HPLC instruments, major

HPLC techniques were initially developed as liquid-liquid chromatographic methods and difficulties in maintaining the stationary phase were resolved by chemically bonding it to the particulate support. Subsequently a whole range of column materials have been developed that enable the basic HPLC instrumentation to be used for the major chromatographic techniques. 

High-performance liquid chromatography (HPLC) is one of the premier analytical techniques widely used in analytical laboratories. Numerous analytical HPLC analyses have been developed for pharmaceutical, chemical, food, cosmetic, and environmental applications. The popularity of HPLC analysis can be attributed to its powerful combination of separation and quantitation capabilities. HPLC instrumentation has reached a state of maturity. The majority of vendors can provide very sophisticated and highly automated systems to meet users needs. To provide a high level of assurance that the data generated from the HPLC analysis are reliable, the performance of the HPLC system should be monitored at regular intervals. In this chapter some of the key performance attributes for a typical HPLC system (consisting of a quaternary pump, an autoinjector, a UV-Vis detector, and a temperature-controlled column compartment) are discussed [1-8]. 

The majority of HPLC instruments use pumps based on alternating pistons. In order to avoid variations in the flow rate that are caused by single piston pumps (solvent in/solvent out), manufacturers have developed pumps that use two pistons. These pumps can be classified into two categories series and parallel. 

HPLC instrumentation and column technology have undergone major advances since the early 1970s, when HPLC made its debut in the field of vitamin analysis. Yet sample preparation in food analysis continues to rely largely on manual wet-chemical techniques, which are time consuming and labor intensive, require considerable analytical skill, and constitute the major source of error in the assay procedure. There is also the serious problem of environmental pollution and the exposure of laboratory personnel to toxic chemicals. 

In HPLC instrumentation troubleshooting, problems can be classified as follows. Major HPLC problems are discussed under this paragraph, while an extended summary of problem causes and remedy actions are tabulated in the respective tables. 

Major manufacturers of HPLC instruments include Waters, Agilent (formerly Hewlett Packard), and Shimadzu, PerkinElmer, Thermo, Beckman, Varian, Hitachi, Jasco, Dionex, Gilson, Scientific Systems (SSI), and Isco. The Internet addresses of these companies can be found in the reference section. HPLC is a mature technology and most manufacturers have highly reliable products with sufficient performance and feature sets to be competitive in the market place. However, there can still be significant differences between the vendors on these performance characteristics on systems (dwell volume, dispersion), pumps (low flow, seal life), autosamplers (carryover, speed, sample capacity, minimum sample volume), and detectors (sensitivity, gradient baseline shift). 

Many major users of HPLC, particularly pharmaceutical companies working in an FDA regulated environment, will log every injection on a column through the column s lifetime and will have specific criteria (called Standard Operating Practices or SOPs) to indicate when a column should be retired. Many HPLC instruments also have bar code readers and the columns have corresponding bar codes to insure that documentation is correct. Major users will also maintain... 

Figure 13.4 is a schematic diagram of a typical HPLC instmment. We will use this figure to discuss the features of the major components, and describe the construction of some of them. We will use this discussion to illustrate some of the major modes of operation of HPLC instruments. We proceed in order of the numbered items of the figure. 

Figure 11.23 presents a schematic diagram of the instrumentation required for HPLC. Six major components needed to perform HPLC are... 

As with gas chromatography, numerous detectors have been developed for use in monitoring HPLC separations. To date, the majority of HPLC detectors are not unique to the method, but are either stand-alone instruments or modified versions of the same. 

Step 5. HPLC on a gel filtration column (Zorbax GF250, Du Pont Instruments) at 20°C, using 10 mM sodium phosphate, pH 6.5, containing 2mM EDTA and 0.1 M Na2SC>4. A major impurity is eluted immediately before the photoprotein peak. The purified photoprotein is stored at -75° C. 

Major advances have occurred over the past few years in equipment for HPLC. Figure 2 presents a block diagram of a basic LC instrument. We shall now discuss individual components. ... 

In most situations analysts can achieve a rapid reasonable separation of compounds using an appropriate standard CE method with generic operating conditions [877]. This eliminates or reduces dramatically the need for method development. Major instrumental error sources in CE are detection, integration and injection. General guidelines for validation of CE methods are available and similar to those of HPLC [878]. Validated CE methods often perform the same as, or better than, the corresponding HPLC methods. 

In the mid-to-late 1990s, SFC became an established technique, although only holding a niche position in the analytical laboratory. The lack of robust instruments and the inflexibility of such systems has led to the gradual decline of SFE-SFC. Only a small group of industrial SFE-SFC practitioners is still active. Also the application area for SFC is not as clearly defined as for GC or HPLC. Nevertheless, polymer additives represent a group of compounds which has met most success in SFE-SFC. The major drawbacks of SFE-SFC are the need for an optimisation procedure for analyte recovery by SFE (Section 3.4.2), and the fair chance of incompatibility with the requirements of the chromatographic column. The mutual interference of SFE and SFC denotes non-ideal hyphenation. 

Aurora Biomolecules dedicates to peptide synthesis (and polyclonal antibody production) for any small quantity purpose. FMOC chemistry (on Perceptive Biosystems Pioneer instruments) is used for peptides synthesis Online monitoring of the coupling efficiencies and HATU activation helps insure that the major component of the synthesis is the correct oligopeptide. Purification is firstly carried out by size exclusion chromatography, and then by HPLC on a PE vision purification workstation. Typically, 20 mg of pure peptide are obtained. The molecular weight of the purified peptide is determined as a final confirmation of quality. 

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