Types of HPLC Columns Used For Analysis

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types of hplc columns

Different Types of HPLC Columns Used For Analysis

we will now discuss the types of HPLC Columns and their applications. HPLC can be distinguished based on the difference in the mobile phase and the stationary phase used. This difference causes the characteristics of the sample that can be analyzed and the preparation required to be different.

IN HPLC, Columns are the main components, For the seperation of sample components columns are responsible.

Columns are the main component in HPLC because the column is responsible for the separation of the sample components.

The sample passes through the column with the mobile phase and separates in its components when it comes out from the column.

Generally, In the high performance liquid chromatography (HPLC) silica gel is filled in columns. because of silica gels particle size & porosity which helps in separation of components.

Also silica gel is an inert and non-reactive material that does not react with mobile phases.

Silica Columns, Therefore can be used to analyze the compounds of different chemical natures.

The material which is filled in the HPLC columns is known as a stationary phase.

Types of HPLC Columns

There are Four types of chromatography columns,

Some of them are described below, on the basis of their composition and method of separation;

1) Normal Phase Columns

2) Reverse Phase Columns

3) Ion Exchange Columns

4) Size Exclusion Columns

1. Normal Phase HPLC

The separation of sample components in the normal phase and the reverse phase is carried out based on polarity.

In this type of HPLC, the stationary phase is polar and the mobile phase is non-polar.

Thus, a sample component that is more polar will interact more strongly with the stationary (polar) phase and thus takes longer to pass through the column compared to a sample component that is less polar.

Normal Phase HPLC: Polar compounds will pass through the column longer than non polar compounds

The stationary phase used usually contains pure silica or groups of organic compounds such as amino or cyano bonded to the silica base.

Whereas the mobile phase is usually a non-polar compound such as hexane or heptane which is slightly mixed with polar compounds such as methanol, ethanol, or isopropanol.

The normal phase column contains silica

Types of HPLC Columns

Types of HPLC Columns

2. Reversed Phase HPLC (Reverse Phase HPLC)

As the name implies, this type of HPLC is the opposite of Normal Phase HPLC.

In this type, the stationary phase used is non-polar and the mobile phase is polar.

Separation occurs because a sample component that is less polar will pass through the column longer than a sample component that is more polar.

Reverse Phase HPLC: Non polar compounds will pass through the column longer than polar compounds

The stationary phase used in this phase is usually a non-polar alkyl hydrocarbon such as a C-8 or C-18 chain bonded to a silica base.

Column C18 or Octadecyl Silica (ODS) is the most popular type used in HPLC.

The mobile phase that is often used in the reverse phase is a mixture of water with other polar solvents such as methanol, acetonitrile, or tetrahydrofuran.

The reverse phase column contains C18 bonded to silica

3. Size-exclusion HPLC

In this type of HPLC, the separation is carried out based on the difference in the molecular size of each component of the sample.

The column used contains the packing material which has a pore size that has been adjusted.

Sample components with smaller molecular sizes will be absorbed into the pores of the packing material so that it takes longer to pass through the column than components with large molecular sizes.

Size Exclusion HPLC: The smaller molecules will be trapped in so that they pass through the column longer

Column size exclusion with a pore size of 300Å

Chromatography This chromatography is also called gel permisation chromatography and can be used to separate or analyze compounds with molecular weights> 2000 daltons.

The stationary phase used can be silica or a polymer which is porous so that the solution can pass through the porous (between particles), or diffuse through the stationary phase.

Solute molecules, which have a much larger BM, will elute first, then medium size molecules, and finally the molecules that are much smaller.

This is because the solute with a large BM does not pass through the porous, but passes between the stationary phase particles.

Thus, in separation with this size exclusion there is no chemical interaction between the solute and the stationary phase like other types of chromatography.

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4.Ion Exchange HPLC ( Ion Exchange HPLC)

This type of HPLC is used for samples that are ionic or can be converted into ionic.

The stationary phase has an ionically charged surface opposite to the sample. The stronger the charge of the sample component, the component will be attracted to the surface of the stationary phase so that it will take longer to pass.

Ion pair chromatography can also be used to separate ionic samples and overcome the inherent problems of ion exchange methods. Ionic samples are covered with ions which have opposite charges.

Ion exchange column with Polyethyleneimine as stationary phase

HPLC application

As mentioned in the previous article, HPLC is often used in the pharmaceutical industry or pharmaceutical laboratories as a sample analysis method.

However, HPLC also has uses in other fields such as in the food and beverage industry, environmental analysis, forensics, and in clinical testing.

Types of HPLC and Their Applications in Analysis

Thermo Scientific’s UHPLC Ultimate 3000

In the pharmaceutical field, HPLC can be used to test drug stability, dissolution testing, and quality control .

Whereas in the food and beverage industry, HPLC can be used in various analyzes such as for analysis of the presence of polycyclic compounds, analysis of sugar and preservative content, or measuring the quality of soft drinks and water.

HPLC ion exchange chromatography uses a stationary phase which can exchange cations or anions with a mobile phase. There are many ion exchangers on the market, however the most widely used is polystyrene resin.

Most ion chromatographic separations are carried out using aqueous media because of their ionization properties. In some cases mixed solvents such as water-alcohol are used as well as organic solvents.

Ion-exchange chromatography with aqueous mobile phase, the peak retention is affected by the total salinity or ionic strength as well as by the pH of the mobile phase.

The increase in salt content in the mobile phase decreases solute retention. This is due to a decrease in the ability of the sample ions to compete with the mobile phase ions for the ion-exchange groups in the resin

In other fields, HPLC can also be used for analysis of antibiotics in blood, analysis of cocaine in urine, and so on


Separation by HPLC can be carried out with normal phase (if the stationary phase is more polar than the mobile phase) or reverse phase (if the stationary phase is less non-polar than the mobile phase).

Based on these two separations, HPLC is often classified into normal phase HPLC and reverse phase HPLC.

In addition to the above classifications, HPLC can also be grouped based on the properties of the stationary phase and / or based on the solute sorption mechanism, with the following types of HPLC:

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1. Adsorption Chromatography

Adsorption Chromatography The principles of adsorption chromatography are well known as in column chromatography and thin layer chromatography.

The adsorption chromatography separation usually uses the normal phase using the silica gel and alumina stationary phases, however about 90% of this chromatography uses silica as the stationary phase.

In silica and alumina there are hydroxy groups that interact with the solution. The silanol groups in silica have different reactivity, therefore the solute can be bonded tightly so that it can cause a taily peak.


2. Bonded

phase chromatography Most of the stationary phase of this chromatography is chemically modified or bonded silica. So far, non-polar hydrocarbons have been used to modify silica such as with octadecylsilane, octasilane, or with phenyl.

The most popular stationary phase is octadecylsilane (ODS or C18) and most of the splits are reversed.

As the mobile phase is a mixture of methanol or acetonitrile with water or with a buffer solution.

For solutes that are weakly acidic or weakly basic, the role of pH is very crucial because if the mobile phase pH is not regulated, the solution will ionize or protonate.

The formation of these ionized species causes their bonds to the stationary phase to be weaker than if the solute is in the form of non-ionized species, so the ionized species will elute more quickly.

3. Ion Exchange Columns

4.  Size Exclusion Columns

5. Reverse Phase Columns

6. Affinity Chromatography

In this case, the separation occurs because of very specific biochemical interactions.

The stationary phase contains molecular groups which can only absorb the sample if there are certain charge-related and steric conditions in the appropriate sample (as in the interaction between antigen and antibody).

This type of chromatography can be used to isolate proteins (enzymes) from very complex mixtures.

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