HPLC standards

[verita]

Hi, I have a problem in finding a good labeled standard for HPLC size exclusion chromatography.
I try to separate monomeric and oligomeric species on a sephadex 200 (10/30) and need to reach at least 600 000 Da. I have thought of making one myself but I don't know how, and since it is not a main part of our work, I would like to keep the costs somewhat resonable

Many thanks
Mats

[Uwe Neue]

What are your detector options? Standard standards for aqueous SEC are dextranes or polyethylene glycols. You can also get sulfonated polystyrene standards

[verita]

Thanks for the reply, I will look at them

I have a regular protein absorbation (260/280?) detector.

How whould I get those standards, and are they customizable up to high molecular weights?

Mats

[Uwe Neue]

Are you doing proteins? If yes, then I assume that you are doing the SEC in a phosphate buffer pH 7, maybe with some salt. You can create a SEC calibration curve with the polydextranes around 210 nm, if your detector has this option.

Alternatively, you can create a calibration curve with cheap proteins, such as ovalbumin etc. The problem with this is that proteins are folded, and your calibration curve will be bumpy. If you do not mind this small problem, you can try to calibrate with a bunch of cheap proteins.

[moino]

there are sets of also large globular (~ spherical) proteins commercially available, e.g.

Sigma-Aldrich MWGF1000-1KT PROTEIN MOLECULAR WEIGHT MARKER KIT

* Albumin, bovine serum, 66,000 MW 1 vial A8531
* Alcohol Dehydrogenase, yeast, 150,000 MW 1 vial A8656
* β-Amylase, sweet potato, 200,000 MW 1 vial A8781
* Apoferritin, horse spleen, 443,000 MW 1 vial A3660
* Blue dextran, 2,000,000 MW 1 vial D4772
* Carbonic Anhydrase, bovine erythrocytes, 29,000 MW 1 vial C7025
* Thyroglobulin, bovine, 669,000 MW 1 vial T9145

or basically the same from GEHealthcare:

* Gel Filtration Low MW Calibration Kit 28-4038-41 343,00 EUR
* Gel Filtration High MW Calibration Kit 28-4038-42 343,00 EUR


There is a good book chapter about SEC that also covers calibration:
Hagel, L. "Gel filtration" in Protein Purification. Principles, High Resolution Methods and Applications (eds. Janson, J.-C. & Ryden, L.)

could be of interest, too:
Fee, C. J. and J. M. V. Alstine (2004). "Prediction of the Viscosity Radius and the Size Exclusion Chromatography Behavior of PEGylated Proteins." Bioconjugate Chemistry 15(6): 1304-1313.

[verita]

Thank you both for your reply. I will check all of it out and make up my mind. The equipment belongs to another group in another department so I don't know all the specs. but I will find out soon enough