What is it?
Pre & Post-Column
What is HPLC?
HPLC stands for High-Performance Liquid Chromatography. It uses the chemical properties of a stationary phase (the column) and a mobile phase (the buffer) to separate complex mixtures of molecules. The column serves to slow the movement of compounds to varying degrees based on their chemical or physical properties.
By varying the properties of the two phases and the runtime parameters, different compounds can be separated in different ways. The column parameters can vary based on particle or pore size, internal diameter, column length and flow rate through the column. The buffer can vary based on composition (differing levels of aqueous or organic solvents) or by changing the solvent concentration over time (isocratic versus gradient).
Samples may have to be prepared in special buffers prior to loading on the machine. Any particulate in the sample has the potential to clog the column, so it is preferable to separate prior to running. Often there is an in-line Solid Phase Extraction column to remove what is not soluble in the sample buffer.
After being separated by HPLC, each compound is detected by UV/Vis and represented in a chromatogram where the y-axis is absorbance intensity (compound amount) and the x-axis is time (how long the compound was retained). In some cases, compounds will be further analyzed by Mass-Spectrometry.
Common types of HPLC
Size-exclusion separates based on compound size. The column consists of trillions of microscopic pools of solvent around the beads. Smaller proteins will be caught in these pools while larger proteins pass through and elute out faster. The longer the time of elution, the smaller the protein collected.
Reverse-Phase separates based on compound polarity. The column is a non-polar material, often silica based, and the mobile phase is a varying amount of aqueous (polar) and organic (non-polar) solvents, often with a highly polar ion added in. The longer the elution time, the higher the over-all polarity.
Ion-exchange separates based on compound charge. In this method, the mobile phase contains chemicals that induce charge on the compound of interest. This can be done by cations, anions, pH or temperature.
Affinity columns separate based on the formation of stable, specific and reversible complexes. This can be done using antibodies, receptors or their respective ligands. By lining the column with antibody, the analyte of interest will be bound and isolated. Later, the mobile phase can be changed so the antibody no longer binds, and the analyte is then eluted and collected.
A. A functioning HPLC system includes a sampler, pump, column, detector and data processor. A degasser and column oven may also be used.
A. There are UV, fluorescence, electrochemical, conductivity, refractive index, evaporative light scattering, chiral, radioactive and mass spectrometry detectors.
A. Check the assay protocol to instructions on the appropriate column. The most commonly used column is C18 which covers a wide range of applications.
A. Filter samples to remove particulate and make sure the pH of the mobile phase is within specifications for the column. If the column will be stored for an extended period of time, flush with methanol or acetonitrile.