LIQUID CHROMATOGRAPHY – MASS SPECTROMETRY
Liquid Chromatography-Mass Spectrometry (LC-MS) is an analytical chemistry instrument that separates chemicals and aids in identifying compounds in a sample. LC-MS is extremely helpful when working with isomers as such compounds can have equivalent mass and different molecular elements. Liquid Chromatography first separates the isomers allowing them to be individually analyzed during Mass Spectrometry. LC-MS is able to analyze complex mixtures and provides high specificity. In fact, with this technology, multiple compounds can be measured in a single analytical run. LC-MS also has the capability to switch ionization polarity modes from positive to negative at a rapid pace. This allows the chemist to test both positive and negative polarities in the same run of the sample.
LC-MS is commonly used to characterize and analyze the following:
- Drug Metabolites
- Polycyclic Aromatic Hydrocarbons
- Organometallic Compounds
- Amino Acids
- Nucleosides & Nucleotides
- Peptides & Proteins
- Bile Acids
- Fatty Acids
There are a number of different LC/MS ionization techniques. Some of the variations in technique include DLI, thermospray, CFFAB, electrospray, atmospheric pressure chemical ionization, SFC/MS, moving belt, and particle beam. We will be primarily focusing on electrospray ionization (ESI). ESI causes very little fragmentation to the molecules as it only transfers small amounts of energy to the analytes. This is a very positive aspect of this testing methodology as it increases the analyst’s ability to identify the particle as a whole. Alternative methods are chosen when dealing with molecules of neutral or low polarity as ESI does not always sufficiently ionize this type of molecule.
The process starts when the liquid sample is inserted into the LC column. The solution is nebulized and the particles are charged through electrospray ionization. Heat and dry nitrogen are used to evaporate the charged droplets and the electrical charge transfers to the analytes. These ionized analytes then travel through small mechanisms and enter the vacuum of the Mass Spectrometer (MS).
The Mass Spectrometer measures the mass to charge ratio (m/z) of the ions. It does this by using radio frequency voltages to accelerate the ions in the mass analyzer. There are multiple types of analyzers but their various functions all result in measuring the m/z of the ions. The mass of each of the elements is distinct and the electromagnetic field sorts them as they travel towards the detector. As the ions hit the detector the signal is processed into mass spectra. The data retrieved thereby contains the identifiable mass of each element as well as the total concentration of each analyte.