Hold-up volume

H = gas phase hold-up (volume of gas/volume of gas -I- liquid), dimensionless... 

At the end of the incubation period the fermentation culture mixture is adjusted to pH 2 with concentrated hydrochloric acid, the solid material present is removed by filtration, and the filter cake is washed with water. The washings are combined with the main filtrate, adjusted to pH 7.0, and 15.5 liters of the filtered culture liquid is introduced into a columnar exchanger d /a" i.d.) packed with 380 ml of carboxylic acid resin which has been preliminarily washed in succession with two liters of an aqueous solution of 37.5 grams of sodium hydroxide and with two liters of water. The column containing paromomycin is washed with two hold-up volumes of water and is eluted with 0.5 N hydrochloric acid. 

Horizontal or vertical tanks are used, depending on the size and duty. Where only a small hold-up volume is required this can be provided by extending the column base, or, for reflux accumulators, by extending the bottom header of the condenser. 

The maximum injection volume depends on the volume of the sample loop in the injection valve. The reproducibility of manual injection depends on the skill of the operator. The use of a small sample loop and an overflow injection of the sample solution so that the loop is fully flushed with sample are basic requirements for quantitative analysis. The highest injection reproducibility can be obtained by an auto-injector with a fixed sample loop. The smallest reasonable injection volume is 1 (A. A nl-scale injection valve can be constructed however, the memory effect at the surface of contact parts affects quantitative analysis compared with the use of a /d-scale injection valve. For a semi-micro system, a low hold-up volume injection valve is desired. The minimum injection volume is 80 nl. For a preparative-scale injection, the sample loop can be easily replaced with a larger-volume loop, such as a 200 jA, instead of the standard 20 /A loop. 

If the correctly sized flow cell and connecting tubing are not used, the high efficiency of a column or high theoretical plate number columns cannot be effectively used. The detector cell volume contributes hold-up volume. The larger is the cell volume, the greater the peak broadening. The cell volume... 

Gritti, F. and Guiochon, G, Influence of the pressure on the properties of chromatographic columns, iii. Retention volume of thiourea, hold-up volume, and compressibility of the C-18-bonded layer, J. Chromatogr. A, 1075, 117, 2005. 

N is the average number of theoretical plates determined under isocratic conditions in mobile phases within the gradient concentration range is the hold-up volume of the column... 

RPC separations of ionic samples usually require ionic additives to the mobile phase, which may cause problems in HPLC/MS operations. Completely ionized solutes are much less retained than the corresponding uncharged species and elute close to the column hold-up volume, often as asymmetric or even split peaks. Weak acids are eluted in order of decreasing constants and weak bases... 

FIGURE 5.4 Effect of the gradient dwell volume, V7>. the elution volume, Vj, in reversed-phase chromatography. Solute neburon, retention equation (Equation 5.7) with parameters a=A, m = 4. Linear gradients 2.125% methanol/min (a) from 57.5% to 100% methanol in water in 20min ( i = 50) (b) from 75% to 100% methanol in water in 11.75 min (k = 10). Vg uncorrected calculated from Equation 5.8, Vg + Vg, Vg, added to Vg uncorrected, Vg corrected calculated from Equation 5.21. (A) A conventional analytical C18 column, hold-up volume y ,= ImL flowrate l.OmL/min. (B) A microbore analytical C18 column, hold-up volume y = 0.1mL flow rate 0.1 mL/min. 

If a column with different diameter, but the same length is used, the column hold-up volume changes in proportion to the second power of the column diameters. To keep the separation time constant, the flow rate of the mobile phase should be adjusted in the proportion of changing V , see Equation 5.28. Another possibility of compensation is by adjusting the gradient time to keep the ratio VJitQ-FJ constant [8,9]. 

The effect of the dwell volume on the retention times of analytes increases with decreasing retention factor at the start of gradient elution and with increasing ratio VpIV, and becomes very significant in the instrumental setup with the dwell volume comparable to or larger than the column hold-up volume, which is more likely to occur in micro- or in capillary LC than in conventional analytical LC (see Figure 5.4) [12]. 

Figure 7.7 CCC chromatogram of the 4-nitrobenzoic elution. Hydrodynamic CCC column 53 mL rotor speed 830 rpm liquid system [C4CiIm][PF5]-acetonitrile-water 40/20/40 %w/w stationary phase IL-rich lower phase, Vg = 38 mL mobile phase acetonitrile-rich upper phase, f = 1 mL/min in the tail-to-head direction injection volume 500 pL of a mixture of 6 ppm of 4-nitrobenzoic acid (retention time 18.595 min) + 2.5 ppm phenylalanine (retention time 15.02 min) as a hold-up volume tracer UV detection 254 nm.

For a highly concentrated product, a large system hold-up volume increases the potential for product loss. For concentration/diafiltration operations, scale-up may require re-optimization of process parameters, especially if membrane capacities are changed. However, every effort should be made to keep recirculation flux constant with similar inlet and outlet pressures. 

Elsewhere the coefficient K (or the values of k) of the compounds does not change from one column to another, therefore the retention volumes, Vr = Fm(1 + k), remain the same as the hold-up volumes. The retention volume of the narrow column (0.3 mm) is therefore equal to that of the broader column (4.6 mm) x 0.0043 (a factor of 235 times less). 

Hold-up volume. V. Volume of mobile phase from the point of injection of the sample and the point of detection. In GC it is measured at the column outlet temperature and pressure and is a measure of the volume of carrier gas required to elute an unretained component (including injector and detector volumes). 

Reproducibility of peak height is also quite dependent on the reproducibility of the sample injection. This is especially important on early and thus normally quite sharp, narrow peaks. On such early peaks the width of the peak is controlled more by the injection time rather than the chromatographic process. A fraction of a second increase in injection time can double the width of these peaks and reduce peak height 50%. The peaks most subject to error in peak height measurement from injection problems are those with retention volumes between one and two times the hold-up volume of the column. Peaks beyond five to ten times the hold-up volume are negligibly affected by injection technique. 

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