Gradient dwell volume

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. 

Microbore columns Typically 0.3 mm ID Many columns available Small particle/high pressure Compatible with NPLC Loop injection, 10-100 nL Small gradient dwell volume Dual LEAP, 18 microplates 8 Individual detectors Diode array No fluorescence... 

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. 

Here, as in Eq. 4, is the column holdup volume and Vr, is the gradient dwell volume. 

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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