Introduction

Mass spectrometry was developed in the search for and discovery of isotopes almost 100 years ago, and has evolved into an incredibly powerful tool for analyzing the mass and structure of compounds. It is widely used in laboratories all across the world for forensic analysis of trace amounts of substances, and also in research to determine the structures of large and complex natural products, such as proteins and nucleic acids. What are the basic principles of mass spectrometry and how are they applied to determine the structure of a compound?

Concepts

• Mass spectrometry

• Ionization
• Atomic theory
• Isotopes
• Natural abundance
• Atomic mass
• Molecular ions
• Fragment ions
• Bonding and molecular structure

Background

The historical development of mass spectrometry can be traced back to the work of the British scientist J. J. Thomson, who obtained precise mass/charge measurements of electrons emanating from the negative electrode in a gas discharge tube. Thomson was awarded the Nobel Prize in Physics in 1906 for the characterization of negatively charged electrons as a universal constituent of matter. Further investigation led Thomson to also study the nature of positively charged streams of atoms generated when electrons are stripped away from a gas under high voltage. When these positive ions were “bent” or deflected in the presence of electric and magnetic fields and then allowed to strike a photographic film, they left curved spots on the film at an angle that depended on their mass-to-charge ratios. In 1912, Thomson found that using neon as the gas source produced two spots. These results implied that neon consisted of or contained two types of atoms having different masses.

Conclusive proof for the existence of isotopes came from the work of Francis Aston, Thomson’s protégé at Cambridge University. Aston built a modified, more accurate version of the gas-discharge apparatus, which he called a mass spectrograph. In 1919 he obtained precise measurements for major and minor isotopes of neon, corresponding to 20 and 22 atomic mass units (amu), respectively. Aston received the Nobel Prize in Chemistry in 1922 for his discovery of isotopes. Advances in instrumentation throughout the 1920s and 30s allowed scientists to measure the masses and natural abundances of isotopes for the known elements in the periodic table. In 1939−40, Alfred Nier of the University of Minnesota, a pioneering figure in the design and construction of even more powerful mass spectrometers, used one of his instruments to separate the U-235 and U-238 isotopes of uranium. Nier supplied microgram samples of the isotopes to Enrico Fermi and his colleagues working on the Manhattan project. The samples were used to prove that U-235 was the isotope responsible for fission.

From these early developments, mass spectrometry has evolved into a powerful method of chemical analysis based on the production, separation, and measurement of charged atoms and molecules according to their masses. While modern mass spectrometers incorporate many advanced features, all mass spectrometers share the following basic design elements (see Figure 1 on page 2). Atoms and molecules in a sample are stripped of electrons and converted to positively charged ions in an ion source, and are then sent through a mass analyzer, where they are separated according to their mass-to-charge ratio (m / z) by the application of electric and magnetic fields. The masses and relative amounts of the separated ions are measured in a detector, and computer processing is used to analyze the results and display them in chart form. The entire process is carried out under high vacuum conditions.

At first restricted to the study of volatile substances, modern mass spectrometry may be used to analyze solids, liquids or gases. Advanced ionization techniques have been developed to permit the formation of gaseous ions from any size molecule and any sample state, even very large molecular weight proteins with masses of thousands of dalton units (Da, where 1 Da = 1 amu). The two most common types of ionization for volatile samples (generally, gases or liquids) are electron ionization (EI) and chemical ionization (CI). In the EI process, a sample is inserted directly into an ionization chamber, where it is exposed to a beam of electrons that have been accelerated to approximately 70 eV of energy. The electron beam strips electrons away from sample molecules (M), producing positively charged radical ions (M⁺). See Equation 1. In the process, the molecular ions M⁺ also absorb some of the excess electron energy, making them susceptible to fragmentation. The molecular or parent ions break apart into different size fragment ions, as discussed below. Because all of the ions will be separated by the mass analyzer and produce signals at various m/z values, the pattern of peaks (signal intensity versus m/z) in the mass spectrum is unique and characteristic of the structure of a compound, much like a fingerprint.

Chemical ionization (CI) is a milder ionization technique. In CI, a reagent gas such as methane (CH₄) is first ionized by electron impact, and the sample M to be analyzed is then exposed to the reagent ions. Collisions and reactions between M and the reagent ions produce molecular ions M⁺ as well as fragment ions. The extent of fragmentation is generally less in CI than in EI, making it easier to associate the heaviest major peak in the mass spectrum with the molecular ion. Fragment ions are produced by bond breakage in the structure of a compound, with certain bonds being more likely to break. For typical organic compounds with many carbon atoms and just a few heteroatoms (X = O, N, Cl, Br, S, etc.), breakage of a C−X bond is common, as is breakage at highly branched carbon atoms.

In almost all cases, a sample molecule will lose only one electron via either CI or EI, producing an ion with a charge z = +1. Because loss of more than one electron from a single molecule is rare, m / z reduces to m, the mass number of an ion. The mass analyzer and detector in a mass spectrometer detect the masses of individual ions. In calculating the mass of a molecular ion or fragment ion it is necessary to add up the actual mass or mass numbers of individual atoms or nuclides in the formula of a molecule, NOT the average atomic mass of an element. Consider methyl chloride (CH₃Cl)—its average molar mass is 50.45 amu. Chlorine exists, however, as a mixture of two isotopes, Cl-35 and Cl-37, with the natural abundance of each equal to 75.8% and 24.2%, respectively. The mass spectrum of CH₃Cl shows two peaks for individual molecular ions. A major peak occurs at m / z = 50 and is designated M+. This is due to CH₃−³⁵Cl. A second peak at m / z = 52 arises from CH₃−³⁷Cl and is designated M+2. The height ratio of the M⁺/M+2 peaks is 3:1, reflecting the natural abundance for the two chlorine isotopes. (The major isotopes of C and H are C-12 and H-1, and their mass numbers are used to determine the mass of the CH₃ fragment, 15 amu.)

Figure 2 shows the mass spectrum and fragmentation pattern of 1-bromobutane, an organic compound with four carbon atoms in a hydrocarbon chain and a bromine atom at the end of the chain. The mass spectrum can be interpreted as follows to identify the structure of 1-bromobutane.

• Two closely spaced peaks at m / z = 136 and 138 appear because bromine has two isotopes, Br-79 and Br-81, with natural abundances of 50.7% and 49.3%, respectively. The height ratio of these peaks, labeled M⁺ and M+2, respectively, is 1:1.

• The tallest or most intense peak in the mass spectrum occurs at m / z = 57. This fragment ion is labeled M−79 and is due to the loss of a Br atom from the M⁺ ion (136 − 79 = 57). The C−Br bond is easily broken, giving rise to the fragment ion CH₃CH₂CH₂CH₂⁺.

• Breakage of a C−C bond in the middle of the hydrocarbon chain leads to the loss of a CH₃CH₂ radical and produces major fragment ion peaks at 107 and 109 labeled M−29 (136 – 29 = 107).

Experiment Overview

The purpose of this guided-inquiry learning activity is to investigate the principles and applications of mass spectrometry. The activity is divided into three parts.

1. Isotopic abundance and the mass spectra of elements.
   
   
2. Ionization methods and fragmentation patterns in mass spectrometry.
   
   
3. Molecular structures and mass spectra of organic compounds.