HPLC (high performance liquid chromatography) Principle

The basic principal of HPLC is the partitioning the analytes between the solid phase and the mobile phase. Since compounds will have different partition coefficients they will be separated on that basis.

HPLC (high performance liquid chromatography) is a chromatography technique where the mobile phase is a liquid and the stationary phase is packed into a stainless steel column at high pressure. It is usually silica particles mainly spherical nowadays. The efficiency is better when the particles are smaller typically 5um. A pump is used to push the solvent through the column and a detector with a flow-through cell used to measure the separated peaks. Usually a computer with integration software collects the data and help quantifying the components. There are huge variations in the sample injection, pump, columns, detectors and software.

HPLC works on the principle that some molecules take longer than others to pass through a chromatography column. This depends on the affinity of the molecule with the mobile phase (liquid or gas) and the stationary phase (solid or liquid). The ones that have more affinity with the stationary phase take longer to pass through and vice versa.

There are different types of columns to separate molecules by different criteria.

There’s molecular exclusion columns, that separate molecules by weight, these columns use tiny beads with canals in them, this causes small molecules to take a longer route and therefore take longer to pass through; normal phase liquid chromatography columns (NPLC), that separate molecules depending on their polarity, these columns are made out of silica with polar functional groups covalently attached, polar molecules take longer to pass through these columns because they are attracted to it through dipole-dipole interactions; reverse phase chromatography columns (RPLC), that do the opposite of NPLC columns, they’re made of silica with non-polar groups equivalently attached, here, non-polar molecules stay behind interacting with the stationary phase through Van Der Waals forces and the hydrophobic effect and take longer to pass through; and ionic exchange columns, that separate molecules by charge, in these columns molecules with formal charges take longer to pass through by electrostatic interactions.

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HPLC is one variation of a “two phase distribution system” consisting of three parts: analytes (the mixture that you want to separate), a stationary phase (some solid material with a known surface chemistry packed inside a metal “column”), and mobile phase (a liquid mixture that we push through the column, usually at relatively high pressure).

The mixture is introduced into the flowing mobile phase and enters the column where it now has a choice between staying in the liquid phase or stopping on the stationary surface (this is the two-phase distribution part). Individual molecules will prefer one phase or the other, depending which phase they are “like.” This process happens continuously through the column, and if the molecules have different preferences for the stationary phase, they will all come out at different times, depending on how many times they stopped on the way through the column. If we push the mobile phase through at a constant rate, they will all come out at different times, and we can “detect” them as individual “peaks.”

[If you don’t have a strong chemistry background, you can think of this distribution process as like a mixture of gasoline and water, or oil and vinegar dressing. In each case, you see two different “layers” because the two parts are not “like” each other. The water (or vinegar) is a hydrophilic phase, and anything that is highly water soluble, like sugars, will go to that phase. The gasoline (or oil) is hydrophobic, and will attract things that are not water soluble, like fats. And, of course, there will be things that are fairly happy in both phases. HPLC is just like lining up a long series of these phases, and passing one of the phases from one bottle to the next, and the next, and so on. By the time you get to the end of the line, you may find that all the separate components in the mixture are now all spread out over different bottles.]

Applications of HPLC are quite diverse. Nearly every pharmaceutical compound, whether over-the-counter or prescription, has been tested for purity and to confirm the amount of drug that is present. The gasoline in your car has been tested to ensure that it has the right proportions of certain types of compounds in it. Want to know the vitamin content in your food? It is probably done by HPLC. This technique has been very successful in helping many industries make and sell better products. This success is due to advances in instruments as well as variation in the chemistry of the stationary phase.

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