Eluent Delivery Systems

Several methods are applied to reduce the separation time. The best way is the selection of a suitable column and an eluent using isocratic elution. However, much skill and knowledge are required to make such a system. A flow rate gradient, step-wise elution, or eluent composition gradients are commonly applied to reduce separation times. 

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The traditional HPLC instrument is composed of two different parts the first part separates the components of the sample and the other part accomplishes the detection of the components separated. The part of the HPLC carrying out the separation contains a column, an injection device and the eluent delivery system (pump with filters, degasser and transfer tubing, eventually a mixer for gradient elution). One or more detectors, a signal output device coupled with appropriate software, are responsible for detection and primary data evaluation. Pumps deliver the eluent or the different components of the eluent into the column with a precise, constant and reproducible flow rate. 

You find yourself for the first time in front of your HPLC equipment, consisting at least of an eluent delivery system (= pump), an injector, a column, a detector and a data evaluation system. If you see several separate devices, you have a modular equipment. If you are in front of a large box, you will work with a compact unit. 

Chromatographic System. The isocratic liquid chromatograph used was a Waters Associates (Milford, MA) Model 24A alc which included a Model 6000A Solvent Delivery System, a Model 401 Differential Refractometer and a Model 440 Absorbance Detector operating at 254 nm and was fitted with a WISP automatic injector. The analog outputs of the UV absorbance detector or differential refractometer were recorded with a Model 730 Data Module (printer, plotter, integrator)(Waters). Eluent flow rate was 1.0 ml/min unless otherwise noted. 

The function of the solvent delivery system is to deliver the mobile phase (eluent) through the chromatograph, accurately and reproducibly. The solvent delivery system comprises the pump, check valves, flow control-... 

An HPLC system, shown schematically in Figure 2.1, consists of a solvent reservoir, which contains the eluent or mobile phse a pump, often called a solvent delivery system an injector through which the sample is introduced into the system without a drop in pressure or change in flow rate the analytical column, which is usually stainless steel and contains the solid packing or stationary phase and a suitable detector to monitor the eluent. 

Sugars. The high performance liquid chromatography (HPLC) method described by Hunt et al. (8) was used to determine the sugars in the kiwifruit and nectars with some modifications (21). A Waters Association Chromatograph equipped with a Model 6000-A solvent delivery system, a Model R401 refractometer detector, a D6K universal injector column was a 30 cm x 4 mm i.d. stainless steel tube packed with u-bondapak-carbohydrate (Waters Associates). The precolumn was packed with CO-PELL PAC (Whatman). The eluent was acetonitril and distilled water (85/15, v/v). 

In GPC instrumentation the pumping system delivers a single eluent throughout the analysis. It may be a single solvent, a blend of solvents, or a solvent plus an additive. See. II.C, but the composition remains constant. Solvent programming, also termed gradient elution, in HPLC requires more eapability from the solvent delivery system to ensure precise flow and repro-dueible solvent eomposition profiles. 

The HPLC was realized with a Waters solvent delivery system. Separations of oligomers were obtained with a Nucleosil 5 pm C-18 reverse phase column from S.F.C.C. (France) (8). The eluent was distilled water filtered through a 0.45 lm Millipore membrane. The starch was dissolved in DMSO and the eluent was DMSO/MeOH (85/15 v/v) in 0.5 M ammonium acetate. The colxamn set was diol silica gel from Merck 2 x Si 1000 Diol, 1 x Si 500 Diol, 1 X Si 100 Diol thermostated at 60 C. The detector was either a differential refractometer from Waters (R 401) or an IOTA (Jobin Yvon). The second on line detector was a light scattering detector (Chromatix CMX 100) or a spectropolarimeter (Perkin Elmer model 241 working at 365 nm with a flow cell of path length 10 cm and a 30 ll volume). The value of [a]D are expressed from [Ct]355 data using a corrective factor. The partition coefficient Kd is expressed as ... 

Figure 13 Schematic diagram of preparative M-RPC. (1, upper part of the stationary chamber 2, lower part of the stationary chamber 3, collector 4, tubes in the collector 5, motor shaft with tube 6, glass rotor 7, stationary phase 8, screw 9, quartz glass cover plate 10, solvent delivery system 11, safety glass 12. mobile phase inlet 13, eluent outlet.) (Reproduced from Ref. 9 with permission.)...

The Rotachrom Model P rotation planar chromatograph consists of four main parts (9) the casing of the instrument with the motor, the electronics, and connections for nitrogen and the eluent outlet the lower part of the stationary chamber the collector system with the chamber types and the U[ r part of the stationary chamber with the solvent delivery system and integrated UV lamps. 

Figure 16 Schematic diagram of Rotachrom model P. (1, upper part of the stationary chamber 2, collector 3, tubes in the collector 4, glass rotor S, stationary phase 6, vapor space 7, fixing screw 8, solvent delivery system 9, safety glass 10, UV lamp (254 nm) 11, UV lamp (366 nm) 12, motor shaft with tube 13, motor 14, lower part of the stationary chamber 15, casing of the instrument 16, front panel for adjusting and controlling units with keyboard 17, eluent outlet.

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