Mass spectrometry and high-performance liquid chromatography are the analytical techniques used in chemistry and biology for identifying the components of the sample, which use different approaches. To make it clear, high-performance liquid chromatography is aimed at separating the individual components of the mixture by means of a laboratory technique called chromatography, whose main principle is to measure an analyte from two phases of molecules, mobile and stationary. The main difference between mass spectrometry from high-performance liquid chromatography is that the first one determines the masses of chemical compounds and identifies the sample elemental composition. Mass spectrometry is also aimed at clarifying the chemical components of molecules (Rubinson & Rubinson 2000). The combination of the analytical techniques mentioned above has allowed scientists to create a modern analytical technique that makes it possible to detect the compounds of sensitive and selective complex mixtures better (Faust 2005).
Use of Mass Spectrometry with High Performance Liquid Chromatography
The use of mass spectrometry with high performance liquid chromatography is based on the main ideas of those two techniques, the principles of physical detection of compounds within mixtures applied by liquid chromatography and the capabilities of mass analysis mass spectrometry is aimed to. To understand the instrumentation of a modern analytical technique, it is necessary to consider which instrumentation peculiarities each of the techniques has obtained. Opposing to the traditional high performance liquid chromatography, its instrumentation in the combination with mass spectrometry is based on the smaller scales used for both diameters internal one and flow rate. The procedure of the use of mass spectrometry with high-performance liquid chromatography can be explained as follows, the compounds should come through the liquid chromatography process of physical detection and then, the chemical components are measured with mass-to-charge ratio, the instrument of mass spectrometry (Potter 1995).
The strategy of the sample preparation is extremely important as in case of mistakes at this stage, the data will be spoiled and unreliable. The bottom-up proteomics process may be used for protein identification by mass spectrometry with high-performance liquid chromatography. The matching process allows the researcher to identify the proteins. The successful identification should be preceded by the peptide spectra and followed by daughter fragments. It is impossible to get good results in case of signal or ion current is poor. The consideration of the lysis process is an essential part of sample preparation. Detergents should not be omitted, and the efficiency of solubility within various classes of proteins should be checked. The cell lysis should be followed by the protein purification stage aimed at reducing chemical presence. The choice of the purification method depends on the reagent qualities. Next, proteins should be prepared for enzymatic digestion by means of solubilization. The effective denaturation should precede the microscale clean-up. This stage is final before the use of mass spectrometry with a high-performance liquid chromatography procedure. At this stage, the detector should be chosen. Within the discussed techniques, Single or Triple Quadrupole, TOF, Ion Trap, or Quadrupole-time of light detectors may be used (Potter 1995). The sample preparation stage usually identifies the outcome of the research.
To understand the advantages of the use of mass spectrometry with high-performance liquid chromatography, it is important to check the main limitations which can be observed in each of these analytical techniques if used separately. The main limitation of performance liquid chromatography is that this technique is unable to detect the components of the mixture definitely. Such identification takes mass spectrometry to do this. At the same time, mass spectrometry can fail to identify the components of a mixture if the analyte of interest is considered to be part of the mixture. Thus, the combination of these two methods is the best way to reduce the limitations of each other and contribute to the unequivocal determination of the compounds of the samples. The disadvantages of this method are numerous as well, as the risk is taken while using mass spectrometry with high-performance liquid chromatography is high. There is a possibility to lose the analyte while extraction, electrospray is not always available, the sensitivity may depend on a number of factors, the problems may appear with chromatography if the stages of the procedure are confused, and the matrix is added to the mobile phase before the column (Faust 2005).
Chemical/Physical Properties of Each Analyte
Having considered the main idea of the use of mass spectrometry with high-performance liquid chromatography, the instrumentation, sample preparation, the advantages and disadvantages of this combined method in comparison with the single use of each technique, it is possible to identify two analytes that can be used for either qualitative or quantitative analysis by this technique. Proteins and ions may be considered as the molecular analytes detected by means of high-performance liquid chromatography combined with the mass spectrometry technique.
Ions may be detected with the help of this method and selected to the certain fragments if required. The main interface used for detecting ions is electrospray or nanospray sources. By the way, an absolutely new approach is under development now, which is going to combine mass spectrometry with high-performance liquid chromatography techniques within ionization procedures. Using either SDS-PAGE gel or HPLC-SCX samples, it is possible to detect proteins from human serum. The coupling of mass spectrometry with high-performance liquid chromatography techniques made it possible to search for protein analytes even within high peptide masses. One of the best methods for identifying protein analytes is considered to be enzymatic digestion. It is significant that the detection of the analytes with the highest unequivocal characteristic became possible only with the idea to combine mass spectrometry with high-performance liquid chromatography and use those as one continuous process, without being separated into two different ones (Rubinson & Rubinson 2000). The use of mass spectrometry and high-performance liquid chromatography as two separate processes have created a number of problems connected with the inability to analyze the mixtures of some substances with high accuracy. Even though the use of combined mass spectrometry with high-performance liquid chromatography still has a number of disadvantages, their separate use does not solve the problems the contemporary analytical technique does.
Therefore, it is possible to conclude that the limitations each of the described analytical techniques have, if used separately can be covered with the help of the use of mass spectrometry and high-performance liquid chromatography is the combination. Nevertheless, the sample preparation should be of the highest quality as in case the problems appear at any stage, especially at lysis one, the results are going to be poor and it will be impossible to detect analytes unequivocally.
Faust, CB 2005, Modern chemical techniques, Royal Society of Chemistry Potter, London.
Potter, GWH 1995, Analysis of biological molecules: An introduction to principles, instrumentation and techniques, Chapman & Hall, London.
Rubinson, KA & Rubinson, JF 2000, Contemporary instrumental analysis, Prince Hall, Upper Saddle River.