OBJECTIVE or GOAL
Determine the system dwell volume to aid in method transfer from one system to another in which the method is using a gradient. (The same measurement technique is used for the original and the target system.)
- ACQUITY UPLC
- Method transfer
- If necessary, add an ultraviolet detector to the system.
- Replace the column with a capillary restrictor. (Restriction should maintain a minimum back pressure of 2000 psi for an ACQUITY system.)
- Prepare Solvent A (100% LC/MS-grade water) and Solvent B (100% LC/MS-grade water with 10 mg/L caffeine).
- Specify a flow rate of 1.00 mL/min.
- Solvent manager gradient at initial conditions is 100% Solvent A.
- Solvent manager gradient at 5 minutes is 100% Solvent A.
- Solvent manager gradient at 25 minutes is 100% Solvent B.
- Solvent manager gradient at 30 minutes is 100% Solvent B.
- Specify the channels to collect the absorbance data, including a trace of the solvent composition percent B.
- Set the ultraviolet detector wavelength to 273 nm.
- Run the method.
- On the chromatogram, do as follows: Determine experimental t1/2 – the absolute time at which the absorbance trace reaches 50% of the absorbance corresponding to 100% B.
- On the chromatogram, do as follows: Determine programmed t1/2 – the absolute time at which the programmed gradient reaches 50%. For the specified gradient, the time is 15 minutes.
- Calculate the time corresponding to dwell volume as follows: tD = t1/2 experimental - t1/2 programmed
- Calculate the dwell volume as follows: VD = tD x F, where F = flow rate in mL/minute.
For additional resources, see Dolan, John W. Gradient Elution, Part IV: Dwell Volume Problems. LCGC North America [Internet]. 2013 Jun.
1. [cited 2018 Oct]; 31(6). Available from http://www.chromatographyonline.com/gradient-elution-part-iv-dwell-volume-problems-0