Chromatography is a technique used to separate and analyze complex mixtures of compounds. The separation process depends on the different affinities of the compounds for a stationary phase and a mobile phase. The stationary phase is usually a solid or a liquid coating on a solid support, while the mobile phase is usually a liquid or a gas.
Solvents are essential components of the mobile phase in liquid chromatography systems. They help to dissolve the sample and transport it through the stationary phase. Here are the steps to apply a solvent in a chromatography system.
Step 1: Choose the right solvent
The choice of solvent depends on the nature of the sample and the stationary phase. The solvent should be able to dissolve the sample and provide sufficient elution strength to separate the compounds of interest. The solvent should also be compatible with the stationary phase and not cause any degradation or swelling.
Step 2: Prepare the solvent
The solvent should be prepared by using high-quality solvents and deionized water. The solvent should be filtered to remove any particulate matter or impurities that can clog the chromatography column. The solvent should be degassed to remove any trapped air bubbles that can interfere with the separation process.
Step 3: Load the sample
The sample should be loaded onto the chromatography column using a syringe or an automated injector. The sample should be dissolved in a small volume of the solvent to ensure complete dissolution and prevent overloading of the column.
Step 4: Apply the solvent
The solvent should be applied to the column at a constant flow rate using a pump. The flow rate should be adjusted based on the nature of the sample and the stationary phase. The solvent should be allowed to pass through the column until the compounds of interest are eluted.
Step 5: Collect the fractions
As the solvent passes through the column, the compounds of interest will be separated and eluted at different times. The eluted fractions should be collected in individual vials for further analysis. The fractions can be analyzed using various techniques, such as UV-Vis spectroscopy, mass spectrometry, or NMR spectroscopy.
In conclusion, applying a solvent in a chromatography system requires careful consideration of the nature of the sample and the stationary phase. The choice of solvent, preparation of the solvent, loading of the sample, application of the solvent, and collection of the fractions are all important steps in achieving a successful separation and analysis of complex mixtures of compounds.
The choice of solvent for the mobile phase in chromatography depends on the nature of the sample and the stationary phase. However, some solvents are more commonly used than others due to their chemical properties and availability. Here are some of the most used solvents in liquid chromatography:
Water is the most used solvent in liquid chromatography. It is a polar solvent that is compatible with most stationary phases and is readily available. Water can be used alone or in combination with other solvents to adjust the polarity of the mobile phase.
Acetonitrile is a polar aprotic solvent that is commonly used in reverse-phase chromatography. It is less polar than water and provides stronger elution strength, making it useful for separating nonpolar and moderately polar compounds.
Methanol is another polar aprotic solvent that is commonly used in reverse-phase chromatography. It has a higher elution strength than acetonitrile and can be used to separate more polar compounds.
Ethanol is a polar protic solvent that is commonly used in normal-phase chromatography. It is less polar than water and can be used to separate polar and moderately polar compounds.
5. Tetrahydrofuran (THF)
THF is a polar aprotic solvent that is commonly used in size exclusion chromatography. It has a low viscosity and good solubility for many organic compounds.
6. Dichloromethane (DCM)
DCM is a nonpolar solvent that is commonly used in normal-phase chromatography. It is useful for separating nonpolar compounds and can be used in combination with other solvents to adjust the polarity of the mobile phase.
Sam Chien joined DKSH in 2022. With more than seven years of experience in commercial and channel management of lab chemicals and life science consumables, he brings extensive experience in these areas. Sam is now the Senior Manager for laboratory consumables to help grow and expand the Life Science portfolio in the APAC region.