I have a waters hplc with its fraction collector II with uv/vis dual wavelength detector. I need to know what might be some good column/methods choices for the separation of proteins in cheese whey and other milk products.
Would a conductivity detector be a better choice than a uv/vis detector?

Can I know if it is possible and scientifically acceptable to determine the concentration of metallothionein using internal or external standard by chromatographic process?If yes how?

The following references may be useful;

Miyairi S, Naganuma A. Metallothionein determination by isocratic HPLC with fluorescence derivatization.Methods Mol Biol. 2002;186:273-83

Miyairi S, Shibata S, Naganuma A. Determination of metallothionein by high-performance liquid chromatography with fluorescence detection using an isocratic solvent system. Anal Biochem. 1998 May 1;258(2):168-75.

Pan AH, Wang ZX, Ru BG. Determination and purification of metallothioneins by high performance liquid chromatography. Biomed Chromatogr. 1991 Sep;5(5):193-7.

There are quite a few published methods available, search Medline for further references. Good luck :o)

What happpened to the discussion on protein separation?

Renee,

No, you won't get much out of conductivty detection of proteins in the presence of normal buffer levels commonly present during protein separations. In fact, I doubt you'll even be able to detect the protein in the presence of buffer even at very high protein concentrations. You are better off sticking with UV detection.

Agree with Chris. Low sensitivity condactivity meters are sometimes used to monitor the gradient in protein ion-exchange. That's it.

Suppose a low ion strength buffer can be used on a column only giving mild electrostatic interactions. I speculate that proteins would give negative peaks and that they could be detected?

As I mentioned above, it's unlikely that one could detect proteins via conductivity detection. My reason for stating this is that proteins are generally characterized by a relatively low charge to mass ratio in solution unless one works well away from the pI. Even if one were to operate at relatively extreme pH conditions, the charge to mass ratio for a protein will still be much smaller than a typical buffer ion charge to mass ratio. In essence, one would be forced to detect proteins providing a small signal on a relatively large background, even if one were to use a relatively dilute buffer. I'm sure the sensitivity would be so low that you would have a difficult time detecting any proteins, even at relatively high concentrations.

Regarding milk proteins, we have achieved a high-resolution separation of milk proteins using a pelicular anion exchange material (ProPac SAX from Dionex). If you are interested in more details about the chromatographic conditions, please e-mail me directly.

Chris,

Isn't low specific conductivity by the protein and reasonable background level ideal conditions for negativ peak detection?

The crux is to find a column that will have suitable capacity and selectivity a mild buffer conditions!


(Negative peak detection may for other reasons be of less interest.)

Low specific conductivity for the protein and reasonable background level for the buffer ion might seem to be ideal conditions for negative peak detection but only if the normality of the protein is a significant fraction of the normality of the buffer ion. But since this is unlikely, you will most likely observe small negative peaks which are difficult to distinguish from the noise. On top of this, as you mention, you will need a retention mechanism which allows for direct competition between the buffer ion and the protein, allowing for use in an extremely low levels of buffer ion. This description rules out ion exchange. I'm not sure there is a retention mechanism which will fit the requirements of this system.

Yes, I agree. It probably rule out conventional ion-exchange.