Chromatography Forum

Dear all,

I am currently trying to determine the LOD for creatine monohydrate. Mobile phase composition: KH2PO4 monobasic adjusted to pH4 using phosphoric acid, isocratic condition. Wavelength: 210 nm. Column: Waterssymmetry shield 4.6 x 250 mm , 5 um.

So far, I am able to get the peak at around RT of 2.9 min as well as 3D spectrum at around 195 nm at higher concentration from 25 ug/ml to 5 ug/ml. But when I tried running at lower concentrations say below 1 ug/ml, I noticed that there isn't any change in peak heights at the different lower concentrations (0.5 ug/ml, 0.1 ug/ml, 0.05 ug/ml etc. ). At the same time, I am also unable to get its respective 3D spectrum. Can anyone know why this is so?

I wanted to find out if the analyte is a basic or acid or neutral compound but not sure how to go about doing it. Is there any particular website that I can refer to?

Also, if I want to know their UV cutoff for both my analyte and the buffer that I am using, where can I locate such informations?

Appreciate if anyone can give me some advise. Thanks.

Tlili,

You might have another compound eluted at the same time or you are maybe too close to the void volume (if not exactly at it).

Have you tried to make a blank injection to see how your chromatogram looks like?

There is a prety good pKa compilation for your and other compounds that can be found at: http://research.chem.psu.edu/brpgroup/p ... lation.pdf

Your mobile phase is quite transparent to UV. Creatine should absorb in UV due to it's carboxylic group so it should adsorb in low UV wavelengths (although I do not have it's structure in mind).

Also: Be careful, phosphate is not a buffer at that pH, and creatine is not very stable, for instance, it may convert to creatinine. Kostas already pointed out that you have a good indication that your peak is not creatine.
For UV info check catalogs, google, Merck Index, Stadtler ......

Just noticed that Stadtler should be Sadtler (Bio-Rad) instead, usually found in university chemistry libraries.

Also, sometimes one finds something there:

http://webbook.nist.gov/chemistry/
http://www.colby.edu/chemistry/cmp/cmp.html