Problem with column Acquity UPLC BEH

<p>We have troubles with our columns’ lifetime (Acquity UPLC BEH C18 1.7 µm 2.1x150mm).</p><p></p><p>The mobile phase is a 25 mM phosphate buffer at pH 3,6 with acetonitrile (85/15). During the run, a gradient go up to 75% acetonitrile and 25% mobile phase (NaH2PO4/ACN 85/15). Column manager is at a temperature of 30 °C. We use a VanGard precolumn.</p><p>The column is cleaned every day for 500 min with water, then 10 min water/acetonitrile (50/50), and then 10 min acetonitrile.</p><p>Before each using, the column is rinsed with water before it is equilibrated with mobile phase.</p><p></p><p>We used two columns Acquity UPLC BEH C18 1.7 µm 2.1x150mm; one had about 300 injections, and the other reached 550 injections. For both of them, the problem is the same: peaks become deformed, with shoulders or splitting.</p><p>We changed column and pre-column frits; we washed the column with water, water/acetonitrile 50/50, and 100% acetonitrile, upside down, without result. The cleaning sequence with water, methanol and THF did not improve the chromatography.</p><p></p><p>What can we do to have longer lifetimes? Can we save our columns, or are they dead?</p>


  • Khalid

    Hello Marylene:

    What is your observation about pressure? Is it increasing once column is ageing? What is your storgae solvent?

    The phosphate buffers are prone to microbial growth. Your mobile phase is highly aqueous in nature and idealy you need to use Acquity UPLC BEH Shielf RP18 Column, providing alternate selectivity compared to straight chain alkyl column and designed to produce exceptional peak shape for basinc compunds.

  • Hello Khalid,

    Thanks for your quick answer

    I don't see any increased pressure, neither cristalization, but peaks are still "double". Maybe it's because of a disability of stationnary phase (storage solvant phase = acetonitril)

    Just another question:  what do you mean by "Acquity UPLC BEH Shielf RP18 Column, providing alternate selectivity"?

    Thanks again


  • Khalid

    Hello Marylene:

    Since column is failing to  perform after a certain Number of injections it is something related to chemsitry/microbiology. I have observed certain cases in the past with the usage of phosphate buffer that microbial contamination plays a role. I assume that you are filtering the mobile phase and sample through a 0.2 micron membrane. Have you tried the ultra cenrifugation for the ready to inject sample after filtering through membrane. some time it works as it will remove the suspended particles. Have you seens some sort of turbidity in ready to inject sample?

    The shield column is desgined for the mobile pahses havgn higher conc. of Aqueous media and usually they give better peak shape for basic compounds.

    Thanks and best Regards


  • sazimi


    Well, you did many extra things to re-generate your columns, 300/500 injections mean columns still very new. Did you inject PQ samples or caffeine? How you assumed that columns are dead? Did you check column end fittings?

    UPLC BEH C18 is Waters standard brand and 15% ACN (organic) in your initial mobile phase is optimal, even 10% organic also okay for BEH C18, yes 95-100% aqueous not good for early eluting (highly polar).

    If you post complete LC conditions, chromatograms and name of compounds then I will try to solve your problem.


  • Hello,

    thanks for your answers!

    We have no PQ samples, we have our own quality controle, and we verify our extract with an internal standard, wich is prazepam.

    We work with drug compounds (screening).

    Our LC conditions are:

    A= phospate buffer (NaH2PO4 25 mM) pH 3.6/ACN 85/15

    B= acetonitril

    flow= 0.45 mL/min

    gradient: initial: 100% A

                 0.9 min: 100% A

                 2.30 min: 89% A; 11% B

                 10.5 min: 25% A; 75% B

                 12.5 min: 25% A; 75% B

                 13 min: 100% A

                 16 min: 100% A.

    Column manager is at a temperature of 30 °C

    We send you two chromatogram with tha same extract, and the same column. You'll see splitting peaks, despite many rinses and frit and VanGard precolumn changes. (We send you only the seven first minutes of the chromatogram)

    Best regards,


  • sazimi


    chromatogram of splitting peaks indicating that your column is fine ! still not clear what do you mean by "same extract" ? are you injecting old sample extract (biological specimen?) several times? I don’t know about your study but sample degradation also possible, normally methods are optimized by mix standards solution, you should have a system suitability mix solution (min. five compounds) for routine check.

    I'm just wondering, that column temp. is just 30C at 0.45 flow, for 150mm BEH C18 column i always recommend min. 50C to avoid high backpressure. I think you will have around 12000-13000 psi pressure

    At this time i'm not going to suggest any change in LC gradient, but can suggest you some tips

    change column temp. to 50C,

    solvent for injection (e.g 85/15 MPA)

    remove vanguard and connect column directly, check column inlet/outlet end-fittings (ferrule and tube-end distance=2mm)

    equilibrate for 15-20min then inject blank first (no need to wash with water for 500min, just 50/50 for 15min at end of the day)

    inject filtered/freshly prepared sample or mix standards (try low volume first i.e 1-2 uL)

    I hope it helps


  • RD

    I suspect that strongly retained "stuff" has polluted the inlet of the column and is binding to adsorption sites.  Thus, the analytes of interest don't "stick" to the column immediately upon injection but  travel a bit down the column before they are retained.  Of course, this is not a perfect process and some molecules stick sooner than others, the result of which is analyte band spread.  When elution proceeds, the molecules that "stuck" first elute first, followed by the others which results in split peaks.

    Another possibility is a small void at the head of the column.

    Either way, I suspect the column can not be regenerated.

    If the first scenario is in play, you may wish to replace your VanGuard more frequently.  The purpose of a guard column is to trap strongly retained sample components and prevent them from reaching & fouling the column.  However, the capacity of a guard column is very low and the trick is knowing when to change it.  If you wait until you see a degradation in your separation, it may be too late as this means that the stuff you were hoping to keep off your column has gotten through.  The best approach for guard column replacement is to monitor the number of injections and replace it when it still has some capacity to retain the "junk".  In your case, if you are getting 300 - 500 injections before seeing a degradation in your separation, you may want to replace the guard column at 200 injections.

    More effective sample cleanup is another option but you'll have to weigh the cost/benefits versus more timely guard column replacement.

    If you have access to local Waters support, I suggest you contact them for assistance.



  • jturpin

    Does your chromatography deteriorate prior to your column rinse/wash procedures?  It typically isn't wise to expose C18 columns to 100% aqueous conditions, particularly for long periods.