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The column is an integral part of HPLC. Column performs the separation of molecules while the analysis. There are several types of columns in HPLC used while analyzing. C18 and C8 are the most common columns used in the pharmaceutical industry. These are widely used in inorganic molecule analysis. Â
Importance of different types of columns
Different types of HPLC columns have unique properties and are suitable for different types of molecules and fixed-dose combinations in the pharmaceutical industry. Normal phase columns are widely used for separating non-polar and moderately polar compounds, while reverse phase columns are ideal for polar compounds. Ion exchange columns are used for charged molecules separation, while the ion exclusion column excludes ions while the separation process. It is very crucial to understand the properties of different HPLC columns for selecting the appropriate column and finding the optimal separation and analysis results.
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Types of HPLC columns:
There are four types of HPLC columns used in liquid chromatography.
- Normal Phase column
- Reverse phase column
- Ion exchange column
- Ion exclusion column.
Normal Phase column:
The normal phase column is packed with extra molar material than the mobile phase.
In normal phase columns, the mobile phase should be methanol, ethanol, acetone, acetonitrile, chloroform, etc. The use of water as a mobile phase is not recommended in normal phase columns because water has a higher polarity in nature.
The eluent is dissolved in less polar material like methanol when it passes through the column and interacts with the high polar stationary phase, thus good separation happens.
Such columns are very popular in pharmaceutical product testing.
Columns containing silica are very efficient for separating non-polar and moderately polar isometric compounds.
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Reverse phase column:
Columns having a non-polar stationary phase are called reverse-phase columns. In this chromatography process, the polar component of eluent drains from the column first, then the non-polar component drains out.
The sample prepared in water results in good separation in this type of column. Aqueous-based mobile phase provides better separation of molecules interacting with stationery non-polar material.
The C-18 column is a reverse-phase column. In the C-18 column, water-based mobile phases are very efficient. Acetonitrile and methanol are also good for the mobile phase.
Reverse phase column example:
- C-18 Reverse phase column
- Alkyl RP-LC column
- C1 RP-LC column
- C30 Reverse phase column
- C4 RP-LC column
- C8 RP-LC column
- PFP reverse phase column
- Phenyl reverse phase column
Ion exchange column:
The Ion exchange column is packed with beads of resin material, selected upon the capability to absorb a particular ion from the aqueous mobile phase when passed through the column. Such a type of packed material helps in the separation of analyte better.
Charged molecules are separated easily in the ion exchange columns.
The stationary phase contains positively charged ions that attract the negatively charged ions from the solution passing through the column.
Types of Ion exchange columns:
Example of an ion exchange column is the PRP-X300 column which is a polymeric column. PRP-X300 column with such material is good for the separation of alcohol and organic acids.
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Ion exclusion column:
In the ion exclusion process, the highly ionized samples are eluted first in the column then weakly ionized samples., weakly ionized samples elutes later. In this process, ions are excluded while separating thus this chromatography is called the ion exclusion process.
Some commonly separated molecules in this chromatography are:
- Acetic Acid
- Acrylamide
- Citric Acid
- Oxalacetic Acid
- Ethanol
- Propane
Who invented the HPLC?
Jack Kirkland is remembered as the father of liquid chromatography(HPLC). Jack Kirkland discovered HPLC with Joseph Huber and Csaba Horvath.
Since the inception of HPLC Jack Kirkland has made significant improvements in HPLC chromatography.
Otherside, some theory says Russian botanist Mikhail Tavet invented chromatography in 1900.
What is RT in HPLC?
The term RT referred to retention time in HPLC analysis. The time between the injection of the sample and the solute passes through the column, making a peak. The RT of a solute is changeable and depends on many chromatography conditions, like column size, the flow rate of the mobile phase, nature of the mobile phase. Â
What is the difference between ODS and BDS columns?
Both ODS and BDS columns are reverse-phase columns.
The ODS column contains an OH functional group in its packing. Whereas in the BDS column deactivated OH groups are used as packing. The ODS column generates high tailing peaks on the graph.
In the use of the BDS column, peak tailing reduces significantly.
The C18 column is an ODS column. This column is packed with Octadecisilyl Group, and bonded with silica gel. The ODS columns have a high theoretical plate number. ODS columns show rapid equilibrium.
In the ODS columns, the elution of molecules occurred very fast.
The hydroxyl group is deactivated in the BDS column. BDS columns are packed with Octadecasilane silica.
BDS column produces very low peak tailing which keeps the tailing factor low.
What is the pore size in the HPLC column?
HPLC columns have different pore sizes. The pore size is used according to the molecule size of the substance to be analyzed in the column. The small molecule size substances are analyzed on 8-12 nm pore size columns.
Usually, 5-10 µm particles are used in pharmaceutical product tests.
A small pore diameter means a larger surface area of packing particles in the column. Larger pore sizes have a small surface area of the packing material of the column.
The surface area of the packing particles is inversely proportional to the pore diameter of the column.
HPLC column classification?
As per USP Chapter 621, the HPLC column should be classified according to its packing materials. USP made characterization of the HPLC column for two main reasons, first, the replacement of column on deterioration due to usage becomes easy if the packing and other specifications are classified under their trade name.
Second, the transfer of the developed method of analysis makes it easy to select columns based on their specification provided by the manufacturer.
Types of columns as per USP?
TYPECOLUMN SPECIFICATIONL1C18 (ODS) bonded to porous Silica 1.5µm to 10µm in diameter.L3Silica (Si) unbonded porous particles. 5um to 10um in diameter.L7C8 (Octyl) bonded to porous Silica 1.5µm to 10µm in diameter.L8NH2 (Aminopropyl Silane) bonded to totally porous Silica 3µm to 10µm in diameter.L9Porous Silica with a chemically bonded, strongly acidic cation-exchange coating (SCX). 3µm to 10µm in diameter.L10CN (Cyano) groups bonded to porous Silica 3µm to 10µm in diameter.L11Phenyl (C13H25) groups bonded to porous Silica 1.5µm to 10µm in diameter.L14Silica with a chemically bonded, strongly basic quaternary ammonium anion-exchange coating (SAX), 5µm to 10µm in diameter. USP-column-type
What are guard columns and what is their use?
The guard column is a device that is used to protect the column, this device is installed between the injector and the column. The work of the guard column is to protect the column from impurities and suspended particles in the mobile phase. The stop to enter them in the column and improve the life span of the column.
HPLC column price?
The price of the HPLC column differs from Rs. 14000 per column to Rs. 40000 per column. That varies from company to company, and the type of column.
The packing material of the column also matters when deciding the price of the column.
Conclusion:
Types of columns in HPLC are the most discussed topic in the chromatography world. HPLC columns are mostly four main types Normal phase, Reverse Phase, and Ion Exchange columns. In the pharmaceutical industry, mostly reverse columns are used for analysis.. This very vast and deep topic when going into detail about each type of column specification. Focusing on types of columns in HPLC used in the pharma industry is limited.
References:
- LCGC: Pore size and particle diameter
- Column Classification: USP-Chapter-621
