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There chromatography (from the Greek χρῶμα, transliterated in khrôma, “color”), is a technique for separating the components of a homogeneous mixture based on the distribution of its components between two phases, one stationary and one moving along a defined direction. Incolumn chromatographythe direction goes from top to bottom and is dictated by a glass tube (the column).
There column chromatography is a particular chromatographic technique based on the property that some substances have, once dissolved in suitable solutions, to interact with a suitably prepared solid matrix. I'll try to explain better what it means what I just wrote!
Column chromatography: what it is
It is nothing more than a chromatographic separation method that takes place in a glass column. This technique exploits the difference in charge, size, bond affinity or other properties of the component to be isolated to create bands with different characteristics.
In the photos on this page, we show you the sequences of thecolumn chromatographyand in particular the extraction of the eluate, that is the "partially purified" substance that comes out of the terminal nozzle of the glass column.
Therecolumn chromatographyit is very useful for the purification of proteins and for the separation of a large number of substances to be examined individually. Sometimes, substances isolated withcolumn chromatography they lose the properties they had inside the mixture. It all depends on the technique ofcolumn chromatographyand from the substance that you want to purify.
Column chromatography: how it works
A glass tube is filled with a porous solid material, defined as "stationary phase". The stationary phase has suitable chemical properties chosen on the basis of the substance to be purified / separated from the mixture. The column is then filled with a buffer solution, also suitably formulated. The buffer solution is called "mobile phase”And contains the mixture you want to separate.
The mixture to be separated is dissolved inmobile phase. The mobile phase then comeslayeredon the top of thecolumnand percolated through thestationary phase(the solid matrix). In this way, the solution diffuses along thecolumnand forms a continuous band along the entire stationary phase. The substances that elute (leave) the column have specific properties based on the order of avoidance.
To understand better, let's take the example of protein purification, even though the mobile phase can be represented by any mixture. We will have the individual proteins elute from the column more or less quickly according to their properties.
According to the type of stationary phase, we have different techniques ofcolumn chromatography, Which:
- Ion exchange chromatography
- Molecular exclusion chromatography
- Affinity chromatography
There is also a more expensive technique that uses high pressure, namely thehigh pressure liquid chromatographyor HPLC.
Ion exchange chromatography
In ion exchange chromatographythe solid matrix (stationary phase) is rich in positive or negative charges. It goes without saying that theion exchange chromatographytakes advantage of the differences in the type and intensity ofnet electric chargeat a certain pH.
In this case, thestationary phaseis a synthetic polymer (resin) containing negatively charged groups (hence we speak ofcation exchangers) or positively charged groups (we speak ofanion scabers).
Returning to the example of protein purification, the difference in charge is linked to the presence of amino acids with certain functional groups. The affinity of each protein for the charged groups ofcolumnit depends on the pH which in turn affects the ionization state of the functional groups of the constituent amino acids.
Incolumn chromatographythe solution preparing for the mobile phase is critical. In our example with proteins, the salt concentration and pH can create a gradient that can favor the extrusion of certain proteins.
Ion chromatography: cation exchange
In the case of the ion chromatographywith cation exchange matrix, the solid matrix has negatively charged groups so proteins with net positive charge will be the last to flow from the column. The first compounds to come out of thecolumnthey will be those with a strong net negative charge (by repulsion from the matrix), then substances with a partial negative charge will come out. The substances to be eluted will move through thecolumnwith a rate dependent on their net charge in the solution used.
Molecular exclusion chromatography
In this case, the mixture (mobile phase) is added in onecolumncontaining apolymerwith cross-links and molecules fromisolatethey will be separated by size. Instinctively one thinks that the smallest molecules will come out of the column first, but in reality it is the exact opposite.
The larger molecules will be forced to travel the shortest path to the end of the column. By diffusion, the smaller molecules will be free to penetrate the pores of the solid matrix, taking longer to elute. The first eluate collected will be the one holding the largest molecules.
Affinity column chromatography
A ligand is present in the stationary phase. As the solution to be separated passes through the column, molecules that have affinity for that ligand will form a specific bond. The substances of interest will be held in the first or last fractions collected based on the type of ligand used.
High pressure liquid chromatography
The techniques ofcolumn chromatographyjust described can have a discrete resolution of the bands. The reason? Each substance tends to diffuse after a certain time.
By using high pressure pumps, the speed increases and the time taken by the molecules to travel the column decreases. In this context, chromatographic matrices capable of withstanding strong pressures are required.
By reducing the transit time through the column, thehigh pressure liquid chromatography(HPLC) limits the spread of the bands and greatly improves the resolution of the collected eluate.