Sample Overloading in SEC???

[rick1112]

Hi

I would like to know how estimate loading capacity (or Overloading amount?) is done in Size exclusion chromatography?? What Happens during “overloadingâ€

[Uwe Neue]

There are multiple effects. First, I assume that you want to get an average molecular weight and a molecular weight distribution of something. Then your injection volume must be consistent between analyte and standards and much smaller than your peak volume. You also want to have a sample of a low concentration to avoid "viscous fingering", which can get you ugly peak distortions For organic polymers, the following rules of thumb apply to keep the sample viscosity low:

MW < 25,000, concentration < 0.25%
MW < 25000-200,000, concentration < 0.1%
MW < 200,000-2,000,000, concentration < 0.05%
MW > 2,000,000, concentration < 0.02%

(from my book)

If you are asking about proteins, and not analytical SEC, you can get away with larger injections. In all cases, the sample must be dissolved properly, or you will see undissolved sample eluting in the void volume.

[rick1112]

Thanks Uwe,

Well yes I am working with polymers (PEG )…there are some points that is still lingering in my mind:-

(a)What is “viscous fingeringâ€

[Uwe Neue]

a. Viscous fingering comes from injecting a highly viscous sample (it is actually a bit more complicated, but you won't have a problem if you inject concentrations as suggested). Viscous fingering can result in strangely distorted peaks that also are irreproducible.
b. Overloading results in peak distortions. Extra peaks are due to incomplete solution of your sample, or they are real.
c. Depends. Stay away from either problem. You want the injection volume to be less than maybe 5% of the peak volume. It is also best to inject the same volume for standards and analyte.
d. The combination of the proposed concentrations and the proposed injection volume from (c.) above will give you all the guidance that you need for the maximum injectable amount.