Determining dwell volume for UPLC

How have other people determined the dwell volume for their Acquity systems? Should I base the flow rate on a minimum backpressure or just choose a default flow rate? What about the ramp for the gradient? One reference I read mentioned a 20 minute gradient. How should I address the two different slopes observed in the data during a gradient run? Initially, it rises steeply, then flattens out a bit and remains steady for the remainder of the gradient change. Which slope do I draw the tangent on?

Answers

  • The flow rate is not terribly important. I would probably select 0.5 mL/min for the ACQUITY system. The ramp of the gradient as well as the concentration of the acetone should be such that you can clearly see the slope. The standard approach uses 0.1% acetone. Also, the gradient run time is not terribly important as long as you get a nice slope from which you can determine the 50% point. We have occasionally included a small gradient delay in the gradient table, and we then subtract the programmed gradient delay from the measured gradient delay.

    The observation that you have a steep rise at the beginning may indicate that you may have a problem with the second pump.

  • Greetings,

    First off, I have performed delay and dwell volume measurements many times on an ACQUITY UPLC system. With the standard 10 uL sample loop and 50 uL mixer installed, a perfectly acceptable estimate is 100 uL for total dwell volume. This estimate should be within +/- 10 uL of your system's, actual, volume. Just the other day we measured one of our systems at 92 uL.

    For the sake of this discussion, let's define delay volume as the initial "lift off" in a gradient or change in mobile phase composition. Further, let's define dwell volume as mid point (or inflection point) of any compositional "step" change. I prefer to measure the dwell volume as I believe it more accurately models the effective solvent change on the column. The "lift off" definition has the problem that the presentation is, usually, graphic and the interpretation tends to be subjective. It depends a great deal on the level of magnification used to examine the data and the "threshold" value chosen to define a departure from baseline.

    In most cases, the measurement of either delay or dwell volume will have similar experimental set ups. Pump A will be primed with some solvent and Pump B will be primed with the same solvent containing a spike that is recognizable by the detector in use. The column will be removed from the system and a union (or small restrictor) is used in place of the column to connect the solvent stabilizer to the inlet of the detector. For example, on a UV based system, I will often use 90% water and 10% ACN as the common solvent. Spiking the "B" solvent with 12 mg/L of caffeine will yield a signal of, approximately, 0.5 AU (273 nm) at 100% B. One of the attached procedures uses methanol with propylparaben as the spike. The principles are the same regardless of the solvents and absorbing compounds used. Often a short length of 0.0025" PEEK tubing must be inserted in place of the column to generate some back pressure. This tubing is included in the measurements. So, it should be short enough for the volume to be, essentially, negligible. Alternately, a small restrictor of fused silica tubing can be created to perform the same function with even less volume. The back pressure should be above 300 psi just to give the check valves something to work against. Flow rate is not, extraordinarily, important, but lower flow rates will offer greater resolution for the measurements.

    Once the system is set up, there are several approaches that will all arrive at very similar results. These may use linear gradients or step responses. I have attached a couple of documented variations. One procedure is to perform a step gradient and allow Empower to create a derived channel for the first derivative of the step response in the absorbance. I then use the apex of the first derivative peak (inflection point) as the reference for the dwell volume. The inflection point will be quite close to the mid point of the step response which is another valid reference of the dwell volume. One advantage of using longer linear gradients to calculate dwell volume (also attached) is that a back regression along the linear response will intersect with the level of the initial baseline. This intersection point is a very good, and less subjective, estimation of the delay (lift off) volume of the system.

    I have also attached a procedure that uses chromatographic information to determine dwell volume. This approach has several advantages and disadvantages. It does not require a specific set up nor does it require the use of a spiked solvent. It does use a column with common chromatographic solvents and, so, more closely reflects actual chromatographic practice. However, it does require some a priori knowledge about the probe compounds and the solvents in use. It is based on the principle of comparing the retention time of an unretained peak against the retention of a r...

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