How to use LC to analyse water extractable protein accurately by using suitable column both quantitatively and qualitatively

How to analyse organic compound in the same homolog series with nearly the same polarity?Can LC be use to detect the non water soluble organic compound without spoilt the column

Are both of the above posts from the same "Anonymnous"? If, so, could you provide more information on what you are trying to accomplish?

Thanks!

-- Tom Jupille / LC Resources Inc.

Just a comment: I often find a second peak very near one of the compunds I expect in field samples. The UV spectra are often not quite the same, and I presume that the compound involved is very much like the furfuraldehyde family member I am expecting. Obviously if it is only 1/10 of the way from one known family member's retention time to the next, and has only slight shifts in its UV absorbance maxima, it is a very similar molecule. If I had the equipment or budget to find out, this speculation could be confirmed (or disproved).

Has anyone else reading this experienced similar "sister peaks" near expected ones? Repeated analysis of such a sample always produces these "sister peaks", and they occur less than 5% of the time. Thus they are not column or chromatography mistakes, and they are never identical in UV spectra.

Happens all the time in degradation studies of pharmaceutical formulations. As a very broad generalization, you need a certain amount of information in order to identify / quantitate related compounds. That information can be chromatographic, spectroscopic/spectrometric, or some combination. The more information you have in one category, the less you need in the other(s). UV spectroscopy doesn't provide much information (compared to, say IR or mass spectrometry).

As you say, if you *really need* to find out what the new peak is, you can. LC-MS is the "bigger hammer" that comes immediately to mind.

One other possibility is that you have different conformation states of the "same" molecule. This is fairly common with proteins, but can occur with smaller molecules as well. If the time required for equilibration between the conformers is long relative to the residence time on the column, you can see two peaks -- often close and similar in UV spectrum. If the equilibration time is fast, you'll see only a single peak representing the "average" of the conformers. In between you can see some funny peak shapes.

I ran into this a number of years ago with a column that could separate the alpha- and beta- anomers of glucose. At high temperature (>65 deg), we got a single sharp peak. At room temperature, we got a broad, misshapen peak. At low temperature (4 deg), we could pull two peaks partially apart.

-- Tom Jupille / LC Resources