Tuesday, September 28, 2010
Since the pump has a defined dead volume, some time delay to reach the half maximal signal obviously occurs. Assume that channel A underdelivers (an internal leak) compared to channel B. As a result, less mobile phase will be delivered by A and, therefore, half maximal of the signal will occur earlier, and the V50 value with decrease (gradient slope becomes slightly steeper than expected). A minor leak in channel B will cause a delay in the half maximal, and the V50 value will be larger. Differences in the gradient slope, when comparing prior and newly acquired UV traces, might not be indicative of a leak but are more likely due to imprecision of the B preparation (% acetone in solvent) and reductions in UV lamp efficiency.
In contrast, the V50 value is invariant regardless of variability in the signal maximum value. Once V50 is established, it can be used as a reference value for a given pump (a gradient fingerprint) under current hardware configuration. Of note, this value is pressure, temperature, and hardware dependent. If a 5% difference is observed in V50 compared to a prior measurement, but the diagnostic test is completed successfully, the authors recommend swapping outlet check valves A and B and repeating the test. To reemphasize, an increase in V50 implies underdelivery of channel B solvent, while a decrease in the V50 value implies underdelivery from channel A. After switching the outlet valves and rechecking the gradient run, increases in the newly obtained V50 value indicate an outlet check valve malfunction; if the V50 value remains unchanged, it is indicative of inlet valve failure in the suspected channel.
Tight control of the V50 value is important to ensure faster hardware diagnostics and troubleshooting, since built-in diagnostic tests are unable to identify minor leaks. Progressive drift of the analyte retention time may be a clear indication of such hardware-related minor leaks. For long 40–60 min gradients (used frequently in proteomics analysis), the performance of each solvent delivery channel should be evaluated to exclude impact of the hardware on retention time instability. If the V50 value has not previously been determined or cannot be routinely monitored, a diagnostic and troubleshooting protocol becomes much more complicated and prolonged. Hence, use of a high-pressure liquid flowmeter may be a faster but more costly alternative for control of system performance.
- Dolan, J. Check valve problems. LC·GC North Amer. 2006, 24, 132–8.
- Rogatsky, E.; Braaten, K.; Cruikshank, G.; Jayatillake, H.; Zheng, B.; Stein, D.T. Flow inconsistency: the evil twin of column switching— hardware aspects. J. Chromatogr. A 2009, 1216, 7721–7.
- Dolan, J. Dwell volume revisited. LC·GC North Amer. 2006, 24, 458–66.
Dr. Rogatsky is Senior Scientist, Clinical Chemistry R&D, and Dr. Stein is Director, Biomarker Analytical Resource Center, Analytical Core Lab, Einstein-Montefiore Institute for Clinical and Translational Research, Albert Einstein College of Medicine, 1300 Morris Park Ave., Golding Bldg., Rm. G02, Bronx, NY 10461, U.S.A.; tel.: 718-430-3391; fax: 718-430-8998; e-mail: firstname.lastname@example.org. Mr. Gallitto, of John F. Kennedy High School, Bellmore, NY, was Dr. Rogatsky’s summer student.