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Calculation for size exclusion chromatography (quantitative analysis)

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I am analyzing a sample using size exclusion chromatography as the separation technique, and UV-VIS is used as the detector. I have some troubles regarding the calculation of the final fractions that I get from the separation. Will these two experiments give the same absorbance results?


Experiment 1: Inject 0.5 ml of sample A into the column

Experiment 2: Inject a mixture (1 ml) of 0.5 ml of sample A + 0.5 ml of solvent that does not have absorbance at the wavelength I am using.


I am aware that the absorbance is of course related to the concentration. However, in this case, both experiments have the same amount of sample A running through the column. Therefore, even though the latter is diluted, should they both give similar absorbance results or one would be 50% less? Please help to clarify.


Thank you very much in advance!

Edited by thalia12

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The injection volume does matter to a degree, as it can affect peak shape. Two things to consider, the first is the effects of the solvent. If it is different from the mobile phase, large volumes of it can distort the chromatographic run to different degrees.

The second is the volume itself. If you are within 5-10% of the column volume you will generally not see a lot of differences. However, with increasing volume your peaks will start to widen. Another thing to consider is that your injector may be more or less accurate at a given volume range.

If you need the highest reproducibility, it is good practice to keep the volume constant (as well as all other parameters).

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