Medical Science

Why should We Do The HPLC Test?

By William Watson

Posted on March 16, 2021

Chromatography may be a process of separating mixtures through different techniques like using moving solvents or by paper or suspension through a medium. The varied components of the mixture travel at different speeds, and this results in the separation. This takes place only at the stationary phase of the mixture when it’s unstable and non-volatile and once they have a relatively high relative molecular mass.

Liquid Chromatography is typically employed across industries to check water samples to detect pollution levels in lakes and rivers. It helps in the analysis of metal ions and organic compounds in solutions. Liquid Chromatography uses liquids that can incorporate hydrophilic, insoluble molecules. HPLC testing is a type of liquid chromatography.

Gas chromatography is employed to detect bombs and is also employed in forensics in many ways. It’s used to analyze fibers on dead bodies and also analyze blood found at a crime scene. In gas, chromatography helium is employed to maneuver a gaseous mixture through a column of absorbent.

Thin-layer Chromatography uses an absorbent on flat glass or plastic plates. This is often an easy and rapid method to see the purity of a compound. It’s used to detect pesticide or Insecticide residue in food. Thin-layer chromatography is additionally utilized in forensics to research the dye composition of fibers.

Paper Chromatography is one of the foremost commonly used types of chromatographic technique. It uses a strip of paper for the stationary phase. Capillarity is employed to tug the solvents up through the paper and separate the solutes.

What Is HPLC, and Why Should We Use HPLC Testing?

HPLC (High-performance Liquid Chromatography) is an effective chromatography wherein the analyte is separated.

In this process, a solvent is pumped along with the sample mixture, also referred to as the mobile phase. It was initially named high liquid chromatography. They’re widely used to research antibiotics as it’s superior to the traditional microbiological assays due to its specificity, sensitivity and analysis time. HPLC testing is employed to separate components of an organic mixture. HPLC may be a new age analytical technique in analytical chemistry. Analytical chemistry is becoming a most sought after career choice in recent times.

Polar compounds will quickly reach the column due to the attraction between the polar solvents and the polar molecules which are present within the mixture. There are several forms of HPLC, and a couple of them are listed below.

• Normal phase HPLC

• Reversed-phase of HPLC.

• Size-exclusion HPLC

• Ion-Exchange HPLC

Normal Phase HPLC

This method separates analytes based on polarity. Therefore, the stationary phase is typically silica, and typical mobile phases are hexane, dichloromethane, chloroform, ether, and mixtures of those. Polar samples are then kept on the polar surface of the column, packing longer than the polar materials.

Reverse Phase HPLC

The stationary phase of the reverse phase of HPLC is nonpolar (hydrophobic) in nature, while the mobile phase of the reverse phase of HPLC may be a polar liquid, like mixtures of water and methanol or acetonitrile. It depends on the principle of hydrophobic interactions; hence the more nonpolar the stationary phase is, the longer it’ll be retained.

Size-exclusion HPLC

The column is crammed with the material having precisely controlled pore sizes, and therefore the particles are separated consistently with their molecular size. Larger molecules are quickly washed when it passes through the column while smaller molecules penetrate inside the pores of the packing particles.

Ion-Exchange HPLC

The stationary phase of the ion exchange HPLC has an ionically charged surface of opposite charge to the sample ions. This system is employed almost exclusively with ionic or ionizable samples.

The stronger the charge on the sample, the longer it’ll fancy eluting. Aqueous buffer is used as the mobile phase where both pH and ionic strength helps to control elution time.