Im doing HPLC analysis for organic compound. I am not able to understand at what time should I inject the sample. The reason being that im starting with 5% A :95% B. If I inject a sample at two different time within the window of example 5%A:95%B I will get two different RT of the same sample. How can i get reproducible RT.?
Dear Savio
Your question is very hard if not impossible to answer without details about:
1) your sample matrix 2) the components you want to separate & quantitate 3) the choice of your column type (e.g. straight phase / reversed phase) 4) the choice of your mobile phases / eluents A and B.
Please study basic introductions to HPLC and Gradient elution like https://www.chromatographyonline.com/view/understanding-gradient-hplc
Succes
You should make the injection before the gradient starts. Most HPLC systems trigger a gradient program upon injection, even for manual injection. The injector creates a signal for the software, which then starts the gradient program. It seems you should find out how to cause this synchronization in your system.
There can't be much of a gradient, starting at 95% B (the strong solvent). As the compound has different retention times based on the injection, the compound is moving down the column, so the retention time will change relative to the start of the gradient..
So I have Waters 1500 series, The problem occurs that gradient starts and then I load and inject the sample. Therefore if I repeat it, the time of my injection may vary, hence the rt might change.
Savio Cardoza , yes, it will. As mentioned, you need to make the gradient start at the injection time. I think the 1500 series are pumps only? You probably have a gradient controller in the system someplace, and you need to connect your injector to the gradient controller, injector, and maybe the pump depending on how the software works.
From the 1500 series pump i/m guide:
"For 1500 Series Pumps with an optional 1500 Series Manual Injector, you need to make an Inject Start output signal connection from the I/O terminal block on the 1500 Series Pump to the appropriate inputs on the Waters 2487 or 2410 Detector."
It seems the LC system isn't configured correctly, or the driving software isn't configured to recognize the injection signal to start the gradient upon injection.
It is best to get someone there to help you and verify everything is configured and wired correctly.
You have not configured the HPLC correctly. As noted by others, the "time of injection" confirms the start of the run. You may have a "start" signal sensor connected to the control module to trigger a contact closure for injection (to tell the controller when to begin the gradient). The older Waters pumps that you have setup are often configured with another module, the "Gradient controller" and this must be correctly configured first, for YOUR method, to run the analysis in a reproducible way. Also, for best performance, program an initial "HOLD" time in the method, before the gradient starts, to allow the solvent (and sample) to reach the column. Then change the composition to run the gradient portion of the method. Please refer to "Practical HPLC Method Development" and other classic basic texts on HPLC for an introduction. *Please ask for help from an experienced local chromatographer at your school and also, review the operating manual for the gradient controller to better understand how these modules were intended to be used. **Set it to start the METHOD, "run" or analysis, at the exact moment of injection.
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