Chromatography is a versatile field with a wide range of applications. It’s accomplished by fractionating a mixture into its molecular components. Chromatography was first used in 1901 by Russian botanist Mikhail Tsvet when he realized the technique could separate plant pigments. It has since become widely developed and utilized for separation analysis in various scientific fields.
All techniques utilize both a mobile and stationary phase. The analyte separates due to interactions with these phases. Liquid chromatography is defined as such because the mobile phase is a liquid solvent. There are many sub categorical chromatographic methods that fall under the umbrella of liquid chromatography. are A few examples of techniques that also utilize a liquid mobile phase include:
- Affinity chromatography
- Size exclusion chromatography
- Ion exchange chromatography
Liquid chromatography can be performed using either columns or planar bed techniques. In both instances, the liquid phase carries the analyte through the stationary phase. For columns, this movement is vertical. For planar techniques, such as paper and thin layer chromatography (TLC), the mobile phase moves laterally through the stationary phase. While liquid chromatography is utilized via both column and planar bed types, column chromatography has a greater variety of uses because columns are available in a wide range of options. There are many sizes available and the columns can be suited for either reusable or single-use purposes.
The stationary phase within the column is comprised of resin or media. The composition of the stationary phase depends on the physical properties of the sample and the solvent. Solids interact as adsorbents with the analyte. Depending on the polarity of the solutes, the absorbent adheres to some molecules more strongly than others; causing the analyte to separate and elute its parts at different rates. Silica gel and alumina are two commonly used adsorbents. Resins containing ionic groups facilitate separation through the exchange of ions with charged groups in the analyte. A stationary phase comprised of polymer beads that contain pores of differing sizes will separate a compound through size exclusion; the different pores slow down the elution of smaller molecules, while larger molecules elute more quickly.
Selection of solute(s) for the mobile phase is critical for proper separation. The mobile phase dissolves the analyte and passes through the stationary phase. As such, the two most important factors to consider when selecting an eluent are:
- the solubility of the sample (if known)
- the polar properties of both the sample and the stationary phase
If the properties of the solvent do not properly interact with the sample and stationary phase, it can cause the sample to elute too quickly or to not adequately differentiate. Sometimes separate solvents with differing polar strengths ranging from low to high are used to gradient the separation of the sample. This causes weakly adsorbed particles to be eluted first, and for for more strongly adhered molecules to be released during additional elutions using stronger polar solvents.
Liquid chromatography techniques are helpful for purification, decontamination, and both qualitative & quantitative analysis of samples. The complex interactions of the sample, the solvent, and the stationary phase are collective components for liquid chromatography procedures. Amongst the other considerations listed above, it is also important to consider the degree of separation desired when deciding which components to employ during the process.
Liquid chromatography is a popular and useful technique in many settings including both research and commercial labs. Anyone in a scientific field can benefit from a basic understanding of chromatography; the versatility and wide development of these techniques can be a fundamental tool for both novices and experts alike.
