High-Performance Liquid Chromatrography (HPLC)

Take Home Point:

Separation and measurement of known volatile and non-volatile components in mixtures 

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What It Provides:

Chromatogram (intensity versus retention time) showing the separation of volatile and non-volatile components within a sample.  The intensity is proportional to the amount of each component within the sample allowing the technique to be quantitative with proper calibration.     

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In a Nutshell:

 

Separation of Volatile and Non-Volatile Mixtures

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HPLC is one of the two main chromatographic techniques with GC being the other.  HPLC lacks the sample volatility and molecular weight requirements of GC, making it capable of analyzing a larger number of compounds.  While GC is typically more robust and easier to use, HPLC is an ideal technique when determining the presence (qualitatively and/or quantitatively) of volatile and/or non-volatile compound(s) within samples that are unable to be analyzed by the former.  HPLC samples can be liquids or solids (must be dissolved in a solvent).  

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In HPLC the liquid sample is injected into a flow of pressurized liquid (i.e. the mobile phase) and proceeds down a column packed with an appropriate adsorbent (i.e. the stationary phase).  As the sample passes through the column, the various compounds within 'partition' between the mobile and stationary phases.  Those compounds having greater preference for the mobile phase will travel through the column faster than those with a greater affinity for the stationary phase allowing their separation to occur.  The analytes exit the column and enter a detector.  A variety of detectors (e.g. RI, UV-Vis) exist with their operation based on the measurement of various physicochemical properties.  The time it takes for a compound to traverse the HPLC system is termed the 'Retention Time'.  Because many HPLC detectors are nondestructive, this technique can also be used to purify a particular analyte or compound.    

 

HPLC is best employed when looking for known compounds within a sample since it doesn't positively identify most analytes.  Typically, HPLC analysis involves comparing retention times of signals from the sample with that from standards containing the compounds of interest analyzed under the exact same conditions.