Delay volume

It is not common practice, but quite viable, to inject the flow marker at a predetermined volume offset from the polymer injection. This can be accomplished either with a second injection at a predetermined time into the run or by having two coordinated injection valves separated by a fixed volume of tubing. This approach can avoid many of the pitfalls described earlier. However, the mathematics of this correction is slightly different from that for a coinjected marker. The proper correction for the delayed volume injection is shown in Eq. (3) ... 

FIGURE 6.15 Evaluation of delay volume in a /rPLC system equipped with a cartridge with a per-column internal cross section of 0.5 mm. 

Monoliths Low backpressure, suited for conventional HPLC Higher separation efficiency by column coupling Rugged against delay volume and extra-column band broadening fast column re-equilibration Reduced maintenance on pumps and injector seals Reduced need for sample pre-treatment... 

However, those who have worked with smaller diameter columns have often experienced lower performance and other difficulties. This is primarily due to extra-column effects the bandspreading in the injector, detector and tubing, or the gradient delay volume of the instrument. Troubleshooting guidelines for sorting out the causes of these difficulties are available in Reference f. With proper care, 2-mm columns can be run on a standard modern HPLC instrument with few difficulties. Smaller i.d. columns require special instrumentation. 

Delay volume is the volume found between the time the gradient is started and when it reaches the column. The corresponding time is the delay time tD (see earlier). By running a gradient from pure methanol to methanol containing 100 ppm of UV-absorbing toluene, the delay volume can be measured. 

Which type of instrument should be used Is the method for one specific model from a specific vendor or should it be used by all models from all vendors This is especially important for HPLC gradient methods, because different instruments may have different delay volumes, ranging from 0.5 up to 8 ml. This can have a tremendous impact on the separation and elution order of the compounds. 

Equipment with different characteristics (e.g., delay volume of an HPLC system) Variations in material and instrument conditions (e.g., in HPLC, mobile phases composition, pH, flow rate of mobile phase)... 

The slope of versus retention volume as well as the polydis-persity for narrow MWD samples was sensitive to the choice of delay volume as was reported by Shortt and Wyatt (iO). In fact, Shortt recommended that one approach to determine the correct value of the delay volume is to adjust its value until an acceptable slope of the radius of gyration (Rg) versus is obtained, because is dependent on the concentration whereas the radius is not. Accordingly, the delay volume used was 0.312 mL. 

For individual samples of pullulans and dextrans, the conformational coefficients of the Rg versus My, plots were 0.45 and 0.32, respectively, and are in reasonable agreement with the values for the whole sample as displayed in Table I. These are sensitive to the value of the delay volume as described earlier. Minimizing the delay volume and using bovine serum albumin, which is monodisperse, to determine the delay volume may be helpful. 

Reproducibility, as defined by ICH, represents the precision obtained between laboratories with the objective of verifying if the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analyzing aliquots from homogeneous lots in different laboratories with different analysts, and by using operational and environmental conditions that may differ from, but are still within the specified, parameters of the method (interlaboratory tests). Various parameters affect reproducibility. These include differences in room environment (temperature and humidity), operators with different experience, equipment with different characteristics (e.g., delay volume of an HPLC system), variations in material and instrument conditions (e.g., in HPLC), mobile phases composition, pH, flow rate of mobile phase, columns from different suppliers or different batches, solvents, reagents, and other material with different quality. 

Gradient systems are tested for the accuracy and precision of the gradient profile (Figure 25.1), the delay volume, and the mixing volume with channels A and B. 

If the test needs to be repeated later on it is necessary to use the original system configuration because the delay volume of the system is calculated from the gradient profile. 

Also known as gradient delay volume, system dwell volume is the liquid holdup volume of the HPLC system from the point of solvent mixing to the head of the column. This includes the additive volumes of the injector, the sample loop, all fluidic connection tubing, and any internal pump volumes of a low-pressure mixing system. The typical dwell volume of a modem HPLC is 0.5-2 mL, but can be as high as 5-7 mL in older systems. 

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