1-Principle of partition chromatography
1-Principle of partition chromatography:
The partition chromatography is a method of separation in which “the components of mixture get distributed into two liquid phases” because of differences in partition coefficients.
It is based on
• Differences in retention factor (k)
• Distribution coefficient (kd)
In this liquid is used both for stationary as well as for the mobile phase.
Fig-above diagram explains about basic principle of partition chromatography. It is best understood by the separation of solutes ,by using partition of the solutes between two liquid phases.
2-Working of partition chromatography:
• The separation of components of mixture in this technique is based on the Nernst’s law that was distributed by Nernst in 1890.
According to this law,
‘when the ratio of components of mixture distributes between immiscible liquid(stationary and mobile phase),a constant is designed which is called as a partition coefficient r distributive coefficient expressed as k’.
Thus function of partition chromatograph based on the principle of liquid/liquid partitioning chromatography.
• In this two immiscible liquid phases are mixed together to form a two-phase system, and are then separated multiple times.
• The individual solutes are isolated based on the different partitioning coefficients of each compound in this two-phase system. One of the liquid phases of the two-phase system is used as a stationary liquid phase and other as mobile phase.
• Stationary phase is fed into the column(rotor)while the latter is spinning at moderate rotational speed. It is retained inside the column by the centrifugal force generated.
• Mobile phase containing the solute to be extracted. It is fed under pressure into column and pumped through the stationary phases.
• Both phases are mixed together. It is at that time that the exchanger of molecules between the two phases occurs.
• The stationary of the solutes is achieved on the basis of specific partitioning coefficients (kd) between the mobile and stationary phases.
• The mobile phase then gradually pour at each outlet thus entering the next cell. The eluted fractions of the mobile and stationary phases are collected over a period of several minutes to several hours.
• These fractions, or eluates, will contain the individual purified solutes.
FIG-explains the mechanism of partition chromatography
• Chromatography jar
• capillary tube
• stationary phase
• mobile phase
The nature of stationary phase and mobile phase is different in different types of partition chromatography as described below:
• Liquid-liquid chromatography
In this the liquid and the stationary phase is attached to a supporting matrix by the physical means. Cellulose,starch or silica matrix act as supporting material for water stationary phase.
• Bonded-phase liquid chromatography
In this the stationary phase is covalently bonded to the matrix. The Silica is derivatized to immobilized stationary phase by using the reaction with an organochlorosilane. In this way,with the elution stationary phase will not change.
• In normal-phase liquid chromatography
In this, the stationary phase is polar (alkylamine) and the mobile phase is non-polar and organic solvent (hexane,heptane,ethylacetate). These solvents are used because they formed an elutrophic series based on their polarity. with increasing polarity. Least polar analyte is eluted first and the most polar analyte is eluted at last due to the performing well ordered elution of analytes.
• In Reverse-phase liquid chromatography
In this, the stationary phase is non-polar and inert. The mobile phase is polar and has hydrophobic interaction.
• Require and nonpolar charges like cation ;anion exchanger
• Separation is based on Nernst’s law.
• Require distribution coefficient(kd) and retention factor (k)