2-MS Interp

Mass spectral interpretation for chromatographers This material is designed to help GC/MS beginners who want to understand a few
mass spectral interpretation basics. It is not intended to be a complete treatment of mass spectral interpretation. Well not be introducing any complex fragmentation or rearrangements.
Friday, August 6, 2010
What is a mass spectrum
100
Lets review the basic components of a simple mass spectrum.
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0 Mass / Charge
A simple mass spectrum
100
Mass spectra are typically represented as a bar type graph. Each line is used to represent a specific M/Z.
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0 Mass / Charge

X axis - Mass / Charge - (M/Z)
100
This format is used because a M/Z of 28 can represent a mass of 28 with a single charge or might be 56 with a charge of 2, ..... A single charge is the most common so the X axis best equates to mass.
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0 Mass / Charge

Y axis - abundance. In a standard spectrum, the is made relative to the largest line (the base peak) so it will run from 0-100%. Absolute abundance are typically used in GC/MS spectra. They will run from 0 to the highest recorded line intensity.
100
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0 Mass / Charge

Types of information Mass of fragments Presence of isotope lines Gaps between lines. Well look as some of the basics
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0 Mass / Charge

The masses in this example should look familiar. This is a spectrum for air - a mixture.
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Nitrogen - N2
15N14N
50
- an isotope peak. Its small because 15N is not very common in nature
0 Mass / Charge

Well we know what most of the lines are due to but what could 16 and 17 be from?
100
N2
50
Water?
O2
CO2
0 Mass / Charge
Fragmentation pattern
Water shows fragmentation where it can loose 1 or 2 H. Youll see that fragmentation patterns are very important in MS interpretation
50
100
18 - H2O 17 - OH 16 - O
0 Mass / Charge
Atomic masses and isotopes

The atomic weights that we find on the periodic table represent an average for all naturally occurring isotopes. Example Chlorine - atomic weight = 35.453 A mixture of 35Cl - 75.77%, 37Cl - 24.23% We must not only need to account for these isotopes, we can exploit them for determining the number and types of elements that are present.
Atomic masses and isotope

To help with some of our calculations, we commonly work with relative abundances of the isotopes. The most abundant isotope is set to 100% and the the other isotopes are normalized to it. For most elements in organic molecules, the most abundant element is also the lightest. Lets look at some of the more common elements.

Mass H C N O F S Cl Br 1.0078 12.0000 14.0031 15.9949 18.9984 31.9720 34.9989 78.9183 RB Mass RB Mass RB Class
100 100 13.0034 100 15.0001 100 100 100 32.9715 100 100 80.9163
A 1.1 A+1 0.37 A+1 17.9992 0.2 A+2 A 0.8 33.9679 4.4 A+2 36.9659 32.5 A+2 98 A+2
RB = relative abundance
Isotope classes
The A, A+1, A+2 class system is used to indicate the types of isotopes present. A - Only a single isotope. This may mean that the element only exists as a single isotope (like fluorine) The abundance of all but one isotope is too small to use. (Example. deuterium - 0.0015%)
Isotope classes
A+1 Two isotopes. Examples. C and N A+2 At least two isotopes with the highest mass isotope being +2 from the lowest mass isotope. Examples. O, S, Cl, Br. In organic MS, the most abundant isotopes are also the lowest mass.
General steps in mass spectral interpretation

Obtain a good spectrum Evaluate the general appearance Find the molecular ion if possible Determine the elemental composition Identify structural features Propose possible structures
Obtaining a good spectrum

With GC/MS, your mass spectra will vary across a peak. In addition, there can be artifacts resulting from the carrier gas and the column. A few simple steps must be taken to insure that the spectrum you evaluate is a good one.

You must have scanned over the entire mass range of the component. At least 2 mass units higher than the molecular ion mass. If chlorine or bromine are present, you should scan 2 x the number of each higher. For high MW samples, you dont typically need to scan below 50.

Most systems have some sort of user based threshold. Lines below this value are not stored. You need your threshold to be low enough to have recorded less intense lines. This is typically about 0.1% relative abundance. Remember, you molecular ion cluster is often one of the least intense line clusters you have.

Averaging and skewing of spectra. We commonly take an average spectrum by averaging several scans across a peak. This can cause some problems. At the peak top, large lines may have saturated the detector. At the start/end of a peak, smaller lines may not have exceeded the threshold.

Threshold too low for some or all of the scans
Proper spectrum
Saturation of the two largest lines

So what can you do. First, make sure you have at least 7 spectra being collected / peak. You need this to obtain any sort of reasonable integration anyway. Produce a series of average spectra The first few scans The last few scans The center scans.

All scans should give similar results. If the threshold is too high, you can simply use the center average. If saturation occurs, avoid the center of the peak - use one side or the other. This test will also indicate if the peak is pure. The presence of new major lines indicates co-elution.

Finally, GC/MS is subject to background problems that you can account for.

Air, water, other contaminates in the carrier gas. Column bleed. The effects of background can be minimized by calculating and subtracting an average background spectrum - near the peak.
Mass defect
Our current system of assigning atomic masses is based on 12C = 12.00000000. As a result of this and the mass lost to binding energy, the mass of every other element is NOT a whole number. With high resolution MS, we can use this mass defect to specifically identify the elemental composition of each line. With unit resolution MS, this can present a problem.
Mass defect
In organic MS, we are dealing primarily with H, C, N, and O. The most common isotopes of H and N both weigh more than their assigned whole number atomic mass. H - 1.0078 N - 14.0031 Hydrogen presents the greatest problem because our compounds contain many.
Mass defect
Our unit resolution MS may miss assign the mass if a peak if it is > 0.5. We can predict how many hydrogen must be present for this to cause a problem. # Hydrogen = 0.5 / (1.0078 - 1) = 64 hydrogen
128 hydrogen would give us a +1 shift.

Mass defect
An alkane would represent the worst case so lets determine how large it must be to hit the 0.5 limit. alkane = CNH2N+2 2N + 2 = 64 N = 31 MW = 31 * 12 + 64 = 434 So its only a real problem at high mass and most systems can correct for it.
Working with the molecular ion

One of the most powerful features of MS is the ability to determine the molecular formula from the molecular ion. The molecular ion must: Be the highest mass ion - exclusive of isotope related ions which also must be measurable. Be an odd electron ion Agree with the rest of the spectrum.

With GC/MS data, even if the molecular ion is present, it may be hard to work with. For now, lets assume that the data is good and we already know what the molecular or parent ion is. Well deal with some of the additional rules. For now, well simply show how to work with the molecular ion to determine the molecular formula.

Lets assume that you have obtained the following mass spectral data: m/e 26 27 28 29 39 abund 3472 20593 48287 79823 14675 m/e 41 42 43 44 45 abund 14044 7575 27536 31440 1026
Which line would be the molecular ion?


A good guess would be 44. Why? m/e 44 is too big compared to 43 m/e 45 is too small compared to 44 Also, remember that most of our elements consist of a mixture of isotopes. Masses greater that the molecular ion are to be expected (hoped for).

Mass H C N O F S Cl Br 1.0078 12.0000 14.0031 15.9949 18.9984 31.9720 34.9989 78.9183 RB 100 100 100 100 100 100 100 100 Mass RB Mass RB Class
A 13.0034 1.1 A+1 15.0001 0.37 A+1 17.9992 0.2 A+2 A 32.9715 0.8 33.9679 4.4 A+2 A+2 36.9659 32.5 80.9163 98 A+2
RB = relative abundance
Normalization
The next step is to normalize your data. Raw GC/MS spectra are typically unnormalized. Standard spectra are normalized to the largest (base) peak which may not be the molecular ion. There are a few simple steps that you can follow that will help.
Normalization
Select a potential molecular ion along with any lines of higher mass. Normalize these lines so that the potential molecular ion is 100. The error of the isotope lines is estimated as being +0.2 absolute or +10% relative. Using these values, you can begin constructing a normalization table.
Normalization
Select a potential molecular ion m/e abund 44 31440 (potential molecular ion) 45 1026 The values can now be plugged into your normalization table. Normalization table - a simple way to track your calculations.
Normalization table
Start by putting the masses and abundances in the first two columns. Then calculate your normalization factor (NF) where NF = 100 / rel. abund of A
m/e A A+1 A+2 44 45 rel. Norm abund abund 31440 1026 NF Cn-1 Cn Cn+1
100 .00318
Normalization table
Next, calculate the normalized abundances for your isotope lines - A+1 in this example, + 10% relative error
m/e A A+1 A+2 44 45 rel. Norm abund abund 31440 1026 NF Cn-1 Cn Cn+1
100 .00318 3.26 +.33
Normalization table
Based on the A+1 peak, there appears to be three carbons. Well look a 3+1 carbons. In the next columns, calculate the mass due to C and the A+1 abundances.
m/e A A+1 A+2 44 45 rel. Norm abund abund 31440 1026 NF Cn-1 24 2.2 Cn 36 3.3 Cn+1 48 4.4
100 .00318 3.26 +.33
Normalization table
Three carbons looks like a good choice. Now we need to account for the remaining mass and come up with a formula.
m/e A A+1 A+2 44 45 rel. Norm abund abund 31440 1026 NF Cn-1 24 2.2 Cn 36 3.3 Cn+1 48 4.4
100 .00318 3.26 +.33
Finishing up
Mass of parent ion Mass of 3 carbon Remaining mass 44 36 8
A mass of 8 is pretty small. Most likely it is due to hydrogen. Potential formula - C3H8 (propane)
Using isotopic abundances

How did I know there were three carbons? No, I didnt just make it up. The key to using the A+1 and A+2 lines is the known abundances of naturally occurring isotopes. In our simple example, all that was present was H and C so lets consider them first.
Carbon - an A+1 element

When we normalize to the molecular ion, the A+1 element can help us determine the number of carbons in our compound. Roughly speaking, for every 100 carbons we measure, one of them will be a 13C. When normalized to the molecular ion, this equates to an increase in the A+1 peak of 1.1 for every carbon.

As the number of carbons increase, the probability of there being a 13C also goes up. This is an additive process. For our propane example H3C - CH2 - CH3 1.1 H3C - CH2 - CH3 H3C - CH2 - CH3 total 1.1 1.1 3.3
For hydrocarbons, the intensity of the A+1 line tells us the # carbon.
2H
has too small of an intensity to be seen.
Other A+1 elements will also contribute to the intensity of this line.

There is also the chance that a molecule might contain more than one 13C. The probability of this happening is pretty rare - #C *(1.1)2 - 0.000364% for 3 C You instrument will not be able to see this. Its less intense than the contribution from deuterium.
Nitrogen - the other A+1 element

Nitrogen will also contribute to the A+1 line intensity (15N). A nitrogen contributes 0.37 to the A+1 line. For organic GC/MS work, we typically need to rely on another method to determine nitrogen (Nitrogen rule). 0.37/N is pretty hard to see. Compounds containing many N are not very volatile so are not often seen.
diethylamine
30
58
44
In this example, you can see that the molecular ion is odd (73) and the other major ions are even.
73
10
30
50
70
mass / charge
This is because the loss of odd mass fragments is most common.
The A+2 elements

Several A+2 elements are seen in organic GC/MS work.
16O
100 32S 100 33S 0.8 35Cl 100 79Br 100
0.2 34S 4.4 37Cl 32.5 81Br 98
18O
18O
is of too small of an abundance to be very useful in GC/MS work.
Sulfur has an A+1 and A+2 isotope so you will need to correct the A+1 line when sulfur is present.
The A+2 elements

Chlorine and bromine are both common in organic compounds. They also produce very characteristic patterns due to the relatively high abundance of there isotopes. Lets look at some of their patterns. You need to be able to identify them if you ever plan of finding the molecular ion of a brominated or chlorinated compound.
Chlorine
Cl Cl2 Cl3 Cl4
By the time you have 4 chlorine, the A+2 line is significantly larger than the A line.
Bromine
Br Br2 Br3 Br4
Bromine, which is almost a 1:1 mixture of 79Br and 81Br also has a distinctive pattern.
Chlorine and Bromine

The cluster patterns of polychlorinated and polybrominated species are useful in helping to identify this type of material.
Some elements, like tin can give patterns similar to polyhalogenated species. So another factor to consider is the weight of the proposed molecular ion.
Chlorine and Bromine

# 1 2 3 4 5 6 minimum weight of molecular ion Cl Br 35 79 70 158 105 267 140 316 175 395 210 474
These do not include the weights for any other elements. If your proposed molecular ion doesn't have this minimum weight, then you should re-think your proposed assignment.
Another example
Dichloromethane - a common GC/MS solvent 49 CH2Cl2
84
Note the Cl2 pattern at 84 and the Cl pattern at 49
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General appearance
Knowing the molecular formula is nice but only part of the job. We would like to know exactly what the molecule is. This requires that we look at the rest of the data. There are a few basic rules and methods that can be used to help interpret simpler mass spectra. Lets review them
General appearance
The overall appearance can often give you an idea as to what type of material you are dealing with. Some factors to consider. Degree of fragmentation Presence of clusters General shape Odd or even major lines in clusters.
General appearance
Lets look at a few examples so you get an idea what were talking about. First, lets look at some hydrocarbons Straight chain Branched Unsaturated Rings
Each factor will have an effect on the overall appearance.

Saturated hydrocarbons
57 This is a classic hydrocarbon pattern 71 43 29 85 99 Note the regular spacing of the clusters at intervals of 14 mass units. Also, the largest line in each cluster is an odd value.
n-hexadecane
5-methylpentadecane
85 141
43
57
85 57 169 71 167 When a branch is present, we see lines associate with fragmentation on either side of the branch producing lines of higher intensity.
Deviations from the normal distribution may indicate the presence of a branch. As branching increases, the number of lines goes up. As a result, this approach is only of limited use.
normal hydrocarbon distribution Presence of a single branch.
Unsaturated hydrocarbons
Alkenes Spectra are similar to alkanes. Favored ionization process is ionization the the pi bond. Results in reduced fragmentation and increased parent peak intensity. Fragments that contained the double bond are offset by -2.
Note the increased intensity of the parent ion.
55 69 41 29 83
The major ions are at 41, 55, 69, 83, 97 ...... For an alkane they would be at 43, 57, 71, 85, 99, .....
97
111
1-hexadecene
Cycloalkanes Compared to alkanes: Reduced fragmentation is also observed. The is an increase in the parent ion. Reason: Fragmentation requires that at least two bonds be broken.
EI
+C - C - C - C - C - C
Aromatics Aromatics are easy to ionize and produce stable molecular ions. This results in reduced fragmentation and abundant parent peaks. Unfortunately, it is difficult or impossible to determine ring substitution patterns +
EI
Hydrocarbon examples
Lets look at a series of simple examples. All will be C6 hydrocarbons The spectra will only show the major lines to make it easier to view the changes. First well look at some linear C6 hydrocarbons. Then well evaluate some cyclic ones.
n-hexane
Note the molecular ion at 86. We have a typical hydrocarbon pattern with 57 being the most intense line.
57 relative abundance
43 41
27 71
86
0
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40
60
80
M/Z
n-hexane vs. 1-hexene
n-hexane Note how the molecular ion for 1-hexene (at 84) is more intense.
0 20 40 60 80
1-hexene
Also, in each cluster, the CnH2n-1 and CnH2n ions are of increased intensity.
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80
All three hexenes

1-hexene
Here you can see that the is not a large difference regardless of where the double bond is. The tendency for the bond to migrate during ionization tends to make the spectra look quite similar.
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80
2-hexene
20
40
60
80
3-hexene
20
40
60
80
1,3-hexadiene
n-hexane
With the addition of a second double bond, you can see that the mass of the molecular ion is reduced by two to 82. In addition, there is less fragmentation.
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80
1-hexene
20
40
60
80
1,3-hexadiene
20
40
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80
cyclohexane
n-hexane For cycloalkanes, we see a large increase in the intensity of the molecular ion. This is because at least two bonds must be broken to form a fragment
0 20 40 60 80
cyclohexane
EI
+CH2-CH2-CH2-CH2-CH2-CH2
0 20 40 60 80
Unsaturated rings
cyclohexene
20
40
60
80
1,3-cyclohexadiene
As the degree of unsaturation increases, the relative intensity of the molecular ion will increase. Fragmentation is also reduced.
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benzene
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80
Aromatic systems
Aromatic species are easy to ionize and tend to produce stable molecular ions.
EI +
In the case of benzene, you have three double bonds and one ring for 4 degrees of unsaturation. With the exception of some ortho isomers, most substitution patterns can not be distinguished.
Some other helpful tools

Some simple tools can be used to help interpret your spectra. The odd electron rules. Used to confirm if a spectral line could be due to a molecular ion. The nitrogen rule. Helpful if an odd number of N are present. Logical losses. Do the other lines make sense based on your molecular ion?
Odd-electron ions
Ionization of our sample occurs by the loss of an electron. The species we form is a radical +
Our original molecule had an even number of electrons. A molecular ion will have an odd number. This is a useful in identifying molecular ions.
Rings and double bonds

Assume that you have identified a spectral line as a possible molecular ion. We can use a simplistic relationship to determine it could be molecular ion. Its based on the valences of the elements involved. Only odd electron ions could be a molecular ion. After determining the formula for an ion, this is a good test to run.

Rings + Double bonds = X - 1/2 Y + 1/2 Z + 1 Where X = # of C and Si Y = # of H, F, Cl, Br and I Z = # of N and P
It the calculated value is an integer, it can only come from an odd electron ion. All molecular ions are odd electron ions but not all odd electron ions are molecular ions.

Examples Assume that you have determined the following formula for specific lines based on isotopic abundances. C3H9NO C5H5N C7H5N2 C8H16Cl Determine if any could be a molecular ion.

C3H9NO R+DB = 3 - 9/2 + 1/2 + 1 C5H5N C7H5N2 R+DB = 5 - 5/2 + 1/2 + 1 R+DB = 7 - 5/2 + 2/2 + 1 =0 =4 = 6.5 =0
Odd electron ion - could be molecular ion. Odd electron ion - could be molecular ion. Even electron ion - not molecular ion. C8H16Cl R+DB = 8 - 17/2 + 1 Even electron ion - not molecular ion.
The nitrogen rule

In organic compounds, the is a relationship between the valance and the mass of the most common isotope for most elements. Even elements have an even valance. Odd elements have an odd valance. This leads to the nitrogen rule. It assumes that we are limiting our elements to C, H, halogens, O and N.
The nitrogen rule
A compound containing only C, H, O or X will have an even molecular weight A compound with an odd number of nitrogens will have an odd molecular weight. A compound with an even number of nitrogens will have an even molecular weight.
So if the parent ion is odd, look for nitrogen.

Logical losses
Only a limited number of neutral fragments of low mass are commonly lost. 1 H 15 CH3 16 NH2 17 OH 19 F 26 CN 27 29 31 35 43 46 C2H3 C2H5 or CHO OCH3 or CH2OH Cl OC2H5 or COOH NO2
These are for single bond cleavage - radical losses.

Logical losses
We can also have neutral losses. It requires that at least two bonds are broken so they are less common. 2 H2 17 H3 N 18 2O H 20 F H 27 HCN 30 CH2O 34 H2S 36 HCl 74 C3H6O2 80 HBr 28 CO/C2H2 44 CO2
We can use these losses to help piece thing together.

Logical loss tables

The last two tables only represented partial lists of logical losses. More complete lists of these losses and common fragments can be obtained from the main menu for this course material. It is advised that you print out copies of these tables before attempting to work the example mass spectra.
Illogical losses
If the losses you encounter make no sense, then you most likely have misidentified the parent ion. Some illogical neutral losses 3 H3 11 B 4 6 9 He 12 BH or C ? 14 N or CH2 Be 24 C2
These simply dont occur!

Logical losses example

Lets see how logical losses can help. First, here is the information needed for the parent ion. M/e Rel. Abund 57 100.00 84 0.10 85 0.40 86 15.51 87 1.00
86 appears to be the parent ion. Its even numbered so we either have no N or an even number.

Now to renormalize and determine the molecular formula.
m/e A A+1 A+2 86 87 rel. Norm abund abund 15.5 1.0 100 6.5 NF 6.5 C5 C6 72 6.6 C7
86 - 72 = 14
C6H14

100
57 43 29
Relative Abundance
90 80 70 60 50 40 30 20 10 0
C2H5 C3H7 C4H9 C5H11
15 M/Z
71
CH3 86
0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 84 88

CH3 - CH2 - CH2 - CH2 - CH2 - CH3
15 29 43 57 71
So n-hexane appears to be the likely material. Mass, elements, lines and losses all make sense.
Another logical loss example - CH2Cl2

49 84
- Cl (35) - CH2Cl (49)
You can also see how we have a Cl2 pattern at 84 and a Cl pattern at 49 This also confirms the loss of a Cl.
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Common ions
A number of organic functional groups result in some common ions being produced. Observing these lines can help - Identify possible functional groups - When developing SIM based GC/MS methods.
Some common ions

Compound type Alkanes Alkenes/cycloalkanes Aliphatic alcohols Aromatics Acids/esters Alkyl amines Chloroalkyl Alkyl silanes Common lines 29, 43, 57, 71, 85, 99 27, 41, 55, 69, 83, 97 31, 45, 59, 73, 87, 101 38, 39, 50-2, 63-5, 75-8 45, 59, 73, 87, 101 30, 44, 58, 72, 86, 100 49, 63, 77, 91, 105 31, 45, 59, 73, 87, 101
Alkyl silanes are a common component of column bleed in capillary column GC/MS data
Conclusion
Remember, weve only covered a few of the rules for working with mass spectra. Weve totally left out any of the mechanisms of ion fragmentation. On the other hand, the assumption was that you were working with a GC/MS system. The chromatographic information will also help solve many problems.

2-MS Interp

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Mass spectral interpretation for chromatographers This material is designed to help GC/MS beginners who want to understand a few

Friday, August 6, 2010

What is a mass spectrum

Lets review the basic components of a simple mass spectrum.

A simple mass spectrum

Friday, August 6, 2010

A simple mass spectrum

Friday, August 6, 2010

A simple mass spectrum

Friday, August 6, 2010

A simple mass spectrum

Friday, August 6, 2010

A simple mass spectrum

Friday, August 6, 2010

A simple mass spectrum

Friday, August 6, 2010

Atomic masses and isotopes

Atomic masses and isotope

Atomic masses and isotope

Friday, August 6, 2010

Friday, August 6, 2010

General steps in mass spectral interpretation

Obtaining a good spectrum

Friday, August 6, 2010

Obtaining a good spectrum

Obtaining a good spectrum

Obtaining a good spectrum

Obtaining a good spectrum

Saturation of the two largest lines

Friday, August 6, 2010

Obtaining a good spectrum

Friday, August 6, 2010

Obtaining a good spectrum

Obtaining a good spectrum

128 hydrogen would give us a +1 shift.

Working with the molecular ion

Working with the molecular ion

Working with the molecular ion

Which line would be the molecular ion?

Working with the molecular ion

Friday, August 6, 2010

Atomic masses and isotope

Friday, August 6, 2010

Friday, August 6, 2010

Friday, August 6, 2010

100 .00318 3.26 +.33

Friday, August 6, 2010

100 .00318 3.26 +.33

Friday, August 6, 2010

100 .00318 3.26 +.33

Friday, August 6, 2010

Friday, August 6, 2010

Using isotopic abundances

Friday, August 6, 2010

Carbon - an A+1 element

Carbon - an A+1 element

has too small of an intensity to be seen.

Friday, August 6, 2010

Carbon - an A+1 element

Friday, August 6, 2010

Nitrogen - the other A+1 element

This is because the loss of odd mass fragments is most common.