Hi,
I am analyzing cocaine with an internal standard of bupivacaine using HPLC with ammonium formate buffer adjusted pH 2.4 using acetic acid and buffer B acetonitrile. Temperature setting 60C. I have performed the cocaine extractions and I believe one of the derivatives is present and co eluting with my internal standard. I also have an issue with the void volume. Is there anyway I can resolve these issue?
Thank you in advanced.
You have not provided your HPLC method so we can not provide any constructive comments. Please provide relevant setting and conditions used with chromatography questions (e.g. Column name and type, dimensions, particle size, exact mobile phase mixture/composition, isocratic or gradient, flow rate, detection type and settings, Rt of sample(s) and std, what is sample 'in', injection volume and injection solution...).
What is your Tzero? What are the Kprimes for your sample(s) and standards?
If you are using an isocratic method, well.... don't. Use a gradient method. An isocratic mode will not allow you a wide enough range of polarity change to obtain separation of many related compounds.
Why are you running at 60 C ? This seems very high as 30 or 40 C should provide plenty of resolution using a Water/ACN (pH 2.5) gradient on a C18 column.
It an xbridge C18 5um column. 75% buffer 25% ACN for 15 mins. Samples are in water. Cocaine at 4.239 and Bupivicaine 5.245. flow rate 1ml/min.
Detector DAD. wavelength 200-400nm. 20uL. 4.6x250mm are the dimension.
"Cocaine at 4.239 and Bupivicaine 5.245".
Based on the retention times you report, your method does not provide enough retention of the samples. You should develop a method which results in a K prime (aka: 'Capacity Factor') of at least 1.5 (>2.0 would be much better) for each compound (though, they must not co-elute). The estimated Tzero time of your column at 1.00 ml/min would be 2.9 minutes so a peak which elutes at 4.3 minutes only has a K prime of 0.48. A peak which elutes at 5.3 minutes would have a K prime of only 0.79. Based on your described method, to reach a K prime of 1.5 your first peak would need to elute at 7.25 minutes. Continue to work on the method to increase retention of all compounds such that they meet the minimum retention characteristics of good methods (or just refer to the published literature for better methods).
For example, if you use a better method (e.g. ~ 30% MeOH and Buffer (pH 2.5) on a similar 4.6 x 250 mm, 5u C18 column at 1.000 ml/min), I would expect Cocaine to be retained with a K prime of > 2.0.
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