HPLC systems 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. 

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... 

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. 

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. 

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. 

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. 

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... 

Other considerations include differences in dwell volumes from the different HPLC systems. The dwell volume should be determined for all the systems in the laboratory and based on these determinations, this should be factored into the calculation of the equilibration time. For example, if the maximum dwell volume of all the systems in a particular laboratory to which the method is transferred to is 2mL and you are running on an instrument at 1 mL/min that has a dwell volume of 1 mL, then you should add an extra minute of equilibration time. This becomes extremely important during method transfers where the instruments in the receiving laboratory may be different. 

Gradient methods in HPLC depend on the dwell volume, Vd, i.e., the volume between the point of mixing eluents A and B and the column inlet, including the loop of the injector. This volume differs from instmment to instmment, so that a method can be transferred only if this volume is well defined. When there is a significant difference in dwell volume between the two systems, retention times and resolution are dramatically different. So one must be state in which range of the method is still valid. 

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. 

The dwell volume is the volume within the HPLC system from the point where the solvents are mixed to the entrance of the column. In a low-pressure system it consists of the volumes of the proportioning valve, the mixer, the pump head, the injector and the connecting capillaries. In a high-pressure system it is smaller because only the mixer, injector and the capillaries add their volumes. 

The injector is part of the dwell volume of the HPLC system (see Section 4.3). This must be kept in mind if the loop volume is changed drastically, e.g. from 50 ptl to 1 ml. It may then be necessary to adjust the gradient profile of a complex separation. 

Gradient profile or solvent composition deviates from the written method —> Check the pump valves for leaks. A common cause are crystallized salts in the valves. The remedy is simple flush with salt-free solvents before the pump is switched off. Caution different HPLC systems have different mixing and dwell volumes. With gradient separations, check the equilibration time between injections and prolong it if necessary. Check the gradient method. 

Measure Dwell Volumes of HPLC and UHPLC Systems... 

To calculate holding time, the dwell volumes of both the HPLC and UHPLC systems need to be determined. There are several ways to measure the dwell volume. The following is an example for an HPLC system. 

Prepare 1% acetone in water as mobile phase B, and water alone as mobile phase A. A column under measurement is removed from the line, and the tubing is connected with a zero dead volume union. A gradient of 10 min is run from 100% A to 100% B at the flow rate of 2 mL/min and held for 10 min at 100% B with detection wavelength at 260 nm. The difference between 5 min and the time at half height between the initial baseline and the plateau times the flow rate is the dwell volume for the HPLC system. The typical dwell volume for most HPLC systems is approximately 1 mL. 

The volume between the solvent mixing point and column inlet is defined as dwell volume, and the corresponding time it takes for the selected composition of mobile phase to reach the column is called dwell time. Although the dwell volume is not the part of the extra-column volume, it is discussed in this section, as it impacts chromatographic performance. Under gradient conditions, the dwell volume for an HPLC system can have a significant effect on separation parameters including retention times (tr) and apparent retention factors fc. ... 

Effect of Dwell Volume on Separation As indicated in Eq. (3.10), the retention time and separation can be impacted by the dwell volume of an HPLC system. The delay time of an analyte due to the dwell volume is inversely proportional to the flow rate used for the separation for a given system, as expressed tdweii = where Vdwell is the dwell volume for a UHPLC or an HPLC system. Figure 3.8 shows gradient separations of phenones using UHPLC and HPLC with a 50 x 2.1 mm column. The first peak elution time is 2.2 min on UHPLC and 4.9 min in HPLC. The... 

Within the dwell time, the sample is subject to an extra isocratic or gradient step that is not reflected in the gradient profile, as shown in Eq. (3.10). A conventional HPLC system generally has a large dwell volume, that is, 500-1000 p.1, whereas on the UHPLC systems, the dwell volume is decreased to about 100-200 p.1. Basic gradient... 

Users sometimes need to run the exact same method on a conventional HPLC system as well as a UHPLC system. In this case, the dwell volume difference must be taken into consideration during the method transfer. As shown in Figure 3.21, when the same gradient was used on both systems, the retention time for the first peak was delayed about 1.3 min due to the 0.88 mL greater dwell volume of the HPLC system. When a 1.3 min hold was applied at the beginning of the gradient for the UHPLC gradient profile, the retention times for the peaks are much more comparable. 

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