Pumps gradient elution

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. 

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 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. 

The analysis of pharmaceutical agents in preparations has been one of the most important applications of modem RPC. A simple gradient maker to be used on the low pressure side of the pump for use in gradient elution of pharmaceuticals has been described (560). For the detection and identification of pharmaceuticals, micro internal reflection infrared spectroscopy (561) and ultraviolet scanning spectroscopy with stopped flow (562) were also employed. 

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