I am using a C 18 Column to estimate flavonoids with mobile phase (50:50) as Methanol and 0.5% ortho phosphoric acid. There is also a shift in pressure.
You should track how much of a shift there is. It is common for shifts in RT if the machine has been running for a long time. As long as all the analytes are showing similarly consistent shifts in RT, it should be fine. Just make sure you have an internal standard for each sample, and you have a good internal control.
Hi Praveen! At first, an obvious question related to pressure: did you filter samples and mobile phase? If it is not the problem, maybe a precipitated formation inside C18...
Good luck!
Equilibriate the column for long time and give delay in each injection. One important point is that MeOH:Water ratio increase in pressure due to exothermic reaction. So you should give more equilibriation time for each injection. You can also try 0.1% ortho phosphoric acid/ACN ratio in C-18 with flow=1ml/min.
I am agree with Mayura Dange, column needs cleaning, in the document attached, pag. 6 is the prodedure. Best regards!
Try to run blank after each sample run. It will help to condition the column.
It clearly seems like a case of column contamination since there is a change in RT along with increase in pressure. Using a column guard would prevent several contaminants. If you are already using a column guard, then consider changing it as it needs to be replaced regularly.
Hi Praveen,
How many injections do you have on the column? Depending on how crude the samples are, it may be time to replace the column. I've seen columns go with as few as 250 injections for ACN precipitated blood to as many as 2500 injections for ethyl acetate extracted serum samples. If you see higher pressures and wider peaks, you may need a new column. Also, I've never had much luck with regenerating columns, you may gain a week or two but you will eventually need to replace the column.
Pressure shifting due to
Possible causes : Air bubbles in pump head,
Leaks in system
Check valves are dirty
Plunger seal is damaged
Plunger is spoilt.
Remedy: Purge the pump
Check for leaks in all the connections, especially new connection.
Clean check valves in ultrasonicator
Replace plunger seal
Replace plunger
Check the system methodically:
Disconnect column from detector, if the pressure problem goes way, the problem is clogging of detector cell or piping to and from flow cell.
If pressure persist, disconnect guard column and main column. If pressure becomes normal, clean/replace main column.
If pressure persist, remove guard column. If pressure is normal, replace guard column.
Continue in this manner from auto-injector (if present) to mixer and finally to pump until find out the high pressure spot.
CHANGING RETENTION TIME DUE TO
Contamination was built up
Equilibration time is insufficient
Inconsistent mobile phase preparation
Inconsistent on-line mobile phase mixing
Selective evaporation of mobile phase component
Unstable column temperature
Leakage
Unstable pumping.
these things may help u
I would also like to add one more thing on this issue. Do check whether you have thermostat attached with column. Thermostats provide constant temperatures in HPLC columns for reproducible analyses.
Effect of Temperature: Retention times change by about 1% to 2% per 1º Celsius.
First of all I really like to encourage you to give more information on your system/method if you have a question. I see this pretty often here at RG and the answers are then (of course) a kind of stabbing around in the dark. OK, so I guess (so this is my 'stabbing') you use 2 bottles (one for MeOH and another one for your aqueous phase) on your HPLC-system and mix your mobile phase online and on-site.
Due to the isocratic mode of your method all the matrix components, which need >50% MeOH to get eluted from your column, accumulate on your stationary phase. This can easily lead to the interferences you described. Try to 'clean' your coumn by successively increasing the MeOH to 95% and hold it for some minutes. After sufficient equilibration try analysis at you 50:50 conditions. If this solved the problem, you could include a blank analysis every 10-20 (or more, depending on your matrix) samples including a MeOH-ramp to 95% for automatic cleaning. I use the same principle and it works.
Alternative reason for the increasing pressure: If the quality of your mobile phase may be questionable, the MeOH may contain Ca, which then gives you some ugly precipitations together with your phosphoric acid. Increasing pressure would be the result. You may mix the mobile phase offline, filter it (UF), and use only this mixture for your analysis. Or use MeOH with higher quality.
Good luck and please tell us what you tried and what solved your problems.
If your HPLC System leaks or suffers from a reduced eluent flow due to air bubbles in the pump heads, not only your analyte peaks but also the injection peak will shift. You can calculate the actual eluent flow easily by collecting the eluent at the detector outlet in a graduated cylinder for a defined time period and then compare it with the flow set in your method.
The problem is orthophosphoric acid. In this case the washing of column with water is very important for the lifetime of the column. At the end of day you must wash the column with water to dissolve traces of orthophostate that accumate in the column. For this reason I suggest you to use a different mix of eluting solvents: water-ACN with 0,1% TFA to avoid raising of pressure and washing of the column. In this other case you can obtain the same efficiency in the separation of flavonoids. For the instance try to reverse your C18 column or to re-generate it to obtain good resolution and efficiency.
Hello, for most flavonoids a solution mixture of water:acetonitril and acetic acid or orthophosphoric acid in a rate of 89:10:1 resulted.
most cases pressure shifting refers to pumps especially when to polar solutions used. in this case the pressure is not constant. if pressure is not constant the best way is to wash the system with isopropanol for 30 min. by this both column contamination and the polarity problems of bump will remove. If it does not work it is better to oppen the pumbes an wash its particle by organic liquides like isooctan.
Shift in RT and increase pressure may caused by column blockage over time. If you keep using then you may see even rapid increase in both. It is suggested to clean the column thoroughly with organic solvent (meOH/ACN) at higher temperature >40 for 30 - 60 minutes until pressure drops to previous numbers (120 bar). Other potential reason for shift in RT and Pressure may be due to column temperature whereas, higher the temperature less the pressure and elutes compounds at low RT and opposite for lower temperature. Also check for guard column if blocked then replace it. Make sure aqueous buffers always filtered before use. Hope it helps.
I agree with all the previous comments and would like to suggest that your pre-column filter can also be clogged (supposing that "shift" means increase in pressure). Also, is this shift only in one or in both pumps? It can help you figuring out whether the pump(s) or the stationary phase is an issue.
To conquer shift of TR of the peak of interest, you use Internal Standard. And determine with peak area, not peak height.
Usually the column will get more and more acclimatized/equilibriated after running the same sample/gradient for several runs. One important thing is that, the profile should be reproducible, if not the RT, most importantly the profile of the sample.
There might also be some other causes for the change in pressure.
The shift in pressure may be due to the presence of air bubble in the tubing or in the column. Remove the column and run the each solvent at high flow rate, such that the small air bubbles will be removed.
Some times a clog in the column could also alter/fluctuation in the pressure. Use 70% MEOH soln or 30% IPA soln for washing the column. Run it for a longer time with slow flow rate. If possible, reverse the column and wash it with 30 % IPA (This will clean the frit of the column, if it is clogged.).
If tR is shortened when continuously using, it is due to blocking of spore of the column. If the tR variation has no definite tendency, it is due to pumping. Check you pump.
You have air bubbles in your system so, to prevent such bubbles mix your mobile phase outside your system and degassed it strongly by ultrasonic then vacuum
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