High pressure issue with Acquity BEH amide column

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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I am using Waters Acquity BEH Amide column (1.7um x 2.1um x 150mm) from the past 4 months (around 80-100 injections) with it's pre-column for sugar analysis. I generally centrifuge my samples and filter it using 0.22u nylon syringe filters. For initial 2-3 months pressure was all fine but now I am facing a very high pressure issue of more than 800bar with 0.1ml/min flow rate at column temperature 50degree. I am sharing my column washing and sample running conditions with you guys.

Washing condition: 50:50 acetonitrile: water, column temperature- 50 degree, with gradual increase in flow rate from 0.1 ml/min to 0.4 ml/min.

Mobile phase composition for sample analysis:
Solvent A = 50:50 Acetonitrile : water with 0.2% triethylamine
Solvent B = 95:5 Acetonitrile : water with 0.2% triethylamine
Flow rate = 0.1 mL/min at column temperature 45 degree.
I am running a gradient method.
I am detecting my samples using ELSD and injection volume is 10uL.

Column details: Waters Acquity BEH Amide column of particle size 1.7um, pore size 130 Angstrom, inner diameter of 2.1 mm and length of the column is 150 mm. The guard column which I am using is Waters Vanguard pre-column of particle size 1.7um, inner diameter 2.1 mm and length is 5mm.

Kindly suggest to me regarding the high pressure issue which I am facing. Any suggestions would be greatly appreciated.
Thanks in advance!
I use the same column but only 100mm. When i run my gradient 95acn to 50acn in water and 10mMAmAc at 0.6ml/min over 8min pressure max is at 7500psi. When i flush buffer out after the run I use 80%acn 20%h20 no buffer at 0.1ml/min, then prrssure keeps increasing and is 11000psi after 20min despite the low flow. At first i thought something was wrong but when I run my method gradient with buffer evertything works fine again. To me it seems that the amac buffer in my method helps keep pressure down but it may also just be the water content, I have not tested enough. I was quite suprised at how sensative the pressure was to small changes in mobilephase with huge changes in pressure.

Sorry that this does not answer you question, just wanted to share that i found this column to work well but was very sensative to mobilephase composition.
per_oxid: Methanol and Acetonitrile mixtures with water (or buffer) are commonly used in chromatography so it is key to understand their properties. For those new to liquid chromatography, mixtures of Water/MeOH and Water/ACN exhibit unusual pressure curves, which are normal. Pressure will max out at 50/50 (%) with Water/MeOH and around 30/70 with mixtures of Water/ACN. The pressure curves are not linear. This is important to visualize and test when learning LC method development as it is due to a reaction between the two liquids (BTW: with methanol/water, it is exothermic, you can feel the heat when mixed. It is endothermic with ACN/water). Viscosity properties alone do not always account for pressure changes.

As you change the concentrations of mobile phase containing either of these two liquids, you should see and learn their characteristic pressure profiles. Making note of these normal changes is important so as to not confuse them with obstructions or clogs in the system.

btw: all columns packed with tiny 1.7u particles will show "sensitivity" to changes in mobile phase composition (esp water based) as it requires tremendous force to move liquids past and/or through those very tiny particles. Large back-pressure changes are often normal.

More Info: "HPLC Solvents, Acetonitrile and Methanol, Key Differences and Properties"; https://hplctips.blogspot.com/2017/09/h ... hanol.html
Thanks, I am quite inexprienced so much to learn.

Is it likely that buffer composition can influence the back pressure significantly also?
Mobile phase related back-pressure changes are mostly due to changes in viscosity or from a change in the composition of the mobile phase.Please refer to a general solvent table for physical properties for more info. LINK: https://www.hplctools.com/lcsolvent.htm

Adding a "salt" to a solution usually does not alter this, but I suppose their may be cases where it could as there are millions of possible mixture combinations. In general though, no. The PRACTICAL concern we have with the use of buffers is that of clogging or precipitation due to poor mixing, lack of filtration or precipitation from mixing non-miscible solutions together (or introducing a buffered solution to an organic solvent that is at high concentration resulting in the buffer failing out of solution and clogging the line, at the point of injection, which is a common newby error).
Thanks for explaining to me.

Sorry for hijacking thread and the original question.
According to my experience, the pressure is increasing quickly during sugar analysis on BEH Amide columns. But the column can be regenerate flushing with acidic solution (0.1% formic or acetic acid). It seems the packing swell in alkaline eluent.
Is there another way to explain this phenomenon? A particular equation may be an optimal way. I'm not very clear on the issues and metrics.
color tunnel
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