High-performance liquid chromatography is a type of chromatography that refers to use in analytical chemistry for identifying, separating, and identifying each molecule in a sample mixture. The types of HPLC such as reversed-phase, normal phase, size-exclusion, and ion exchange HPLC are used to isolate the compounds, of these, RP-HPLC is the most commonly used. The selection of columns for HPLC analysis is mainly based on the solubility of compounds in solvents. HPLC samples are normally organic molecules that differ in properties and structures such as polarity, size, pH, and functionality. Some molecules have ionic functionality; they often need modification in ionic strength or pH of the mobile phase to the symmetrical shape of the peak.
There are different types of columns used in HPLC, but the C18 is the most widely used stationary phase in reversed-phase HPLC. C18 (Octyldecylsilane) is hydrophobic in nature that has a carbon chain bonded to the particles of silica within the column. It has 18 carbons that bonded to silica, which is more and a longer carbon chain than the C8 (8 carbons) and C4 (4 carbons). Since it has a more carbon chain, it has a larger surface area that gives a further time of interaction between analytes and bonded phases. As a result, the solutes are gradually separated from the column with more separation. In such a phase non-polar compounds will interact while they travel through the stationary phase. Therefore, if the sample mixture containing two compounds and if one compound is more hydrophobic, then the less hydrophobic compound would elute more rapidly than more hydrophobic compounds.
Both C18 and C8 are RP-HPLC columns and refer to the alkyl chain of the bonded phase. C18 columns can available in several sizes such as length, diameter, hydrophobicity, particle size, and the ability to separate acidicand basic components. Generally, the columns are available in the 30 mm to 250 mm in length, 3µ to 5µ in pore size, and 01 to 05 mm in diameter. The C18 HPLC column is mainly used in pharmaceutical analysis, environmental science, chemical analysis as well as some industrial applications, for qualitative and quantitative analysis of samples. C18, C8, C30, C4, C1, alkyl reversed-phase column, and phenyl columns are the different types of columns used in RP-HPLC. C18 is the most widely used.
Silica gel:
Silica gel is the most commonly used packing material or as a stationary phase in different types of chromatography such as HPLC, TLC, column chromatography, and HPTLC. It is slightly acidic and polar, it has two types such as spherical and irregular shape. The silica gel used in liquid chromatography has a pore on its surface, which gives a larger surface area than pores without. The 5μm is the most widely used HPLC column.
Commonly asked questions on HPLC columns are as follows.
Why c18 column is used in HPLC?
The C18 is the most commonly used column in reverse phase HPLC since it can isolate a large range of components as compared to other types of columns.
How many types of HPLC columns are there?
A variety of HPLC columns are available for liquid chromatography, depending on the composition and separation mechanism i.e. reversed-phase, normal phase, ion exchange, and size exclusion chromatography.
What is the difference between c8 and c18 HPLC columns?
The difference between C8 and C18 is that the column packing in C8 has 8 carbons bonded to silica while C18 has 18 carbon atoms bonded to silica.
Is c18 column polar or nonpolar?
The column is packed with very small particles of silica gel. The surface of silica used in the column is polar as it is covered with silanols.
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