Pumps and gradient elution

All HPLC systems include at least one pump to force the mobile phase through the column whose packing is fairly compact. The result of this is a pressure increase at the injector which can attain 20 000kPa (200 bars) depending upon the flow rate imposed upon the mobile phase, its viscosity, and the size of the particles of the stationary phase. 

The closed part is filled with heptane. Compressibility of this liquid is enough to compensate for the pulsations of the dual piston pump with piston volume around 100 p.L. 

When several analyses are to be done successively, one must avoid the use of gradient elution, by seeking a practical compromise by means of a single eluent of fixed composition. This reduces post-analytical time after each analysis because the re-equilibration of the two phases to their initial composition, after a gradient elution, requires the passage of a volume of at least ten times the dead volume of the mobile phase. 

The column is a straight stainless steel calibrated tube which measures between 3 and 15 cm in length and whose the inside wall is sometimes coated with an inert material such as glass or PEEK . Stationary phase is held in the column 

The stationary phase (SP) in contact with the mobile phase (MP) is the second medium with which the compounds initially dissolved in the mobile phase will interact. On the column there will be as many particular associations of the three constituents [MP/compound/SP] as there are analytes in the sample. 

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The Micromeritics 7000 liquid chromatograph (Fig.3.30) also uses a reciprocating pumping system. The pump has several unique features. It delivers pulseless flow without a pulse damper, it can operate from an unlimited reservoir, it requires only a single pump for gradient elution and it is capable of operating at constant pressure or constant flow-rate. 

Recently, Alexander et al. [44] reported the use of an automated separation system developed for micro-LC and CEC using both isocratic and gradient elutions. The complete system is shown in Fig. 2.15. An enlarged view of the coupling of the column to the injection valve presents Fig. 2.16. The mobile phase was delivered by two micro-LC pumps at a flow rate of 30 pL/min to a post injection splitter that houses the column inlet. In the CEC mode, pressure was not applied (no restriction on... 

Fig. 2.15. Schematic automated isocratic and gradient elution nemo-liquid chromatograph/ capillary electrochromatograph according Alexander et al. (reproduced from Ref. [44] with permission of the publisher). 1, high-voltage power supply (negative polarity) 2, platinum electrode 3, outlet reservoir vial 4, UV detector with on-column flow cell 5, nanocolumn 6, two-position switching valve 7, jack stand 8, fused-silica make-up adapter (split device) 9, ground cable 10, internal loop micro-injection valve 11, plexiglas compartment 12, autosampler 13, dynamic mixer 14, micro-LC pumps.

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. 

The splitting technique is useful for both isocratic and gradient elution. The flow rate of the pump can be set to approx 1 mL/min, yielding a 10-12-fold lower, but constant flow rate over the microbore column because of the splitter A typical LC setup employing a splitter to reduce the flow rate through the microbore column is depicted in Fig. 1 and descnbed in Note 1 of Chapter 14. 

Refractive index detectors. These bulk property detectors are based on the change of refractive index of the eluant from the column with respect to pure mobile phase. Although they are widely used, the refractive index detectors suffer from several disadvantages — lack of high sensitivity, lack of suitability for gradient elution, and the need for strict temperature control ( + 0.001 °C) to operate at their highest sensitivity. A pulseless pump, or a reciprocating pump equipped with a pulse dampener, must also be employed. The effect of these limitations may to some extent be overcome by the use of differential systems in which the column eluant is compared with a reference flow of pure mobile phase. The two chief types of RI detector are as follows. 

The most important part of this type of interface, from a number of points of view, is the pinhole which, in conjunction with the pumping capacity of the mass spectrometer, controls the flow of eluate into the mass spectrometer. This flow, and therefore the properties of the spray being introduced into the mass spectrometer, is affected by a change in the viscosity of the mobile phase. The use of gradient elution has therefore to be approached with some caution as the sensitivity of the mass spectrometer can change significantly during the course of an analysis. 

In the pneumatic pumping system, the pressure (and not the flow rate) is maintained constant as variations in chromatographic conditions occur. Thus, a change in mobile phase viscosity (e.g. gradient elution) or column back pressure will result in a change in flow rate for these types of pumps. The gas displacement pump in which a solvent is delivered to the column by gas pressure is an example of such a pneumatic pump. The gas displacement system is among the least expensive pumps available and is found in several low cost instruments. While the pump is nonpulsating and hence, produces low noise levels with the detectors in current use, its flow stability and reproducibility are only adequate. In addition, its upper pressure limit is only 2000 psi which may be too low in certain applications. 

The amount of current that flows is dependent not only on the condition of the electrode, but also on temperature, pH, and ionic strength of the solvent. Therefore, careful control of the conditions of detection is essential. A reduction of the slope of the baseline in gradient elution is often performed by post-column addition of a solution of strong alkali. Flow is also an important variable,58 and pump fluctuations may be an important source of noise.59 At very high flow rates, amperometric response can decrease depending on... 

Implementation of SFC has initially been hampered by instrumental problems, such as back-pressure regulation, need for syringe pumps, consistent flow-rates, pressure and density gradient control, modifier gradient elution, small volume injection (nL), poor reproducibility of injection, and miniaturised detection. These difficulties, which limited sensitivity, precision or reproducibility in industrial applications, were eventually overcome. Because instrumentation for SFC is quite complex and expensive, the technique is still not widely accepted. At the present time few SFC instrument manufacturers are active. Berger and Wilson [239] have described packed SFC instrumentation equipped with FID, UV/VIS and NPD, which can also be employed for open-tubular SFC in a pressure-control mode. Column technology has been largely borrowed from GC (for the open-tubular format) or from HPLC (for the packed format). Open-tubular coated capillaries (50-100 irn i.d.), packed capillaries (100-500 p,m i.d.), and packed columns (1 -4.6 mm i.d.) have been used for SFC (Table 4.27). 

Where binary, ternary or quaternary gradient elution (p. 91) is required, a microprocessor controlled low-pressure gradient former is the most suitable (Figure 4.31(c)). The solvents from separate reservoirs are fed to a mixing chamber via a multiport valve, the operation of which is preprogrammed via the microprocessor, and the mixed solvent is then pumped to the column. For the best reproducibility of solvent gradients small volume pumps (< 100 gl) are essential. 

Step 4 Clean autosampler sample loop and trap column 2 using 90% organic solvent from pumps 1 and 2 and then condition trap column 2 and sample loop with 95 to 100% water from pump 1. Continue to run gradient elution of sample from trap column 1 and analytical column using pumps 3 and 4 until the end. Switching valve A = on switching valve B = off. 

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