Dwell volume measurement

In 2001, Valko et al. reduced the column length to only 50 mm and increased the flow rate to 2mLmin [42]. The gradient time was diminished to 2.5 min with a gradient cycle time of 5 min. Measurement of CHI and evaluation of log P were excellent with a 3-fold improved productivity. In these conditions, the system dwell volume (Vd) becomes essential and only dedicated chromatographic devices with Vjy lower than 0.8 mL can be used [42]. Special attention should be paid to the injected volume, which must remain lower than 3 pL to avoid any overloading or extra-column volume contributions. 

FIGURE 7 The absorbance gradient trace used to measure the system dwell volume of a Waters Alliance system. Inset shows the intersection point marking the gradient onset. 

The system dwell volume can be measured using a UV detector as follows ... 

Measure the intersection point of the extrapolated absorbance trace with the baseline to record the dwell time, as shown in Figure 4.5. Multiply dwell time by flow rate to obtain the dwell volume. 

Required for systems using Fast LC or <2-mm i.d. Dwell volume Perform linear gradient with 0.1% acetone/water in 10 min at lmL/min without column and measure the intersection of baseline with extrapolate gradient profile (see Fig. 4.5) [Pg.228]

Measure Dwell Volumes of HPLC and UHPLC Systems... 

To calculate holding time, the dwell volumes of both the HPLC and UHPLC systems need to be determined. There are several ways to measure the dwell volume. The following is an example for an HPLC system. 

Prepare 1% acetone in water as mobile phase B, and water alone as mobile phase A. A column under measurement is removed from the line, and the tubing is connected with a zero dead volume union. A gradient of 10 min is run from 100% A to 100% B at the flow rate of 2 mL/min and held for 10 min at 100% B with detection wavelength at 260 nm. The difference between 5 min and the time at half height between the initial baseline and the plateau times the flow rate is the dwell volume for the HPLC system. The typical dwell volume for most HPLC systems is approximately 1 mL. 

The conservation of momentum or Newton s second law applies to a particle or fixed set of particles, namely a system. The velocity used must always be defined relative to a fixed or inertial reference plane. The Earth is a sufficient inertial reference. Therefore, any control volume associated with accelerating aircraft or rockets must account for any differences associated with how the velocities are measured or described. We will not dwell on these differences, since we will not consider such noninertial applications. 

Fluorescence Correlation Spectroscopy and Fluorescence Burst Analysis. Several nanoscopic chemical imaging approaches work very well for measurements of chemical kinetics, interactions, and mobility in solution. Fluorescence correlation spectroscopy (FCS) measures the temporal fluctuations of fluorescent markers as molecules diffuse or flow in solution through a femtoliter focal volume.54 Their average diffusive dwell times reveal their diffusion coefficients, and additional faster fluctuations can reveal chemical reactions and their kinetics if the reaction provides fluorescence modulation. Cross-correlation of the fluorescence of two distinguishable fluorophore types can very effectively reveal chemical binding kinetics and equilibria at nanomolar concentrations. 

Total system Total extra-column volume Connections Gradient dwell time Keep small Should be smooth, use the same measure (metric or inch) Keep short by keeping the system volume before the mixing chamber small... 

The dwell time is best measured under realistic conditions, for example, with %B = 0-100% in 15 min. Instead of the column a capillary with preferably small internal diameter is used, in order to build up a realistic back pressure while increasing the volume as little as possible. Eluent A is water, eluent B water with a few parts per thousand of acetone. When the UV absorbance is set to zero, eluent B should have a maximum absorption of slightly less than 1 AU. The gradient is... 

In contrast to conventional NMR measurements, in flowing systems the sample has a defined dwell time x in the NMR coil. This dwell time is defined by the ratio of detection volume to flow rate and affects the relaxation times Tj and Ti as well as the signal intensity and signal half width ... 

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