i am new in HPLC running. while running HPLC i am watching same peak patter when i run blank or i run sample. please guide me how i can solve this issue.
You should rinse your HPLC system and look for the quality of the solvents you run in it.
What is the size of these peaks, they may very well be noise. Filter and sonicate your aqueous mobile phase. Check the quality of your non-aqueous mobile phase. Give a run without injection, you should get a Stright line, if not you may have some issue with the mobile phase or the lines. If you have a fowled column, you can get some column bleed, check with a different or new column. Check the labelling of your blank and sample, you may be running 'sample' both times. Try these and let us know how it goes. All the best.
You need to provide way more details on your HPLC setup and run conditions. Most of the time there will be a "blip" near the beginning of the run caused by the injection valve switching. If you run gradient, then most of the time you will see another peak towards the end where the gradient switches back. These are all normal behaviors of the system and there isn't much you can do about them.
Another type of background peaks come from contamination. Depend on what you need HPLC for and how deep your pocket is, you can clean/replace the flow path components, or just live with the background peaks, mark out the peaks in your blank and remove them from your sample reports.
Ambreen Latif's is an inexperienced user of HPLC without any formal training, their question is understandably unclear. Ambreen Latif, ff you inject a "blank" sample into the HPLC system (A blank is the mobile phase solution, at the exact same volume as your sample) and you observe a peak eluting at the same retention time as your "sample", then you either have Carry-over Contamination or NO RETENTION of the sample (NO retention equals no chromatography is occurring and the method is invalid). What is the kPrime of the peak?
BTW: Check the condition of the injector too in case this is caused by carryover (install a new rotor seal).
*HPLC operation takes many years of full-time, professional experience just to learn the basics. HPLC is not a technique that you can just "figure out" on your own. If you wish to use an HPLC system for your project, please first contact a local professional chromatographer at your school (ask your teacher for help) so they can assist you with the project and allow you to develop a method and collect scientifically useful data. This process will allow you to learn about the technique and also learn how to work with others in the field who are experienced.
In case you are experiencing "carry-over", here is a link to an article that may assist you in troubleshooting and solving it.
- "Carry-Over (Carryover) Contamination in HPLC and LC-MS Systems"; https://hplctips.blogspot.com/2015/02/carry-over-carryover-contamination-in.html
I think your question is unclear. First, know that in every HPLC analysis, there are certain conditions in which you have set your HPLC and it takes a lot of effort with constant trying different conditions before you can achieve your aim. So please make your question clear enough for us to help you.
If you are observing the same peak pattern when running a blank or a sample on HPLC, it indicates that there may be an issue with the separation or detection of your analytes. You try these steps to address the problem itemized:
1. Check your mobile phase: Ensure that your mobile phase composition is correct and properly prepared. Verify the pH, concentration, and compatibility of your mobile phase components. Incorrect mobile phase composition can lead to poor separation and identical peak patterns.
2. Check your column: Make sure your HPLC column is properly installed and in good condition. Check for any signs of damage or contamination. Columns that are not properly equilibrated or columns that have reached the end of their useful life can result in poor separation and similar peak patterns.
3. Optimize your chromatographic conditions: Adjust your gradient program, flow rate, and temperature as needed to optimize the separation. Fine-tuning these parameters can sometimes help resolve similar peak patterns.
4. Verify sample preparation: Ensure that your samples are properly prepared and do not contain any interfering substances. Contaminants or matrix effects in the samples can cause similar peak patterns.
5. Check the detector settings: Confirm that your detector is properly calibrated and set to the appropriate wavelength or detection mode. Incorrect detector settings can lead to misinterpretation of peak patterns.
6. Consider using a different column or method: If none of the above steps resolve the issue, it may be necessary to try a different HPLC column or method to improve the separation and obtain distinct peak patterns.
I hope these tips are valuable assistance in resolving the issue.
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