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Technique:
Liquid chromatography (LC or HPLC) is an analytical technique used for the separation of thermally labile compounds. HPLC is similar to GC, but uses a liquid as the mobile phase.
Application:
HPLC is important in both the study of pharmaceuticals and in the identification of explosives.
The Science Behind:
HPLC uses a liquid under high pressure as the mobile phase or carrier for the sample. The mobile phase transports the injected sample through a column coated with a stationary phase. The individual components of the sample interact differently with the stationary phase, causing separation. The pressure of the mobile phase is typically between 2000 and 3000 psi (which is why this technique was called High Pressure Liquid Chromatography). The separated individual components are measured by a variety of available detectors. These detectors measure the amount of the component as it exits the column and the data is collected via a computer. The analyst processes this data to determine the identification and amount of the component under study.
There are three main classifications of HPLC: size exclusion, ion-exchange and adsorption, with adsorption having two modes; normal phase and reversed phase.
• Ion-Exchange - A reversible chemical reaction between ions and a polymeric resin.
• Size Exclusion - Physical separation molecules of various sizes.
• Adsorption - The chemical interaction of molecules binding together by surface attraction.
• • Normal & Reverse Phase - Refers to the differences in polarity of the mobile/stationary phases. Normal phase analysis uses a polar stationary phase while reverse phase uses a non-polar stationary phase.
Historically, the standard detector was the UV/Vis, fixed wavelength.
In UV/Vis, the mobile phase continuously flows through a cell and a single wavelength of light is passed through the cell.
As the compound passes through the cell, it will cause a variation in the intensity of the single wavelength of light, which is measured.
Specific wavelengths of light are used for different compounds to maximize sensitivity.
• Size Exclusion - Physical separation molecules of various sizes.
• Adsorption - The chemical interaction of molecules binding together by surface attraction.
• • Normal & Reverse Phase - Refers to the differences in polarity of the mobile/stationary phases. Normal phase analysis uses a polar stationary phase while reverse phase uses a non-polar stationary phase.
Detectors utilized by Armstrong include variable wavelength Photodiode Array, Fluorescence, Electrical Conductivity and UV/Vis. There are three subtypes of UV/Vis detectors: fixed wavelength, variable wavelength and photodiode array.
• Photodiode Array - Used to detect a range of wavelengths simultaneously.
• UV/Vis - Very sensitive to single wavelengths of light in the ultraviolet to visible range.
• Fluorescence - Measures the ability of a sample to fluoresce after absorbing one wavelength of light emitting a different wavelength of light.
• Electrical Conductivity - Measures the resistance to an electrical current passing through the sample.
• UV/Vis - Very sensitive to single wavelengths of light in the ultraviolet to visible range.
• Fluorescence - Measures the ability of a sample to fluoresce after absorbing one wavelength of light emitting a different wavelength of light.
• Electrical Conductivity - Measures the resistance to an electrical current passing through the sample.
Other References:
Wikipedia Definition of HPLC
Excellent HPLC e-Textbook
Column - Thin tube, filled with the stationary phase, selected to optimize analysis for speed or detection limits.
Detector - A device used to measure or record changes in a system.
Liquid - Each type of HPLC analysis has its own solvent requirements but water, acetonitrile and methanol are the most common solvents used.
Mobile Phase - The moving part of all chromatography, in this case, typically a mixture of liquids. Modern instrumentation allows for changing the liquid mixture during the analysis, greatly increasing the efficiency of the analysis.
Stationary Phase - The portion of chromatography that interacts with the sample, separating components of the sample from each other.
Thermally Labile - To decompose or break down under temperature.
UV/Vis - Parts of the electro- magnetic spectrum. UV stands for ultra-violet or beyond violet, a section of the light spectrum most humans are unable to see. Near UV is used in most laboratory operations (200 nanometers (nm) to 380 nm). Vis stands for visible, which is the section of the light spectrum most humans are able to see (400 nm to 700 nm).
Detector - A device used to measure or record changes in a system.
Liquid - Each type of HPLC analysis has its own solvent requirements but water, acetonitrile and methanol are the most common solvents used.
Mobile Phase - The moving part of all chromatography, in this case, typically a mixture of liquids. Modern instrumentation allows for changing the liquid mixture during the analysis, greatly increasing the efficiency of the analysis.
Stationary Phase - The portion of chromatography that interacts with the sample, separating components of the sample from each other.
Thermally Labile - To decompose or break down under temperature.
UV/Vis - Parts of the electro- magnetic spectrum. UV stands for ultra-violet or beyond violet, a section of the light spectrum most humans are unable to see. Near UV is used in most laboratory operations (200 nanometers (nm) to 380 nm). Vis stands for visible, which is the section of the light spectrum most humans are able to see (400 nm to 700 nm).