What is column chromatography? Do you have any idea about this? Well, there are lots of things that you should know about this. Before you can finally understand, how it used and its importance, you have to learn the principle, procedure as well as the applications involved in column chromatography. Likewise, you should be aware of what is this all about.
Well, it is the preparative strategy that is used in purifying the compounds. The process may depend on the hydrophobicity or polarity of the compounds. When it comes to column chromatography, the molecules mixture is segregated depending on the differentials partition between the stationary phase and mobile phase. The different column size can be used for this particular technique.
Column chromatography is a primary form of the chromatography procedure that was developed and introduced throughout the early chromatography discovery phase. From it, the theory of chromatography is portrayed. These are applied in the latest and most advanced settings of chromatography. Other forms of the methods relevant to chromatography were introduced along with column chromatography as the module and through slight variations.
When it comes to column chromatography, the solid adsorbent and stationary phase is set inside the vertical glass column & mobile phase. A fluid is added on the top & flows down over the column by either external pressure or gravity. Column chromatography is normally used as the purification technique. It separates desired compounds from the mixture.
The blend to be examined by using column chromatography is useful to the column’s top. The fluid solvent or the eluent is passed over the column through gravity or air pressure application. The equilibrium is created in between the solute that was wrapped up by the absorbent & the solvent that flows down over the column. As the components present in the blend have various interactions with mobile phases and stationary, they would be carried together with a mobile period to changeable degrees and the separation would be attained. The individual parts or elutants are collected while the solvent trickles from the base of a column.
Column chromatography is divided into 2 categories depending on the way that the solvent flows and goes to the column. When the solvent is permitted to flow down because of gravity or percolation, this process is magnitude column chromatography. When the solvent flows down because of a positive pressure of air, it is flash chromatography. It is an incredible method that is recently used in the research laboratories for organic chemistry. The word “flash chromatography” came from the concept of a professor. It was called “flash” as it could be performed instantly.
Column chromatography is primarily a form of techniques that are used in adsorption chromatography. Here, the components separation will depend on the point of absorption up to the stationary stage. In this situation, the stationary stage is the solid material that is packed in the vertical column that is made with metal or glass.
The Principles behind Column Chromatography
When a blend of mobile point and sample to be segregated are established from the column’s top, the individual parts of the mixture will move with various rates. Those components that have lower affinity & adsorption up to the stationary stage move quicker and are eluted out in the first place. Those which have great adsorption attraction move slower and will be eluted out after those with lower affinity. The molecules of solute adsorb to column on a reverse way. The movement rate of all components is provided as the following:
R – it is the rate of the motion of a particular component or the rate of the movement of a mobile stage. This is the ratio between distance reached by solute and the distance reached by solvent.
The Procedure Involved in Column Chromatography
The process involved in column chromatography requires the presence of some important factors. This process needs the vertical column which is commonly the glass column that has a knob installed on the base end. This is a burette-shaped tube column with no readings or graduations.
When it comes to the stationary phase, its particles must be with uniform size & shape with no contamination. The best solvents to use in the mobile phase are those which are chromatography-grade. Either one solvent or blend of solvents will be required for the segregation process. Asbestos pad or cotton wool is used to plug the column’s exit at the base and from there you have to hold the stationary phase column and allow the sample and solvent only to pass.
The Procedure, Precautions and Applications Needed in Column Chromatography
The material of stationary phase will be moistened with the mobile phase & it will be packed inside the column using either an asbestos pad or cotton at the base. The sample or extract material which must be segregated from the top portion of the packed stationary stage with another asbestos pad or cotton that should be placed in between.
The mobile stage will be poured to the column through the sample. This time, you will need to use a beaker which must be placed in the base of the column close to the ending. This will be responsible in collecting the elute.
There are some things that you should keep in mind. These things are the precautions that must be considered throughout the process. The precautions are the following:
- You have to store the column within a dust-free and clean place.
- You should keep the column untouched unless the separation process is already complete.
- You have to avoid gaps in the stationary stage packing.
Likewise, there are some applications that must be considered as well. These are the following:
- Column chromatography is the most suitable technique to segregate the active principle away from the plant materials.
- The compound separation process should be considered after the organic synthesis. This way, you will be able to obtain the molecule you desired.
- Column chromatography must be also considered when it comes to purifying or separating the organic compound mixtures such as the glycosides and alkaloids.