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Chromatographic concepts

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Why is Chromatography an important tool in analytical chemistry?

 

The main purpose of a chemical analysis is to determine the amount of a certain molecule or element in a sample. Here we call that species the analyte. 

 

To determine the amount of a certain analyte we must be able to distinguish that analyte from the other molecules or elements in the sample. In analytical chemistry there are several ways to do this: extraction, precipitation, evaporation, selective spectroscopical properties, by the sample preparation procedure etc. In most of these procedures the analyte is physically separated from the rest of the sample. When the separation has been achieved, the amount of the analyte is measured. 

 

Chromatography has become the most imoprtant technique to physically separate analytes from each other. The reason is that the chromatographic principle can easily be adapted to many different molecules or ions. It also offers good possibilities to measure the amount of analyte of interest in the sample. Furthermore the amount of a great number of analytes in a sample can be determined at the same time.

 

How are species separated in chromatography?

 

Look at the following animation and try to figure out why the red circles are separated from the blue.

 

Animation of a separation

 

 If you found it difficult to see you can look for the answer here.

 

Conclusion

 

Separation is achieved because different analytes are adsorbed to the stationay phase for different periods of time. The time it takes for an analyte to migrate through the column is called the retention time.

 

The next important question is:

 

How large difference in retention times is needed to obtain a separation?

 

From the animation it is clear that for a particular analyte all individual molecules do not exit the column at the same time. They exit the column in a time distribution. For a good chromatographic run it is close to a normal distribution. A separation between two analytes is therefore obtained when the two distributions or peaks do not overlap in time. The narrower the peaks, the smaller the difference in mean retention is needed to obtain a separation. The measure of the width of the ditstribution divided by the length of the column, is called the column plate height

 

Conclusion

 

The chromatographic separation between two analytes depends on two parameters:

- differences in retention time.

- the width of the peaks or the plate height.

 

Resolution is a number that quantifies how well analytes are separated from each other and depends on these two parameters only.

 

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