Dwell volume, gradient elution

In gradient-elution NPC where Equation 5.10 applies, the elution volumes affected by the gradient dwell volume can be calculated using Equation 5.22 [57] ... 

Figure 5.4A and B compares the uncorrected elution volumes, the elution volumes corrected by simple addition of and the elution volumes calculated using Equation 5.21 in RP gradient elution on a conventional and on a micro-bore C18 column. The effect of the gradient dwell volume is more important for separations on short columns and especially on narrow-bore columns with i.d. 2 mm. 

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. 

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]. 

The contribution of the initial isocratic elution step to the total retention volume of the solute is equal to Fr. The part of the column hold-up volume F ,i through which the solute has migrated at the end of the isocratic step, i.e., at the time when it is taken by the front of the gradient is related to the total column hold-up volume in the same proportion as the gradient dwell volume is to the (hypothetical) elution volume from the column under initial isocratic conditions with the retention volume of the solute, Ai, and for the gradient-elution step thus remains only available the hold-up volume F ,2 = F , - F,n. ... 

Vd is the so-called gradient dwell volume [i.e., the volume of the mobile phase contained in the instrument parts (mixer, filter, and tubing) between the pump and the column]. In an ideal case, linear concentration gradients in RPLC correspond to linear solvent strength (LSS) gradients according to the model developed by Snyder and Dolan " hence, Eq. 4 describes the retention data in LSS gradient elution. 

Gradients can be used with equal ease for either ionization technique. In most cases, cycle time for system reequilibration (determined by the overall system dead volume) provides the practical limitation to their usage. If, for example, a particular HPLC pump/autosampler combination has 1.0 mL of dead volume (or dwell volume, the volume of all plumbing between where the solvents are mixed and the column head) and is operating at a flow rate of 1.0 mL/min (typical for APCI), then the lag time between when the gradient is initiated and when the correct solvent composition reaches the pump head is 1 min (1.0 mL/(1.0 mL/ min)). If the flow rate is only 0.2 mL/min (typical for electrospray), then the lag time will be 5 min. This means that a typical gradient run would require 5 min to initiate reequilibration plus whatever time is required for elution and final reequilibration (usually 10 to 20 column volumes). This is clearly an unacceptable time delay. 

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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