System dwell volume

Also known as gradient delay volume, system dwell volume is the liquid holdup volume of the HPLC system from the point of solvent mixing to the head of the column. This includes the additive volumes of the injector, the sample loop, all fluidic connection tubing, and any internal pump volumes of a low-pressure mixing system. The typical dwell volume of a modem HPLC is 0.5-2 mL, but can be as high as 5-7 mL in older systems. 

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

Modern pumps have more features and better reliability and performance than earlier models because of better designs in seals, pistons, and check valves as well as innovations such as dual-piston in-series and piston seal wash.2 Performance at low rates can be improved by variable stroke mechanism, micro pistons, or active check valves. The fluidic components in more inert pumps for bio-purification or ion-chromatography are often constructed from titanium or polyetheretherketone (PEEK). Low-pressure mixing quaternary pumps have become standard equipment in research laboratories whereas high-pressure mixing pumps are popular for LC/MS, HTS, and micro LC applications. 

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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. 

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. 

Gradient methods have the reputation of being slow because of the need for column equilibration between injections. However, by optimizing column configuration, instrumentation, and method parameters, run times below 10 minutes are readily achievable. Column equilibration requires that a sufficient volume of mobile phase has been flushed through the system to return the column to the initial mobile phase conditions at the start of the gradient. Both the system dwell volume and the column volume must be taken into account. Equilibration time, is described by the following equation ... 

Note that the delay time needed to accoimt for the system dwell volume was ignored, although it can readily be introduced by adding it to fjnj. 

High-performance liquid chromatography (HPLC) is a versatile analytical technique using sophisticated equipment refined over several decades. An in-depth understanding of the working principles and trends is useful for more effective application of the technique. This chapter provides the reader with a concise overview of HPLC instrumentation, operating principles, recent advances, and modern trends. The focus is on the analytical scale HPLC systems and modules (pump, injector, and detectors). System dwell volume... 

Major manufacturers of HPLC instruments include Waters, Agilent (formerly Hewlett Packard), and Shimadzu, PerkinElmer, Thermo, Beckman, Varian, Hitachi, Jasco, Dionex, Gilson, Scientific Systems (SSI), and Isco. The Internet addresses of these companies can be found in the reference section. HPLC is a mature technology and most manufacturers have highly reliable products with sufficient performance and feature sets to be competitive in the market place. However, there can still be significant differences between the vendors on these performance characteristics on systems (dwell volume, dispersion), pumps (low flow, seal life), autosamplers (carryover, speed, sample capacity, minimum sample volume), and detectors (sensitivity, gradient baseline shift). 

This chapter provides an overview of modern HPLC equipment, including the operating principles and trends of pumps, injectors, detectors, data systems, and specialized applications systems. System dwell volume and instrumental bandwidth are discussed, with their impacts on shorter and smaller diameter column applications. The most important performance characteristics are flow precision and compositional accuracy for the pump, sampling precision and carryover for the autosampler, and sensitivity for the detector. Manufacturers and selection criteria for HPLC equipment are reviewed. 

Operational specifications Pump precision of retention time <0.5% RSD Composition accuracy <1% absolute Detector noise, <+2.5 x 10"5 AU Auto sampler precision <0.5% RSD, <0.1 carryover System dwell volume <1 mL Instrumental bandwidth <40 pL (4o)... 

When converting an HPLC method to UHPLC, one should express system dwell volumes and gradient segments in terms of column volume and maintain the same numbers of column volume for UHPLC and HPLC. 

Dong also reported decreased detector sensitivity (signal to noise) due to higher baseline noise than was seen in HPLC applications. This was traced to poor mixing of aqueous and organic phases in the 100 p,L static mixer of the UHPLC employed in the study. Larger volume mixers alleviate this issue, but contribute additional system dwell volume, with all the associated drawbacks listed earlier. 

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