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HPLC Troubleshooting: Precipitation of Buffers in Gradient Elution Mode

Team TFS
Team TFS

Have you ever seen pressure fluctuations or retention time shifts in your HPLC that go away as suddenly as they appeared? Do you use buffers in your HPLC application? If you answered yes, read this blog post for tips on how to overcome and prevent the issue in the future!


HPLC Troubleshooting Tips.jpgThe problem of salt precipitation in HPLC applications is common with phosphate buffers or biological buffers (TRIS, HIPS, MOPS, etc.) in a solvent mix of high or rapidly increasing organic content. 


Knowing the solubility of a given buffer in the respective mobile phase composition is a simple way to overcome this issue. The precipitation point of phosphate buffer salts are established with regard to the percentage of acetonitrile, methanol or tetrahydrofuran in the mobile phase composition.


A good rule-of-thumb to go by is:

  • Phosphate buffers start to precipitate at 80% methanol
  • Potassium phosphate buffers start to precipitate at 70% acetonitrile
  • Ammonium phosphate buffers begin to precipitate at 85% organic content in the mobile phase


Hence, you should always stop a gradient elution method at that specific percentage of organic content mentioned above to prevent the precipitation of phosphate buffer.


However, it is not this old story I want to share here. From my point of view, the main takeaway is written between the lines: Phosphate buffers precipitate when getting in contact with 100% organic solvents — which is exactly what may happen in your LC pump during the solvent aspiration and proportioning phase.


Buffer precipitation in LPG pumps


In a low-pressure gradient (LPG) pump type, a proportioning valve is used to aspirate the desired solvent mix.


If 50% solvent A and 50% solvent B are programmed in the method, for example, the proportioning valve “picks” discrete fractions of solvent A and B to fill the piston chamber with the desired mixture.

HPLC Gradient Pumps.jpg


However, these fractions themselves consist of 100% A or 100% B, respectively, with a border area in-between these two solvent fractions.


By the laws of diffusion, the fractions become more and more indiscrete, but the phosphate buffer solution first mixes with 100% organic content in the border area between both fractions.


Both fractions then converge, but the organic content stays higher than 80% for  enough time that buffer salts begin to crystallize — as we just learned that buffer salts will precipitate under these high organic eluent conditions.


The slower the flow rate, the more time the solvents have to build up a bigger border area, which means more buffers salt out. Typically, the proportioning valve is majorly affected by buffer crystals — as well as the inlet check valve, piston and seals, and outlet check valve — until the solvent fractions are adequately mixed in the mixer device.


Buffer precipitation in HPG pumps


In a high-pressure gradient (HPG) pump type, a flow stream of solvent A converges with a flow stream of solvent B in a Tee-piece after the two pump heads.


This mixing type means that 100% A continuously meets 100% B in the Tee-piece. However, the mixer device is typically placed directly after that T-piece, so buffer salt precipitation may occur for a short time in the Tee-piece and purge unit.


But unlike LPG pumps, no other parts, like the piston seals or check valves, are affected.


Best practices to prevent buffer precipitation 


If you are not sure exactly when to cut off the increasing gradient, you can incubate the buffers with the various gradient mixtures for 10 minutes, then check for precipitates.


At the very least, you should filter the mixture to see if there are any crystals. But a spectral measurement of the turbidity of the buffer-eluent mixture should be carried out with a UV-Vis spectrophotometer to confirm the presence of minute turbidities or precipitates.


With this measurement, long-term effects can be prevented, such as the precipitation of tiniest amounts of buffer, which can grow from a very small layer to a veritable deposit on surfaces in your HPLC pump.


How to overcome buffer precipitation


If buffer salt shows up, the organic content is too high and must be reduced until no crystals are seen anymore. This way, you ensure that buffer does not crystallize or precipitate out of solution when applying the gradient in the LC system.


As a best practice, during method development, use a mixture of buffer solution and the highest organic content being applicable (70 - 80% organic solvent) for channel B (hence, the "organic solvent") while channel A delivers 100% buffer solution (hence, the "aqueous solvent").


This way, the buffer solution never encounters 100% organic content in the border, even when the pump delivers 100% solvent B. This approach should help prevent all bad outcomes to your data and instrument.


After applying gradients with buffer and organic solvents, you should always flush the column with the same ratio of water and organic before preparing for storage. Never leave a column or HPLC system with buffer inside the components!


Bringing it all together


Extra care must be taken in gradient elution mode with buffers, which means do not exceed a certain percentage of organic content in the mobile phase. If you need a high organic eluent composition, then lower the buffer salt concentration to prevent buffer salt precipitation and retention time shifts.






1 Comment
Team TFS
Team TFS

Nice blog Michael and yes, Chemistry comes first before Separations to happen😊. However legacy Pharmacopeia methods do use such high salt concentration buffers and robust pumping technique is the only solution. What say?