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  • Difference Between LC, LC-MS, LC-MS-MS, and HR-MS

    Liquid Chromatography (LC)

    Liquid chromatography is a powerful analytical technique used to separate and identify individual components in a complex mixture. It works by passing a mixture of compounds through a column packed with a stationary phase, which interacts differently with each component in the mixture. This results in the separation of the individual components based on their unique physical and chemical properties, such as size, charge, hydrophobicity, and polarity.

    LC is widely used in various fields, including pharmaceuticals, biotechnology, environmental monitoring, food and beverage industry, and forensics. Some common applications of LC include:

    • Drug discovery and development: LC is used to separate and identify individual components in a drug candidate or drug formulation. It is also used to quantify the amount of drug present in a sample.
    • Environmental monitoring: LC is used to analyze water, soil, and air samples for the presence of pollutants and contaminants.
    • Food and beverage industry: LC is used to analyze the composition of food and beverage products, including the presence of additives and contaminants.
    • Forensic science: LC is used to identify and quantify drugs of abuse, as well as to analyze blood and urine samples for the presence of toxic compounds.

    Overall, liquid chromatography is a versatile analytical technique that can be tailored to meet the needs of many different industries and applications.

    Mass Spectrometry (MS) and Liquid Chromatography-Mass Spectrometer (LC-MS)

    Liquid chromatography–mass spectrometry (LC-MS) is an analytical technique that has the capabilities and that combines liquid chromatography (HPLC) with the capabilities of mass spectrometry (MS) with mass analysis. Mass spectrometry gives spectral information which helps to identify each separated component and liquid chromatography separates mixtures with many components.

    Mass spectrometry is an advanced substantial analytical technique that converts ions in vacuum space to their mass-to-charge ratio (m/z) and quantifies the magnitude of each ion and produces mass spectra. These mass spectra give vital details about molecular mass, structure, identity, and quantity of sample which include qualitative and quantitative information. When the component exists in the complex form then only MS is not doing the separation because of identical molecular weight and fragmented pattern, hence MS with HPLC as a separation process is the most suitable combination.

    Moreover, in a combination of liquid chromatography with mass spectrometry (mass spectra, molecular mass, and structural information) LC provides qualitative information about retention time and quantifying analyte or constituent by using an LC detector. Hence, a combination of LCMS provides exceptional separation resolution of LC with outstanding qualitative measurements of MS. LC-MS is used in various applications, e.g., pharmaceuticals, biochemicals, food, water, soil, forensic, environmental, petrochemicals, cosmetics, industrial, clinical research, and atomic physics.

    Liquid Chromatography with tandem mass spectrometry (LC-MS-MS)

    LC-MS-MS instrument is the two mass spectrometry detectors connected to the HPLC instrument. In LC-MS-MS ions are isolated in the first MS enter in collision cell and are fragmented, this results in the formation of ions called product ions which are separated in the second mass analyzer and detected.

    This instrument is very useful for complex sample matrices, a very low amount of components,s, and when the resolution is inadequate for both chromatography and its mass-to-charge ratio. Therefore, this technique provides a higher selectivity, specificity, and sensitivity which provides supplementary unique data on the mass and structure of the selected required component. It is very much useful in new chemical entity development, identification, and quantitation study of metabolites, polysaccharides, and proteins.

    High-Resolution Mass Spectrometry (HRMS)

    High-resolution mass spectrometry (HRMS) is a spectrometer having the capability of high resolution, high mass accuracy measurement and determination of elemental compositions, etc. The mass can be measured in many decimal places. Normal MS is supposed to measure nominal mass and HRMS can measure exact mass so precisely that it can detect minute differences in mass between two complexes whereas normal MS is indistinguishable.

    Earlier a large number of conventional quadrupole and ion trap mass spectrometers were used. Now instruments such as Fourier-transform ion cyclotron resonance (FTICR), modern orbitrap, and Q-TOF systems are used.

    There are three types of HRMS:

    1. Time-of-flight MS (TOFMS),
    2. Orbitrap MS, and
    3. Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry.

    Overall HRMS, the mass accuracy is below 5 ppm. The mass resolution specifies a capability to discriminate peaks, and a more resolution produces more separation. Considering the qualitative analysis TOFMS is exceptional on the basis of mass accuracy and more resolution.

    This is a very significant technique for determining the structure of molecules and for finding the peptide mass data that states the sequence of amino acids.

    FAQ’s:

    What is Liquid Chromatography (LC)?

    Liquid chromatography (LC) is a technique in which the mobile phase is liquid and is carried out in a column or plane, presently high-performance liquid chromatography (HPLC) is used where the sample is injected at high pressure (mobile phase) through a column in which is packed with small packing particles. 

    What is the advantage of LC-MS?

    LC-MS is suitable for metabolomics as it has a wide range of coverage of chemicals. This technique can be used to analyze organic, inorganic, and biochemicals which are often found in samples of biological origin and environment.

    What is high-resolution mass spectrometry (HRMS)?

    High-resolution mass spectrometry (HRMS) has the capability of high resolution and high mass accuracy measurement, determination of elemental compositions, etc. The mass can be measured in many decimal places. Normal MS is supposed to measure nominal mass and HRMS can measure exact mass so precisely that it can detect minute differences in mass between two complexes whereas normal MS is indistinguishable.

    References

    1. De Hoffmann, J. Charette and V.Stoobant. “Mass Spectrometry, Principal & Applications,” John Wiley & Sons,
    2. Breitkopf, SC, et al. “A relative quantitative positive/negative ion switching method for untargeted lipidomics via high-resolution LC-MS/MS from any biological source,” Metabolomics,
    3. Taste, M, et al. “Applications of Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap-based high-resolution mass spectrometry in metabolomics and lipidomics,” Int. J. Mol. Science,
    4. Kumar, P. R., & Rini, R. (2016). LCMS-A REVIEW AND A RECENT UPDATE.
    5. de Hoffmann, Edmond; Stroobant, Vincent (2002). Mass Spectrometry (Principles and Applications) (2nd ed.). Wiley. pp. 157–158.
    6. Parasuraman, S., Anish, R., Balamurugan, S., Muralidharan, S., Kumar, K. J., & Vijayan, V. (2014). An Overview of Liquid Chromatography-Mass Spectroscopy Instrumentation. Pharmaceutical Methods.