow full
characterisation of the VOC profile.

of sample matrix with physically large

sorbents, such as PDMS, is the key to the
sensitivity advantage over the
aforementioned techniques.
Sorptive extraction is complemented by
thermal desorption (TD), which
concentrates the sample, making ppt
detection possible, and provides efficient
injection directly into the GC.

at conventional SIM detection levels

whilst still producing classical spectra
that are compatible with both
proprietary mass spectral libraries and
commercial databases (e.g. NIST).
The high sensitivity BenchTOF-dx
(figure 2) may be used in combination
with GC for routine screening of the
chemical profile of a sample. In this
application, four commercially available
beers were selected and their chemical
profiles analysed to test the ability of the
TOF-MS to determine both high and low
level compounds.

Figure 1. Long, hollow, PDMS-coated SPE-tD

cartridges

MS capability
Recording full spectral information using
GC quadrupole MS (or MS-MS) in scan
mode leads to the identification of a
large range of compounds in a sample,
however it does not provide the required
sensitivity for the identification of tracelevel compounds. Therefore, any
important flavour compounds are
unlikely to be detected. Conversely,
selected (or single) ion monitoring (SIM)
conditions provide the sensitivity for
trace-level identification but preclude
the gathering of spectral information.

Figure 2. BenchTOF-dx mass spectrometer

Using Gas Chromatography Tandem Mass Spectrometry to

Improve the Reliability and Accuracy of Organic Acid Analyses
in Urine
Company: Bruker

High/low analysis
To ensure trace-level components in a
particularly complex matrix are precisely
measured (i.e. for quantification), a highly
concentrated sample needs to be
introduced to the GC/MS; however this
may exceed the capacity of a highly
resolving column and result in detector
overload. By contrast, desorbing a
smaller sample to the column will allow
correct characterization & measurement
of the major components, but then the
low-level compounds will be lost.

Organic acids are water-soluble compounds containing one or

more carboxyl groups, as well as other functional groups (-keto,
-hydroxy). They occur as physiologic intermediates in a variety of metabolic
pathways. The organic acidurias are a group of disorders in which one or
more of these pathways are blocked, resulting in a deficiency of normal
products and an abnormal accumulation of intermediate metabolites
(organic acids) in the body. These excess metabolites are excreted in
the urine and can be analysed by capillary gas chromatography-mass
spectrometry (GC-MS). Herein we describe a tandem GC/MS approach for
analysis of fifteen organic acids in newborn urine.
Sorptive extraction using SPE-tD
cartridges (figure 1) is a simple yet highly
sensitive technique used for extracting
organic compounds from aqueous
matrices. The enrichment factor possible
from thorough mixing of large volumes

Advances in time-of-flight (TOF)

technology have resulted in the
development of a TOF-MS with the
ability to record full spectral information

National Scientific Mass Spec Certified Vials

Industrys first and only pre-cleaned,

low particle, low background
chromatography vial

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Application Note: ANBT11

Comprehensive analysis of crude oil by two-dimensional GC

(GCxGC) and time-of-ight (TOF) MS
Introduction
Crude oil is the generic term for the
unrened ammable liquid that is mined
from the ground. It contains vast
amounts of organic compounds ranging
from light hydrocarbons to complex
biomolecules, derived from the remains
of ancient marine organisms and
bacteria.

One-dimensional gas
chromatography
Within the complexity of crude oil, the
compounds of most interest to the
petroleum industry are relatively volatile
(boiling points generally below 400C)
and non-polar, therefore separations are
predominantly performed by GC with a

non-polar column. The resulting

chromatograms are highly convoluted
and usually characterised by a matrix of
unresolved material that appears as a
signicant background hump beneath
the partially resolved non-polar
compound peaks (Figure 1).
A background subtraction/library search
method is able to identify the non-polar
compound peaks, but for those lost in
the matrix, there is little hope for a
positive identication. Incomplete
characterisation limits the understanding
of the oil samples geochemistry and
provides no insight into issues that might
arise during extraction, transport, or
rening. In addition, as the more easily
extracted light crude oils become
depleted, oil companies are moving to

reservoirs containing heavier crudes,

bituminous shales and tar sands. These
contain higher proportions of involatile,
polar compounds, exacerbating the
problem of incomplete analysis1 and
such constituents are thought to
contribute to the process of
solidication of oil in transmission
pipes. Others might have relevance to
the environmental impact of oil, a factor
that will gain further attention with the
introduction of programs such as
European REACH (Registration Evaluation
Authorisation and Restriction of
Chemicals), which focuses on the
identication of chemicals in order to
assess their toxicological impact.

Prediction of Negative Ion Mass Fragmentation of

Chloramphenicol
Company: ACD Labs

Comprehensive (2D) gas

chromatography
Comprehensive two-dimensional
chromatography (GCxGC) is a technique
which has benetted the petrochemical
industry signicantly due to its ability to
separate very complex mixtures2,3. This
technique requires a conventional nonpolar column to be connected to a short
length of a polar column with a GCxGC
modulator. The modulator collects time
slices of efuent from the rst column
(typically 5 seconds wide) and re-injects
them onto the short polar column. The
result is a separation that details both
polar and non-polar elements along two
planes. With two levels of separation, the
complexity of the matrix is pulled apart,
increasing the resolution and allowing
the identication of far more
components.

The majority of experimental mass spectra are acquired in

positive ionization mode, due to an abundance of compounds
which have basic moieties to ionize and that generally offer
structurally informative ions if they are fragmented. However, particularly
using atmospheric pressure ionization techniques, there remains a large
amount of chemical space for which deprotonation is the preferred route of
ionization. Historically this has not been studied as deeply as protonation
and positive ionization mass spectrometry, consequently the availability
of public domain knowledge for how these compounds fragment is more
limited.
Figure 1. GC/MS total ion chromatogram of a sample of lubricating oil (a distillate fraction of a crude
oil)

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See for yourself. Download our technical poster,

read our whitepaper and get your free sample pack.
Visit www.nationalscientific.com/theonlyvial
www.nationalscientific.com
800-332-3331 865-717-1986 (international)

Products
Analytical Studio Reviewer Software
Company: Agilent
Manufacturers description: Implementing a fast, efficient, high-throughput data review process can be
critical to successful discovery of lead candidates in fields such as pharmaceuticals and agrichemicals.
Available from Agilent is the Analytical Studio Reviewer software designed to provide fast, accurate,
and flexible analysis of large volumes of LC/MS data. Combined with its Easy Access software, an Agilent
1200 Series LC, and an Agilent 6100 Series Single Quadrupole LC/MS, the Analytical Studio Reviewer
software meets the need to acquire, process, and review high-quality data as quickly and accurately as
possible, claims the company. Reported features of the software include:
An intuitive user interface with multiple display options, from stacking or overlaying large numbers of
chromatograms for simultaneous review to zooming in on a single chromatogram
A tree view control allows the user to easily examine the contents of the original summary results file
without having to open the file in a separate application
It accepts summary results from a wide variety of platforms, including the Agilent ChemStation and
any system using the RPT file format
Customized batch reporting allows the user to choose which samples to report on while reviewing the
data. Reports can be printed in customizable Microsoft Word or Adobe formats
Chromatogram and spectra data images can be captured and shared with colleagues in presentations
or e-mails, as well as exported to an external search engine for literature comparison
It provides the ability to edit automatically processed data and regenerate a report in the same format,
while logging changes and allowing the user to annotate the reasons for the edits
Summary values (e.g., purity, area percentage and area absolute) can be quickly reviewed to make
such changes as deleting peaks and chromatograms, and revising sample meta data such as sample
name and well location
Modified results can be exported into a new output file to reflect the results determined by the user,
preventing inconsistencies between the final result file and the overall conclusions
For more information, visit www.agilent.com

Learning
Modules

Lecture-style sessions by John Dolan and Tom Jupille, LC Resources,

in simple to use slide-by-slide instruction, together with a
comprehensive handout supporting the learning experience.

ule
/ mod
$147
onth
for 1-m access
ited
unlim

HPLC Troubleshooting & Preventive Maintenance

1: Principles of HPLC Troubleshooting
This 60-minute e-learning module covers problem isolation, important measurements,
tricks of the trade and general principles. Click here to learn more>>

LC IsoLink
Company: Thermo Scientific
Manufacturers description: The LC IsoLink is a high-sensitivity interface
connecting HPLC with isotope ratio MS for the reproducible and
accurate on-line determination of C/12C isotope ratios.
According to the company, it has been built to directly address
the analysis of polar and thermo-labile compounds. All organic
compounds eluting from an HPLC column are analysed while
maintaining the chromatographic resolution. Reported features
include:
Isotope ratio data on an HPLC time scale
Integrity of chromatographic resolution
High-sensitivity sample analysis in the low nano-mole range
Accurate and reproducible results
Direct injection of international reference materials
Maintenance-free operation
High throughput
Capability for full automation
Recommended application areas are metabolism studies (e.g., tracer
experiments with amino acids, lipids, carbohydrates; nucleotide
analysis for DNA synthesis rates), food and food ingredients (e.g.,
authentication of honey and fruit juices, determining origin of
flavours and nutrients, analysis of phospholipids, carbohydrates,
amino acids and vitamins), pharmaceuticals (e.g., authenticity control
of drugs, investigation of agents and additives), doping control (e.g.,
analysis of steroids and hormones), and biogeochemistry (e.g., history
of biomolecules in sediments and soils).
For more information, visit www.thermo.com

2: Troubleshooting HPLC Pumps and Autosamplers

This 60-minute e-learning module covers reservoir and degassing, pumps and autosamplers.
Click here to learn more>>
3: Physical Problems with HPLC Columns
This 60-minute e-learning module covers plugging the trit or column, mechanical shock,
when the column is not the problem, adsorbed sample impurities and chemical attack on the
silica or bonded phase. Click here to learn more>>
4: Chemical Problems with HPLC Columns
This 70-minute e-learning module covers column contamination, column regeneration,
minimizing chemical attack, minimizing performance problems and maximizing column lifetime.
Click here to learn more>>
5: HPLC Detectors
This 55-minute e-learning module covers variable wavelength and diode array detectors, mass
spectrometers and MS/MS, fluorescence, EC, RI and ELSD detectors. Click here to learn more>>
6: HPLC Quantification, Integration, and Data Systems
This 65-minute e-learning module covers measuring peak area, calibration curves and reproducibility.
Click here to learn more>>

Fundamentals of Gradient HPLC

This 85-minute e-learning module covers when it is appropriate to use gradient HPLC, similarities
between gradient and isocratic methods, differences between gradient and isocratic methods,
hardware issues relating to dwell volume, hardware issues relating to baselines.
Click here to learn more>>

Introduction to LC-MS and LC-MS/MS

This 75-minute e-learning module covers a overview of LC-MS and LC-MS/MS, quadrupole physics,
instrumentation, ion formation and elements of LC-MS/MS analysis. Click here to learn more>>

Performance Qualification of HPLC Equipment

This 80-minute online learning seminar covers an overview of HPLC qualification, pump and detector checks,
online mixing checks and chromatographic checks. Click here to learn more>>

www.sepscience.com

MS Masterclass
US$1600
3 days

LC/MS Fundamentals & Applications

8-10 November, 2010 ( 3-day course)
Furama City Centre- Singapore

Who Should Attend

Researchers, practitioners, technicians, and others who are currently using
LC, LC/MS, or plan to use LC/MS in the future, and those dealing with data
produced by LC/MS. This course is designed to provide a practical overview.
Chromatographers just embarking on the technique will gain insight on
how to select the appropriate instrument for different applications, and
those currently using LC/MS and its data will develop an appreciation for,
and an understanding of, the complexities of the data generated.
How Youll Benefit from This Course:
1. Understand the operational fundamentals of liquid chromatography,
mass spectrometry, and the commercially available LC/MS interfaces.
2. Receive an overview of LC/MS applications in pharmaceutical, chemical,
and biotechnology industries plus information on forensic and
toxicological applications
3. Learn how to modify existing HPLC methods to optimize results when
moving to LC/MS.
4. Find out how to use LC/MS to obtain qualitative information about your
samples; from simple molecular weight determinations to structural
analysis through spectral interpretation.

Next month:

Adduct Ion Formation in Electrospray Part 2 in which

we will discuss a real application development
problem involving formation of some seemingly
unusual potassium adduct species.

Course Topics:
1. Introduction to LC/MS-overview and historical perspective
2. Review of atoms, isotopes, molecules and ions
3. What is LC?-- history, fundamental parameters, columns/solvent systems,
concerns related to MS
4. What is MS?-- history, mass analyser types, detectors, vacuum systems;
MS/MS and CAD
5. LC/MS Interfaces: historical interfaces, particle beam, atmospheric
pressure chemical ionization, electrospray and atmospheric pressure
photoionization interfaces
6. Applications review: LC considerations for MS, drugs and metabolites,
pharmaceuticals, environmental applications, natural products, protein/
peptide analysis
7. Bibliography of LC/MS; glossary of correct and incorrect terms

CLICK HERE TO
REGISTER ONLINE

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member who would benefit from this monthly
publication then send us their details below.

Recommend

Read the latest

Clinical Edition

Volume 2, Issue 13
September 2010

Cd

Peak Tailing in GC Trace Analysis

Jaap de Zeeuw

Synthetic Oligonucleotides The Next Big

Challenge for Separation Sciences
George Okafo, Mike Webb and Nigel Richardson

Exploring new sampling techniques to provide

simplified material emissions assessment to the
manufacturing industry
Caroline Widdowson

Proteomic analysis to define cellular changes induced by

antineoplastic agents
Tandem mass spectrometry to understand Helicobacter pylori
cell-surface glycans

Synthetic Oligonucleotides
The Next Big Challenge for
Separation Sciences
www.sepscience.com

Synthetic Oligonucleotides
The Next Big Challenge for Separation Sciences

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