Overview

Mass spectrometry (MS) is a proven analytical method used to glean information about the chemical structure of a chemical sample. MS is applied to fields as disparate as airport security, food and wine analysis, drug and explosives analysis, as well as most fields of chemical and biological research. MS works by ionizing, or bestowing a net charge, on a sample of molecules and then sorting the ions based on their mass-to-charge ratio. Since the particle has a one electron negative charge or one proton positive charge, the mass spectrometer can make use of electrical and/or magnetic fields to essentially sort molecules by their masses. The charged molecules are then guided by electromagnetic attraction or repulsion to a detector mechanism. A typical mass spectrum (shown below) plots the different mass-to-charge ratios (m/z) against their abundances (occurrence of a certain ion divided by the occurrence of the most plentiful ion) within the sample.

Aspiring chemists and biologists, as well as anyone interested in gaining a greater understanding of these fields, can benefit from a greater understanding of MS. This Instructable will provide a set of instructions for the reading and comprehension of a simple mass spectrum of a halogenated alkane (haloalkane), or a compound containing only hydrogen, carbon and a halogen. Haloalkanes will be analyzed both because of their wide use as chemical solvents and because understanding of their spectra forms a good baseline for future learning in the MS field. The above information will be explained in more detail in the coming steps to share insight into how a mass spectrometrist can use a mass spectrum to determine the identity of an unknown chemical sample.

Materials: Four-function calculator, notebook, Periodic Table of Elements (optional)

Time Investment: 10 to 15 minutes

Experience Required: High school level knowledge of chemistry (i.e. atoms, elements, Periodic Table)

Step 1: Step 1: Identify the Molecular Ion

The molecular ion represents the entire molecule in question, prior to any fragmentation. Each analyte molecule is given a charge of one, so the molecular ion m/z value represents the molecules total mass. Ionization, specifically electron impact (EI) ionization, is used to remove an electron from an analyte molecule so that it can be analyzed by the electrical and magnetic fields of the mass spectrometer. EI, however, is a “hard” ionization source that can cause molecules to fragment, or break into multiple pieces. It is therefore important to first identify the molecular (complete) ion.

1. The molecular ion is typically represented on the mass spectrum as the peak with the highest m/z ratio. Find and record this value in your notebook.

Example: In the EI mass spectrum of water (shown above) a large peak is seen at m/z value 18. Water has a weight of 18 atomic mass units, or Daltons, so the peak at m/z 18 represents the molecular ion. The smaller peak at m/z 17 represents a water molecule in which a hydrogen is removed by fragmentation.

Step 2: Step 2: Identify Major Fragmentation Clusters

The molecular ion reflects the complete weight of an analyte molecule, but, considering the fact that there are dozens of stable elements, the molecule’s weight alone will not reveal its identity. Fortunately, individual molecules have relatively unique EI fragmentation patterns. The different peaks on a mass spectrum reveal the compounds identity, so, as shown below, a mass spectrometrist should identify all major spectral peaks. A major peak is the most abundant peak within a cluster of smaller peaks. For this introductory Instructable, the largest (most abundant) peak in each cluster will represent the entire cluster.

1. Use a highlighter to identify the most abundant peak in each cluster.

2. Determine the numerical m/z value for each of these major peaks and write all of them down in your notebook.

-Note that the molecular ion is not necessarily the most abundant peak. The most abundant peak is, by naming convention, the base peak.

Example: Major peaks at m/z 15, 29, 43, 57, and 184 (see above sample spectrum).

Step 3: Step 3: Determine the ∆m for Each Major Peak

Smaller peaks clustered around each major peak are largely present due to differences in which of the two fragments retains the ion, gain or loss of protons, and naturally occurring elemental isotopes. For a simple alkane or halogenated alkane the ∆m (mass difference between two singly charged ions) values will typically involve the gain or loss of about 14 or 15 Daltons between major peak clusters. These mass differences represent the gain or loss of a methyl (-CH3) or methylene (-CH2-) from the larger ion. Some long or branched molecules exhibit advanced fragmentation patterns, so you should assume the presence of a halogen only if there is a 17 Dalton or greater ∆m value between major peaks in a haloalkane.

1. Use the simplified “mass spectrum” from Step 2 to determine the mass difference (∆m) between each peak and the next peak on the spectrum.

2. Use your calculator and list of major m/z peaks to determine numerical ∆m values. Write these values down in your notebook.

Example: ∆m values between major peaks (starting from the left, m/z 15) are 14, 14, 14, and 127 Daltons.

Step 4: Step 4: Identify Any Heteroatoms

Mass differences between major peaks will often exceed the 14 or 15 mass units representing a methyl or methylene carbon. A ∆m value greater than 16 Daltons indicates the presence of a heteroatom, or atom that is not carbon or hydrogen (for our study, a halogen). Common halogens (Group 17 on the Periodic Table) include fluorine, chlorine, bromine and iodine. These can essentially replace one hydrogen-to-carbon bond of an alkane and dramatically alter its chemical and physical properties. Luckily, singularly halogenated alkanes have easily identifiable mass spectra (see iodobutane spectra below).

1. Check your list of mass differences created in Step 3. If one of these ∆m values is larger than 16 mass units, use the Periodic Table to determine which halogen the molecule contains.

2. Use the Periodic Table (or the table above, for convenience) to determine if the halogen is element F, Cl, Br, or I. Write this halogen down in your notebook.

Example: The ∆m value of 127 Daltons from Step 3 indicates the presence of an iodine atom in the example spectra (see previous table).

Step 5: Step 5: Identify Remainder of Molecule

Now that the halogen has been identified, the length of the carbon chain itself can be determined. The general chemical formula of an alkane is CnH2n+2, but, since the halogen effectively replaces one of the iodobutane hydrogens, the modified alkane formula is CnH2n+1X (where X is a halogen).

1. Look to the largest peak on your mass spectrum that does not include the halogen (molecular ion mass minus halogen mass).

2. Use this mass, algebra and the masses of carbon and hydrogen to solve for n (see example).

3. Add this number back into the chemical formula (CnH2n+1) and append the halogen for the complete chemical formula. Write this down in your notebook.

Example: The largest non-halogenated m/z value from Step 2 is 57. By plugging this into the equation above (CnH2n+1X) and substituting the atomic masses of carbon (12 Da.) and hydrogen (1 Da.) (12n + 1(2n+1) = 57; see Periodic Table for masses), the example alkane formula can be determined to be C4H9X. The previously determined halogen (iodine) can then be added to this formula for a final identification of C4H9I.

Step 6: Step 6: Name the Molecule

1. Combine the name from the number of carbons with the halogen prefix from the tables above to give the total chemical name. Write this down in your notebook.

Example: C4H9I = butane (C4) + iodo- (I) = iodobutane

Step 7: Step 7: Check Your Work

The National Institute of Standards and Technology (NIST) database contains a collection of standardized mass spectra. This database can be used to confirm a naming decision made based on a mass spectrum.

1. Simply use a Google search of “[chemical name] NIST EI Spectra” and go to the http://webbook.nist.gov option to check your work. The two spectra (provided and NIST standard) should match closely. A close match indicates correct identification.

1. If the major peaks of the NIST mass spectrum do not match your teaching spectra:

· Recheck your naming of the chemical formula (Step 6)

· Recheck your identification of major peaks for a missed cluster of peaks (Step 2)

Example: See NIST EI spectra of iodobutane (above). This closely matches the example spectra in Steps 2 and 3.

Further Information

You now have a basic knowledge of the landmarks and meaning of a simple mass spectrum. If this topic interests you, consider exploring the following intermediate topics:

· MS analysis of oxygen (and other non-halogen heteroatom) containing compounds, such as alcohols, ethers and carboxylic acids

· MS identification of hydrocarbon isomers (molecules with the same chemical formula but containing different bond patterns)

· MS application to protein analysis (proteomics)

The following link will take interested readers to a good intermediate level primer on mass spectrometry (courtesy of Thermo Fisher Scientific):

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-mass-spectrometry.html