Types of HPLC | Their Principle and Application
1. Based on modes of chromatography :
a) Normal phase mode
b) Reverse phase mode
2. Based on principle of separation :
d) Ion exchange chromatography
e) Ion pair chromatography
f) Size exclusion / gel permeation chromatography
g) Affinity chromatography
h) Chrial chromatography
3) Based on Elution method followed:
i) Isocratic separation
j) Gradient separation
4) Based on the type of analysis :
k) Quantitative analysis
l) Qualitative analysis
5) Based on the scale of operation:
m) Preparative type
n) Analytical type
Let us see the above types of HPLC in more detail
1.Based on modes of chromatography
Here based on polarity of the stationary and mobile phase the HPLC chromatography is either of
i. Polar : Polar (stationary phase : Mobile phase)
ii. Polar : Nonpolar
iii. Nonpolar: Nonpolar
2. The stationary phase in the column used
Based on the nature of stationary phase used it can be either normal phase or reverse phase HPLC.
1. Normal phase chromatography: Here the stationary phase of the column is made of polar compounds like silica gel, alumina, etc. When the process is run, the nonpolar compounds are eluted first. The polar compounds in the sample have higher affinity to the stationary phase, and so they are retained longer in the column than non-polar ones. Hence polar compounds are eluted latter. This is normal type because more common substances can be analyzed. But it is not highly used on routine basis as drugs, food and other biological are non polar in nature.
2. Reverse phase HPLC: Here exactly the opposite of normal phase happens. The stationary phase is made of non-polar compounds like octadecyl silane, C18, C8 type of organic compounds. The mobile phase used is polar. So compounds of high polarity or eluted first while those of low polarity or no polarity are eluted last.
Most of the applications in HPLC require the evaluation of drugs, biochemical molecules and other substances used by humans and they are polar (water soluble) in nature. So, reverse phase HPLC is widely used.
Based on principle of separation
Here the HPLC is categorized based on the principle used in separation.
a) Adsorption chromatography: The principle of separation is adsorption mode. Separation of components occurs due the differences in the affinity of compounds towards the stationary phase. This is routinely employed principle in HPLC chromatography
b) Ion exchange chromatography: In this HPLC type, the separation occurs due to reversible exchange of functional groups. This principle can be better understood by going through the article ion exchange chromatography.
An ion exchange resin is employed to separate the sample mixture containing charged ions. For anion an anion exchange resin is used and to separate cations a cation exchange resin is used. It is used specifically for separation and estimation of acidic and basic compounds.
c) Ion pair chromatography
In this type of hplc a revese phase coloumn (nonpolar) is temporarily converted into ion exchange. This is done by using ion pairing agents like pentane, hexane, heptane or octane sulphonic acids salts.
d) Size exclusion chromatography: Here the components of sample are separated as per their size. The coloumn used is made of soft gels like agarose, dextran, polyacrylamde. Besides these semi rigid gels like alkyl dextran, poly styrene are also used.
e) Affinity hplc chromatography relies on the affinity of sample with specific stationary phase.
f) Chiral chromatography: This methods is used to separate optically active isomers in a sample. The chemically bonded silica gel is used as stationary phase.
Based on scale of operation
HPLC technique can also be used for different scales and is as
1. Analysis Mode: The procedure is done to estimate different types of molecules and their individual quantities in the mixture using the help of a detector.
2. Preparative mode: Here the intention of the process is to separate large amounts of a particular molecule from a mixture. The molecule or substance eluted is of the highest purity. The column size, the sample size is comparatively large than that of analytical mode.
HPLC advantages and disadvantages
Based on Elution method followed: As we know by now, the separation of mixture occurs due to flow of mobile phase through the HPLC column. This mobile phase is either a single solvents or a mixture of solvents. Based on this there are two types of hplc techniques like
a) Isocratic separation: Here a single solvent is run through out the operation. So, all the operation is under the influence of that one solvent.
b) Gradient separation: Here are two or more solvents are allowed to pass through the column. This is intended to separate components from the column at different rates.
4) Based on the type of analysis
a) Quantitative analysis: This is intended to identify the compounds and determine the impurities in a sample
b) Qualitative analysis: Here the quantity of individual compound in the mixture is estimated.