High-performance liquid chromatography (HPLC) is a separation technique that can be used for the analysis of organic molecules and ions. HPLC is based on mechanisms of adsorption, partition and ion exchange, depending on the type of stationary phase used. HPLC involves a solid stationary phase, normally packed inside a stainless-steel column, and a liquid mobile phase. Separation of the components of a solution results from the difference in the relative distribution ratios of the solutes between the two phases.
Modern high performance liquid chromatography or HPLC has its roots in this separation, the first form of liquid chromatography. The chromatographic process has been significantly improved over the last hundred years, yielding greater separation efficiency, versatility and speed.
Chromatography separates a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation. A Russian botanist named Mikhail S. Tswett used a rudimentary form of chromatographic separation to purify mixtures of plant pigments into the pure constituents. He separated the pigments based on their interaction with a stationary phase, which is essential to any chromatographic separation. The stationary phase he used was powdered chalk and aluminia, the mobile phase in his separation was the solvent. After the solid stationary phase was packed into a glass column (essentially a long, hollow, glass tube) he poured the mixture of plant pigments and solvent in the top of the column. He then poured additional solvent into the column until the samples were eluted at the bottom of the column. The result of this process most crucial to his investigation was that the plant pigments separated into bands of pure components as they passed through the stationary phase.
High Performance Liquid Chromatography has brought desirable advantages in the field of food analysis. Food matrices are generally complex and extraction of analytes is not an easy task. To further complicate matters both desirable and undesirable components are often found in trace levels and classical extraction and analysis does not provide the required levels of accuracy and precision. HPLC offers viable solutions due to vast choice of stationary phases and mobile phase options.
The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature the separation is performed at.
Modern high performance liquid chromatography or HPLC has its roots in this separation, the first form of liquid chromatography. The chromatographic process has been significantly improved over the last hundred years, yielding greater separation efficiency, versatility and speed.
Chromatography separates a sample into its constituent parts because of the difference in the relative affinities of different molecules for the mobile phase and the stationary phase used in the separation. A Russian botanist named Mikhail S. Tswett used a rudimentary form of chromatographic separation to purify mixtures of plant pigments into the pure constituents. He separated the pigments based on their interaction with a stationary phase, which is essential to any chromatographic separation. The stationary phase he used was powdered chalk and aluminia, the mobile phase in his separation was the solvent. After the solid stationary phase was packed into a glass column (essentially a long, hollow, glass tube) he poured the mixture of plant pigments and solvent in the top of the column. He then poured additional solvent into the column until the samples were eluted at the bottom of the column. The result of this process most crucial to his investigation was that the plant pigments separated into bands of pure components as they passed through the stationary phase.
High Performance Liquid Chromatography has brought desirable advantages in the field of food analysis. Food matrices are generally complex and extraction of analytes is not an easy task. To further complicate matters both desirable and undesirable components are often found in trace levels and classical extraction and analysis does not provide the required levels of accuracy and precision. HPLC offers viable solutions due to vast choice of stationary phases and mobile phase options.
The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The sampler brings the sample mixture into the mobile phase stream which carries it into the column. The pumps deliver the desired flow and composition of the mobile phase through the column. The detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components. A digital microprocessor and user software control the HPLC instrument and provide data analysis. Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase. Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most HPLC instruments also have a column oven that allows for adjusting the temperature the separation is performed at.
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Kaira G. Tafoya has spent the past 30 years helping entrepreneurs and organizations with their laboratory start-up endeavors. He now uses his experience and expertise to help groups choose the best laboratory compliance consultant for them. If you would like to learn more about Toxicology Lab Start Up Experts he suggests you click here to learn more.
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