This section will try to introduce new users of mass spectrometry to why an understanding of isotopes is important even if the desired information is just molecular weight (MW).

In this discussion, it is assumed that reader already knows what is an isotope.

Let us look at a simple molecule such as citronellol. Citronellol is the principle constituent of citronella oil and is also present in other oils such lemon, lemon grass and Melissa. Citronellol is composed of C10H18O. If its MW is obtained from a chemical catalog or a reference book, such as the Merck Index, the MW shown will be 154.4 u. But if a mass spectroscopist is asked what the MW of citronellol is that person will more than likely tell you that it is 154.1 u. Who is right? Both answers are correct. The first MW is based on average elemental weights and the second MW is based on mono isotopic MW of the most abundant isotope of an element. You can use this web linked table to calculate these MW yourself.

Now that you see both answers are correct, the question to ask is which one should you use? To answer this question let us look at the mass spectrum of citronellol below.

Citronellol

You can see that there are three peaks in the MW region and the most abundant ion has the mass of 154.1 u. The 154.1 u peak is produced by citronellol ions that are entirely composed of ten carbon 12, eighteen hydrogens, and one oxygen 16. The middle peak is principally composed of nine carbon 12, one carbon 13, eighteen hydrogens and one oxygen 16. Of course, there are molecules that help comprise the second peak that are composed of ten carbon 12, one deuterium, seventeen hydrogens, and one oxygen 16. In addition, molecules made up of ten carbon 12, eighteen hydrogens , and one oxygen 17. The ratios of these molecules combinations can be calculated statically from the relative abundances found in the table.

The third peak is principally composed of eight carbon 12 and two carbon 13, eighteen hydrogens and one oxygen 16 with minor contributions from all other combinations of isotopes that can be combined to make a MW of 156 u.

Since the major peak in the mass spectrum is 154.1 u it becomes easy to see why the mass spectroscopist would give a MW based on mono isotopic abundances.

Is Average MW Close Enough For Me? (Not Always)

Another interesting molecule is chlorophyll-a from plants. This is makes green plants green. It is composed of C55H72MgN4O5. It has an average MW of 893.5 u and a mono isotopic MW of 892.5 u. This is a large difference at this MW. Again, to understand what is going on let us look at the mass spectrum.

Chlorophyll a

It is observed that the mono isotopic peak is still the most abundant but its relative abundance to the other peaks has significantly decreased as compared to citronellol. The reason for the peak ratio difference is due to the law of averages. As more atoms make up a molecule, the higher the probability is that the a carbon 13 will be present or any other minor isotopes of the elements that make up the compound. Also Mg has a significant percentage of minor isotopes.

In general, if your compound is below 1000 u and you can see your mono isotope, you should use mono isotopic abundances to calculate your MW for comparing against your mass spectrum.

Isotope Ratios Serve As Composition Indicators.

The MW isotopic ratios can be very diagnostic especially when looking at compounds that contain elements that have very distinctive patterns. One such example is 4-chloro-3,5-xylenol (C8H9ClO) which is a topical antiseptic and a urinary antiseptic. The mono isotopic MW (MWmono) is 156.0 u and the average MW (MWave) is 156.6 u.

4-chloro-3,5-xylenol

One observes the distinctive pattern associated with one chlorine. The two major peaks are located 1 u apart from each other and the ratio is 3:1. Bromine also produces a similar pattern but the ratios of the two peaks are 1:1.

Zinc stearate [Zn(C18H35O2)2, MWave 632.4 u, MWmono 630.5 u] is used in the manufacturing of cosmetic and pharmaceutical powders and ointments. It is also used in many other areas such as in lacquers as a flatting and sanding agent. The zinc in this compound produces a pattern that would lead one to immediately think the compound contains a metal.

Zinc stearate

Similar types of distributions are common among the metals.

Average Versus Mono isotopic Calculations For High MW Compounds

As a general rule below 1000 u use mono isotopic abundances, but what about above 1000 u?

The following spectrum is of mellitin (C131H228N38O32, MWmono 2845.7 u, MWave 2847.0 u) or bee venom.

mellitin

As can be seen the MWmono is no longer the most intense peak in the spectrum. With the number of atoms mellitin contains, the probability of a mellitin molecule containing one or more minor isotopes has statistically increased beyond that of not contain any minor isotopes.

Lets take this one step further and look at human P450 3A4 (C2578H4046N650O710S25, MWmono 56271.3 u, MWave 56308 u).

human P450 3A4

Looking at the above mass spectrum, it is observed that the law of averages dictates that probability of having a molecule of human P450 3A4 composed of just the major isotopes (MWmono 56271 u) is comparable to winning the lottery. The MWave 56308 u is the only way to compare what you expect and what is measured by mass spectrometry. The MWave is at the apex of the near Gaussian curve as defined by the tips of the peaks.

In summery, as a general rule use MWmono until the MWmono peak is not visible and then use MWave.

Also see "Resolution, How It Affects My Mass Spectrum."

The above spectra were produced using Bruker software. If you are interested in what your compound's isotopic pattern would look like without obtaining a mass spectrum, bring the its composition to the Mass Spectrometry Facility to have it generated by computer.