We are trying to standardize a method for analysis of TAGs using Reverse phase C18 column and RI detector in an Agilent HPLC using literature based reports. Since RI detector in our HPLC is not working below 35oC the detector and column temperature was set at 35oC. As per literature Acetonitrile: acetone, we are using as mobile phase. The problem we are facing is at ratio of 1:1 acetonitrile: acetone we are not able to achieve a stable base line despite of running the system at prolong low flow rates, daily column washing by changing the polarity gradient of both solvents, changing the flow rates and occasional detector purging. When we tried to run with different mobile phase ratios like 70:30 and 60:40 or 55:45 Acetonitrile: acetone (when concentration of acetonitrile was more) the baseline we achieved was abnormally and unbelievably straight and 100% noiseless. But when we tried to run the samples (TAG Standard) in those conditions not a single peak nor a single noise appeared which indicated that samples/analytes are probably not getting detected somehow. Hence we had to change the mobile phase ratio again into 50:50 Acetonitrile: Acetone because most of the reports suggested either 1:1 ratio of both or acetone on the higher side for TAG analysis in RP-HPLC. Also more polar acetonitrile at higher ratio was although supporting correct baseline, but was not supporting analyte detection. However, at 1:1 ratio we again lost the baseline stability (extremely noisy baseline). Can anybody suggest suitable conditions in terms of mobile phase or anything else for TAG analysis with C18 column and RI detector? Someone's kind help will be extremely appreciated.
Batul Diwan You can try test I told you in the earlier post. Your observations " abnormally straight and 100% noiseless baseline " does indicate some problem with the detector rather. Again are you manually mixing the mobile phases or the HPLC is mixing it? Do you have the manual of your RI detector? What is the make? They always have a section on troubleshooting problems.
One simple solution, instead of randomly trying experiments is to repeat a bench mark experiment. I am sure, RI must have been used before and someone is incharge of it. Try a simple published method or something which someone else has tried before, and which does not involve acetone or acetonitrile. If you can reproduce the result, this will establish that there is no problem in the instrument and the detector. Otherwise you will keep randomly changing the ratios of ACN and acetone will keep seeing this odd behavior without any clue to what is going on. The safest path is to go from a known to an unknown method.
And there is no need put too much trust in published methods here and there. Choose papers from good established journals and read at least 5-10 papers before trying a new approach.
RI detectors are somewhat problematic. Baseline drift and noise is a common issue. First of all read this excellent article "Avoiding Refractive Index Detector Problems". http://www.chromatographyonline.com/avoiding-refractive-index-detector-problems-0?id=&sk=&date=&pageID=4
1. Are you mixing the mobile phases or the instrument? Try a hand mixed mobile phase to rule out pump problems.
2. Degass the mobile phase under vacuum and sonication. Just sonication does not do anything to remove dissolved gases.
I feel it is acetone which could be causing the problem at 35 C (it has a very low boiling point). Just try a control experiment. Run pure acetonitrile and see how the baseline looks like. Stop the flow and see what happens.
Next run pure acetone at 35C and monitor the baseline. Stop the flow and check again.
RI detectors are also insensitive too. Unless you are injecting a 'pure' oil, they are limited to ~0.1% wgt/wgt. Thus, most chemists use an ELSD detector.
You are also constrained by the RI detector to perform 'isocratic' chromatography with just 1 mobile phase, and not 'gradient'.
M. Farooq Wahab Thankyou sir for giving attention to our problem. Yesterday we also tried to increase the Acetone concentration in order to check whether really acetone is the problem or is it the detector. To our surprise at 48:52 Acetonitrile :Acetone ratio (acetone in higher amount) we again achieved that abnormally straight and 100% noiseless baseline. However when we injected sample at those conditions again their was no analyte detection as previously observed. But as soon as we did detector purging this abnormally straight baseline became heavily noisy. We observed this behavior from last few days that this abnormally straight baseline suddenly becomes heavily noisy as soon as we purge the RI detector which upon changing the mobile phase Acetone : Acetonitrile ratio straightens again. What could be the problem, how can we detect the analyte, not even pure standards are getting detected.
Batul Diwan You can try test I told you in the earlier post. Your observations " abnormally straight and 100% noiseless baseline " does indicate some problem with the detector rather. Again are you manually mixing the mobile phases or the HPLC is mixing it? Do you have the manual of your RI detector? What is the make? They always have a section on troubleshooting problems.
One simple solution, instead of randomly trying experiments is to repeat a bench mark experiment. I am sure, RI must have been used before and someone is incharge of it. Try a simple published method or something which someone else has tried before, and which does not involve acetone or acetonitrile. If you can reproduce the result, this will establish that there is no problem in the instrument and the detector. Otherwise you will keep randomly changing the ratios of ACN and acetone will keep seeing this odd behavior without any clue to what is going on. The safest path is to go from a known to an unknown method.
And there is no need put too much trust in published methods here and there. Choose papers from good established journals and read at least 5-10 papers before trying a new approach.
I would second the reply from M. Farooq Wahab , if you are getting no signal at all, there is an issue with the detector. Is your lamp still in good condition and providing enough intensity? And as Bruce Neagle mentioned, RI detectors aren't that good for sensitivity, I usually only used them for production scale analysis when I had a lot of material to work with, and they don't work with gradients at all.
M. Farooq Wahab sure sir, Ill go through more literature. However the procedure we were following was derived through a published research paper where RI detector and C18 column was used. Everything right from mobile phase, flow rate detector, column was tried to replicate as reported in a paper. When nothing worked then we started changed the conditions but still things didn't work out. Yes as per your suggestion we will try for mobile phase other than acetone : acetonitrile. Thankyou so much for the valuable suggestions.
I agree Dr. Chris A Singleton Chris, I guess RI detector can also be the problem, people generally use ELSD detector for such analysis.
Grzegorz BoczkajDr. Grzegorz : we are purging/ flushing our RI reference cell with the mobile phase.
Dr Balaji: Thankyou very much sir for the suggesting this article, I have already read this .
Chris A SingletonChris A SingletonChris A Singleton
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