Dwell volumes differences

Users sometimes need to run the exact same method on a conventional HPLC system as well as a UHPLC system. In this case, the dwell volume difference must be taken into consideration during the method transfer. As shown in Figure 3.21, when the same gradient was used on both systems, the retention time for the first peak was delayed about 1.3 min due to the 0.88 mL greater dwell volume of the HPLC system. When a 1.3 min hold was applied at the beginning of the gradient for the UHPLC gradient profile, the retention times for the peaks are much more comparable. 

Manufacturers publish their product s performance characteristics as specifications, which are often used by the customer for comparison during the selection process. Table 1 shows the specifications of an Agilent 1100 Series Quaternary Pump, which is quite representative of other high-end analytical pumps. Note pulsation is particularly detrimental to the performance of flow-sensitive detectors (e.g., mass spectrometer, refractive index detector). Differences in dwell volumes and composition accuracy between HPLC systems might cause problems during method transfers. 

Isocratic method. Whenever possible, use isocratic HPLC condition, as this is affected less by the variation in flow rate, temperature, and dwell volume. If gradient HPLC conditions have to be used, a simple linear gradient is preferred over multistep gradients. Complicated gradient conditions are more susceptible to differences between HPLC instruments (e.g., flow rate, dwell volume). 

The volume between the point at which solvents are mixed and the beginning of the column is called the dwell volume. The dwell time, tD, is the time required for the gradient to reach the column. Dwell volumes range from 0.5 to 10 mL in different systems. For Figure 25-13. the dwell volume is 5 mL, and the flow rate is 1.0 mL/min. Therefore, the dwell time is 5 min. A solvent change initiated at 8 min does not reach the column until 13 min. 

Differences in dwell volumes between different systems are an important reason why conditions for gradient separations on one chromatograph do not necessarily transfer to another. It is helpful to state the dwell volume for your system when you report a gradient separation. One way to compensate for dwell volume is to inject sample at the time fD instead of at t = 0. 

What is the difference between dead volume and dwell volume How do each of these volumes affect a chromatogram ... 

Other considerations include differences in dwell volumes from the different HPLC systems. The dwell volume should be determined for all the systems in the laboratory and based on these determinations, this should be factored into the calculation of the equilibration time. For example, if the maximum dwell volume of all the systems in a particular laboratory to which the method is transferred to is 2mL and you are running on an instrument at 1 mL/min that has a dwell volume of 1 mL, then you should add an extra minute of equilibration time. This becomes extremely important during method transfers where the instruments in the receiving laboratory may be different. 

Gradient methods in HPLC depend on the dwell volume, Vd, i.e., the volume between the point of mixing eluents A and B and the column inlet, including the loop of the injector. This volume differs from instmment to instmment, so that a method can be transferred only if this volume is well defined. When there is a significant difference in dwell volume between the two systems, retention times and resolution are dramatically different. So one must be state in which range of the method is still valid. 

Method transfer from one laboratory to another one (from development to routine, from manufacturer to customer and so on) can be difficult because HPLC separations are influenced by many parameters. At the new place the resolution of a critical peak parr can be worse than required or the whole chromatogram looks different. In order to prevent such surprises, whenever possible, it is necessary to describe every detail of the method column dimensions, stationary phase (maybe even the batch number), preparation of the mobile phase (the order the individual components are mixed can be critical), temperature, volume flow rate, extra-column volumes of the instrument, the dwell volume in the case of gradient separations (see Section 4.3) as well as detection and integration parameters. It can be useful to designate alternative stationary phases, i.e. materials which are located close to each other in representations such as Figure 10.9. The true temperature in a column oven must be verified because it can differ from the requested one Method transfer also includes the detailed description of sampling, storage and sample preparation. 

Major manufacturers of HPLC instruments include Waters, Agilent (formerly Hewlett Packard), and Shimadzu, PerkinElmer, Thermo, Beckman, Varian, Hitachi, Jasco, Dionex, Gilson, Scientific Systems (SSI), and Isco. The Internet addresses of these companies can be found in the reference section. HPLC is a mature technology and most manufacturers have highly reliable products with sufficient performance and feature sets to be competitive in the market place. However, there can still be significant differences between the vendors on these performance characteristics on systems (dwell volume, dispersion), pumps (low flow, seal life), autosamplers (carryover, speed, sample capacity, minimum sample volume), and detectors (sensitivity, gradient baseline shift). 

Gradient profile or solvent composition deviates from the written method —> Check the pump valves for leaks. A common cause are crystallized salts in the valves. The remedy is simple flush with salt-free solvents before the pump is switched off. Caution different HPLC systems have different mixing and dwell volumes. With gradient separations, check the equilibration time between injections and prolong it if necessary. Check the gradient method. 

Even if it is slowly becoming boring, this can but need not be. Bear in mind that a different Dwell Volume may result in a change in the chromatogram. 

To avoid difficulties when a gradient HPLC method is transferred between the instruments with different Vd values and to improve the precision of predictive calculations of the gradient elution data, the correction for the gradient dwell volume should be accounted for in calculations, using equations such as Eqs. 4, 6, or 8, as appropriate. ° ... 

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