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The plot of elution volume along with the absorbance is known as chromatogram as given in Figure 29.4. The volume or time it takes for a analyte to come out from the column is known as retention volume or time. The chromatogram may have separate peaks (A and B) or peaks (C and D) with overlapping base, these peaks are called fused peaks.

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Column Chromatography animation JHU, Organic Chemistry Lab

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Column Chromatography: In column chromatography, a stationary phase is filled into a cylindrical tube made up of glasss or steel. The mixture of analyte is loaded on the top and it runs from top to bottom. . How Kd is exploited in column chromatography ? Assume two molecules, X and Y with a Kd value of 1 and 9 and they are traveling through a column with water as mobile phase as given in Figure 29.3. As they will travel, X and Y will partition between stationary phase and mobile phase. As there is a huge difference in Kd, Y will be associated with the matrix and remain on the top of the column where as X will move along the water. At the end of chromatography, X will come out first whereas Y will come out last.

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Principle of Seperation: How a physical or chemical property will allow to isolate a particular substance?The mixture of compound 1 and 3 is shown in Figure 29.2 and assume if we are using boiling point as a criteria to isolate them. As we will heat the mixture there will two phase forms, one liquid phase and other is vapor phase. The molecules of compound 1 and 3 will distribute between these two phases and as the temp is near to boiling point of compound 1, more amount of 1 will be present in vapor phase than liquid phase. Where as more number of compound 3 will be in liquid phase. Eventually as this process will continue, at the end two molecules will get separated from each other. The distribution coefficient (Kd) to describe the distribution of compound 1 between two phase A and B is as follows:

Concentration in Phase A ⁼ Concentration in Phase B

Similarly one can also exploit other physical & chemical parameters as well. With each and every physical and chemical parameter the molecule present in the mixture will distribute as per their behavior in each parameter.

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ВИДЕО ПРО ХРОМАТОГРАФИЮ

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ВИДЕО ПРО ХРОМАТОГРАФИЮ

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Антон Клейман

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Хроматография

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PHYSICAL AND CHEMICAL PROPERTIES OF CHROMATOGRAPHY

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PHYSICAL AND CHEMICAL PROPERTIES OF CHROMATOGRAPHY

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Physical Properties

1.             Molecular weight

2.             Boiling point (in case both are liquid, as in this case)

3.             Freezing point

4.             Crystallization

5.             Solubility

6.             Density

Chemical Properties

1.             Functional Group, for example, phenol has –OH where as aniline has NH2.

2.             Reactivity towards other reagent to form complex

Now for example you have a mixture of compound 1 (benzene) and compound 3 (Aniline) and you would like to purify benzene rather than aniline. In this situation, you can take the physical and chemical properties of benzene into the account and isolate it from the mixture.

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INTRODUCTION
The molecules present in biological system or in synthetic chemistry are produced through a series of reactions involving intermediates.Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase.