What’s the Relation between Filter Paper and Paper Chromatography?
Paper chromatography is partition chromatography with filter paper as an inert support. With a strong affinity of water, the filter paper fiber can absorb much water and some are combined with the hydroxyl group of the cellulose in the form of hydrogen bonds, which is difficult to remove under normal conditions, while the affinity of filter paper fibers to organic solvents is weak.
The stationary phase is generally the water adsorbed on the paper fiber, and the mobile phase is an organic solvent that is not compatible with water; the filter paper can also absorb other substances as a stationary phase, such as buffer, formamide, etc. In the experiment of extracting pigments related to photosynthesis, paper chromatography is used to extract pigments with different solubility. In environmental analysis and testing, filter paper chromatography is sometimes used to separate sample components. It is used for some low-precision analysis, such as 3,4-benzopyrene. But it is not as common as GC and HPLC.
Background of filter paper and paper chromatography
First, we must understand what partition chromatography is. Partition chromatography is a method of separating substances by using the different partition coefficients of each component in a mixture in two different solvents. The partition coefficient refers to the ratio of the concentration of a solute in two solvents when the dissolution of a solute in two immiscible solvents reaches equilibrium.
Different substances have different distribution coefficients because of their different solubility in various solvents. The most widely used porous support in partition chromatography is filter paper, followed by silica gel, diatomaceous earth, cellulose powder, starch, and microporous polyethylene powder.
Theoretical basis of filter paper and paper chromatography
In actual operation, one end of the spotted laboratory filter paper is immersed under the liquid surface of the mobile phase. Due to capillary action, the organic phase, namely the mobile phase, begins to penetrate and expand from one end of the filter paper to the other end. When the mobile phase (organic phase) is spotted along with the filter paper, the solute on the sample point is continuously distributed between the water and the organic phase.
Part of the sample leaves the origin and moves with the mobile phase into the solute-free zone, where it is redistributed. Some solute from the mobile phase enters the stationary phase. With the continuous movement of the mobile phase, various solute components in the sample have different distribution coefficients and moving speeds.
Therefore, the various parts are continuously distributed according to their respective distribution coefficients and flow along with the flow. The direction of phase flow moves so that the components in the sample are separated and purified.
When paper chromatography separates the mixture, two effects occur: the first is the distribution of the solute between the water bound to the fiber and the organic phase of the flow filter paper (ie liquid-liquid separation); The second is the absorption of solutes by filter paper fibers and the distribution of different distribution ratios of solutes dissolved in the mobile phase (ie solid-liquid distribution), even though the separation of the mixture is the result of the combined action of these two factors.
Quantitative calculation of filter paper and paper chromatography
The relative mobility (Rf) can be used to express the migration of a substance:
Rf=The distance the component moves/the distance the solvent front moves = The distance from the origin to the center of the component spot/the distance from the origin to the solvent front.
In the case of constant experimental conditions such as filter paper, solvent, temperature, etc., the Rf value of each substance is unchanged, and it does not change with the change of the moving distance of the solvent. The relationship between Rf and distribution coefficient K: Rf=1/(1+αK).
α is a constant determined by the nature of the filter paper. It can be seen that the greater the K value, the greater the tendency of the solute to be distributed to the stationary phase, and the smaller the Rf value; conversely, the smaller the K value, the greater the tendency to distribute to the mobile phase and the greater the Rf value. Rf value is an important indicator of qualitative analysis.
When the sample contains a lot of solutes or the Rf of some components in single-phase paper chromatography is relatively close, and it is not easy to be clearly separated, two-way paper chromatography can be used. This method is to spread the filter paper in one direction in a special solvent system, and then dry it, then turn to 90, spread it in another solvent system, and take out the filter paper after the solvent reaches the required distance. Dry and develop color to obtain two-dimensional chromatography.
Using this method, if the solute cannot be completely separated in the first solvent, it can be completely separated by chromatography in the second solvent, which greatly improves the separation effect. Paper chromatography can also be combined with zone electrophoresis to obtain a more effective separation method. This method is called fingerprint spectroscopy.