When determining the structure of a compound or detecting what compounds are present, one of the very best methods is mass spectrometry (MS).

When measuring a known compound, mass spectrometry can be used to determine with a high degree of confidence whether or not it is a target compound. Even if it is an unknown compound, mass spectrometry can be used to measure its molecular weight. As a result, the structure of the molecule can be deduced.

In both organic chemistry and biochemistry, MS is the analytical instrument used by a very large number of researchers. Therefore, you must understand how to utilize mass spectrometry.

Aside from understanding the principles of MS, you also need to learn how to read spectral data. This will allow you to measure the molecular weight of your target compound and estimate the structural formula. How to use mass spectrometry will be explained here.

Mass spectrometry is a method of measuring the molecular weight of a compound

Among the analytical methods, what is measured by mass spectrometry? Mass spectrometry measures the molecular weight of a compound.

  • An unknown natural compound is extracted.
  • Want to know the contaminant of the substances that are mixed in food.
  • Unknown if the target compound was produced.

Because these situations are so common, mass spectrometry is used. Once the molecular weight is determined by MS, it is possible to guess what substances are contained in the sample, even if it is not known what substances are contained.

Of course, it is necessary to set and measure a sample with only one target compound in it, not multiple compounds. For this reason, the substance is isolated in advance by chromatography. After that, the molecular weight of the target compound is obtained by measuring it.

Utilizing a Magnetic Field to Measure the Mass of a Compound

How does MS give us the molecular weight of the substance being measured? This is because we use magnetic fields to measure the ionized material.

There are several ways to ionize a substance. Once ionized, the molecule becomes charged. In mass spectrometry, the molecule becomes a positive ion.

A magnetic field is then applied. As the ions pass through the magnetic field, they curve. Different strengths of the magnetic field will cause the ions to curve to different degrees. To be more precise, the higher the molecular weight (heavier substances), the more powerful the magnetic field must be to curve the ions.

The same is true for humans and magnetic fields, as we cannot move heavy objects without applying a strong force.

So, MS changes the force of the magnetic field little by little to see if the detector can measure it. If the magnetic field isn’t optimal, it won’t reach the detector because it will hit a wall along the way, and nothing will be observed. On the other hand, if the magnetic field is optimal, the molecules can be observed.

The molecular weight of the target compound can be measured by gradually changing the magnetic field in order to see if ionized molecules are detected.

The MS spectrum obtained in this way is called a mass spectrum. By looking at the spectral data observed by mass spectrometry, it is possible to infer what kind of substance the target compound is.

In many cases, electron ionization is used in mass spectrometry

There are many different ways of doing these mass spectrometry methods. The following types are available

  • Electron ionization (EI)
  • Chemical ionization (CI)
  • Fast atom bombardment (FAB)
  • Matrix Assisted Laser Desorption/Ionization (MALDI)
  • Electrospray ionization (ESI)

Unless you are a student studying for an exam, there is no point in understanding and remembering these detailed features. For this reason, I will not describe the detailed characteristics of each analytical method. In any case, it is good to understand that there are several methods of mass spectrometry.

The most common of the MS methods is electron ionization (EI). This technique is used to measure molecular weight by heating the sample to ionize it. The sample to be measured is ionized by bombarding it with electrons. Since only the target compound is measured, it is simple and easy to analyze data.

However, the electron ionization (EI) method cannot measure heat-sensitive compounds or high-molecular compounds such as proteins. In such cases, other analysis methods are used to measure molecular weight.

Spectral Data and Readings in MS: Mass-to-Charge Ratio (m/z) and Charge (z)

So, when you measure the mass spectrum with MS, what does the reading look like? When you do mass spectrometry, you won’t detect a single peak. Mass spectra allow you to observe multiple peaks.

For example, when acetic acid is measured with MS, the data in the mass spectrum is as follows.

As you can see, multiple peaks can be observed.

When the instrument ionizes a molecule, it does not necessarily ionize the molecule in its acetic acid state. The cleavage of the molecule produces the molecules shown in the figure above. As a result, these molecules can be detected in the mass spectrum.

The horizontal axis is expressed in terms of the mass-to-charge ratio (m/z). The m is the mass of the ion and the z represents the charge. In short, let’s understand that m/z describes the molecular weight.

On the other hand, the vertical axis is ionic strength. The most common peak in the mass spectrum is 100%. In the previous figure, the molecule with the highest peak (m/z 43) has 100% ion intensity on the vertical axis. Compared to this intensity, the ratio of how much was detected in the mass spectrum is shown on the vertical axis.

Using acetic acid as an example, we have explained how to read the mass spectrum. In the mass spectrum, not only the molecular weight of the target compound, but also several other molecules created by electron bombardment are produced, from which the structural formula can be inferred.

Radical Cation-Induced Cleavage Reaction Separates the Molecules

So what kinds of molecules can be detected in the mass spectrum? Mass spectrometry uses a high-energy beam of electrons to eject electrons present in a molecule and create radical cations.

As the electrons fly away, unpaired electrons are created. If the positively charged radical cation is detected without causing any particular reaction, the molecular weight of the target molecule can be measured.

However, radical cations are highly reactive. For example, with acetic acid, as noted earlier, multiple molecules can be detected.

In many cases, unshared electron pairs (electrons not involved in the bonding process) are scattered outward when a high-energy electron beam is applied by MS. For example, when nitrogen and oxygen atoms are in the structural formula, the unshared electron pairs of these atoms are ejected, and radical cations are easily formed.

For this reason, the presence of amino (NH2) and carbonyl (C=O) groups in the structural formula makes it easier to break the bond in that part of the molecule.

Other atoms such as chlorine, bromine, and iodine atoms bound to the benzene ring tend to fly away when they are hit by high-energy electrons.

Note that radical cleavage does not necessarily occur, and other reaction mechanisms can break the bonds between molecules. Sometimes the shape of the molecule can change dramatically due to a rearrangement reaction. The most common reaction, however, is that of radical cleavage. Radical cleavage results in multiple peaks in the mass spectrum.

Isotopes Such As Chlorine and Bromine are Detected

When making measurements with MS, the other thing to consider is the isotope. For example, the most common carbon atom is 12C. However, there is a small amount of 13C carbon. These isotopes have different masses. Therefore, they have different peaks that are detected by mass spectrometry.

13C is a small percentage. However, chlorine and bromine have a high percentage of isotopes. The ratio is as follows.

  • 35Cl : 37Cl = 3 : 1
  • 79Br : 81Br = 1 : 1

Therefore, isotope peaks are clearly detected in cases where chlorine or bromine atoms are present in the structural formula. For example, we have the following.

In the case of chlorine atoms, the isotope ratio is 3:1, as described above. Therefore, the ionic strength on the vertical axis also shows a spectral peak at a ratio of 3:1.

Isotope Peaks of Carbon Atoms Are Observed at M+1

As explained earlier, the probability of 13C’s existence is low compared to 12C. 13C exists at an existence ratio of 1.1%. So, can we ignore 13C when analyzing with MS? In mass spectra, it is not always sufficient to ignore 13C.

When the molecular weight is low, 13C is almost not observed because the ratio of 13C is only 1.1%. However, as the molecular weight increases and the number of carbon atoms increases, the 13C isotope peak becomes more and more visible. The original molecular weight is represented by M, and the 13C isotope peak is represented by M+1.

With n carbon atoms, the intensity ratio of M+ to [M+1]+ is as follows

  • M+ : [M+1]+ = 100 : 1.1 x n

The presence of n carbon atoms increases the probability of a 13C carbon atom being observed. For example, the following is a mass spectrum of 4-acetyl biphenyls.

The molecular weight of 4-acetyl biphenyl is 196. However, in the mass spectrum, you can see a smaller peak next to the main peak. This peak is not meaningless, because 13C was observed as an isotope. This molecule has 14 carbon atoms, so a spectral peak of [M+1]+ is observed at 15.4% intensity.

  • 1.1% x 14 = 15.4%

There are few opportunities to be aware of the isotopes when doing research. However, in mass spectra, we need to be strongly aware of the isotopes of carbon atoms.

Mass spectra important in structural analysis

One of the most widely used analytical methods in many studies is mass spectrometry. It allows you to measure the molecular weight of a target compound and estimate what it contains as an unknown compound. It is a widely used analytical method in both organic chemistry and biochemistry.

It is not only the molecular weight that can be determined. As radical cations are produced, they cleave within the molecule and other peaks can also be observed. From these peaks it is possible to deduce the structural formula of the molecule.

The principle is simple, as the strength of the magnetic field is used to measure the molecular weight. However, there are several analysis methods, and the electron ionization (EI) method, the most commonly used, has the disadvantage that it cannot measure molecules that decompose by heat. So, when doing MS, we have to choose the best method.

In addition, it is important to consider isotopes. With an understanding of these characteristics, you should use mass spectrometry in your scientific experiments.