Instrumentation isocratic system

Liquid chromatography has a number of different configurations with regard to technical (instrumental) as well as separation modes. The HPLC system can be operated in either isocratic mode, i.e. the same mobile phase composition throughout the chromatographic ran, or by gradient elution (GE), i.e. the mobile phase composition varies with run time. The choice of operation... 

Some LC/MS users adhere to isocratic separation because of the myths around gradient elution (it is complex to develop and transfer between instruments and laboratories, it is inherently slower than isocratic methods because of re-equilibration, and other reasons summarized by Carr and Schelling6). A researcher may have a very good reason to use an isocratic method, for example, for a well defined mixture containing only a few compounds. The isocratic method would certainly not be useful in an open access LC/MS system processing varying samples from injection to injection. 

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. 

From both theory and experimental evidence, raising the temperature by 10°C decreases the retention time by about 20% in isocratic chromatography and decreases the backpressure by 10% to 20% because of a reduction in the viscosity of the mobile phase. This can help to overcome the instrument limitations associated with running shorter columns packed with smaller particles, i.e., the pressure limitations of current HPLC systems. However, since the majority of reversed-phase columns available are silica-based, operating at temperatures above... 

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. 

A standard instrumental system for isocratic elution consists of ... 

Ion chromatography instruments have the same components as those found in HPLC (see Fig. 4.1). They can exist as individual components or as an integrated instrument. The components of the system are made out of inert materials because the mobile phase is composed of acids or alkaline entities that can be highly corrosive. Instruments that operate in the isocratic mode are used more often than those allowing gradient elution. 

A CEC instrument basically consists of a system for injection (pressure driven or electrokinetic), a column in which the separation takes place, a detector and a high voltage supply (Fig. 16.1). The most commonly used detector so far has been UV with transmission through the capillary outside of the packed bed. Laser induced fluorescence detection has been employed in several studies. Also, mass-spectrometry has been used. Normally, isocratic CEC is performed, but approaches to gradient CEC have been reported [29]. However, special equipment must be employed in most cases. 

Whether background correction needs to be applied depends on the separation system employed. If the instrument is balanced properly, then, for isocratic separations, the solvent background will be eliminated by the built-in automatic subtraction of the solvent spectrum, as present at the beginning of the analysis, from all recorded spectra. For gradient separations, background corrections will have to be applied after the analysis. 

The present operating procedure describes the procedure and the documentation for the performance qualification (PQ) of HPLC systems It can be used for both isocratic and gradient systems with UV detection and it is independent of the instrument manufacturer. The procedure includes the tests of pump, autosampler, UV detector and column oven. It can be put into practice immediately. 

The measuring instruments for flow and pressure are connected and the pump is switched on according to the isocratic working conditions. The system is run for 10-15 min. Afterwards the displays of the instruments as well as the pressure displayed at the pump are documented during 6 min at 1 min intervals. 

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. 

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