![]() The sample is separated into bands into individual components that can be further analyzed in mass spectrometry. ![]() This can be based on polarities, meaning that if a component of the specimen has a different polarity compared to the mobile phase, it will migrate down the chromatograph column faster. Liquid chromatography separates samples based on interactions with the mobile and stationary phases. Notably, a gas chromatography sample must be volatile, meaning it must enter the gas phase so it does not break down while in the mass spectrometer apparatus. After separation, the gases enter the mass spectrometer for analysis. A sample is diluted and vaporized in the chromatograph, where it is separated. Gas chromatography separates components of a mixture of gases and filters the passage of these molecules based on physical characteristics like shape, size, molecular weight, and boiling point. The two types of chromatography procedures that are used to prepare the sample are gas chromatography and liquid chromatography. Thus, samples are either in the liquid or gaseous phase by utilizing chromatography techniques. Prior to using the mass spectrometer, a sample must be prepared for it to be ionized. Additionally, if there is a peak at m/z = 87, this would be classified as the m+1 peak because all atoms have various isotopes. For example, if the specimen in the mass spectrometer was hexane, the m/z would be 86 since the molecular weight of hexane is 86 g/mol. The molecular ion peak is known as the parent peak because it corresponds to the molecular weight of the sample. This ion is set to 100% on the y-axis for its relative intensity, and all the remaining ion peaks are generated relative to this value. The mass-to-charge ratio (m/z) is on the "x-axis," while the relative intensity is on the "y-axis." For a given sample, the most abundant ion in the sample molecule is known as the base peak. Ī chart will be generated to analyze the mass spectrometer's results. After the ions are separated, the detector will quantify the ions. The neutral species in the mass spectrometer goes undetected because it can be 1) absorbed by the apparatus or 2) removed by a vacuum. Of note, only the cationic fragments are separated. Next, the ionized molecule breaks apart into smaller fragments, then separated according to their mass-to-charge ratio in the mass analyzer. Protons can also be added to create a positive electrical charge. This positive charge is achieved by removing a valence electron. The sample molecule entered into the mass spectrometer will first get a positive charge from an ionization source. This requires three main components, which includes:ĭetector: ions are measured and displayed on the mass spectrum chart.Ītoms and molecules must first be ionized before they can be accelerated through the mass spectrometer and detected. Mass spectrometers convert molecules into ions which are then manipulated by electric and magnetic fields. ![]() The goals of derivatization vary, depending on the application, but typically include (1) increased volatility, (2) greater thermal stability, (3) modified chromatographic properties, (4) greater ionization efficiency, (5) favorable fragmentation properties, or a combination of these. Derivatization usually involves the addition of some well-defined functional group. Derivatization is the process of chemically modifyingthe target compounds to be more favorably analyzed by MS. This typically involves one or more of the following steps: (1) protein precipitation followed by centrifugation or filtration, (2) solid-phase extraction, (3) liquid-liquid extraction, (4) affinity enrichment, or (5) derivatization. Sample preparation is critical to successful MS, particularly when dealing with complex matrices, such as are commonly encountered in clinical chemistry. ![]() In these cases, the m/z value will be a fraction of the ion's mass. However, when larger molecules such as proteins or peptides are measured, they typically carry multiple ionic charges, and therefore the z value is an integer greater than 1. For small molecules (<1000 Da), there is typically only a single charge therefore, the m/z value is the same as the mass of the molecular ion. Most mass spectrometry data are presented in units of the mass-to-charge ratio, or m/z, where m is the molecular weight of the ion (in daltons) and z is the number of charges present on the measured molecule. In addition, because of its ability to identify and quantify proteins, MS is an essential analytical tool in the field of proteomics. When coupled with gas or liquid chromatographs, mass spectrometers allow the expansion of analytical capabilities to various clinical applications. Mass spectrometry (MS) is a powerful qualitative and quantitative analytical technique used to identify and quantify a wide range of clinically relevant analytes.
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