I'm currently working at a lab implementing a methodology to analyze folic acid using a Select B column and using a phosphate buffer (0.03M), but I've been seeing the pressure go up when I try to flush the system only with relatively high polar solvents. The thing is that when I use acetonitrile the flow can be as high as 1.000ml/min and have a relatively tamed pressure, but be raised exponentially with a combination as little as 80:20 ACN:Water even at a flow rate of 0.200ml/min. I've been looking for answers in several chromatography forums but they don't seem to have much info on clogs of this nature. What could be the source of the clogging that water can't seem to be used to clean the column, and how can it be fixed.
Some additional facts, I tried a solvent of intermediate polarity like methanol and after a while pressure seems to go down, so much that allows me to up the flow rate from 0.200ml/min to 0.500ml/min. The mobile phase that was used prior to the clogging was adjusted at pH 3 and I was using a gradient that used 99:1 (buffer:methanol) that at some parts is 80:20 (buffer methanol).
Thanks for the assistance.
Training in HPLC, at a basic level, takes years in the lab. I think that first of all you are not aware of the different viscosity properties of liquids used and their impact on the back-pressure of the HPLC system. The mobile phase solution (or mixture) plus flow rate through the same support (column) result in an observed back-pressure. This back-pressure will change as you change mobile phase mixtures and is normal, not always related to any clogs or partial obstructions (those should be obvious). Take a look at the Solvent Properties Table on this link to see viscosity values for individual solvents used in HPLC [ https://www.hplctools.com/tablescharts.htm ].
Additionally, something that new users are often unaware of is that many common HPLC mobile phase MIXTURES of Methanol/Water and ACN/Water can show even higher back-pressures than measured for either of the two solvents, alone. Examples of this are: (1) MeOH and Water mixtures. If you record the pressure, then run a gradient of Water vs MeOH (5% MeOH to 95% MeOH over time) you will see a Gaussian curve plot (pressure). The pressure max occurs at 50/50 mixture! (2) A similar profile can be seen with Waters vs ACN, but the max shifts to ~ 70 - 75%. These are due to a chemical reaction between the two solvents. In any case, if you have not had training in HPLC operation, the effects can confuse many users, but are normal in routine work.
Finally, if you believe you have a partial plug in your HPLC system or RP column, first remove the RP column and replace it with a restriction capillary [https://hplctips.blogspot.com/2018/04/the-hplc-restriction-capillary.html]. Run a known liquid through the system to generate enough back-pressure to stabilize the HPLC pump(s) properly (often > 700 psi), then monitor the output. If that is OK, place a RP column back inline and starting with pure water, at low flow rate, run steady for ~ 30 to 60 minutes (monitor baseline and pressure). *Flow rate and time depend on column dimensions used. If buffer is still in the system, then this will wash it out. If this step is satisfactory, reduce the amount of water and slowly increase the amt of methanol used over time, stopping at different ratios to monitor. This basic first step / flushing procedure should help you determine if any clog exists in the system or column.
If you have other HPLC questions, please include:
- the exact column details (Name and type, Length x ID, particle size);
- the make and model of HPLC system you are using plus its configuration (which modules your has, e.g. DAD, AS, Quat pump etc);
- the exact composition, flow rates and detection system settings used for any tests, plus results.
- do you filter all samples before injection? You should be using an appropriate filter.
- do you make up FRESH mobile phase solutions EACH day, then filter them before use? You should be doing this.
- do you flush down the HPLC system starting with pure water, to remove your 30mM buffer, then re-equil the system in a mixture of pure water (no buffer) plus organic solvent (e.g. MeOH) to prevent bacterial growth each day? This is critical to prevent contamination, fouling and clogs.
"HPLC Solvents, Acetonitrile and Methanol, Key Differences and Properties"; https://hplctips.blogspot.com/2017/09/hplc-solvents-acetonitrile-and-methanol.html
"Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline)"; https://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html
"Tips and Advice for Priming your HPLC PUMP (or similar pumps, FPLC or UHPLC Pump)"; https://hplctips.blogspot.com/2020/09/tips-and-advice-for-priming-your-hplc.html
William Letter many thanks for your prompt response. I resolved the issue but I'll still go back to you with your questions and I'll make sure to include them in future reports.
1. RP-Select B (5micrometers) LiChrospher 60 (i think it's 250mm x 4mm)
2. It's got quadpump, UV simple detector, it's a hitachi one (I'm at the office but it's rather old 2003)
3. the composition of the MP was 99:1 (Buffer a 30mM Phosphate one pH 3 : Methanol) All where passed through a filter of 0.45micrometers, the settings were 1.2 mL/min flow, 10 microliters, samples were diluted in water and the peak was found at aprox. 12min. (There's gradient that only lowers the concentration of Buffer to 80 from min 8 - 12 min.
4. Samples are always filtered through a 0.2micromolar membrane
5. the solutions were "fresh" since the validation must cover a lot of days and the autosampler allows for the equipment to work usupervised during the night (when no one's there) I prepared enough buffer to let the pump running. Post phase cleaning consisted of methanol - metanol:water 50:50 and finally water over the spawn of 3 hours.
6. yes I do. the post phase sequence will finally wash everything with water.
The issue was that I'm working on an extraction from a matrix that has mostly quelated iron (this is susceptible to changes in pH so when it's on basic conditions most precipitates leaving the rest in a clear solution). I didn't consider the efect that having a basic sample running on an acid MP could cause so I had to flush the column with acid to allow the soluble crystals in basic solution that where created to elute. I guess my issus is the post phase cleaning of the column and I didn't foresaw what happened. I'll have that in mind the next time I work on the HPLC.
Thanks for the reply.
Best Regards.
Precipitation will certainly result in problems, so it sounds like you are on the correct path to the solution... That HPLC system is really a giant, expensive filter with no tolerance for particulate (as you now know). Be sure to prepare fresh HPLC Mobile phase solution EVERY day, then filter and use. Flush the system down every day with clean solution, no buffers. Good luck with the project and thank you for the additional information too.
William Letter , thanks for your informative explanation notes.Really appreciated it.
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