Dwell volumes

The CHI index is reportedly a relevant parameter in quantitative structure-activity relationship (QSAR) studies [41]. With this approach, log P could be determined in the range -0.45more than 25000 compounds with excellent reproducibility (within 2 index units) and reported in a GlaxoSmithKline database [11]. Two main drawbacks were identified using this approach (i) the assumptions used in Ref [7], i.e. that S is constant for all compounds and that the system dwell volume is excluded in calculations, yield some discrepancies in the resulting log P, and (ii) the set of gradient calibration... 

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. 

An overview of HPLC instrumentation, operating principles, and recent advances or trends that are pertinent to pharmaceutical analysis is provided in Chapter 3 for the novice and the more experienced analyst. Modern liquid chromatographs have excellent performance and reliability because of the decades of refinements driven by technical advances and competition between manufacturers in a two billion-dollar-plus equipment market. References to HPLC textbooks, reference books, review articles, and training software have been provided in this chapter. Rather than summarizing the current literature, the goal is to provide the reader with a concise overview of HPLC instrumentation, operating principles, and recent advances or trends that lead to better analytical performance. Two often-neglected system parameters—dwell volume and instrumental bandwidth—are discussed in more detail because of their impact on fast LC and small-bore LC applications. 

Gradient parameters (flow rate, gradient time, peak capacity, and dwell volume)... 

Today s HPLC pumps have sophisticated designs honed by decades of incremental improvements. They are also more reliable and easier to maintain than their predecessors. Short seal life and check valve malfunctioning seen in the early models are no longer problems. In this section, the principle of pump operation is described with an emphasis on advances leading to higher reliability and performance. A discussion of system dwell volume is included. 

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. 

Manufacturers publish their product s performance characteristics as specifications, which are often used by the customer for comparison during the selection process. Table 1 shows the specifications of an Agilent 1100 Series Quaternary Pump, which is quite representative of other high-end analytical pumps. Note pulsation is particularly detrimental to the performance of flow-sensitive detectors (e.g., mass spectrometer, refractive index detector). Differences in dwell volumes and composition accuracy between HPLC systems might cause problems during method transfers. 

Significant time is saved in this calibration procedure by minimizing sample preparation and the number of calibrated test apparatus wherein only a balance, a 5-mL volumetric flask, a stopwatch, a thermal probe, two standards, one HPLC column, and one mobile phase are used. The use of MS Word template forms (see Figure 10) saves considerable time by eliminating any notebook entries and also improves the consistency of calibration records. Two important system parameters, dwell volume, and... 

To fully utilize small particles (i.e., less than 2 pm) packed in columns greater than 50 mm in length, an HPLC system must be developed that can operate at high pressures. For the best results using columns with 1-2-pm particles, extra-column effects, dwell volumes, and detectors must be optimized for optimum performance. Additionally, commercial... 

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]. 

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... 

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