G'day all,
I'm currently developing a routine method to separate and analyze the organophosphate pesticide chlorpyrifos and it's major metabolite 3,5,6-trichloro-2-pyridinol (TCP). I'm using a PDA detector at 280 nm, a cyano column and a 50:50 AcCN : 0.1 M Acetic acid:Acetate buffer (pH apprx. 4.60) at a flow rate of 0.4 ml/min. The injection volume is 100µL. I get good separation and the TCP gives me a nice sharp peak the chlorpyrifos gives me a broad tailing peak with poor sensitivity. Has anyone got any suggestions as to how I can improve the peak shape and sensitivity?. I'm sorry if I have left any info out I'm pretty new to chromatography.
Thanks

What are the approximate retention factors of both compounds and is the sample dissolved in? What is the sensitivity on your PDA (or the peak height response)?

The retention time of the chlorpyrifos is appx. 5 min and the TCP is apprx. 3 min. The peak height response for chlorpyrifos is less than 5 mAbs whilst for TCP it's more than 3 times that. Sorry I should have said that this is pure stds (1 ppm) in milli-Q water.
Thanks

Hmm - I am still missing information. What is your column - length and diameter? If you are using a 4.6 mm x 150 mm column, the first peak is unretained (elution volume 1.2 mL). The second peak may tail because your column may be shot, because of the solvent in which the sample is dissolved, or plainly because you are using a column that is not suitable for this analysis (CN columns can be weird).

If indeed you are using a 4.6 x 150 mm column, you need to rework the method until both peaks are retained.

Sorry, the column is a 150 x 3.0 mm 3µ particle size. I can get hold of a C18 and a C12 column, do you think it would be better to give these a try?

Thanks

Tristan,

Your flow rate (0.4 mL/min) is pretty low. How about trying to increase the flow rate to sharpen up the peak? You could leave it at 0.4 until the first peak elutes, then increase it to sharpen up the second peak. This may also improve your response.

No, don't play with the flow. The peak will become narrower, but it will tail nearly as much as before, and the response will decrease.

Since you have a C18 and a C12 (?) column, I would get one of them and see if the peak shape gets better. If your columns are of the modern high purity silica types, tailing is a rare event, and you should get good peaks. You will need to increase the amount of organic to get about the same retention, but do not change the buffer - this is OK.

I have run both TCPY and chlorpyrifos by LC/MS, along with other pesticides & metabolites. Those two separate easily and have good peak shape on a base-deactivated C18 with water - methanol gradient, 0.2% (v/v) acetic acid (approx 35 mM) in both mobile phases. Running from about 5 to 95% B, both of these elute in the later half. I think water - acetonitrile should work just as well if those are your only two analytes.

Thank you all very much for your suggestions they are very much appreciated.

Tristan