High Performance Liquid Chromatography (HPLC) is a type of column chromatography designed to separate and classify components of a sample based on its interactions with a specific column. HPLC is distinct because it utilizes a more pressurized environment to propel the liquid samples through the densely-packed column more efficiently. Three types of HPLC columns are in common use today; they differ in the nature of the packing material coating the column and interacting with the sample.
Experimenters use Ion Exchange Chromatography when separating ionizable samples, or samples that can become charged. The stationary phase, which is the solid material lining the column and interacting with the sample, possesses a charge opposite that of the sample of interest, so that the sample will be attracted to the walls of the column. This attraction slows down the elution time, or time it takes to pass all the way down the column in the mobile phase, of the sample. The more charged the sample particles are, the more attracted they will be to the stationary phase, resulting in a slower elution time. The elution times represent the strength of the charge of the particles, and thus are the primary means of classification of the different compounds within the sample.
As its name indicates, Adsorption Chromatography functions via an adsorbent stationary phase. Adsorption, not to be confused with absorption, involves the temporary binding of the sample particles to the stationary phase of the column. The degree to which a sample particle may adsorb to the stationary phase is determined by the relative polarity, or charge, that the particle may possess at a given time. This varies from ion-exchange chromatography in that the particles are not explicitly charged, rather they are slightly positive or negative at times as the particle rotates and vibrates. As the particle moves down the column, it adsorbs and de-adsorbs to varying degrees based on the level of polarity it possesses. Elution times provide a measure of level of polarity of the sample particles.
Size-Exclusion Chromatography utilizes a different separation technique than that of Ion Exchange or Adsorption Chromatography. This column type is less common than the other two, but is useful when separating large biomolecules like proteins or carbohydrates. The stationary phase is made up of uncharged particles, which are size-controlled and porous. When the sample is fired through the column, the smaller sized particles are temporarily absorbed into the column lining, whereas the larger particles pass straight through and elute first. The stationary phase is designed so that the smaller the particle, the deeper into the column lining it penetrates, meaning that the smallest particles will take the longest amount of time to re-surface and elute. Samples can be classified according to elution time, which corresponds directly to particle size.
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