3 HPLC Manufacturers in 2023

What Is a HPLC?

HPLC (High Performance Liquid Chromatography) is a method of separating and detecting individual compounds in a sample using the interaction between a column and a sample. It can be easily measured and can detect trace components, so it is used in various industries, mainly pharmaceuticals, biochemistry, food and environment.

Since the peak area of ​​HPLC is proportional to the sample concentration, it is also possible to quantify the concentration of components contained in the sample. Since the separation behavior of samples in HPLC differs depending on the column and mobile phase, it is necessary to design appropriate analysis conditions.

How to Use HPLCs and Their Applications

High Performance Liquid Chromatography (HPLCs) are analytical methods that use the interaction between a column and a sample to separate each component in a sample. The method of use is very simple, and analysis can be performed by directly injecting the sample solution or by placing the sample solution in the autosampler and performing batch processing.

HPLCs are used in a variety of industries. For example, they are used in the pharmaceutical field to analyze trace amounts of impurities and active ingredients, in the food, beverage, and environmental fields to analyze nutritional ingredients, functional ingredients, additives, and pesticide residues, and in biochemistry to analyze proteins and nucleic acid-related substances.

Principles of HPLCs and Columns Used

HPLCs and gas chromatography (GC) are types of chromatography. Chromatography compounds are a method of separating contained in an analyte by passing them through a column or other media while allowing them to adsorb to the column, etc. The medium through which the sample components flow, such as solvent in HPLCs or gas in GC, is called the "mobile phase," while that to which the sample is adsorbed, such as a column, is called the "stationary phase".

The type and magnitude of interaction between the sample components and the column vary depending on the type of column. For example, in an ODS column, an alkyl chain (octadecyl group) is modified on the column, and the sample is adsorbed by hydrophobic interaction.

On the other hand, silica gel columns have silanol groups on the surface that adsorb polar compounds. Other types of columns are also available, including columns modified with phenyl groups, cyano groups, and amino groups.

Concentration Calculation Using HPLCs Peak Area

The peak area of HPLCs are proportional to the concentration of a sample. However, when a UV detector is used, the peak area varies depending on the absorbance coefficient (ease of light absorption) even if the sample concentration is the same. Therefore, when analyzing concentrations using HPLCs, it is necessary to prepare standards to compare the areas.

One method for analyzing concentrations is the "external standard method". In this method, multiple standard samples with known concentrations are prepared for HPLCs analysis, and the peak areas are determined. Since the concentration of each sample is known, an equation for determining the concentration from the area value can be obtained by plotting the area value and concentration.

The second method is the "internal standard method". In this method, another compound that is chemically and physically stable is added as an internal standard to a standard sample with a known concentration. After the addition, HPLCs analysis is performed to determine the ratio of the peak area of the standard sample to that of the internal standard. A calibration curve can be obtained by using the ratio of the amount of the added internal standard as the abscissa and the ratio of the peak areas as the ordinate.

HPLCs Detectors and Limits of Detection

A variety of instruments are available as HPLCs detectors. Examples include ultraviolet-visible (UV-Vis) detectors, fluorescence detectors, and differential refractive index detectors (RID). The detection limits of these detectors vary widely, depending on the sample. For example, the UV-Vis detector has a detection limit of approximately 10 picograms (pg) and the fluorescence detector 0.1 pg.

The most sensitive is the mass spectrometer (MS), which is estimated to have a detection sensitivity of 0.01 pg. However, the detection limit depends on the type and concentration of compounds in the sample and the degree of separation. In some cases, derivatization Optimization of HPLCs analysis, including sample pretreatment, is necessary for high-sensitivity detection.