Column selection is the first and most important step for the development of an analytical method, whether it is gas chromatography or high-performance liquid chromatography. It is not possible to develop a rugged, robust, and reproducible method without high-performance and stable HPLC columns. Significantly, columns are reproducible and stable to avoid errors with irrelevant sample retention time during the development of the HPLC method.
The RP-HPLC column is selected based on the nature of the molecule and the information about the solute. Reversed-phase chromatography is the types of HPLC which have broad columns range such as dimethyl silane (C2), butylsilane (C4), octylsalane (C8), octadecylsalen (C18), cyanopropyl, BDS phenyl, and nitro, etc. A C18 or C8 column is specially made with less acidic, high-quality pure silica and is particularly suitable for the isolation of basic analytes. They are normally appropriate for all the samples and are strongly recommended by the chromatographers.
The following few points give guidance in the selection of column for RP-HPLC:
- Almost all HPLC columns are made of steel. The major advantage of steel is that it is relatively inert to corrosion and pressure resistance.
- It is significant for a better separation of solutes that there is no scoring, roughness, inside a chromatographic column.
- Silica gel is the most popular stationary phase in high-performance liquid chromatography, which consists of Si atoms and oxygen atoms and it carries OH groups (silanol groups). It is available in different particle sizes and is used within a pH range of 1.00 to 8.00.
- Polymer-based columns are also used in reversed-phase HPLC, providing higher pH stability than silica gel columns. It can be used for the isolation of strongly acidic or basic components.
- Column packing materials of various types and sizes are available according to the nature of the compound and HPLC method.
2. Coreshell particles have a solid core, and the outer shell is porous, resulting in improved efficiency in the separation. One major disadvantage of coreshell particles is that it has low load-ability compared to porous materials.
3. The monolithic phase column consists of a piece of porous material, like a silica gel. A disadvantage of monolithic phases is that the availability of selectivity is limited in contrast to fully porous materials.
- The longer columns generally give a better separation because of the higher theoretical plate numbers. If the particle size decreases, then surface area available for coating increases. 5-μm particle size HPLC columns offer the best solution in terms of reproducibility efficiency, and reliability.
The C18 is the most common reversed-phase column may be used in several analyses. To decide which stationary phase or column is suitable, the C18 column is considered the first choice at the beginning of method development. Since it has longer carbon chains (typically 18 carbons) than other column and provides a wide surface area which offers an additional time of reaction between the bonded phases. The very-polar molecule does not interact with the C18 material and is eluted at the dead time, in such cases you can use C4, CN, diol, or phenyl phases. Conversely, if the molecules are very un-polar you can use silica, CN, or NH2 phase.
While doing the HPLC method development, there are some aspects to be considered: separation scheme, mobile phase, type of HPLC column, the diameter of the column (such as particle size, pore size, internal diameter, diameter, and carbon content), analytes of interest, pKa value, pH of the solvent system, detector, and injector, etc. Method development has different conditions in each case; each chromatographer should consider these factors when performing the HPLC analysis.
Commonly asked questions on the HPLC column are as follows.
How many types of HPLC columns are there?
There are four main types of HPLC columns-reversed phase HPLC column, normal phase HPLC column, the size-exclusion HPLC column, and the ion-exchange HPLC column.
Is c18 column polar or non-polar?
The C18 column is used in the reversed-phase chromatography; it’s packed with non-polar material as compared to the polar stationary phase.
What is the difference between HPLC and RP-HPLC?
HPLC is the type of chromatography in which RP-HPLC is the most commonly used modes of HPLC. HPLC stands for high-performance liquid chromatography and RP-HPLC stand for reversed-phase high-performance liquid chromatography.
What is the difference between c8 and c18 HPLC columns?
Both C8 and C18 are the types of columns used in HPLC, the major difference between C8 and C18 is that C18 has 18 carbon atoms while C8 has only 8 carbon atoms.
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