What is HPLC?
HPLC is short for High Performance Liquid Chromatography. High Performance Liquid Chromatography (HPLC) is the technique of separating the components from each other by leaving the column at different times, as a result of the different interactions of the components dissolved in a liquid with the stationary phase in the column, usually on a solid support, moving at different speeds in the column. The "chromatogram" resulting from this separation is referred to as the result of chromatography.
The main parts of HPLC are: mobile phase, degasser, pump, sampler, column-column furnace and chromatography software.
- Mobile phase is a mixture of solvents with various physical and chemical properties that carries the sample through the stationary phase (column).
- The purpose of the degassser unit is to remove air bubbles and dissolved air that may occur in the mobile phase.
- Pump draws the mobile phase from the deaerator, then sends it to the sampling (autosampling) and column unit, allowing the mobile phase to move with high pressure within the HPLC system.
- The main purpose of the sampler is to send the samples to the column and detector. Samplers are available in two forms, manual and autosampler. In manual samplers, the samples are drawn with the help of a syringe and sent to the system with the valve. In autosamplers, on the other hand, the device is designed to perform all these operations on its own.
- In column unit, the column separates the substances according to their physical and chemical properties. The column furnace is used to keep the column providing this separation at a constant temperature and to maintain it. The columns (stationary phase) are usually silica or polymer-based filled.
Types of HPLC Methods
- In normal phase chromatography, the stationary phase is polar and the mobile phase is non-polar or low polarity solvents. Polar compounds are more dispersible in the stationary phase with similar properties and will hold longer than non-polar compounds, so the non-polar compounds in the mixture are separated from the column earlier. In normal phase chromatography, the retention time is long in the low polarity mobile phase and short in the high polarity mobile phase.
- In reversed phase chromatography, the stationary phase is nonpolar and the mobile phase is polar. Analytes with higher polarity exit the column first, since polar compounds in the mixture show similar properties to the polar mobile phase. In reversed phase chromatography, the retention time is long in the polar mobile phase and short in the apolar mobile phase.
- The purpose of ion exchange chromatography is to separate ionic compounds. For positively charged cations, fillers with functional groups with negative charges are preferred, while columns with functional groups with positively charged functional groups are preferred for anions.
- Size exclusion chromatography is preferred for analyzing samples with large differences in molecular weights. In this method, there is a physical interaction between the column filler and the analytes. The high molecular weight component leaves the column earlier because it cannot fit through the pores in the column filler.
- Special chiral columns are used in the Chiral Separation technique. The aim is to convert isomers with the same physical properties into diastereomers by interacting with the stationary phase.
High Performance Liquid Chromatography (HPLC) systems are widely used in food industry, pharmaceuticals, agricultural products, biochemicals, pollutants, industrial studies and etc.
What is LC-MS (Liquid Chromatography Mass Spectrometer)?
Liquid Chromatography - Mass Spectrometry (LC-MS) is an analytical technique that combines the physical separation technique of liquid chromatography (HPLC) with mass spectrometry (MS), where mass analysis methods are applied.
Liquid chromatography separates multicomponent mixtures, while mass spectrometry establishes the structural identity of individual components with a high level of molecular specificity and detection sensitivity.
The LC-MS technique is widely used in the analysis of biochemical, organic and inorganic compounds of environmental and biological origin, in a wide variety of fields such as biotechnology, food processing and pharmaceutical, agrochemical and cosmetic industries.