Hi I was separating carbohydrates by RP-HPLC. I used to get the solvent peak before the analytes and it was normal. All of a sudden, the solvent peak got inversed. I don't know why? Can anyone help me in this?
I suggest that its not at all a problem on getting the solvent peak before r after the analyte peak which depends upon the elution factor of the solvent u ve used .so tell which solvent u have used ,and explain briefly about the inverse peak .since while handling HPLC there was no option to get sudden changes .so check once again what are all u done
@ Jose ours is a agilent 1260 HPLC. quarternary gradient and we use refractive index detector
Hi
Check the purity of your solvent and run the pure solvent to see the solvent front and compare it with your problem
The inverse peak is due to the absorption of mobile phase is more than your desired compound. So check your mobile phase.
As well said by Robin, you should verify your mobile phase components.
Have nice elutions!
Carlos de Souza Teixeira
Mobile phase plays a very crucial role in RI detector. If you are working on carbohydrates then you can go also for ELSD detector. ACN:Water (78:22) amino column on ELSD detector.
Dinesh -
Did the area of your analyte, as measured by your calibration standards, change? If not, don't worry about it. We have a similar issue which seems to be related to the ambient temperature - on warm days, we get positive peaks for the solvent front, on cooler days, negative. The lab is not air-conditioned, and while the column is thermostatted, the detector is not.
I agree with john, you use the refractive index detector that mean you measure the difference between refractive of the mobile phase and analyte, and refractive of materials is very depended than temperature. that means may be refractive of your analyts is less than the refractive of the mobile phase then all peaks appear negative.
Yes you are right Ebrahim. The negative peaks is due to lesser refractive index of analyte than mobile phase. So you have optimize the mobile phase means you have to choose that mobile phase whose refractive index lesser than your desired analyte. So play with the mobile phase.
gud luck!
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