Instrumental considerations

There are four major sources of extracolumn dispersion (i) dispersion due to the injection volume, (ii) dispersion due to the volume of the detector cell, (iii) dispersion due to the detector response time, and (iv) dispersion resulting from the volume in the connecting tubing between the injector and the column and also between the column and the detector. Thus, extracolumn dispersion takes place between the injector and the detector, only, and the system volume contributed by the solvent delivery system does not contribute to dispersion. The total permitted extracolumn dispersion (variance) is shared, albeit unequally, between those dispersion sources. A commonly accepted criterion for the instrumental contribution to zone broadening, suggested by Klinkenberg,17 is that it should not exceed 10% of the column variance. 

Commercially available HPLC instrumentation was originally designed for use with standard-bore columns (4.6 mm I.D.). Detector flow cells were optimized for maximum sensitivity with these analytical columns, injectors were designed to introduce microliter quantities of sample, and pumps were designed to be accurate and reproducible in the milliliter flow-rate ranges commonly employed with standard-bore columns. However, these instruments are not well suited for use with small-bore columns, as the dispersion introduced by the large volumes is detrimental to the separation. In addition, the reproducibility and accuracy of the pumping system at the low flow rates required are questionable. 

Because of the interest in narrow-bore and microbore columns, instrument manufacturers now have developed solvent delivery systems that are capable of accurately pumping at the low flow rates typically required for microbore applications ( 10 / L/min). In addition, injectors have been designed that are capable of introducing the smaller sample volumes, and detector cells are available that are small enough to monitor the reduced sample volumes passing through the detector. Thus narrow-bore and microbore applications are possible with readily available instrumentation, and reports may be found in the literature. 

Ideally, a sample is introduced into a chromatograph as a perfect plug. In practice, this is not the case, and diffusion occurs because of the injector. For narrow-bore and microbore applications, injectors capable of introducing the required sample volumes are commercially available and optimized to reduce dispersion. This is not the case for capillary LC, and homemade injection systems include the sample tube technique, in-column injection, stopped-flow injection, pressure pulse-driven stopped-flow injection (PSI), groove injection, split injection, heart-cut injection, and the moving injection technique (MIT). Of the injection techniques, only the split injector, MIT and PSI approaches can introduce subnanoliter sample volumes accu- 

If the sample is dissolved in a solvent that is weaker than the mobile phase, then the sample can be enriched on the head of the column without penetrating into the column bed. This compression effect is particularly important for capillary LC applications, since it permits significantly larger injection volumes. A substantial increase in sensitivity results, and conventional autosamplers with 20-jnl loops can be used.16 However, sample solubility and recovery, miscibility of the sample with the mobile phase, and the maximum tolerable loss in column efficiency and resolution must all be assessed experimentally for optimum on-column focusing.16 

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This technique assumes a Gaussian spreading function and thus does not take into account skewness or kurtosis resulting from instrumental considerations. It can, however, be modified to accommodate these corrections. The particle size averages reported here have been derived usino the technique as proposed by Husain, Vlachopoulos, and Hamielec 23). 

The short (UV) wavelength limit of the optical range is imposed by instrumental considerations (spectrophotometers do not usually work at wavelengths shorter than about 200 nm) and by the validity of the macroscopic Maxwell equations. These equations assume a continuous medium in other words, that there is a large number of ions within a volume of. The long (IR) wavelength limit of the optical range is basically imposed by experimental considerations (spectrophotometers work up to about 3000 nm). 

CE has been included as a distinct analytical technique in a general monograph in the Ph.Eur., JP, and USP. These monographs have been harmonized and at present only some minor differences exist between the different pharmacopoeias. They give an overview of the general principles, instrumental considerations, and the different separation modes. Also, attention is paid to quantification and system suitability aspects. 

Johansson, I. M., Huang, E. C., Henion, J. D., and Zweigenbaum, J. (1991). Capillary electrophoresis-atmospheric pressure ionization mass spectrometry for the characterization of peptides. Instrumental considerations for mass spectrometric detection. /. Chromatogr. 554, 311 — 327. 

Common characteristics of all viable process Raman instmments include the ability to measure at multiple locations, simultaneously or sequentially, and in a variety of electrically classified or challenging environments with no special utilities. The instrument must be reasonably rugged and compact, and not require much attention, servicing, or calibration [19]. Modem process Raman instruments essentially are turnkey systems. It is no longer necessary to understand the optical properties and other instrnment details to suc-cessfnlly nse the techniqne. The general design options will be described briefly, bnt more information is available elsewhere [20,21]. This section will focus primarily on special instrumentation considerations for process installations. 

Kinetic measurements by EPR are difficult, primarily because of the instrumental considerations mentioned in Sections V.B and V.C. Nonetheless, the specificity of EPR toward paramagnetic species has prompted workers to measure reaction rates of radicals generated by both in situ and external electrolysis, using both static and flowing solutions. 

In the first place column characteristics will often be determined by practical conditions, such as availability of columns and materials and instrumental considerations. 

As an example, we assume a gradient from 100% water to 100% methanol in 20 minutes, on a column with a t0 value of 1.5 min. Now a solute that elutes with a retention time t = 15 min (t is the retention time under gradient conditions) is expected to yield fc=3 at the composition that was reached at the column inlet at r = 15 - 2x1.5 =12 min, which is 60% methanol, 40% water. Assuming that there is no delay time due to instrumental considerations, this is the composition at the start of the column, but not at the end. One and a half minutes (t0) later, this composition will have reached the end of the column. 

Instrument Considerations when Using Ultra-High Pressures... 

N. M. Djordjevic, P. W. J. Eowler, and F. Houdiere, High temperature and temperature programming in high-performance liquid chromatography instrumental considerations, 7. Microcol. Sep. 11 (1999), 403-413. 

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