Liquid chromatography–mass spectrometry
Thermo Fisher Scientific
|Related||Gas chromatography–mass spectrometry|
Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS). LC-MS is a powerful technique that has very high sensitivity and selectivity and so is useful in many applications. Its application is oriented towards the separation, general detection and potential identification of chemicals of particular masses in the presence of other chemicals (i.e., in complex mixtures), e.g., natural products from natural-products extracts, and pure substances from mixtures of chemical intermediates. Preparative LC-MS systems can be used for rapid mass-directed purification of specific substances from such mixtures that are important in basic research, and pharmaceutical, agrochemical, food, and other industries.
Present day liquid chromatography generally utilizes very small particles packed and operating at relatively high pressure, and is referred to as high performance liquid chromatography (HPLC); modern LC-MS methods use HPLC instrumentation, essentially exclusively, for sample introduction. In HPLC, the sample is forced by a liquid at high pressure (the mobile phase) through a column that is packed with a stationary phase generally composed of irregularly or spherically shaped particles chosen or derivatized to accomplish particular types of separations. HPLC methods are historically divided into two different sub-classes based on stationary phases and the corresponding required polarity of the mobile phase. Use of octadecylsilyl (C18) and related organic-modified particles as stationary phase with pure or pH-adjusted water-organic mixtures such as water-acetonitrile and water-methanol are used in techniques termed reversed phase liquid chromatography (RP-LC). Use of materials such as silica gel as stationary phase with neat or mixed organic mixtures are used in techniques termed normal phase liquid chromatography (NP-LC). RP-LC is most often used as the means to introduce samples into the MS, in LC-MS instrumentation.
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When standard bore (4.6 mm) columns are used the flow is often split ~10:1. This can be beneficial by allowing the use of other techniques in tandem such as MS and UV detection. However splitting the flow to UV will decrease the sensitivity of spectrophotometric detectors. The mass spectrometry on the other hand will give improved sensitivity at flow rates of 200 μL/min or less.
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of charged particles. It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. MS works by ionizing chemical compounds to generate charged molecules or molecule fragments and measuring their mass-to-charge ratios. In a typical MS procedure:
A sample is loaded onto the MS instrument and undergoes vaporization.
The components of the sample are ionized by one of a variety of methods (e.g., by impacting them with an electron beam), which results in the formation of charged particles (ions).
The ions are separated according to their mass-to-charge ratio in an analyzer by electromagnetic fields
The ions are detected, usually by a quantitative method.
The ion signal is processed into mass spectra.
Additionally, MS instruments consist of three modules:
An ion source, which can convert gas phase sample molecules into ions (or, in the case of electrospray ionization, move ions that exist in solution into the gas phase)
A mass analyzer, which sorts the ions by their masses by applying electromagnetic fields
A detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present
The technique has both qualitative and quantitative uses. These include identifying unknown compounds, determining the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation. Other uses include quantifying the amount of a compound in a sample or studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum). MS is now in very common use in analytical laboratories that study physical, chemical, or biological properties of a great variety of compounds.
Understandably the interface between a liquid phase technique which continuously flows liquid, and a gas phase technique carried out in a vacuum was difficult for a long time. The advent of electrospray ionization changed this. The interface is most often an electrospray ion source or variant such as a nanospray source; however atmospheric pressure chemical ionization interface is also used. Various deposition and drying techniques have also been used such as using moving belts; however the most common of these is off-line MALDI deposition. A new approach still under development called Direct-EI LC-MS interface, couples a nano HPLC system and an electron ionization equipped mass spectrometer.
LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals and is thus the most frequently used technique in the field of bioanalysis. These studies give information about how quickly a drug will be cleared from the hepatic blood flow, and organs of the body. MS is used for this due to high sensitivity and exceptional specificity compared to UV (as long as the analyte can be suitably ionised), and short analysis time.
The major advantage MS has is the use of tandem MS-MS. The detector may be programmed to select certain ions to fragment. The process is essentially a selection technique, but is in fact more complex. The measured quantity is the sum of molecule fragments chosen by the operator. As long as there are no interferences or ion suppression, the LC separation can be quite quick. It is common now to have analysis times of 1 minute or less by MS-MS detection, compared to over 10 mins with UV detection.
LC-MS is also used in proteomics where again components of a complex mixture must be detected and identified in some manner. The bottom-up proteomics LC-MS approach to proteomics generally involves protease digestion and denaturation (usually trypsin as a protease, urea to denature tertiary structure and iodoacetamide to cap cysteine residues) followed by LC-MS with peptide mass fingerprinting or LC-MS/MS (tandem MS) to derive sequence of individual peptides. LC-MS/MS is most commonly used for proteomic analysis of complex samples where peptide masses may overlap even with a high-resolution mass spectrometer. Samples of complex biological fluids like human serum may be run in a modern LC-MS/MS system and result in over 1000 proteins being identified, provided that the sample was first separated on an SDS-PAGE gel or HPLC-SCX.
LC-MS is frequently used in drug development at many different stages including peptide mapping, glycoprotein mapping, natural products dereplication, bioaffinity screening, in vivo drug screening, metabolic stability screening, metabolite identification, impurity identification, quantitative bioanalysis, and quality control.
- Gas chromatography–mass spectrometry
- Capillary electrophoresis–mass spectrometry
- Ion-mobility spectrometry–mass spectrometry
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