Introduction: How to Read a Simple Mass Spectrum
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
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FAQs
How do you interpret a mass spectrum? ›
We start interpreting the mass spectrum by looking at the peak of furthest to the right. This is the
How do you read LC MS data? ›Interpreting the Chromatograms
The chromatograms show components as functions of their retention time and mass to charge ratio by the mass relative abundance, meaning the total output from a full LC-MS is a graph with two horizontal axes. The focus of the graph can be altered depending on the researcher's goal.
They will gain a certain amount of energy. What that means is that the large mass. And K equals one-
What does M Z stand for? ›01. What does m/z mean? M stands for mass and Z stands for charge number of ions. In mass analysis, an electron is taken from molecules to create single charged ions. If two electrons are removed, double charged ions are produced.
What is mass spectrometry for dummies? ›Mass spectrometry is an analytical tool useful for measuring the mass-to-charge ratio (m/z) of one or more molecules present in a sample. These measurements can often be used to calculate the exact molecular weight of the sample components as well.
What does M+ peak mean? ›The M+ peak is usually the highest intensity peak in the cluster of peaks at highest m/z.
What do the peaks on the mass spectrum represent? ›The mass spectrum will contain peaks that represent fragment ions as well as the molecular ion (see Figure 1.3). Interpretation of a mass spectrum identifies, confirms, or determines the quantity of a specific compound.
What is M Z ratio in mass spectra? ›In mass spectroscopy, the mass-to-charge ratio (symbols: m/z, m/e) of a cation is equal to the mass of the cation divided by its charge. Since the charge of cation formed in the mass spectrometer is almost always +1, the mass-to-charge ratio of a cation is usually equal to the mass of the cation.
What is q1 and q3 in mass spec? ›In the product scan, the first quadrupole Q1 is set to select an ion of a known mass, which is fragmented in q2. The third quadrupole Q3 is then set to scan the entire m/z range, giving information on the sizes of the fragments made.
What does LC-MS measure? ›Liquid chromatography-mass spectrometry (LC–MS) is a powerful analytical technique used for separation, identification, and quantification of both unknown and known compounds as well as to elucidate the structure and chemical properties of different molecules.
What is the difference in LC-MS and LC-MS MS? ›
LC-MS instruments are basically HPLC units with a mass spectrometry detector attached to it whereas LC-MS/MS is HPLC with two mass spectrometry detectors.
What are the two elements in mass spectrometer? ›A mass spectrometer consists of three components: an ion source, a mass analyzer, and a detector.
How do you calculate protein mass in MZ? ›Its mass can be determined by taking the monoisotopic mass (which is the very first m/z in the envelope), multiply that by 16 and substract 16: (946.00384*16)-16 = 15120.06 Da.
Why only positive ions are detected in mass spectrometry? ›Because mass spectrometry measures the mass of charged particles, only ions will be detected, and neutral molecules will not be seen. Ions are created by giving electrons to a molecule (producing a negatively charged ion) or taking electrons away from a molecule (producing a positively charged ion).
How do you pronounce MZ? ›How to pronounce mz - Vocab Today - YouTube
Is mass spectrometry hard to learn? ›In a recent article published in BioTechniques, titled: Democratizing Mass Spectrometry, author Jeffery Perkel indicates that; mass spectrometry is often seen as a complicated and highly technical technique that requires years of experience to master.
What are the 5 processes of mass spectrometry? ›In a mass spectrometry experiment, the process sequence of analysis works in five stages, including sample introduction, analyte ionization, mass analysis, ion detection, and data processing.
How do you find the number of carbons from mass spec? ›If you measure the peak height of the M+1 peak as a percentage of the peak height of the M+ peak, that gives you the number of carbon atoms in the compound.
What does base peak tell you? ›The vertical axis denotes the relative abundance of ions. The most intensive peak in a spectrum is called the "Base Peak", whose intensity is taken as 100 percent. This ion exists most abundantly in the ion source and represents the most stable ion, which is useful for identifying the compound.
How do you predict base peak? ›Mass Spectroscopy: The Difference Between M+ (Parent) Peak vs Base ...
What is m 2 in mass spectrometry? ›
M+2: In mass spectrometry the molecular ion whose mass is two higher than M. Molecular ion region of the mass spectrum of 1-chlorodecane (C10H21Cl). The relative abundance of M is due to 12C101H2135Cl.
How do you predict mass? ›Divide the object's weight by the acceleration of gravity to find the mass. You'll need to convert the weight units to Newtons. For example, 1 kg = 9.807 N. If you're measuring the mass of an object on Earth, divide the weight in Newtons by the acceleration of gravity on Earth (9.8 meters/second2) to get mass.
What are fragments in mass spectrometry? ›In mass spectrometry, fragmentation is the dissociation of energetically unstable molecular ions formed from passing the molecules in the ionization chamber of a mass spectrometer. The fragments of a molecule cause a unique pattern in the mass spectrum.
What is the difference between base peak and molecular ion peak? ›The base peak is always the tallest peak in the spectrum. The base peak represents the most common and most abundant ion. The molecular ion peak is the peak that represents the heaviest ion. This peak has the highest m/z ratio on the mass spectrum but it does not necessarily represent the most abundant ion.
Is amu same as M z? ›The mass spectrometer measures the mass to charge ratio of individual ions commonly written as m/z where m is the mass in amu and z is the charge in units of the charge of a proton.
What is m2 peak? ›mass spectra - the M+2 peak. MASS SPECTRA - THE M+2 PEAK. This page explains how the M+2 peak in a mass spectrum arises from the presence of chlorine or bromine atoms in an organic compound. It also deals briefly with the origin of the M+4 peak in compounds containing two chlorine atoms.
How many peaks will the mass spectrum have? ›The 5 peaks in the mass spectrum shows that there are 5 isotopes of zirconium - with relative isotopic masses of 90, 91, 92, 94 and 96 on the 12C scale. This time, the relative abundances are given as percentages.
What is the difference between single quadrupole and triple quadrupole? ›Single quadrupole systems contain only one mass filtering quadrupole while triple quadrupole systems consist of three quadrupoles. Q1 and Q3 are working as mass filters while Q2 is acting as collision cell. Quadrupoles can be used in scanning or filtering mode.
What is SIM mode in LCMS? ›Selected Ion Monitoring (SIM) allows the mass spectrometer to detect specific compounds with very high sensitivity. In SIM mode, the instrument is set to gather data at masses of interest, instead of stepping the mass filter over a wide range of masses.
What is QqQ in mass spectrometry? ›A triple-quadrupole mass spectrometer, also known as QqQ, is a tandem MS method in which the first and third quadrupoles act as mass filters and the second causes fragmentation of the analyte through interaction with a collision gas—it is a radiofrequency-only quadrupole, and can be used in either SIM or scan mode.
Why is LC-MS important? ›
Liquid chromatography mass spectrometry (LC-MS) is the main method for detecting drugs and their major metabolites in vivo, and can provide high enough sensitivity, specificity, and molecular structural information for the qualitative assay of drugs and their metabolites (Shi et al., 2011).
How accurate is liquid chromatography mass spectrometry? ›The intra- and interday precisions were <5.2%, the accuracy varied from -4.1 to 4.5%.
Is LC-MS quantitative or qualitative? ›LC-MS is a versatile and highly sensitive analytical technique for the measurement of small molecular weight compounds in a diverse range of sample types. It uses a series of mass detection systems to provide both quantitative and qualitative analyses.
Which one is better GC-MS or LC-MS? ›This is because LC–MS-MS offers several advantages over GC–MS such as quicker and less extensive extraction procedures and the ability to identify and measure a broader range of compounds.
Is LC-MS more sensitive than GC-MS? ›First and foremost, GC/MS methods tend to need a higher sample volume than LC/MS/MS methods. GC/MS is a less sensitive instrument, which means more sample is needed to achieve lower limits of quantitation (LOQs). Many LC/MS/MS methods only require 50-200 μL of sample.
Is LC-MS same as HPLC? ›Compared to HPLC, the described LC-MS was faster, more sensitive and specific. Unlike HPLC, LC-MS could be applied to analyze incompletely resolved mixtures. The absolute detection limits for LC-MS and HPLC were 0.2-0.5 and 10-25 ng, respectively.
How does a mass spectrometer work step by step? ›The four stages of mass spectrometry are – ionization, acceleration, deflection, and detection. The sample is vaporized before being passed into an ionization chamber where it is bombarded by a stream of electrons emitted by an electrically heated metal coil.
What kind of information can be determined from a mass spectrum? ›A mass spectrum is a type of plot of the ion signal as a function of the mass-to-charge ratio. These spectra are used to determine the elemental or isotopic signature of a sample, the masses of particles and of molecules, and to elucidate the chemical identity or structure of molecules and other chemical compounds.
What do the peaks in mass spectrometry mean? ›A mass spectrum will usually be presented as a vertical bar graph, in which each bar represents an ion having a specific mass-to-charge ratio (m/z) and the length of the bar indicates the relative abundance of the ion. The most intense ion is assigned an abundance of 100, and it is referred to as the base peak.
What do the heights of the peaks represent in the mass spectrum? ›Molecular ions arriving at the detector produce a peak in the mass spectrum at the molecular mass. The peak height is proportional to the number of ions arriving in a given time.
What are the 5 processes of mass spectrometry? ›
In a mass spectrometry experiment, the process sequence of analysis works in five stages, including sample introduction, analyte ionization, mass analysis, ion detection, and data processing.
What is the first step in the process of mass spectrometry? ›The first step in the mass spectrometric analysis of compounds is the production of gas phase ions of the compound, basically by electron ionization. This molecular ion undergoes fragmentation. Each primary product ion derived from the molecular ion, in turn, undergoes fragmentation, and so on.
What is M Z ratio? ›m/z (mass-to-charge ratio): In mass spectrometry the ratio of an ion's mass (m) in atomic mass units (amu) to its formal charge (z). Formal charge is usually +1. The units for m/z are usually not included. Fragmentation.
How many peaks will the mass spectrum have? ›The 5 peaks in the mass spectrum shows that there are 5 isotopes of zirconium - with relative isotopic masses of 90, 91, 92, 94 and 96 on the 12C scale. This time, the relative abundances are given as percentages.
How do you determine the number of carbons from mass spec? ›If you measure the peak height of the M+1 peak as a percentage of the peak height of the M+ peak, that gives you the number of carbon atoms in the compound.
What does base peak tell us? ›The vertical axis denotes the relative abundance of ions. The most intensive peak in a spectrum is called the "Base Peak", whose intensity is taken as 100 percent. This ion exists most abundantly in the ion source and represents the most stable ion, which is useful for identifying the compound.
What is m2 peak? ›mass spectra - the M+2 peak. MASS SPECTRA - THE M+2 PEAK. This page explains how the M+2 peak in a mass spectrum arises from the presence of chlorine or bromine atoms in an organic compound. It also deals briefly with the origin of the M+4 peak in compounds containing two chlorine atoms.
What are the different peaks in mass spectrum? ›The two most important peaks in any mass spectrum are the base peak and the molecular ion peak. The base beak is the largest peak in the spectrum. In the case of methanol, the base peak is the peak at m/z 31 corresponding to the CH3O+ fragment.
How do I find my mass spectrum RAM? ›2.1 Calculating relative atomic mass (SL) - YouTube
What is a parent peak in mass spectrometry? ›The molecular peak , the parent peak, corresponds to the M+ molecular ion in pure compounds. This is the one that has the largest, easily identifiable m/z ratio. The existence of the molecular ion in the spectrum allows accurate determination of molecular mass.
What is the difference between base peak and molecular ion peak? ›
The base peak is always the tallest peak in the spectrum. The base peak represents the most common and most abundant ion. The molecular ion peak is the peak that represents the heaviest ion. This peak has the highest m/z ratio on the mass spectrum but it does not necessarily represent the most abundant ion.