Integrated HPLC systems

FIGURE 2 Examples of modular and integrated HPLC systems. Upper row (I to r) modular systems Agilent 1100 Series, Jasco LC-2000 Series Lower row (I to r) integrated systems Waters Alliance, Shimadzu LC-2010. 

The validation of materials upon entering the compound repository is assured by various analytical and separation techniques. Mass spectrometry is often featured for this application. A recent example of this by Pharmacia researchers [98] details an integrated HPLC system with detectors, including UV, ELSD, and CLND in addition to TOF-MS. More references on the use of hyphenated methods for library-quality assessment can be found in [91]—[97] in an earlier section, where library characterization is described. 

Figure 4.2. Examples of modular and integrated HPLC systems. Modular systems (a) Agilent 1100 Series, (b) Jasco low-pressure mixing system. Integrated systems (c) Waters Alliance, (d) Shimadzu 2010.

Whilst for the analysis of plant material for cannabinoids both GC and HPLC are commonly used, in analytical procedures the employment of GC-based methods prevails for human forensic samples. Nonetheless, the usage of HPLC becomes more and more of interest in this field especially in combination with MS [115-120]. Besides the usage of deuterated samples as internal standards Fisher et al. [121] describe the use of a dibrominated THC-COOH (see 7.5). The usage of Thermospray-MS and electrochemical detection provide good performance and can replace the still-used conventional UV detector. Another advantage in the employment of HPLC rather than GC could be the integration of SPE cartridges, which are needed for sample preparation in the HPLC-system. 

The equipment used in 2DLC is the same as in HPLC with the addition of valves and integrated control of both HPLC systems. Most chromatography data systems allow... 

Wagner, K., Miliotis, T., Marko-Varga, G., Bischoff, R., Unger, K.K. (2002). An automated online multidimensional HPLC system for protein and peptide mapping with integrated sample preparation. Anal. Chem. 74, 809-820. 

FIGURE 9.4 Schematic diagram of the on-line comprehensive two-dimensional HPLC system including an integrated sample preparation step. 

Their method included a Waters 2795 Alliance HT (high throughput) HPLC system with an integrated autosampler. The stationary phase was a Supelco C18 column (250 x 4.6 mm, 5 fim). The mobile phase consisted of solvent A (water containing 2mM ammonium acetate and 0.1% formic acid) and solvent B (methanol containing 2mM ammonium acetate and 0.1% formic acid). The mobile phase was delivered at a flow rate of 0.6 mL/min in a step gradient mode 50% solvent B from 0 to 0.4 min and 100% solvent B from 0.4 to 0.8 min. 

A simple system is comprised of an isocratic pump, a manual injector, a UV detector, and a strip-chart recorder. A schematic diagram of an HPLC instrument is shown in Fig. 15.4. This simple configuration is rarely used in most modern laboratories. A typical HPLC system is likely to consist of a multi-solvent pump, an autosampler, an on-line degasser, a column oven, and a UV/Vis or photodiode array detector all connected to and controlled by a data-handling workstation. Examples of modular and integrated systems are shown in Fig. 15.5. Some of the important instrumental requirements are summarized in Table 15.2. 

HPLC systems can be classified as modular or integrated. In a modular system, separate modules are stacked and connected to function as a unit, whereas in an integrated system, modules are built inside a single housing and often share a common controller board. These built-in modules cannot function outside the system solvent lines and electrical wires are inside the housing. Modular systems are considered easily serviceable since internal components are easily accessible, and the malfunctioning module can be swapped. Integrated systems provide... 

There are typically six components in an HPLC system (1) solvent reservoirs (2) a pumping or solvent management system (3) an injector, which can be either manual or automated (4) a column (5) a detector (6) a data recorder, which can be an integrator or a computer system. 

Elevated temperatures up to 100°C have also been used with integrated HPLC chips that include both a column and an electrochemical detector [83]. This system used a resistive heater and a mobile phase preheater and was used to separate o-phthaldialdehyde amino acid derivatives. 

Often, treatment of samples with fluorescence labeling agent reacts with primary and secondary amines to give a fluorescent compound. This is especially important for detecting amino acids in protein hydrolyzates. Fluorescence detectors may also be integrated into a high performance liquid chromatographic (HPLC) system. 

The performance of an HPLC system can be evaluated by examining the key functions of the various modules that comprise the system, followed by holistic testing that challenges the performance of the HPLC components as an integrated system. The holistic testing is commonly referred to as the performance qualification (PQ). The holistic test can be as simple as running a frequently used HPLC method in the laboratory. Modular testing of various components, which is... 

HPLC system consisting of a buffer pump, preferably with degasser, autosampler with cooling system, fluorescence detector (excitation 384 nm, emission 516 nm) and an integration and data handling system. 

In 1993, the incidence of antimicrobials in car tanker milk and the suitability of different tests for the detection of antimicrobials on the MRL level has been examined in Northern Germany using an integrated detection system (14). This system comprised microbial inhibitor tests for screening, immunochemical tests for preliminary confirmation, and high-performance liquid chromatography (HPLC) for final confirmation either in a parallel or a subsequent fashion in case of positive or questionable screening results. 

HPLC systems are composed of several components with defined functions that represent independent yet interrelated entities, such as those one might find in a hi-fi audio system. The components can be, and often are. inserted in a common frame to form an integrated unit (Fig. 3.1). 

Modern HPLC systems commonly include computer software that automatically integrates peak areas. For older systems, manual integration may be necessary. 

MT Tena, MD Luque de Castro, M Valcarcel. Total and individual determination of carbamate pesticides by use of an integrated flow-injection/HPLC system. J Chromatogr 33 449-453, 1992. 

HPLC systems operate on a master-slave arrangement in setting up for automation. One module sets the timing and initiates the process, and the remaining modules accept a signal and follow the leader. In a gradient HPLC system, the master module can be a microprocessor-based controller, a computer software-based controller, an autosampler, or an integrator. 

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