- Last update
- Save as PDF
- Page ID
- 13929
\( \newcommand{\vecs}[1]{\overset { \scriptstyle \rightharpoonup} {\mathbf{#1}}}\) \( \newcommand{\vecd}[1]{\overset{-\!- \!\rightharpoonup}{\vphantom{a}\smash{#1}}} \)\(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{ span}}\) \( \newcommand{\kernel}{\mathrm{null}\,}\) \( \newcommand{\range}{\mathrm{rango}\,}\) \( \newcommand{\RealPart }{\mathrm{Re}}\) \( \newcommand{\ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argument}{\mathrm{Arg}}\) \( \newcommand{\ norma}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm {span}}\) \(\newcommand{\id}{\mathrm{id}}\) \( \newcommand{\Span}{\mathrm{span}}\) \( \newcommand{\kernel}{\ mathrm{nulo}\,}\) \( \newcommand{\rango}{\mathrm{rango}\,}\) \( \newcommand{\RealPart}{\mathrm{Re}}\) \( \newcommand{ \ImaginaryPart}{\mathrm{Im}}\) \( \newcommand{\Argumento}{\mathrm{Arg}}\) \( \newcommand{\norm}[1]{\| #1 \|}\) \( \newcommand{\inner}[2]{\langle #1, #2 \rangle}\) \( \newcommand{\Span}{\mathrm{s pan}}\)\( \nuevocomando{\AA}{\unicode[.8,0]{x212B}}\)
Mass spectrometry (MS) is a powerful analytical technique that is widely used by chemists, biologists, medical researchers, and environmental and forensic scientists, among others. In MS we look at the mass of a molecule or different fragments of that molecule.
The basics of a mass spectrometry experiment
There are many different types of EM instruments, but they all share the same three key components. First, there is an ionization source where the molecule acquires a positive electrical charge either by removing an electron or adding a proton. Depending on the ionization method used, the ionized molecule may or may not break up into a population of smaller fragments. In the image below, some of the sample molecules remain whole while others are broken down into smaller pieces.
Then there is a mass analyzer where the cationic fragments are separated by mass.
Finally there is a detector that detects and quantifies the separated ions.
One of the most common types of MS techniques used in the organic laboratory iselectronic ionization. In the ionization source, the sample molecule is bombarded with a high-energy electron beam, which deprives the molecule of a valence electron, resulting in aRadical cation. Because a large amount of energy is transferred through this bombardment process, the radical cation quickly begins to break down into smaller fragments, some of which are positively charged and some of which are neutral. Neutral fragments are adsorbed to the chamber walls or removed using a vacuum source. In the mass parser component, the positively charged fragments and any remaining unfragmented fragmentsmolecular ionsThey are accelerated through a tube by an electric field.
This tube is curved and the ions are deflected by a strong magnetic field. Ions with different mass-to-charge (m/z) ratios are biased to different degrees, resulting in a "ranking" of ions per mass (virtually all ions have charges z = +1, so ranking by mass to charge mass ratio is the same as a mass classification). A detector at the end of the curved flight tube captures and quantifies the classified ions.
Look at the mass spectrum
Below is the typical result of an electron ionization MS experiment (MS data in this section are from theSpectral Database of Organic Compounds, a free web-based service provided by AIST in Japan.
The sample is acetone. On the horizontal axis is the value m/z (as we have already said, the charge z is almost always +1, so in practice it is equal to the mass). The vertical axis plots the relative abundance of each detected ion. The most frequently occurring ion is named on this scaleBasis-Pico, is set to 100% and all other peaks are plotted relative to this value. For acetone, the base peak is at m/z = 43; We will discuss the formation of this fragment a little later. The molecular weight of acetone is 58, so we can identify the peak at m/z = 58 as correspondingmolecular ion peak, ÖDad lace. Note that there is a small peak at m/z = 59: this is calledImage M+1. How can there be an ion whose mass is greater than the molecular ion? Very simple: A small fraction, about 1.1% of all carbon atoms in nature are actually carbon atoms.13C instead12C.O. isotopes13The C isotope is obviously heavier than12C per 1 unit mass. Also, about 0.015% of all hydrogen atoms are actually deuterium, that2Isotope H. Therefore, the M+1 peak represents the few acetone molecules in the sample that contained a13The neck2H
Molecules with many oxygen atoms sometimes show a small onePico M+2(units 2 m/z larger than the original peak) in their mass spectra due to the presence of a small amount of18O (the most common oxygen isotope issixteenEITHER). Because there are two abundant chlorine isotopes (about 75%35Cl 25%37Cl) and bromine (about 50%79Bre 50%81Br), chlorinated and brominated compounds have very large and discernible M+2 peaks. Fragments containing both Br isotopes can be seen in the mass spectrum for ethyl bromide:
Much of the utility of MS electron ionization stems from the fact that the radical cations generated in the electron bombardment process tend to decay in a predictable manner. A detailed analysis of typical fragmentation patterns of different functional groups is beyond the scope of this text, but some representative examples are worth considering even if we don't try to understand the exact process by which fragmentation occurs. For example, we have seen that the base peak in the mass spectrum for acetone is m/z=43. This is the result of cleavage at the "alpha" position; in other words, on the carbon-carbon bond adjacent to the carbonyl. Alpha cleavage leads to the formation of an acylium ion (representing the base peak at m/z = 43) and a methyl radical, which is neutral and therefore not detected.
After the main peak and the base peak, the next largest peak is at m/z=15 with a relative abundance of 23%. This is expected to be the result of the formation of a methyl cation in addition to an acyl moiety (which is neutral and not detected).
A common fragmentation pattern for larger carbonyl compounds is reportedMcLafferty rearrangement:
The mass spectrum of 2-hexanone shows a "McLafferty fragment" at m/z = 58, while the propene fragment is not observed since it is a neutral species (remember that only cationic fragments are observed in MS ). The base peak in this spectrum is again an acylium ion.
When alcohols are subjected to MS electron ionization, the molecular ion is very unstable and therefore no main peak is usually detected. Often the base peak is from an "oxonium" ion.
Other functional groups also have predictable fragmentation patterns. By carefully analyzing the fragmentation information provided by a mass spectrum, a skilled spectrometer can often "put the puzzle together" and make some very reliable predictions about the structure of the original sample.
You can see many other real examples of mass spectra belowSpectral Database of Organic Compounds
Exercise 4.1:Using the fragmentation patterns of acetone as a guide, predict the signals you would find in the mass spectra of:
a) 2-Butanon; b) 3-Hexanon; c) Cyclopentanon.
Task 4.2:Predict some signals you would expect to see in a mass spectrum of 2-chloropropane.
Exercise 4.3:The mass spectrum of an aldehyde shows a major peak atm/z= 58 and a base peak atm/z= 29. Propose a structure and identify the two species whosem/zValues were enumerated.(
solutions
Gas chromatography - mass spectrometry
Mass spectrometry is often used in conjunction with a separation technique called gas chromatography (GC). The combined GC-MS method is very useful when dealing with a sample that is a mixture of two or more different compounds, since the different compounds are separated from each other before subjecting them individually to MS analysis. We won't go into detail about gas chromatography here, but if you take an organic laboratory course you may have the opportunity to try GC and will almost certainly be introduced to the conceptually analogous techniques of thin layer and column chromatography. Suffice it to say that in GC, a very small amount of liquid sample is vaporized, injected into a long coiled metal column, and forced through the column with helium gas. Along the way, different compounds in the sample adhere differently to the column walls and therefore move at different speeds and exit the column separately. In GC-MS, each purified compound is sent directly from the end of the GC column to the MS instrument, so we end up with a separate mass spectrum for each of the compounds in the original mixed sample. Since the MS spectrum of a compound is a very reliable and reproducible "fingerprint", we can instruct the instrument to search an MS database and identify each compound in the sample.
Gas chromatography-mass spectrometry (GC-MS) scheme.
(Picture ofWikipedia, the free encyclopedia)
The extremely high sensitivity of modern GC-MS instruments enables the detection and identification of traces of very small organic compounds. Environmental chemists are increasingly using GC-MS to detect the presence of harmful organic contaminants in food and water samples. Airport security checkpoints also use high-speed GC-MS instruments to check for bomb-making chemical residues in checked baggage.
Protein mass spectrometry: applications in proteomics
Electron ionization mass spectrometry is not very useful for analyzing biomolecules in general: their high polarity makes them difficult to localize in the vapor phase, the first step in EIMS. Biomolecule mass spectrometry has undergone a revolution over the last few decades with the development of many new ionization and separation techniques. In general, the strategy involves the analysis of biomoleculessoft ionization, in which much less energy (compared to techniques such as EIMS) is transferred to the molecule to be analyzed during the ionization process. Soft ionization usually adds protons instead of removing electrons: cations formed in this way are significantly lower in energy than radical cations formed by removing an electron. The result of soft ionization is that little or no fragmentation occurs, so the measured mass is that of an intact molecule. Typically, large biomolecules are broken down into smaller fragments using chemical or enzymatic methods, then their mass is determined by “soft” MS.
Recent developments in soft ionization MS technology have facilitated the detection and identification of proteins present at very low levels in biological samples. InElektrospray-Ionisation(ESI) the protein sample in solution is sprayed into a tube and the molecules are induced by an electric field to accept additional protons from the solvent. Another common method of "soft ionization" is "matrix-assisted laser desorption ionization" (Maldi). The protein sample is adsorbed on a solid matrix and protonated with a laser.
Typically, both electrospray ionization and MALDI are used in conjunction with a time-of-flight (TOF) mass analyzer component.
Proteins are accelerated through a column by an electrode, and the separation is achieved because lighter ions move faster than heavier ions for the same overall charge. In this way, the many proteins in a complex biological sample (e.g. blood plasma, urine, etc.) can be separated and their individual masses determined very precisely. The modern MS protein is extremely sensitive: recently scientists were able to prove the presence of MSTyranosaurus rexProtein in a fossilized skeleton! (Science2007, 316, 277).
Soft ionization mass spectrometry has become an increasingly important tool in the field ofProteomik. Traditionally, protein biochemists have tended to study the structure and function of individual proteins. Proteomics researchers, on the other hand, want to learn more about how the multitude of proteins in a living system interact with each other and how they react to changes in the organism's state. An important area of proteomics is the search for "biomarker" proteins of human diseases, i.e. proteins that are present in larger quantities in the tissue of a sick person than in a healthy person. Detection of a known biomarker for a disease, such as diabetes or cancer, in a healthy individual can provide physicians with an early warning that the patient may be particularly susceptible to the disease, allowing preventative measures to be taken to prevent or delay its onset.
In a 2005 study, MALDI-TOF mass spectrometry was used to compare fluid samples from lung transplant recipients who experienced tissue rejection with samples from recipients who did not. Three peptides (short proteins) were found at elevated levels specifically in tissue exudate samples. It is hoped that these peptides could serve as biomarkers to identify patients who are at higher risk of rejecting their transplanted lungs. (Proteomik2005, 5, 1705).