Chromatographic process and instrumentation

Unlike the situation in most other chromatographic techniques, the stationary phase is not packed into a column, but is coated as a thin layer on to a glass, plastic or metal plate. The samples in solution are applied as spots or bands on to the bottom edge of the plate, which is then placed in a closed tank containing the mobile phase (usually a mixed solvent system), and the solvent is drawn up the plate by capillary action thereby effecting the separation. Two basic separation modes are used in polymer analysis. 

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The advantages of HPLC over classical chromatographic methods stem from the employment of a precision instrument that utilizes high-performance columns with concomitantly high analytical speed and resolution and affords total control over the chromatographic process and sensitivity of analysis. In a way, the recent emergence of capillary electrophoresis (CE) follows the same patterns electrophoresis, a well-established and widely used method of biopolymer analysis, is carried out... 

This section describes the fundamental relationships that are required to understand the rationale behind optimization of LC separations and to make predictions based on a theoretical understanding. For experienced users, it is an opportunity to add to their knowledge, as well as being a timely revision lesson on some of the more fundamental aspects. Beginners should refer to a text book that addresses HPLC knowledge from the scratch [1], since the understanding of the chromatographic process and the functional parts of the HPLC instrument is a premise. 

Figure A2.1 Waters ProMonix On-Line HPLC analyzer. The upper compartment door contains a keypad for programming and operation of the analyzer. The upper window allows viewing of indicator lights and a liquid crystal display that provides the operator with analyzer interface, programmed parameters, and instrument status results. The lower chamber contains the pumps, valves, injector, and detector(s) required for the chromatographic separation. The sample conditioning plate for online process monitoring is to the right of the analyzer. This is a typical process HPLC. (From Cotter, R.L. and Li, J.B., Lab Rob Autom., 1, 251,1989. With permission of VCH Publishers.)...

Principles and Characteristics High-performance thin-layer chromatography (HPTLC), also known as planar chromatography, is an analytical technique with separation power and reproducibility superior to conventional TLC, which was first used in 1938 [7] and modified in 1958 [8]. HPTLC is based on the use of precoated TLC plates with small particle sizes (3-5 xm) and precise instruments for each step of the chromatographic process. 

Vibrational spectroscopy, in the form of mid-IR, NIR and Raman spectroscopy has been featured extensively in industrial analyses, both quality control (QC), process monitoring applications and held-portable applications [1-6]. The latter has been aided by the need for advanced instrumentation for homeland security and related HazMat applications. Next to chromatography, it is the most widely purchased classihcation of instrumentation for these measurements and analyses. Spectroscopic methods in general are favored because they are relatively straightforward to apply and to implement, are rapid in terms of providing results, and are often more economical in terms of service, support and maintenance. Furthermore, a single spectrometer or spectral analyzer, in a near-line application, may serve many functions, whereas chromatographs (gas and liquid) tend to be dedicated to only a few methods at best. 

It is beyond the scope of this chapter to discuss and explain how the requirements can be implemented in analytical laboratories. This has been described in a six-article series published in Biopharm [16-21]. We elaborate here on the validation aspect of the rule. Part 11 requires that computer systems used to acquire, evaluate, transmit, and store electronic records should be validated. This is not new, as processes and steps to validate such systems were described earlier in the chapter. FDA s expectations for validation have been described in the Part 11 draft guidance on validation [4]. This guidance makes it very clear that functions as required by Part 11 should be validated in addition to functions that are required to perform an application such as chromatographic instrument control, data acquisition, and evaluation. Specific functions as required by Part 11 are as follows ... 

Because analytical chromatography is used inherently in quantitative analysis, it becomes crucial to precisely measure the areas of the peak. Therefore, the substances to be determined must be well separated. In order to achieve this, the analysis has to be optimised using all the resources of the instrumentation and, when possible, software that can simulate the results of temperature modifications, phases and other physical parameters. This optimisation process requires that the chromatographic process is well understood. 

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