By Jorim on Monday, December 8, 2003 - 06:18 am:
I have to do a determination of sodium salicylate with HPLC with a C-18 column. I don't have the possibility to use another column or gradient hplc (schoolstuff). I'm interested in suggestions for eluent and flow or complete SOP’s ;)
Ps. Another technique like spectrometric is not a problem, it’s even better. :-)
By Consumer Products Guy on Monday, December 8, 2003 - 07:03 am:
Jorim - you sound inexperienced as this is an easy assay. But we all need to learn sometime. We use RP-18 columns with RP-18 guard columns.
Solvent A = 4.0 % acetic acid in HPLC H2O
Solvent B = ACN or Methanol
Flow = 1.00 ml/min (4.6 mm i.d. columns) or 0.25 ml/min (2.1 mm i.d. columns)
% Solvent B = 40
Wavelength = 304 nm
Run time = 8 min.
Column temperature = 30 *C
Salicylic acid will elute at about 2.5 - 4 minutes using these conditions. Use either pure salicylic acid or pure sodium salicylate as your standard. If you don't acidify, the sodium salicylate will not be retained on your RP-18 column. Samples can be made up in water, methanol, or acidified (acetic) versions.
By Jorim on Monday, December 8, 2003 - 12:10 pm:
I'am indeed quite inexperienced with HLPC.
Correct me if I'am wrong. As eluent I have to use a mixture of solvent A (60%) and B (40%) without using a 'eluent gradient'??
By Consumer Products Guy on Monday, December 8, 2003 - 01:23 pm:
We normally use isocratic (straight mixtures) here when at all possible, as in this case. I'd start with 60% solvent A and 40% solvent B; if peak comes out too soon, increase A and decrease B. Opposite if peak takes too long. The two mobile phases can either be mixed by modern instrumentation (recommended) or pre-mixed and drawn from one reservoir. Remember, gradients are OK, but you need to re-equilibrate the column (approx 10 column volumes) at initial conditions before making next injection.