HPLC mobile phases, characteristics

Figure 11.6.5 The UV characteristics of daidzein (dashed line), genistein (solid line), and glycitein (dotted line) in HPLC mobile phases (A 1 % acetic acid in water, B 100% acetonitrile, A/B = 85 15) at 25°C monitored at 260 nm.

Equipment with different characteristics (e.g., delay volume of an HPLC system) Variations in material and instrument conditions (e.g., in HPLC, mobile phases composition, pH, flow rate of mobile phase)... 

Reproducibility, as defined by ICH, represents the precision obtained between laboratories with the objective of verifying if the method will provide the same results in different laboratories. The reproducibility of an analytical method is determined by analyzing aliquots from homogeneous lots in different laboratories with different analysts, and by using operational and environmental conditions that may differ from, but are still within the specified, parameters of the method (interlaboratory tests). Various parameters affect reproducibility. These include differences in room environment (temperature and humidity), operators with different experience, equipment with different characteristics (e.g., delay volume of an HPLC system), variations in material and instrument conditions (e.g., in HPLC), mobile phases composition, pH, flow rate of mobile phase, columns from different suppliers or different batches, solvents, reagents, and other material with different quality. 

Ideally, solvents used as HPLC mobile phases should have these characteristics ... 

The development of a chromatographic procedure for an unknown sample (mixture) requires the selection of a variety of experimental conditions (type and composition of the mobile phase, characteristics of the column and the stationary phase, temperature, flow-rate, pressure, type of gradient, etc.). This problem was traditionally solved in an empirical way and with the aid of the vast literature on similar situations already dealt with. The last few years have seen attempts at the rationalization and automation of the optimization of chromatographic processes which have resulted In Interesting systematic approaches of great use. The monographs by Berridge [56], devoted to HPLC, and that by Shoenmakers [57], which deals with both HPLC and GC, represent the most systematic and complete compilations in this field at present. 

Infrared absorbance detectors. For completeness we should note that IR detectors for HPLC do exist. As is the case for GC-IR (Section 12.8.2) a continuous rapid response HPLC-IR detector benefits from FTIR spectral acquisition and computerized data file storage. Such spectra as may be obtained have spectral peak widths more characteristic of a liquid matrix, instead of the more information rich, sharp gas phase GC-IR peaks. The major drawback to IR detection in HPLC is that most of the commonly useful HPLC mobile phases absorb strongly in many areas of the IR spectral region. Thus HPLC-IR can be used for only a very limited set of analytes. 

In an extension of this work, Konig reports the determination of surfactants in toothpaste by HPLC (37). Here, one takes advantage of the knowledge that usually only one of a limited number of surfactants is present. The toothpaste is dissolved directly in the HPLC mobile phase, filtering out the abrasive and other insoluble matter. The characteristic retention time and peak pattern under a few standard HPLC conditions allow both identification and quantification of the surfactant. 

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

Qualitative (identification) applications depend upon the comparison of the retention characteristics of the unknown with those of reference materials. In the case of gas chromatography, this characteristic is known as the retention index and, although collections of data on popular stationary phases exist, it is unlikely that any compound has a unique retention index and unequivocal identification can be effected. In liquid chromatography, the situation is more complex because there is a much larger number of combinations of stationary and mobile phases in use, and large collections of retention characteristics on any single system do not exist. In addition, HPLC is a less efficient separation... 

Unlike gas chromatography, in which the mobile phase, i.e. the carrier gas, plays no part in the separation mechanism, in HPLC it is the relative interaction of an analyte with both the mobile and stationary phases that determines its retention characteristics. Hence, it is the varying degrees of interaction of different analytes with the mobile and stationary phases which determines whether or not they will be separated by a particular HPLC system. 

A general approach to the problem of identification, should more definitive detectors not be available, is to change the chromatographic system , which in the case of HPLC is usually the mobile phase, and redetermine the retention parameter. The change obtained is often more characteristic of a single analyte than is the capacity factor with either of the mobile phases. 

How then can this problem be addressed From a chromatographic standpoint, the usual method is to change either the stationary phase or, more usually in the case of HPLC, the mobile phase, and look for a change in the retention characteristics. The change observed is usually more characteristic of a single analyte than is the actual retention on any individual chromatographic single system. Even this, however, does not always provide an unequivocal identification. 

The need for a more definitive identification of HPLC eluates than that provided by retention times alone has been discussed previously, as have the incompatibilities between the operating characteristics of liquid chromatography and mass spectrometry. The combination of the two techniques was originally achieved by the physical isolation of fractions as they eluted from an HPLC column, followed by the removal of the mobile phase, usually by evaporation, and transfer of the analyte(s) into the mass spectrometer by using an appropriate probe. 

We first note the very large differences in column performance for the two methods. Effective plates per second represents the speed characteristics of a column (e.g., the number of plates that can be generated in a given time interval) (13). As can be seen, HPLC is 100 to 1000 times faster than classTcal LC. (We shall discuss the differences between PLB and PB in the next section.) This improved performance arises mainly from the use of significantly smaller particle sizes in HPLC. Moreover, in classical LC, the mobile phase is delivered to the column by gravity feed, hence, the very low mobile phase velocities. In HPLC, it is desireable to improve performance... 

Microbore HPLC-FTIR detection limits are about 10 times lower than analytical-scale HPLC-FTIR detection limits. The lowest reported LC-FTIR detection limits are approximately 100-1000 times higher than the best GC-FTIR detection limits. The main characteristics of flow-cell HPLC-FTIR are summarised in Table 7.44. Because of mobile-phase interferences, flow-cell HPLC-FTIR is considered as a powerful tool only for the specific detection of major components but is otherwise a method of limited potential, and SFE-SFC-FTTR has been proposed as an alternative [391]. 

The function of the detector in hplc is to monitor the mobile phase emerging from the column. The output of the detector is an electrical signal that is proportional to some property of the mobile phase and/or the solutes. Refractive index, for example, is a property of both the solutes and the mobile phase. A detector that measures such a property is called a bulk property detector. Alternatively, if the property is possessed essentially by the solute, such as absorption of uv/visible radiation or electrochemical activity, the detector is called a solute property detector. Quite a large number of devices, some of them rather complicated and tempremental, have been used as hplc detectors, but only a few have become generally useful, and we will examine five such types. Before doing this, it is helpful to have an idea of the sort of characteristics that are required of a detector. 

You have the task of purchasing some n-hexane for use in three different applications (i) pesticide analysis by gas chromatography, (ii) as a solvent to extract some non-polar high-boiling (200-300°C) oils from a soil sample, and (iii) as a mobile phase for HPLC analysis with UV detection. List and contrast the performance characteristics you need to take into account for purchasing the appropriate grade of hexane in each case. n-Hexane boils at about 70°C. Will any of your choices of hexane be suitable for use for HPLC analysis with fluorescence detection Explain your decision. 

The ideal HPLC detector should have the same characteristics as those required for GC detectors, i.e. rapid and reproducible response to solutes, a wide range of linear response, high sensitivity and stability of operation. No truly universal HPLC detector has yet been developed but the two most widely applicable types are those based on the absorption of UV or visible radiation by the solute species and those which monitor refractive index differences between solutes dissolved in the mobile phase and the pure mobile phase. Other detectors which are more selective in their response rely on such solute properties as fluorescence, electrical conductivity, diffusion currents (amperometric) and radioactivity. The characteristics of the various types of detector are summarized in Table 4.14. 

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