Pumps for HPLC

The driving force producing mobile phase flow in GC is simply the cylinder of compressed gas dehvered via a pressure regulator, although modern electronic flow controls introduce a useful level of sophistication (Hinshaw 2002b). However, HPLC pumps must provide a continuous constant flow of the eluent through the injector, colnmn and detector, and are a crucial component of any HPLC system (Kazakevich 1996). 

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. 

Constant-flow pumping systems are of two basic types, reciprocating piston and positive displacement (syringe) pumps. Both can provide reprodncible elntion times and 

Syringe pumps are generally larger scale versions of manual syringes (Section 2.4.4) and consist of a cylinder containing the mobile phase that is expelled by a piston 

Recently, a different approach to HPLC pumping was developed (Jensen 2004). Pressure in the system is generated by connecting laboratory air or nitrogen to a pneumatic amplifier that produces an amplification factor for pressure values up to 36 for example, a nitrogen supply at 100 psi can be amplified to deliver 

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In attempting to design an ideal pump for HPLC, the following features should be built into it. 

The most commonly used pump for HPLC is the reciprocating pump. This has a small cylindrical piston chamber that is alternately filled with mobile phase and emptied via back-and-forth movement of the piston. This produces a pulsed flow that must be damped. Reciprocating pumps have a number of advantages. They have a small internal volume, are capable of high output pressures, and they can readily be used for gradient elution. They provide constant flow rates, independent of solvent viscosity or column backpressure. Other pumps used are motor-driven syringe pumps and pneumatic (constant-pressure) pumps. 

J. Toei and N. Baba, Flow Injection Analysis for Glucose Using Multi-Function Pump for HPLC [in Japanese]. J. Flow Injection Anal., 2(2) (1985) 151. 

Fig. 5 Construction of a reciprocal high-pressure pump for HPLC.

The electrospray ionization tandem mass spectrometer was obtained from Finni-gan MAT. Pumps for HPLC were purchased from Applied Biosystems (Cat. No. 140B, dual-syringe pump), and a sample injector was obtained from Rheodyne (Cat. No. 8125, 5- 1 loop injector). 

The FIA system constructed consisted of the enzyme-immobilized column integrated with gold electrodes on a silicon chip, a pump for HPLC (minimum flow rate OIL min ), an injector for HPLC (sample size 0 2 or 0 5 L, Rheodyne), a potentiostat (handmade), and a data processor Commercially available flange-type fittings were used for the connection of the biosensing device with other components of the FIA system... 

Allene (prodiene) [463-49-0] M 40.1, m -146 , b -32 . Frozen in liquid nitrogen, evacuated, then thawed out. This cycle was repeated several times, then the allene was frozen in a methyl cyclohexane-liquid nitrogen bath and pumped for some time. Also purified by HPLC. [Cripps and Kiefer Org Synth 42 12 1962.]... 

The types of pumps used for HPLC can be divided into two categories constant-pressure pumps (e.g. the inexpensive gas-displacement pump) and the constant-volume type (e.g. the reciprocating and syringe pumps). The most commonly used pumps in HPLC are the single- or multi-head reciprocating type. The former delivers the flow as a series of pulses which must be damped... 

A microreactor was also applied to this reaction. The slit interdigital micromixer was purchased from IMM (Mainz, Germany). The width of the interdigital channels is 25 pm. HPLC pumps were used to feed the two reaction solutions. One is a mixture of Boc-AMP and 1.2 molar equivalents of r-BocaO. The other is a 50% aqueous KOH solution. The microreactor was immersed in a temperature controlled cooling bath at 15 °C. The product was quenched with an acid, and samples were taken for HPLC analysis. 

Besides differences in column design, we need to note that instrimentation is quite different for HPLC relative to the classical mode. Pumps, injectors and detectors are all important components in the achievement of high performance. In addition, automated injection is available which could be important in the clinical chemistry field. 

Figure4.62 Experimental set-up for liquid/liquid experiments (a) reservoir for the substrate in n-heptane (b) water reservoir (c, d) high-pressure liquid pumps (e) HPLC injection valve with sample loop for catalyst injection (f) micro mixer ...

FIGURE 16.5 Schematic of instrumental setup for 2D micro-RPLC-CZE. A split injection/ flow system is used to deliver a nanoliter per second flow rate to the micro-RP-HPLC column from the gradient LC pump. The HPLC microcolumn has 50 pm i.d. and 76 cm length, and the electrophoresis capillary has 17 pm i.d., L — 25 cm, and/= 15 cm. The valve is air-actuated and controls the flow of flush buffer (reprinted with permission from Analytical Chemistry). 

A six-port valve was used in both manual and semi-automated SPME interfaces and PEEK tubing used to connect the HPLC system to the SPME probe. A Cohesive HTLC 2300 with dual pumps along with a Sciex API 3000 mass spectrometer was used for LC/MS/MS and a Symmetry Shield RP-18 (5 ji, 50 x 2.1 mm) for HPLC. A quaternary pump with flow switching was used for desorption chamber flushing along with MS make-up flow and a binary pump for LC/MS/MS. Acetoni-trile/0.1% acetic acid in water (90 10, solvent B) and 10 90 acetonitrile/0.1% aqueous acetic acid (solvent A) were used, with 10% B for 0.5 min ramped to 90% B in 2 min and held at this concentration for 1.5 min before returning to 10% B for 1 min at a flow rate of 0.5 mL/min. 

The pumps specifically designed for HPLC are able to provide constant flow of the mobile phase against column pressure up to 10,000 psi. However, it is important to recognize that most HPLC separations are run at pressures lower than 6,000 psi. A comparison of various pumps used for HPLC is given in Table 15.3. 

The gradient elution method for HPLC is the method in which the mobile phase composition is changed in some preprogrammed way in the middle of the run. The device that accomplishes this is called the gradient programmer and is placed between the mobile phase reservoir and the pump. It is useful in experiments in which altering the mobile phase composition assists with the resolution of the mixture. 

The pump is a very delicate and sensitive part of HPLC unit therefore, all buffer solutions should be removed carefully after use either by pumping water (HPLC-grade) or an appropriate solvent (HPLC-grade) for several minutes. 

This is a more recently developed technique which is a hybrid between HPLC and capillary electrophoresis. The capillary is packed with HPLC media and the mobile phases are aqueous buffers. A voltage is applied to generate an electroendosmotic flow and the analytes separate by interaction with the stationary phase and electrophoretic forces no pump being required as for HPLC. Improved separation efficiencies have been reported. 

The following standard operating procedures are recommended for HPLC pump operation ... 

This chapter reviews the principles and strategies used for HPLC system calibration that includes the pump, the detector, the autosampler, and the column oven. A case study is used to illustrate the development of the calibration procedures for all system modules and the rationale of setting up acceptance criteria that balance productivity and compliance. 

A second approach to on-line SPE is to use an SPE extraction column that can be used for hundreds of samples. In the simplest of systems, two pumps (either HPLC systems or stand-alone pumps) are connected to an extraction column and an analytical column via 6 or 10 ports, and these are further linked to an MS system. The pump that is connected in-line with the autosampler loads the sample under high flow rate (3 to 5mL/min). The large molecules from the matrix are not retained by the SPE sorbent and are diverted to waste. The analytes of interest are retained by the sorbent. The valve then switches so that the second pump with the elution solvent is now in-line with the SPE column and elutes the analytes onto the analytical column for HPLC/MS analysis. This type of system has proved useful for the analysis of small molecules in a variety of sample matrices such as plasma and urine. While it is relatively straightforward to plumb this type of system with components already in the laboratory, commercial systems are available from such companies... 

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