High-performance liquid chromatography (HPLC; formerly referred to as high-pressure liquid chromatography), is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column.
HPLC has been used for medical (e.g. detecting vitamin D levels in blood serum), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and manufacturing (e.g. during the production process of pharmaceutical and biological products) purposes.
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.
HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50-350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1-4.6 mm diameter, and 30-250 mm length. Also HPLC columns are made with smaller sorbent particles (2-50 micrometre in average particle size). This gives HPLC superior resolving power when separating mixtures, which makes it a popular chromatographic technique.
Increasingly, food analysis methods are built around high-performance liquid chromatography (HPLC), which has proven to be an optimal technology for detecting and/or quantifying the vast majority of food analytes. These methods employ a stepwise approach that first removes the sample matrix, then isolates the analytes of interest and individually resolves them on a chromatographic column. The efficiency of the separation depends on, among other things, the differential interaction of analytes of interest with both mobile and column stationary phases. Of course, classifying food analytes according to their relative volatility and polarity are factors that must be considered when selecting an appropriate analytical method for their determination. Keeping track of the increasing diversity of food products with their ever-expanding profusion of additives is a daunting analytical challenge; one that must be continually met if we are to ensure the quality and safety of our food supply. HPLC is powerfully current because it's fundamental principle-selection through differential molecular interaction-is based on fundamental variations in classes of properties across all chemical species. This selection principle continues to be a rich source of methodological innovation for analytical separation, detection and quantification.
HPLC has been used for medical (e.g. detecting vitamin D levels in blood serum), legal (e.g. detecting performance enhancement drugs in urine), research (e.g. separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and manufacturing (e.g. during the production process of pharmaceutical and biological products) purposes.
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.
HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational pressures are significantly higher (50-350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1-4.6 mm diameter, and 30-250 mm length. Also HPLC columns are made with smaller sorbent particles (2-50 micrometre in average particle size). This gives HPLC superior resolving power when separating mixtures, which makes it a popular chromatographic technique.
Increasingly, food analysis methods are built around high-performance liquid chromatography (HPLC), which has proven to be an optimal technology for detecting and/or quantifying the vast majority of food analytes. These methods employ a stepwise approach that first removes the sample matrix, then isolates the analytes of interest and individually resolves them on a chromatographic column. The efficiency of the separation depends on, among other things, the differential interaction of analytes of interest with both mobile and column stationary phases. Of course, classifying food analytes according to their relative volatility and polarity are factors that must be considered when selecting an appropriate analytical method for their determination. Keeping track of the increasing diversity of food products with their ever-expanding profusion of additives is a daunting analytical challenge; one that must be continually met if we are to ensure the quality and safety of our food supply. HPLC is powerfully current because it's fundamental principle-selection through differential molecular interaction-is based on fundamental variations in classes of properties across all chemical species. This selection principle continues to be a rich source of methodological innovation for analytical separation, detection and quantification.
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