We need to quantify certain proteins on UPLC. The protein mixtures are seperated very well and the calibration curves of standards are also good. The problem is for some samples the protein peaks split and the minor peaks can make up to 10% of the major peak area. The column is Aquity UPLC Protein BEH C4, 300A, 1.7 um, flow rate 0.2 ; MPA is 0.1%TFA in water, MPB is 0.1% TFA in IPA:ACN(70:30). The gradient starts from 70% MPA at time 0 and gets 30% at time 13min. All 3 proteins elute before 13 minutes. temperature is 40 degree C.
One of my question is that if the protein will denature in this condition? If the denatured protein will cause the peak split? I searched articles and didn't find related research. If anyone can give some ideas or discussion?
- Please ignore Kou's suggestion as it is not related to your questions. He likes to hijack threads and keep posting the same article, even when it is not relevant. It also suggests using a column type and particle size which are outdated and not applicable to your method. Do not use 10u particles (the 1980's are over).
- Denature it? Perhaps, as you have a lot of ACN and IPA in your mobile phase. No, it will not cause peak splitting as that is a chromatographic effect caused by your method/technique.
- Terminology: "UPLC"? I think you mean "UHPLC" or better yet just call it what it is, HPLC. "UPLC" is a Waters Trademark only.
- Split peaks? You provided no chromatogram so we have no idea (btw: split peaks are often caused by sample overload), but I can provide you with a short article which will explain to you what the most common causes of split peaks are and how to solve them. You should be able to use the information to troubleshoot the problem. Here is the link to the article; "HPLC Peak Splitting. Common Reasons For It", https://hplctips.blogspot.com/2016/09/hplc-peak-splitting-common-reasons-for.html
- Training: Is there someone at your company who is experienced in chromatography? Get some local help with your method and of course more training as these are very fundamental questions and you will get them solved quickly if you have someone to help you on-site (not via the web). It takes many years to learn the basics, but if you have high quality resources available to you the information you will learn will also be of high quality (and you will make fewer mistakes).
- Standards elute well- this rules out the column and system as potential causes for peak splitting
- How are you dissolving your protein? If they are being dissolved in something like DMSO, DMF, or another "strong" solvent, one could observe peak splitting or otherwise poor peak shape. Are you dissolving the standards in the same solvent, and injecting the same volume of this mixture? I have observed that one may use these dissolution solvents in low injection volumes with chromatography failure at larger injection volumes. What are you using for standards?
- I may not rule out denaturation at this point as a cause for peak splitting, but I would look at the point above first.- see under section 6.2.3 of this link: https://books.google.com/books?id=Nlq-2bGZor0C&pg=PA271&lpg=PA271&dq=protein+peak+splitting+denaturing&source=bl&ots=MFPptcKqin&sig=sybwVmO4KlfGtcTfGOmZw58MaVE&hl=en&sa=X&ved=0ahUKEwjir6nrvI7XAhUF4yYKHRXPDEA4ChDoAQglMAA#v=onepage&q=protein%20peak%20splitting%20denaturing&f=false
Thank you Jack for the discussion. You remind me something.
- The calibration standards and QC standards are all freshly made and results are good. Calibration standards are desolved in water and Qc standards are desolved in the same buffer as samples. The buffer is NaHCO3+NaCl, PH 7-7.5, very neutral environment. The samples had already stayed in refridgetor for ~2 weeks, which may cause the problem. I should do a stability test.
- The book contains a lot of information. I was looking for a book like this to tell about challenges of Intact protein chromatography.
This is all very interesting... I have dealt with such HPLC phenomena quite a bit, hence a couple questions/comments. (1) What standards are you using, are they closely related to your analyte? I am asking this because (2) To me it sounds like either (a) less likely - a column/instrument problem; or much more likely (b) the behavior of your analyte is pH-dependent, possibly solvent dependent and/or concentration dependent. Concentration dependency can vary with pH, too.Thus, if standards are chemically quite different from the analyte, they can elute without peak splitting while the analyte may be responding to the pH differently, causing the splitting you observe. Also, pH 7.5 is not a "very neutral environment", at least in HPLC terms... You will find out all about it, with a little experimentation - please let us know about your findings. Good luck.
Yun, please review my earlier suggestions found in the link supplied:
[ "HPLC Peak Splitting. Common Reasons For It", https://hplctips.blogspot.com/2016/09/hplc-peak-splitting-common-reasons-for.html ]
- Esp. #2 in the list as it is most likely related to your issue.
Most troubling is your statement about the samples used being 2 weeks old (sitting in the refrigerator). This may or may not have anything to do with the problem, but must be investigated as many compound types do indeed change based on time alone.
Hi Jacek, you have a very good point. The standards are the standard proteins, the same as my analytes. For example, if I look at BSA, I use BSA standard. My unknown samples have concentrations within the range of calibration standards. I agree with you that the PH maybe a problem. The sample matrix is more complex than the QC buffer. The sample pH may need to be adjusted. Thanks for your comments.
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