High Performance Liquid Chromatography (HPLC) is an analytical technique used for the separation of compounds soluble in a particular solvent. Liquid chromatography was initially discovered as an analytical technique in the early twentieth century and was first used as a method of separating colored compounds. This is where the name chromatography chroma means color, graphy means writing, was derived. All chromatographic separations, including HPLC operate under the same basic principle; every compound interacts with other chemical species in a characteristic manner.
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.
High Performance Liquid Chromatography provides reliable quantitative precision and accuracy along with a high linear dynamic range to allow determination of API and related substances in a single run. A convenient method for sample preparation for solid dosage forms is dispersion in water or aqueous media modified with acetonitrile or methanol .HPLC offers several possibilities for separation of chiral molecules into their respective enantiomers. These include precolumn derivatization to form diastereomers. Alternately, specialty columns prepared with cyclodextrins or special chiral moieties as stationary phases maybe used .In short HPLC, particularly reverse phase HPLC is the most popular choice for quantitative analysis in the pharmaceutical industry. HPLC has contributed to analytical solutions in diverse fields such as pharmaceuticals, foods, life sciences, environment, forensics, etc. In the present module we shall discuss some application areas in pharmaceuticals and foods.
HPLC can be used in both qualitative and quantitative applications that is for both compound identification and quantification. Normal phase HPLC is only rarely used now, almost all HPLC separation can be performed in reverse phase. Reverse phase HPLC (RPLC) is ineffective in for only a few separation types; it cannot separate inorganic ions (they can be separated by ion exchange chromatography). It cannot separate polysaccharides (they are too hydrophilic for any solid phase adsorption to occur), nor polynucleotides (they adsorb irreversibly to the reverse phase packing). Lastly, incredibly hydrophobic compounds cannot be separated effectively by RPLC (there is little selectivity). Aside from these few exceptions, RPLC is used for the separation of almost all other compound varieties.
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.
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.
High Performance Liquid Chromatography provides reliable quantitative precision and accuracy along with a high linear dynamic range to allow determination of API and related substances in a single run. A convenient method for sample preparation for solid dosage forms is dispersion in water or aqueous media modified with acetonitrile or methanol .HPLC offers several possibilities for separation of chiral molecules into their respective enantiomers. These include precolumn derivatization to form diastereomers. Alternately, specialty columns prepared with cyclodextrins or special chiral moieties as stationary phases maybe used .In short HPLC, particularly reverse phase HPLC is the most popular choice for quantitative analysis in the pharmaceutical industry. HPLC has contributed to analytical solutions in diverse fields such as pharmaceuticals, foods, life sciences, environment, forensics, etc. In the present module we shall discuss some application areas in pharmaceuticals and foods.
HPLC can be used in both qualitative and quantitative applications that is for both compound identification and quantification. Normal phase HPLC is only rarely used now, almost all HPLC separation can be performed in reverse phase. Reverse phase HPLC (RPLC) is ineffective in for only a few separation types; it cannot separate inorganic ions (they can be separated by ion exchange chromatography). It cannot separate polysaccharides (they are too hydrophilic for any solid phase adsorption to occur), nor polynucleotides (they adsorb irreversibly to the reverse phase packing). Lastly, incredibly hydrophobic compounds cannot be separated effectively by RPLC (there is little selectivity). Aside from these few exceptions, RPLC is used for the separation of almost all other compound varieties.
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.
About the Author:
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 Top Toxicology Lab Start Up Experts in Dallas he suggests you click here to learn more.
0 comments:
Post a Comment