What is the exact mass of my chemical?

A previous page showed spectra of Glucose-6-phosphate which has a molecular formula C₆H₁₃O₉P. This gives it a molecular mass of (6×12)+(13×1)+(9×16)+31 = 260.

We saw it as an ion following loss of H⁺ at a mass of 259.0. Here, as another example is (somewhat fragmented) NAD:

NAD has the formula C₂₁H₂₇N₇O₁₄P₂, giving it a molecular mass of 663.

The big peak in the spectrum above is a fragment. Concentrate for the moment on the smaller peak. There is a discrepancy! The peak has appeared at 662.2. We would have expected 662.0 (this is negative ion chromatography, so there should have been loss of H⁺). But even our fairly inexact mass spectrometer has identified 662.2.

Worse still, the chemical catalogue lists NAD as having a mass of 663.4. How can this be?

There are two completely different reasons why masses are not integers, and there are two different sorts of "exact mass" that can occur. The next two paragraphs explain what is happening:

Non-integer masses caused by isotopes

This is the cause that is familiar from chemistry. Most elements exist as isotopes, differing in mass. Usually the heavy isotopes are fairly rare. For instance, about 1% of carbon weighs 13, not 12. However, in some cases they are common (chlorine, for instance, is about three-quarters 35 and one quarter 37). The usual way to deal with this is to use relative atomic masses quoted to suit the mixture of isotopes found in the natural world. Hence everyone knows the mass of chlorine is 35.5 (approximately). This is because 35.5=[(3/4)×35]+[(1/4)×37].

The exact mass of NAD quoted on the bottle (and in the catalogue) is intended to allow you to weigh out exactly one mole of NAD, so it is the mass taking into account the heavy isotopes.

If a mass spectrometer had very bad mass resolution, it would also measure this average mass of isotopes. However, being an exact sort of instrument, real mass spectrometers will almost always see all the isotopes separately. NAD containing carbon-13 is a whole Dalton heavier and shows up as a separate peak. This is just visible on the spectrum above, but much clearer examples are available in our page on isotope effects. This sort of exact mass therefore has no relevance in mass spectrometry.

Really exact masses

Protons do not weigh exactly 1.000, and nor do neutrons. Worse still, when they combine in a nucleus some of their mass disappears to create the energy needed to bind the nucleus together (Einstein's E=mc² energy). If you want to know more about this sort of thing and are a human with a sense of fun, I really, really strongly recommend The University of Colorado site. We define the atomic weight of carbon-12 as exactly 12.000. In general, small nuclei need less glue, so they have smaller mass-losses, and weigh a bit more than they should. Hydrogen thus weighs 1.007825 (Data from SIS (Scientific instrument services), who got it from the CRC Handbook and other references). Nitrogen weights 14.003074, and Oxygen weighs 15.994915. Thus water (H₂O) does not weigh the same as an ammonium ion (NH₄⁺), even though it ought.

Simple mass spectrometers such as quadrupole instruments tend to be accurate to about 0.1amu, which is not enough to distinguish between isomers that nominally weigh the same, but actually differ in exact mass because of the mass defects described above. Ion cyclotrons and time-of-flight instruments often can. They are therefore capable of guessing the empirical formula of an ion from its exact mass. More information is available on this and on isotope effects.