Monday, 19 October 2009

Help on: Retention time shift in HPLC analysis


Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

"I am using 0.1 % TFA in water + 100 % Acetonitrile as mobile phase in gradient RP-HPLC. But since last month I have problem of RT shift. I am not able to rectify it. Please suggest solution for the same. "

"Some of the most common problems which can cause a shift in retention time are temperature, mobile phase composition and equilibration of the column. I shall discuss each of these first and then move on to other potential causes of your problem.
Is your column in a column oven and maintained at a constant temperature throughout your analysis? Temperature is a variable in HPLC analytical methods (we often use it to adjust retention during the method development process) and changes in temperature can result in shifts in retention time. A method that runs at ‘ambient’ temperature can be subject to large variations in the room temperature of a laboratory. So even if the method says ambient it is best to control the temperature.
Mobile phase composition:
How do you create the gradient for your method? If you premix your mobile phases of 0.1% TFA in water and 100% acetonitrile to result in one reservoir which corresponds to the method starting conditions and another which corresponds to the end conditions then it is possible that changes in the volatile components of these mixtures due to evaporation could lead to shifting retention times. However, if you use online mixing and simply have 0.1% TFA in one bottle and acetonitrile in the other then this cannot be the source of the problem.
Column equilibration:
Generally, when using RP-HPLC methods the column can be equilibrated relatively quickly. Check that you have allowed enough time at the end of each injection before the next injection. Typically 10 column volumes will be sufficient (refer to the previous post on column equilibration). An exception to this is when using ion-pairing reagents, these can take a significantly longer time to equilibrate due to the adsorption of the ion-pairing reagent on the surface of the stationary phase. Although TFA can act as an ion pairing reagent I have never had an issue with equilibration at the concentration you are using.

If you can rule out these common causes then we need to look more closely at both the HPLC method that you are using and the problem that you are experiencing. Some questions to consider:
You say your problem started last month, was the method working well before this, and for how long?
If the method has been trouble free for a significant length of time then it indicates that the problem is more likely to be due to a problem with the HPLC system or the column. Consider carefully, are you certain that you haven’t changed any of the method parameters? Review the method thoroughly. Run the method on a different column (preferably new) and see if the drifting retention time is observed.
If the method hasn’t been proven to work then the problem could be due to the method parameters, e.g. sample preparation could be inadequate leading to progressive contamination of the column, or the column and analytes may not be completely compatible.
Are all the peaks in the chromatogram moving (including the solvent front) or is it restricted to certain peaks only?
If all the peaks, including the solvent front, are shifting then the problem may be due to system leaks, or air bubbles in the pump. If the solvent front does not move then the change may be linked to the mobile phase and method conditions. If the shift in retention time is only observed for certain peaks then it may be due to a change in the column which only effect certain types of functional groups , e.g. subtle pH change.
What is the nature of the retention time shift, changing within a run, changing between runs, increasing with time, decreasing with time, no pattern in the drift?
The way in which the retention time is shifting will provide extra information on potential causes and may support a diagnosis based on the discussion above.
A note regarding your mobile phase. TFA does not actually have much buffering capacity, it is very convenient for acidifying the mobile phase when you need a volatile buffer. Depending on the parameters of your gradient it may be that there is very little acid at the end of your gradient. You can add TFA to the organic portion of your mobile phase, i.e. 0.1% TFA in acetonitrile and thus the amount of TFA present throughout the gradient remains constant. This may result in some improvement in your chromatography, however I don’t think that this would explain the shift in retention time that you are experiencing.
Please use the comments if you would like to continue this dialogue and provide some more information which may help to diagnose your problem.”

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Help on: Measurement of urinary citrate levels by HPLC


Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

Question:"We would like to measure 24 hrs urinary citrate levels in humans for our upcoming project. How can HPLC help us?"

"To utilise HPLC for the measurement of urinary citrate you need to consider the nature of the analyte, in this case the citrate molecule. The relatively polar nature of this molecule makes it suitable for analysis by either ion exchange HPLC [1] or reversed phase partition HPLC. Reversed phase partition HPLC is usually preferred due to its availability in most laboratories.

Examples in the literature report the use of C18 columns for the analysis of citrate [2,3]. A low pH aqueous mobile phase was used combined with a column preconditioning step consisting of 100% methanol, this organic solvent rinse was repeated between injections to ensure that the chromatography was acceptable. The reason for using a 100% aqueous mobile phase is to obtain satisfactory retention of the relatively polar citrate molecule.

I suggest that you make use of an aqua column which can perform in 100% aqueous conditions, and should adequately retain the citrate molecule. The low pH of the mobile phase means that citrate is in its unionised form and maximises the retention on a RP column. It is important to control the pH adequately since citrate has 3 pKa values at 3.1, 4.7 and 5.4, changes in pH around these values may have an effect on the retention of the analyte and thus the reproducibility of the chromatography.

The citrate molecule contains a weak chromophore which will enable the use of UV detection. A low wavelength such as 210 nm will be suitable. LC-MS may also be used.

The other issue to consider when analysing samples of urine is the matrix and how much sample preparation is required. Khaskhali et al. [2] found that deproteinisation of the urine sample was adequate."

References:1. Kristina L. Penniston, Stephen Y. Nakada, Ross P. Holmes, Dean G. Assimos. Journal of Endourology. March 2008, 22(3): 567-570. ‘Quantitative Assessment of Citric Acid in Lemon Juice, Lime Juice, and Commercially-Available Fruit Juice Products’
2. M. Hassan Khaskhali, M. Iqbal Bhanger and F. D. Khand Journal of Chromatography B Volume 675, Issue 1, 12 January 1996, Pages 147-151. ‘Simultaneous determination of oxalic and citric acids in urine by high-performance liquid chromatography ‘
3. Keevil B. G. ; Owen L. ; Thorton S. ; Kavanagh J. ; Annals of clinical biochemistry 2005, vol. 42 (5), pp. 357-363 ‘Measurement of citrate in urine using liquid chromatography tandem mass spectrometry : comparison with an enzymatic method'

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Friday, 16 October 2009

Lab Tech Guy on Agilent HPLC purge valve frits

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