Isocratic Pumping Systems

A simple system is comprised of an isocratic pump, a manual injector, a UV detector, and a strip-chart recorder. A schematic diagram of an HPLC instrument is shown in Fig. 15.4. This simple configuration is rarely used in most modern laboratories. A typical HPLC system is likely to consist of a multi-solvent pump, an autosampler, an on-line degasser, a column oven, and a UV/Vis or photodiode array detector all connected to and controlled by a data-handling workstation. Examples of modular and integrated systems are shown in Fig. 15.5. Some of the important instrumental requirements are summarized in Table 15.2. 

Regarding OQ validation, if one has only an isocratic pump available, it is recommended that one does not perform a detector linearity test at this time. However, this test can be subsequently performed as part of either the PQ validation or individual method validation, both of which typically test the performance of the system as a whole (holistically). 

Apparatus Pumping systems used in these studies for high-performance columns were a Varian 8500 syringe pump and a Varian 5000 isocratic pump. An Altex IlOA was employed for the con-trolled-pore glass (CPG) columns. Waters Associates model 401 refractometers were used on all instruments. Stagnant mobile phase was kept in the reference side of the refractometer. Samples were injected with a Rheodyne 70-10 injection valve using a 20yl loop (lOOyl for CPG columns). 

Micro-HPLC operation sets special demands on the gradient instrumentation. As the internal column diameter, d, decreases, lower flow rates should be used at comparable mean linear mobile phase velocities, u = 0.2-0.3 mm/s. At a constant operating pressure, the flow rate decreases proportionally to the second power of the column inner diameter, so that narrow-bore LC columns with 1mm i.d. require flow rates in the range of 30-100pL/min, micro-columns with i.d. 0.3-0.5mm, flow rates in between 1 and lOpL/min, and columns with 0.075-0.1 mm i.d. flow rates in the range of hundreds nL/min. Special miniaturized pump systems are required to deliver accurately mobile phase at very low flow rates in isocratic LC. 

Chromatography conditions. HPLC was performed using a Milton Roy HPLC system (Riviera, FL, USA), consisting of a Constametric 3000 Series isocratic pump, a Rheodyne injector (Rheodyne L.P., Cotati, CA, USA), and a... 

Preparative separations in the grams per injection level are different. Separations are run isocratic in 1- to 3-in columns with large pore, fully porous packings (35-60jUm). An analytical, two-pump system can just barely reach the 20-mL/min flow rates needed to run a 1-in column. Special preparative HPLC systems deliver flow rates of 50-500 mL/min to handle the larger bore columns. A stream splitter is used to send part of the flow through a refractive index detector with a flow cell designed for concentrated solutions. 

Isocratic elution is commonly used for the elution of analytes from the column. In isocratic elution, the mobile phase is kept constant throughout the analysis. The mobile phase can be a single solvent or a solution of two or more miscible solvents. The major requirements of isocratic pumps are accuracy and smoothness of flow. Because the pump delivers only one solvent system, simple, inexpensive pulse dampeners and rudimentary flow or pressure feedback control circuits can be used. The basic setup of an isocratic system is illustrated in Figure 3.10. 

Moreover, low-pressure systems often use three or four solvents. This multiple-solvent blending might also be useful for the optimization of both isocratic and gradient elution methods. This is an advantage that the high-pressure system also has when not using a gradient system, the operator has two independent isocratic pumps. 

HPLC system (isocratic pump UV detector helpful, but not required). 

There are available microprocessor controlled solvent delivery modules which can generate the required gradient profile, be it stepwise, linear, convex, concave or simply isocratic. Up to four solvents can be selected. A detailed discussion of the instrumentation is presented in Chapter 6. The pumping systems and accessories are equally adaptable to use with open tubular chromatography as with high pressure systems. 

This active flow control unit is plugged to an isocratic or gradient pumping system. It divides the total pump flow Ftomi into exactly equal split... 

The most common requirements of an HPLC pumping system are delivery of a constant flow of mobile phase in both isocratic and gradient modes in the range from a few mL.min to 10(xL.min , with an inlet pressure generally np to 5000 psi ( 35 MPa) thongh considerably higher valnes (up to 15 000 psi) are required for columns packed with ultra-small particles (Section 3.5.7) moreover, pressure pulses from piston-driven pnmps must be no larger than 1% of the total flow rate for normal and reverse phase separations. 

Figure 11.36 Schematic diagram of the dual-column extraction system on-line via a Valeo 10-port switching valve with the chiral LC-MS/MS system. The components were as follows PI, a Leap Technologies autosampler with two HPLC pumps delivering mobile phases A and B to the extraction column EC-1 or EC-2 (Waters Oasis HLB 25 gm, 1 x 50 mm) P2, HPLC pump system delivering isocratic elution mobile phase through the extraction column to the chiral analytical column full bold arrows, pathway for mobile phase A (Table) used to load plasma sample onto extraction column dashed arrows, pathway for mobile phase (neither A nor B) used to elute analytes from extraction column to the chiral analytical column F, in-line filter G in-line guard column MS, a triple-quadrupole instrument in MRM mode. Reproduced from Xia, J. Chromatogr. B 788, 317, copyright (2003) with permission from Elsevier.

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