Can pressure influence retention (Acquity)?

[Mattias]

Hi,

I have had some problem with clogged tubings (post column) on our Acquity. I have still been able to run the method, giving a total pressure drop of 12000 psi.

I have now changed to new tubings and the pressure drop (using the same column) is now 8000 psi. Same mobile phases and conditions.

The problem is that the separation does not look the same after the shift of tubings. First of all, the main peak comes out after 14 minutes - before the shift of tubing it was at about 20 minutes. The separation of the impurities does also look different, and two peaks have shifted elution order.

Can the pressure itself be a separation parameter in UPLC?

[Alex Buske]

Yes, it can, I was also surprised when i read it in LCGC Europe in April.

[Mattias]

I didn't know this was a known fact! I will look up that article.

thanks,
Mattias

[Bluesman75]

This may be the article you are looking for http://chromatographyonline.findanalyti ... ail/725571.

[Klaus I.]

I'm also surprised that the friction heat can cause such problems, it's really amazing.

But I'm not sure that this is the effect that Mattias have observed. Maybe I'm biased, because the last times my colleagues often complains about clogged capillaries after the column. Can it also be possible that the column have lost much of its particles?

It would be very interesting if Mattias will attach a restrictor capillary after his column to have again 12000 psi and compares the chromatogram with the other two chromatograms.

[pharmaguy]

Prof. Tanaka did a talk at HPLC 2011 on this topic, in his examples, increasing pressure after the column with a capillary gave results very similar to what you would find by lowering the temperature of the column.

[Mattias]

I'm also surprised that the friction heat can cause such problems, it's really amazing.

But I'm not sure that this is the effect that Mattias have observed. Maybe I'm biased, because the last times my colleagues often complains about clogged capillaries after the column. Can it also be possible that the column have lost much of its particles?

It would be very interesting if Mattias will attach a restrictor capillary after his column to have again 12000 psi and compares the chromatogram with the other two chromatograms.

Hi again,

The big shift in retention time of the main peak was an artefact. For some reason the time axis in my chromatograms are not correct, all of a sudden. I watched when a chromatogram was collected yesterday (Empower), and one minute in the chromatogram turned out to be close to two minutes in real time (making the chromatograms shorter). I suspect that this is due to some "hotfix" of Windows that our IT-department installed yesterday.

Anyhow, the shift of selectivity is still an effect of something else. It seems as the pressure and the flow does not have a linear relationship in UPLC. The pressure at 0.5 ml/min is not 200% of the pressure at 0.25 ml/min - it is more like 170%. I guess this has to do with friction heat, which also should influence the selectivity of the separation (?).

The detector cell can only take 1000 psi, so I cannot install a 4000 psi restrictor after the cell. Would it be OK to install a restrictor between the column and the detector?

[AA]

Would it be OK to install a restrictor between the column and the detector?

Sure, you can put as much restriction as you want here (a column is a restrictor), of course depending on what you use as a restrictor, you will change RT and peak shape (peaks will be broader) due to the volume in the restrictor tubing.

[Hollow]

It seems as the pressure and the flow does not have a linear relationship in UPLC. The pressure at 0.5 ml/min is not 200% of the pressure at 0.25 ml/min - it is more like 170%. I guess this has to do with friction heat, which also should influence the selectivity of the separation (?).

Normally the flow-pressure linearity is also valid in UPLC, but the frictional heat could have some effect though. It is possible that the pressure will equilibrate at some lower value than expected after some time.

But in the first period after the flow change the linearity should be pretty good. Otherwise there could be some small leak, which can be difficult to be seen at low flowrates.

I often check the pressure-linearity after installing a column. If the linearity is ok up to the needed working level (r2 >0.990), than I'll be sure that the column is connected well.
If at one point the pressure deviate from the linearity, I can be sure that the
sealing got weakened and a leak could be observed soon.

[Tom Waeghe]

Well, it won't solve this current problem as you've resolved it as being due to data system issues. Nonetheless, here are some additional references folks may want to take a look at regarding the influence of pressure on retention and on selectivity.

Journal of Chromatography A
Volume 1209, Issues 1-2, 31 October 2008, Pages 195-205
Investigation of the effect of pressure on retention of small molecules using reversed-phase ultra-high-pressure liquid chromatography
Morgane M. Fallas, Uwe D. Neue, Mark R. Hadley and David V. McCalley

Abstract
The effect of inlet pressure on the retention of a series of low molecular weight acids, bases and neutrals, was investigated at constant temperature in reversed-phase liquid chromatography using a commercial ultra-high-pressure system (Waters UPLC instrument). For neutral compounds, relatively small increases in retention factor of up to 12% for a pressure increase of 500 bar were noted; the largest values were obtained for polar solutes, or solutes of higher molecular weight. Ionisable acids and bases gave much larger increases in retention with pressure, in some cases as high as 50% for a pressure increase of 500 bar. [/b]Thus, such compounds could show increases in retention factor approaching 100% over the pressure range available in the commercial UPLC instrument. Due to these differential increases, significant selectivity effects can be obtained for mixtures of different types of solute merely by changing the pressure.

Journal of Chromatography A, 1090 (2005) 16–38
Michel Martin, Georges Guiochon
Effects of high pressure in liquid chromatography
(Abstract too long to repeat here, but very good article)

Journal of Chromatography A, 1070 (2005) 13–22
Influence of pressure on the properties of chromatographic columns II. The column hold-up volume
Fabrice Gritti, Michel Martin, Georges Guiochon

Journal of Chromatography A Article in Press,
Practical Implications of the “Tanaka” Stationary Phase Characterization Methodology using Ultra High Performance
Liquid Chromatographic Conditions
Melvin R. Euerby, Matthew James, Patrik Petersson

Abstract
The practical implications of performing column characterization protocols (i.e. Tanaka) and their resultant chromatographic selectivity parameters using small dimension columns (i.e. 50 × 2.1 mm I.D.) at high pressures have been critically compared to those obtained using conventional LC methodology. Retention factors should be corrected for the system extra column volume even when determined on ultra high performance liquid chromatographic (UHPLC) systems with low system volumes.

An increase in pressure resulted in a general increase in the retention factor for most analytes, the degree being dependent on the physico/chemical properties of each analyte and the chromatographic conditions employed. However, analytes chromatographed at pH values close to their pKa values exhibited a substantial decrease in retention factor. Performing the Tanaka and extended column characterization procedures at pressures that would be encountered during the characterization of small particle sizes packed into 50 × 2.1 mm I.D. column formats at a constant linear velocity according to standard protocols, resulted in comparable chromatographic selectivity parameters to those determined using standard HPLC systems and column formats. However, due to the wide structural diversity of analytes employed in other popular column characterization protocols, it is imperative to demonstrate comparable results when small columns packed with small particle sizes are chromatographed at increased pressure and compared to standard column formats – otherwise erroneous comparisons and conclusions may be made.



LC-GC online, April 2011
Selectivity in Reversed-Phase LC Separations, Part IV: Pressure Selectivity
John W. Dolan

Journal of Chromatography A, 1136 (2006) 192–201
Experimental evidence of the influence of the surface chemistry of the packing material on the column pressure drop in reverse-phase liquid chromatography
Fabrice Gritti, Georges Guiochon

The effect is very pronounced for larger molecules like peptides, proteins etc.

Anal. Chem. 2005, 77, 3425-3430
Separation of Peptides from Myoglobin EnzymaticDigests by RPLC. Influence of the Mobile-Phase Composition and the Pressure on the Retention and Separation
Nicola Marchetti and Georges Guiochon

Cheers,

Tom Waeghe

[Klaus I.]

Anyhow, the shift of selectivity is still an effect of something else. It seems as the pressure and the flow does not have a linear relationship in UPLC. The pressure at 0.5 ml/min is not 200% of the pressure at 0.25 ml/min - it is more like 170%. I guess this has to do with friction heat, which also should influence the selectivity of the separation (?).

The detector cell can only take 1000 psi, so I cannot install a 4000 psi restrictor after the cell. Would it be OK to install a restrictor between the column and the detector?

Yes, the restrictor should be installed between column and detector.

I have also sometimes observed a non-linear relationship of the pressure. But not in dependency from the flow rate, i observed this with different column lengths. E.g. a 10 cm UPLC colum has about 170 % of the pressure of a 5 cm column.

Which column temperature do you use normally and do you use a solvent pre-heater (heat-exchanger capillary just before the column)?

[remesquaddie]

Hi,

I have had some problem with clogged tubings (post column) on our Acquity. I have still been able to run the method, giving a total pressure drop of 12000 psi.

I have now changed to new tubings and the pressure drop (using the same column) is now 8000 psi. Same mobile phases and conditions.

The problem is that the separation does not look the same after the shift of tubings. First of all, the main peak comes out after 14 minutes - before the shift of tubing it was at about 20 minutes. The separation of the impurities does also look different, and two peaks have shifted elution order.

Can the pressure itself be a separation parameter in UPLC?

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I suspect the pump is NOT pumping the correct amount of flow, because of the excessive back pressure. If flow IS short, that explains the RT delay.

[Mattias]

Thanks for all your answers!

I need to investigate this further, since the method is not robust if it is so dependant on the backpressure.

Can anyone recommend a low volume pressure restrictor giving 3-4000 psi at 0.5 ml/min? I have tried using a meter of red PEEK, but that didn't give much extra backpressure.

[Andy Alpert]

While evaluating a new UHPLC machine for analysis of hemoglobin protein variants, we installed a piece of narrow-bore capillary tubing between the column and detector. The added volume was negligeable and so there was no effect on peak shape; the only effect was to raise the backpressure from about 6,000 psi to 13,000 psi. Result: The proteins eluted about 30% earlier in the gradient. That runs counter to the above speculation that the effect of pressure is implemented merely by the pumps running slower. Some of the references above cite increases in retention with pressure. That depends on the particular mode involved. It's true of reversed-phase. In the case of HILIC, retention tends to decrease with increasing pressure. That was true as well in our experiment with hemoglobins, which involved ion-exchange.

[AA]

Can anyone recommend a low volume pressure restrictor giving 3-4000 psi at 0.5 ml/min?

You can purchase lengths of 25um and 50um Peeksil tubing from Supelco, these can make pretty good restrictors. They are 1/16 o.d. so you can use your typical fittings.