Instrument: Dionex ICS1100 ion chromatography system; Dionex Ultimate 3000 system
HPLC is different from standard LC in that the beads are smaller and more regular. They pack more tightly into the column and require higher pressures to force the liquid flow through the column.
Because beads are more regular, the performance of the column is much improved and this allows high resolution separation of chemicals or biomolecules that have very similar properties.
Eluted substances can be detected by a range of different detectors, the most common being UV absorbance, but mass spectrometry is also widely used.
Columns packed with beads functionalised in different ways are available to allow selective binding of chemicals with a range of different properties.
The most common type of HPLC column is reverse phase, and involves hydrophobic beads binding hydrophobic molecules through hydrophobic interactions. HPLC columns that function via hydrophillic or charge-charge interactions are also used widely for specific molecular subsets. An example of this is ion chromatography, which is based on ionic interactions.
Size exclusion columns, which separate molecules according to size, are also available for HPLC.
Typically a UV detector at either 280nm or 214nm is used to detect compounds that elute.
In general, HPLC works well for molecules that are at least partly hydrophobic and molecules with a high degree of hydrophilicity tend not to be retained on HPLC columns. Hence inorganic compounds are not typically amenable.
HPLC can be used, for instance, for separation of mixtures of pesticides, pharmaceutical preparations, metabolites from blood/plasma, peptides and many other things.
Because HPLC is a solution-phase technique, analytes need to be either in solution or capable of being solubilised by one of the compatible solvents, such as water, methanol, acetonitrile or similar organic solvents.
Particulates must be removed from solutions prior to introducing them to HPLC columns.
Although HPLC systems that operate at large preparative scale exist, JBL Science operate instruments at semi-preparative scale and lower, hence < 10mg of sample is optimal and analytes present at levels as low as pg or even fg levels can be detected.