method determination nereistoxin thiosultap

[aflores]

I am try analyze thiosultap (formula:thiosulfuric acid (H2S2O3) S,S′-[2-(dimethylamino)-1,3-propanediyl] ester). The certification´s patron mobil phase is ACN:H20(+0.5% H3PO4) 90:10, but the chromatogram is not good. ¿Anyone know any method¿. I think is better a mobil phase with buffer to control the pH.

[Mark Tracy]

0.5% phosphoric acid is enough acid to protonate everything is sight. Your poor peak shape may be due to a mismatch between mobile phase and sample solvent. Or your column could be bad or just the wrong column for this job.

[KC]

What do you mean by chromatogram is not good?
Seeing as you are running isocratically I will assume you have a flat baseline and your peak just has poor shape. As Mark mentioned, what is the solvent composition of your sample? A rule of thumb I try to use is that your sample composition should have organic strength equivalent to or lesser than your mobile phase, be the same type (ie ACN in your case) and never shoot 100% solvent unless I absolutely have to or my injection volume is very small. What type of column are you using and what is the retention time of your analyte? If I have to run isocratically with 90% organic to see my analyte then I will typically think my stationary phase is too retentive and I have the wrong column (especially if your retention time under these conditions is greater than about 10-12 min). However, if you are using say a C18 and your compound has a retention time of 4 min and it is an ugly blob, back off on the ACN strength, your compound is just smearing and screaming through the column and you are not allowing it to do its job, you may very well have the right column (you have checked another column and seen the same response, right?). Based on whatever your retention time is, I may also suggest a gradient; they will inevitably sharpen up a poor peak shape.

[aflores]

Thank you for your answers and sorry for my english.
My column is a C18 and the retention time is about two minutes. the flow is 0,5ml/min, and the spectra change with the concentration. A low concentration there are one maximun, a 191.8 (200 ppm) and two maximum, 196-197 y 255 a high concentration (800 ppm). If a change the concentration of phosphoric at 1% the maxium peak vary light. I think perhaps the detector uv lamp is old or the flow cell is dirty (the sensibility is low). I will change the lamp first and the column. I disolve the sample (is a pesticide formulate) in the mobil phase. First in water (ultrasonic for five minutes) and later in ACN (the sample is polar and not disolve well in the mixture).

[KC]

If you are using a C18 column with isocratic mobile phase of 90% organic and you have a 2 min retention time then I would say your mobile phase is way to strong. what does it look like if you run 50:50 mobile phase?

[KC]

I would hold off on changing the lamp and reduce, not increase the phosphoric concentration (maybe 0.1%). I would get my analyte out to a retention time of 6-8 min and evaluate what you have before going into flow cells and lamps.

[tom jupille]

aflores, to expand a bit on KC's suggestions:

The internal volume of a reversed-phase HPLC column is about 0.5 x L x dc, where L is the column length and dc is the column diameter (both in cm). For a 4.6-mm id column, this works out to about 0.1 mL per cm of column length. If you have a 100 x 4.6-mm column running a flow rate of 0.5 mL/min, it will take about 2 minutes to flush the column (this is called the "dead time" of the column, and usually symbolized by t0).

As a rule, good quantitation requires that your retention time be between 3 and 11 times t0.

[SIELC_Tech]

You will have hard time retaining thiosultap on reverse phase column. It is very polar molecule at any pH with one amino group and two sulfuric acid fragments: http://www.alanwood.net/pesticides/thiosultap.html

You have few choices in this case: RP with ion-pairing reagent, ion-exchange, HILIC and mixed mode.
Here is mixed mode application for similar molecule-taurine (aminoethanesulfonic acid):

http://www.sielc.com/pdf/SIELC_Taurine.pdf

Provide me with your email and I will send you couple applications on compounds with few sulfuric acid fragments.

regards,

Vlad