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Ion Composition Elucidation (ICE) 

ICE - an important analytical tool for human risk assessment and regulatory proceedings.

Frequently Asked Questions from the Public and Scientific Communities

How can ICE benefit the Public? What is a Relative Abundance?
What is Mass Spectrometry? Of What Use is a Relative Abundance?
What is Gas Chromatography? What is ICE?
What Analytical Problem does ICE Address? What is MPPSIRD?
What is an Exact Mass? What is the Profile Generation Model (PGM)?
Of What Use is an Exact Mass? Can I see a Typical ICE Experiment?
What is a Possible Composition? What is Mass Resolution?
What is a Double Focusing Mass Spectrometer?

How can ICE benefit the Public?

Pollutants in the air, water, and food can cause acute or chronic illnesses.  When epidemiological studies reveal elevated incidences of illness in a small geographical area, the public often demands that the cause be found and eliminated. If the causative agent is one or more chemicals (as opposed to a biological organism), the chemicals must be identified before toxicological studies can be made to determine which chemicals are responsible for the observed illness. Such environmental pollution can provide a very difficult analytical problem. When no history of the pollution is available, no assumptions concerning the types of compounds present nor the elements they contain can be made.

Ion Composition Elucidation (ICE) is an important new tool in the chemist's analytical arsenal for identifying chemicals. ICE has been used to characterize a Superfund site and to identify trace amounts of isomeric compounds (compounds having the same number of atoms of each element, but with different structures) in an extract of water from a municipal well near Toms River, NJ where an increased incidence of childhood cancer had been observed.

Ion compositions determined for 51 compounds in a tar-like sample from a Superfund site revealed a family of benzothiazole based compounds, which are used in the rubber and dye industries. ICE provided a preponderance of scientific evidence to identify the source of the pollution and to assign responsibility in a legal proceeding had one been necessary. For more details see the poster "Characterizing Hazardous Waste Constituents: A New Tool"  [PDF, Poster - 1 pp., 484 KB, About PDFor the publication

Grange, A.H.; Brumley, W.C.
"Identification of Ions Produced from Components in a Complex Mixture by Determination of Exact Masses and Relative Abundances Using Mass Peak Profiling"
LC-GC 1996, 14, 478-486.

Ion compositions were determined for the molecular ion (the molecule minus one electron) and 10 fragment ions formed from unidentified pollutants in the NJ well water. This information narrowed the possible compound identities to a small number. A quick search of the chemical literature then provided tentative identifications for the isomeric compounds, which were confirmed by comparison of mass spectra and retention times for standards obtained from the corporation with an interest in our study. This corporation then performed toxicological tests on larger amounts of the compounds under scrutiny of Region 2 of the EPA. For more details see the poster "Well Pollutants Identified with a New Mass Spectrometric Technique" [PDF, Poster - 1 pp., 3.7 MB, About PDFor the publication

Grange, A.H.; Sovocool, G.W.; Donnelly, J.R.; Genicola, F.A.; Gurka, D.F.
"Identification of Pollutants in a Municipal Well Using High Resolution Mass Spectrometry"
Rapid Commun. Mass Spectrom.,1998,12,1161-1169.

What is Mass Spectrometry?

Numerous text books answer this question in great detail. Two classic texts are F.W. McLafferty and F. Turecek Interpretation of Mass Spectra, 4th ed. Mill Valley, CA: Univ. Science Books, 1993 and K. Biemann, Mass Spectrometry: Organic Chemical Applications, McGraw-Hill, New York, 1962. Here, however, only a few fundamental points are needed to understand how ICE works. Everything we see is composed of molecules.  A pure compound consists of only one type of molecule, which is in turn composed of a combination of atoms. It is the types of atoms (elements) and the number of each in the molecule that Ion Composition Elucidation determines. Most items and environmental samples are made up of mixtures of compounds. To identify a compound using a double-focusing mass spectrometer, molecules of that compound are first ionized. The ions are manipulated by an electrostatic field and then by a magnetic field to determine the ions' mass-to-charge ratios, which are measures of the mass of the molecule. When electron impact is used to create ions, excess energy imparted to the molecular ions (the molecule minus one electron) causes some of these ions to fragment into smaller ions and neutral losses from the molecular ion. The abundances of these ions are recorded as the mass spectrum, an example of which is seen below. The mass spectrum is a histogram of ion abundances on the vertical axis vs the mass-to-charge ratios (m/z) on the horizontal axis. When a mass spectrum displays numerous fragment ions, it can often be matched to entries in a mass spectral library to provide a tentative identification of a compound.

Figure 1 - For further information contact grange.andrew@epa.gov

A Mass Spectrum

 What is Gas Chromatography?

A gas chromatograph is most often used to separate compounds found in extracts of environmental samples. A gas chromatograph is a temperature controlled oven that contains an internally coated capillary column typically 30 meters long with a 0.25 millimeter internal diameter onto which 1 or 2 L (1 or 2 millionths of a liter) of a sample extract is injected. Ideally, each compound is retained by the coating to a different extent as they travel the length of the column and each compound exits alone into the ion source of the mass spectrometer after a retention time specific to that compound. If one plots the ion abundance vs time for the compound's molecular ion or other ions characteristic of the compound, a chromatographic peak is observed.

Figure 2 - For further information contact grange.andrew@epa.gov

The Total Ion Chromatogram is a plot of the sum of ion abundances for all m/z ratios vs
the retention time. Multiple chromatographic peaks indicate elution of numerous compounds.

What Analytical Problem does ICE Address?

Pollutants in our air, water, and food might be harmful, benign, or beneficial. But before toxicological studies can be made on such pollutants, these compounds must first be identified and the amounts people are exposed to must be determined. ICE is a new tool for helping to identify the numerous compounds that are overlooked by traditional analytical methods that target lists of a few hundred compounds for which mass spectra are found in data system libraries or for which pure standards are commercially available.  Other compounds are usually ignored, since identifying each compound would be a research project in itself.  Consequently, most researchers select a set of compounds consumed in large quantities and use purchased standards to develop an analytical method to extract and analyze them before collecting samples. Even if numerous researchers each studied a different set of compounds, most pollutants would remain unidentified and unquantified. Thousands of high production volume chemicals [PDF, 65 pp., size not available, About PDF] and their degradation products must end up somewhere!

How big is this problem? A portion of a total ion chromatogram below for an extract of 12 liters of lake water used as a drinking water supply displays evidence of the presence of dozens of compounds.

Figure 3 - For further information contact grange.andrew@epa.gov

Each visible chromatographic peak corresponds to at least one compound and the peaks due to compounds present at lower levels may be masked by the observed peaks.

ICE is a high mass resolution mass spectrometric technique that provides the scan speed, sensitivity, selectivity, and stability needed to determine the ion compositions of ions observed in the mass spectra. Knowledge of these compositions reduces the number of possible compounds that could produce a given mass spectrum to a small number, thereby making searches of the chemical and commercial literature practical.  ICE provides the means to identify many of the mystery compounds responsible for the myriad of peaks in the complex total ion chromatogram from an environmental sample. An excellent example of compound identification is provided in the poster Well Pollutants Identified with a New Mass Spectrometric Technique.  [PDF, Poster - 1 pp., 3.7 MB, About PDF

What is an Exact Mass?

Molecules are composed of atoms. An ion is a molecule or a fragment of a molecule which has fewer or more electrons than the electrically neutral molecule or fragment. (All ions considered at this web site lack a single electron to provide a positively charged ion.) Mass spectrometers manipulate ions with electric and/or magnetic fields to measure their masses. The exact mass of an ion is the sum of the masses of its atoms.

Most mass spectrometers (quadrupole and ion trap based instruments) measure nominal masses, rounded to the nearest whole mass. This limitation results in multiple possible compositions for a given measured mass.  For example, a m/z ratio of 28 could be due to ions from three common compounds with dissimilar properties:  N2 (nitrogen gas), the majority of earth's atmosphere; CO (carbon monoxide), a poisonous product of incomplete combustion; or C2H4 (ethylene), a simple hydrocarbon used in chemical syntheses.

Composition
Nominal Mass
Exact Mass
CO
28
27.99491
N2
28
28.00615
C2H4
28
28.03130
Mass spectrometers capable of high mass resolution can easily distinguish between these three possible compositions.

Of What Use is an Exact Mass?

Exact masses are of no use when mass spectrometers that provide unit mass resolution are used.  As illustrated below, mass peak profiles for the three ions with a nominal mass of 133 amu are not resolved.

Figure  - For further information contact grange.andrew@epa.gov

However, if a double focusing mass spectrometer is used at a mass resolution of 10,000, the three mass peak profiles are greatly narrowed and are baseline resolved from each other. The exact mass measured at 10,000 resolution would determine which of the three compositions is correct. Thus, measurement of exact masses reduces the number of possible compositions for an ion in a mass spectrum, which in turn reduces the number of compounds from which the ion could have been produced.

What is a Possible Composition?

For an exact mass measurement, all compositions of the elements considered (e.g. C, H, N, O, F, P, S, and Si) are possible that have exact masses between the error limits of the measurement. For example, 14 possible compositions corresponded to a measured exact mass of 236.12908 atomic mass units (amu) using a mass resolution of 10,000, which provided an error limit of 6 parts per million (ppm). Liberal error limits are used in the Profile Generation Model to ensure that the correct composition is not overlooked, even when the data obtained suffers from minor mass interferences.

m/z = 236.12908 6 ppm
#
Composition
Exact Mass
Difference
(ppm)
#
Composition
Exact Mass
Difference
(ppm)
1 C6H18N7OS 236.12936
+1.2
  8 C10H26P2Si 236.12791
-5.0
2 C7H21N4FSi2 236.12888
-0.8
  9 C10H23NFSSi 236.13045
+5.8
3 C7H20N4O3S i 236.13047
+5.9
  10 C10H20NOF2Si 236.12822
-3.6
4 C8H25N2PSi2 236.12939
+1.3
  11 C10H19NO4F 236.12981
+3.1
5 C8H17N4O3F 236.12847
-2.6
  12 C11H20NF2S 236.12845
-2.7
6 C9H23NOFSi2 236.13022
+4.8
  13 C12H22NSi2 236.12908
-0.0
7 C9H21N2O3P 236.12898
-0.4
  14 C13H18NO3 236.12867
-1.7

At least one third of the mass of the ion was assumed to be due to carbon atoms to reduce the number of possible compositions. The number of possible compositions increases very rapidly as higher-mass ions are considered and approximately doubles if the error limits are doubled or an additional element is considered.

What is Mass Resolution?

Mass resolution is a measure of how well mass peak profiles with similar masses can be separated from each other. The "10% valley" definition of mass resolution cited for double focusing mass spectrometers is as follows. For two mass peak profiles of equal ion abundance with a minimum between them of 10% of the maximum profile height, the mass resolution is their average mass divided by the mass difference between them. In the figure, the tails of two profiles overlap at 5% of the profile maxima to provide a 10% valley between the two profiles.

Figure - For further information contact grange.andrew@epa.gov

Mass peak profiles become narrower as the mass resolution increases, which is why separation of the profiles occurs. In the example below, three ions with m/z ratios of 281 were separated using a mass resolution of 5000.

Figure  - For further information contact grange.andrew@epa.gov

Each ion is from a compound from a different source: an ever-present calibrant ion (steady signal), the coating within a gas chromatograph column (gradually increasing signal in concert with the temperature ramp within the oven), and an analyte (the chromatographic peak). High mass resolution provides separation of signals by mass to complement the separation in time imperfectly afforded by gas chromatography.

What is a Relative Abundance?

A relative abundance is the ratio of one mass peak profile’s area or height to that of another. When using Ion Composition Elucidation (ICE), the abundances considered are those of the +1 and +2 profiles relative to the profile of the ion containing none of the higher isotopes of elements such as 13C, 15N, 18O, or 34S.  The figure below shows partial profiles for m/z 198 and its +1 and +2 profiles.

Figure  - For further information contact grange.andrew@epa.gov 

The relative abundances were calculated as ratios of the sums of the areas used to plot the partial profiles x 100%. The partial profiles for two ions from perfluorokerosene, the ever-present calibrant, were used to calibrate the three exact masses also shown on the figure.

Of What Use is a Relative Abundance?

The relative abundance of the +1 profile arises primarily, but not entirely, from the presence of a single 13C atom in place of a 12C atom in the ion.  Each C atom has a 1.1% chance of being a 13C atom.  An ion with 10 C atoms would have a +1 profile abundance of 11% due to 13C atoms alone.  In fact, the relative abundance of the +1 profile has long been used to estimate the number of C atoms in an ion.  Likewise the number of Cl, Br, or S atoms has been estimated from the relative abundance of the +2 profile. Mass Peak Profiling from Selected Ion Recording Data (MPPSIRD) provides more accurate relative abundances than conventional full scanning. Hence, relative abundances become even more useful for determining the numbers of atoms of different elements in an ion. Note that in the absence of mass interferences, one can use low mass resolution to obtain accurate relative abundances. This is important when trace levels of compounds are studied and the +2 relative abundance is less than 1%.

Relative abundances have been used to determine ion compositions without even considering exact masses of ions! Mass calibrants were not required!  The poster Determination of Elemental Compositions by High Resolution Mass Spectrometry without Mass Calibrants  [PDF, Poster - 1 pp., 696 KB, About PDF] and a journal article describe this work.

Grange, A.H.; Sovocool, G.W.
“Determination of Elemental Compositions by High Resolution Mass Spectrometry without Mass Calibrants” 
Rapid Commun. Mass Spectrom., 1999, 13, 673-686.

What is ICE?

ICE is the easily remembered acronym for Ion Composition Elucidation, which states "what" is done by this new analytical technique. ICE has two aspects. Mass Peak Profiling from Selected Ion Recording Data (MPPSIRD) to acquire data and a Profile Generation Model (PGM) to automatically interpret that data. MPPSIRD indicates "how" the data is acquired while the PGM deals with "planning" of experiments and the "utility" of the data by comparing measured and calculated values for each possible composition.

What is MPPSIRD?

MPPSIRD is the acronym for Mass Peak Profiling from Selected Ion Recording Data. Ion chromatograms are recorded for several m/z ratios across a single mass peak profile. The area under a chromatographic peak observed in each of the ion chromatograms is plotted vs the m/z ratio and lines are drawn between these points to provide the mass peak profile. The exact mass of 332.08471 atomic mass units (amu) in the figure was calculated as the weighted average of the nine points that delineate the profile. Similarly, multiple m/z ratios are often monitored over the top portions of up to three analyte profiles during the same experiment to provide partial, rather than full profiles.

Figure - For further information contact grange.andrew@epa.gov
MPPSIRD is the
data acquisition aspect of ICE!

What is the Profile Generation Model (PGM)?

The profile generation model written in QuickBASIC 4.5 helps plan data acquisitions and automatically interprets the data obtained.  For a low-mass ion (up to 150 amu) ICE usually determines its composition from its exact mass.  For higher-mass ions, multiple possible compositions remain and the PGM then distinguishes between compositions based on the exact masses of the mass peak profiles higher in mass by +1 and +2 amu that arise from heavier isotopes of elements.  For example, a carbon atom (C) can be 13C, which is 1.1% as abundant as 12C, an oxygen atom (O) can be 16O (99.762%), 17O (0.038%), or 18O (0.200%), a nitrogen atom (N) can be 14N (99.634%) or 15N (0.366%), and a sulfur atom (S) can be 32S (95.02%), 33S (0.75%), or 34S (4.21%).  The PGM also uses the abundances of the +1 and +2 profiles relative to the ion's abundance (relative abundances) to determine an ion's composition.

To understand how +1 and +2 profiles can distinguish between ions with the same nominal mass (rounded to the nearest whole number), consider three ions that provide m/z 133 molecular ions in their mass spectra: C7H7N3, C8H7NO+, and C9H11N+. The figure below shows the composite +1 profiles and the individual contributions made to them by each ion containing an atom of a +1 isotope. The PGM represents each +1 ion as a Gaussian distribution (normal curve) with the appropriate exact mass and relative abundance. The composite +1 profile is the sum of the points on the individual curves. At the mass resolution of 10,000 depicted, both the exact masses and relative abundances are sufficiently different that any one of the three ions could be distinguished from the others.

Figure - For further information contact grange.andrew@epa.gov

In the tabular output from the PGM below, for an ion with m/z 236 each "X" indicates the measured and calculated values are inconsistent for the composition in the same row.  Only for C13H18NO3+ are all three measured and calculated exact masses and two measured relative abundances consistent with the calculated values.

Table - For further information contact grange.andrew@epa.gov

Can I see a Typical ICE Experiment?

Typically, two cycles of data acquisition using MPPSIRD followed by automated data interpretation using the PGM are required to determine an ion composition. The following example illustrates determination of the composition of the m/z 151 observed in the low resolution mass spectrum shown below.

Fi

1.  MPPSIRD is used to survey a broad mass range about a target ion with a mass resolution of 3000.

Figure - For further information contact grange.andrew@epa.gov

One profile defined by three points due to the target ion was seen. The coarse exact mass provided by this profile was used as the center mass for the next experiment.

2. A narrower mass range was monitored with 10,000 resolution and 10 ppm mass increments between the points.

Figure - For further information contact grange.andrew@epa.gov

The profile was delineated by 10 points and an exact mass accurate within 6 ppm was obtained.

The exact mass was entered into the Profile Generation Model, which output a list of two possible compositions based on the measured exact mass and its 6 ppm error limit.

Figure - For further information contact grange.andrew@epa.gov

A hypothetical composition was chosen (in blue) to provide center masses for the partial profiles to be obtained in the next MPPSIRD experiment.

4.  Partial profiles were obtained using 10,000 resolution with 10 ppm mass increments.

Figure - For further information contact grange.andrew@epa.gov

The partial profiles provided the exact masses of the m/z 151 and its +1 and +2 profiles.  They also provided the abundances of the +1 and +2 profiles relative to the m/z 151 profile.  These five measured values were entered into the Profile Generation Model, which prepared a table of the possible compositions listed for the previous cycle along with calculated exact masses and relative abundances for the partial profiles.

Figure - For further information contact grange.andrew@epa.gov

 An "X" next to a value indicated disagreement between the measured and calculated values and the composition in that row was rejected.  Thus, the correct composition is C10H15O for the ion observed in the low resolution mass spectrum.

What is a Double Focusing Mass Spectrometer?

Like all mass spectrometers, a double focusing mass spectrometer has an ion source, a mass analyzer, and a detector.  In place of a quadrupole or ion trap mass analyzer, an electrostatic sector separates ions based on their kinetic energy (1/2mv2) and a large magnet separates ions based on their momentum (mv).  Ions having many m/z ratios pass through an entrance slit into the mass analyzer while at any instant, only ions within a very narrow spread of m/z ratio are focused onto and pass through the exit slit to the detector.  Hence, the ions have been "double focused" for both kinetic energy and momentum.

Relatively few double focusing mass spectrometers are available compared to quadrupole and ion trap mass spectrometers due to their high cost (about $500,000 vs $100,000), their large size (an entire room vs a bench top), and their difficulty of use (since research flexibility is emphasized rather than specific production capabilities for which many variables are permanently set).

Definitions

By definition, an atomic mass unit (amu) is 1/12 of the mass of a 12C atom.

A part per million (ppm) is a number divided by one million (106).  For example, 6 ppm of 236.12908 amu is 236.12908 / 106 x 6 = 0.00142 amu.

A mass interference is a mass peak profile that overlaps the analyte ion profile or its +1 or +2 profile. Higher mass resolution reduces the number of mass interferences.

The weighted average of the areas used to plot a profile is the sum of the products of each area times the m/z ratio monitored divided by the sum of the areas.

Note: No aspect of the materials provided by EPA employees on this web site should be construed as representing thinking or positions regarding policy.  This point is codified in the mission of the Office of Research and Development (ORD), which sponsors this web site.  This web site is simply a repository for ICE development and related research.

 

Analytical Environmental Chemistry
ICE Home Page

Environmental Sciences | Office of Research & Development
National Exposure Research Laboratory
Author: Andrew Grange
Email: grange.andrew@epa.gov


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