Tips for Choosing the Right HPLC Reagents for Use in a Laboratory

High- Performance Liquid Chromatography (HPLC) is a laboratory method used to separate different components in a mixture. This also used to quantify, identify, and compare these components. HPLC works on the simple concept of flow rates and adsorbents. Each component in a mixture is adsorbed at different levels of the HPLC column. The HPLC column is filled with adsorbent material, through which a pressurized liquid is passed. This pressurized mixture basically contain a solvent or reagent in which the mixture is dissolved. There are many type of reagents available in the market like acetic acid, ammonia, phosphoric acid, sulphuric acid, sodium hydroxide, methylethylketone, etc. Now, choosing the right HPLC reagents or solvents from the vast options available could be a very crucial decision and factor in this process. Therefore, here are few tips and factors to consider while choosing the solvent for High performance liquid chromatography.

Purity:

The reagents used for High performance liquid chromatography should be extremely pure. Even the slightest trace of impurity could interfere greatly with the results. The reagents used in High performance chromatography should not cause any avoidance or interference even at high detection wavelengths. Therefore, only use the reagents of high grade for high performance chromatography. There HPLC grade reagents special available in the market.

Type of the reagent:

There are two types of HPLC reagents available. There liquid reagents and gas reagents. So first decide which type you require based on the mixture you are using, as it has to easily dissolve in this reagent. The volatility of the reagent is also a deciding factor, since the more volatile reagents tend to evaporate.

Type of HPLC:

There are many types of high performance liquid chromatography, such as Partition chromatography, Normal–phase chromatography, Displacement chromatography, Reversed-phase chromatography (RPC), size-exclusion chromatography, ion-exchange chromatography, bioaffinity chromatography, and aqueous normal-phase chromatography. Each of these chromatography techniques have specific requirements and thus the chosen reagent should meet the requirements of that particular HPLC technique. For example, the bioreagents should contain minimum or no enzymes for optimum results.

UV Absorbance:

The reagents used in high performance chromatography or high pressure chromatography (HPLC) should exhibit low UV absorbance even at critical ranges.

Low Residue:

The reagents used in high performance liquid chromatography (HPLC) show leave low residue after evaporation.

Buffers:

The type of reagent used in high performance liquid chromatography (HPLC) also determines the type of buffer used. Some volatile reagents evaporate quicker thereby causing a change in the pH of the mobile phase, therefore a buffer is required to maintain the pH. The reagent used in the high performance liquid chromatography should be compatible with the buffer used for obtaining optimum result.

Adsorbent:

The reagents used in the high performance liquid chromatography must be compatible with the adsorbent used. It should be able to flow properly through the absorbent, thereby allowing the components to be adsorbed at different levels. This is possible only when the solvent flows through the adsorbent at different flow rate as each component gets adsorbed.

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