Relevance of HETP and asymmetry in SEC resp. affinity chroma

Discussions about gel permeation chromatography / gel filtration chromatography / size exclusion chromatography

4 posts Page 1 of 1
Hello!

Can anyone tell me, which value of asymmetry and HETP (column performance test) is more relevant for the following chromatography methods:

1. Size exclusion chromatography
2. Affinity chromatography
3. Ion exchange chromatography

As I understand, the asymmetry is in size exclusion chromatography more relevant than the HETP value.
For affinity and ion exchange chromatography the HETP value is the more important value.

Do you agree and could you explain your answers?

Many thanks in advance!
Assuming you are talking about purifying proteins in all three cases, it's an "apples and oranges" comparison.

To grossly oversimplify, the plate count primarily characterizes how well-packed the column is (given the particle size, flow rate, and molecular weight). The tailing factor characterizes the presence of secondary interactions. Both are affected by overload.

One could argue that plate count is irrelevant in affinity chromatography because of the high selectivity (verging on specificity). A biochemist friend of mine one said that he loves affinity because you only need *one* theoretical platen "it just has to be the right one!".
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
Tank you for your answer.

Perhaps, I explained my question a little complicated...
First of all, yes, I am talking about protein purification in preparative chromatography.
We are performing column performance tests after packing our columns (minimum diameter 35 cm), so we use the same column long time with different column materials. As we pack the columns ourselves, we have to do a performance test as a release criterion for the subsequent production run. The results of HETP and peak asymmetry are the release parameters for the column. So my question is, which parameter is the more important one at SEC, IEC, RP-HPLC and affinity chromatography?
So what you are checking is the *physical* properties of the column (i.e., how well-packed). In that situation, both parameters are useful for all techniques. A low plate number would indicate a loosely packed column, while a high tailing factor would indicate excess extra-column volume or a head space. To be useful, you need to have "reference" values for a good column with good probes (no micro heterogeneity).
-- Tom Jupille
LC Resources / Separation Science Associates
tjupille@lcresources.com
+ 1 (925) 297-5374
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