SEC Inclusion Compound Needed

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Sami Tuomivaara
Sami Tuomivaara's picture
SEC Inclusion Compound Needed

Hi Everyone,

I've been looking for a molecule, to be used as a inclusion marker in size exclusion chromatography experiment, that fulfills the following criteria:

(i) Water soluble
(ii) Low mw, preferably less than 200 Da so as to be included as much as possible in the gel
(iii) Neutral, so that its properties do no change as a function of buffer pH and concentration
(iv) Doesn't interact with B(OH)3 buffer pH 6-10, so 1,2 cis-diols are not permitted
(iv) There is a straightforward way to assay it from aqueous chromatography fractions in pure water or various concentrations and pHs of borate buffer. I'm using a large gravity flow column and currently I can't do online detection.

The amount of this compound I want to load with my sample depends on how sensitive assay I can find for it... Hopefully in low mg regime.

So the idea is to see whether this inclusion standard elutes at the same position (volume) in water and various borate buffers or does the borate change the gel properties.

I welcome all comments...
Best regards

Dr. Analytical
Dr. Analytical's picture

Thanks for posting on  What an interesting problem!

There are many molecules to choose from.  One question would be: what analytical tools do you have for determining this compound?  UV/VIS/RI/GC/LC?
The polyethylene glycols (PEGs) come to mind, and I don't think their diol structure would be a concern.  They are available in a variety of different MWs.
An aromatic compound would be easy to detect by absorbance, but finding one that is not pH sensitive will be more difficult.

Acetone absorbs somewhat in the UV range, so if you have a spectrophotometer with UV capabilities, that would be a good choice.  Try preparing a series of dilutions, and check absorbance at 220, 250, and 300 nm.

Depending on what gel you are using, you may see some changes with buffer concentration.  In theory it is not supposed to happen, but we know that it does.

Sami Tuomivaara
Sami Tuomivaara's picture
Dr. Analytical,

Dr. Analytical,

Thanks for the reply. I have PEG 200 and I considered using it for exactly the reason you mentioned but I couldn't find very sensitive assay for it... If there is, please let me know.

Actually I'm using PEG 8000 as an exclusion standard and I can assay it by precipitation it quite easily with KI/I2, unfortunately, I couldn't find a way to precipitate low mw PEGs.

I think using another chromatography (GC/LC) step to assay the fractions is too much work, I want to avoid that unless I absolutely cannot find other way... I have access to microplate reader (Bio-Rad 680 with handful of pre-set wavelengths) and I just like colorimetric assays.... ;-). I can also use variable wavelength spec....

I could maybe use TLC aas well... any ideas how to detect... iodine vapor... sulfuric acid...

Since the buffer absorbs at low UV, where PEGs absorb (200 nm and lower), this doesn't work for them, but I'll check this for acetone...

Thanks again and cheers