HPLC systems automated

Advances in understanding solute interachons in liquid-liquid systems in a nonequilibrium environment brought reversed-phase (RP)-HPLC into the forefront of lipophilicity determinahon. The development and manufacturing of rigid, reproducible and well-characterized stationary phases and columns, as well as the accessibility and high level of automation of modern HPLC systems, have made RP-HPLC the method of choice for many laboratories. 

An HPLC system equipped with an ultraviolet/diode-array detector (UV/DAD) and automated column switching system is used. 

High Performance Liquid Chromatographic (HPLC) Analysis. A Waters HPLC system (two Waters 501 pumps, automated gradient controller, 712 WISP, and 745 Data module) with a Shimadzu RF-535 fluorescence detector or a Waters 484 UV detector, and a 0.5 pm filter and a Rainin 30 x 4.6 mm Spheri-5 RP-18 guard column followed by a Waters 30 x 3.9 cm (10 pm particle size) p-Bondapak C18 column was used. The mobile phase consisted of a 45% aqueous solution (composed of 0.25% triethylamine, 0.9% phosphoric acid, and 0.01% sodium octyl sulfate) and 55% methanol for prazosin analysis or 40% aqueous solution and 60% methanol for naltrexone. The flow rate was 1.0 mL/min. Prazosin was measured by a fluorescence detector at 384 nm after excitation at 340 nm (8) and in vitro release samples of naltrexone were analyzed by UV detection at 254 nm. 

HPLC log P techniques, first described by Mirrlees et al. [374] and Unger et al., [375], are probably the most frequently used methods for determining log/1. The directly measured retention parameters are hydrophobicity indices, and need to be converted to a log P scale through the use of standards. The newest variants, breadths of scope, and limitations have been described in the literature [292-298]. A commercial automated HPLC system based on an extension of the approach described by Slater et al. [150] has just introduced by Sirius (www. sirius-analytical. com). 

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. 

Fig. 7. Illustration of an automated reagent introduction, reaction and analysis set-up based on the incorporation of a micro-fluidic device into a conventional HPLC system...

Adequate resolution of the components of a mixture in the shortest possible time is nearly always a principal goal. Establishing the optimum conditions by trial and error is inefficient and relies heavily on the expertise of the analyst. The development of computer-controlled HPLC systems has enabled systematic automated optimization techniques, based on statistical experimental design and mathematical resolution functions, to be exploited. The basic choices of column (stationary phase) and detector are made first followed by an investigation of the mobile phase composition and possibly other parameters. This can be done manually but computer-controlled optimization has the advantage of releasing the analyst for other... 

To enhance automation capacity, a direct transfer of the acceptor phase to a HPLC system can be arranged by setting up a pre-column that allows the injection of as much volume of analyte as possible (Figure 1.33). Pneumatically or electrically actuated valves controlled by a computer provide... 

A six-port valve was used in both manual and semi-automated SPME interfaces and PEEK tubing used to connect the HPLC system to the SPME probe. A Cohesive HTLC 2300 with dual pumps along with a Sciex API 3000 mass spectrometer was used for LC/MS/MS and a Symmetry Shield RP-18 (5 ji, 50 x 2.1 mm) for HPLC. A quaternary pump with flow switching was used for desorption chamber flushing along with MS make-up flow and a binary pump for LC/MS/MS. Acetoni-trile/0.1% acetic acid in water (90 10, solvent B) and 10 90 acetonitrile/0.1% aqueous acetic acid (solvent A) were used, with 10% B for 0.5 min ramped to 90% B in 2 min and held at this concentration for 1.5 min before returning to 10% B for 1 min at a flow rate of 0.5 mL/min. 

Most manufacturers of dissolution testing devices offer semi-automated systems that can perform sampling, filtration, and UV reading or data collection. These systems automate only a single test at a time. Fully automated systems typically automate entire processes including media preparation, media dispensing, tablet or capsule drop, sample removal, filtration, sample collection or analysis (via direct connection to spectrophotometers or HPLCs), and wash cycles. A fully automated system allows automatic performance of a series of tests to fully utilize unused night and weekend instrument availability. 

The Gilson Aspec automatic sample preparation system is a fully automated system for solid-phase extraction on disposable columns and online HPLC analysis. The Aspec system offers total automation and total control of the entire sample preparation process including clean-up and concentration. In addition, Aspec can automatically inject prepared samples into on-line HPLC systems. 

Other techniques to improve throughput are instrumentation based and may involve multiple HPLC systems. The simplest method involves the automated use of solid phase extraction cartridges for sample cleanup followed by direct injection into the mass spectrometer [114], Coupling of multiple HPLC systems to one mass spectrometer allows one column to equilibrate and separate while another column to flow into the mass spectrometer. Multiple HPLC systems may be configured such that the mass spectrometer is only exposed to each serial HPLC eluent as the analyte of interest is eluted [115,116]. Although multiple H P LC-based methods may increase throughput, they also typically decrease sensitivity and may confound data workup and interpretation. 

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. 

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]. 

Finally, in Part III, we will talk about putting the system to work on real-world applications. We will look at systematic methods development, both manual and automated, and the logic behind many of the separations that others have made. We will discuss how to interface the HPLC system to computers and robotic workstations. I will also give you my best guesses as to the direction in which HPLC columns, systems, detectors, and liquid chromato-graphy/mass spectrometer (LC/MS) systems will be going. 

There are four basic system types. Type I are basic isocratic systems used for simple, routine analysis in a QA/QC environment often for fingerprinting mixtures or final product for impurity/yield checking. Type II systems are flexible research gradient systems used for methods development, complex gradients, and dial-mix isocratics for routine analysis and standards preparation. They fit the most common need for an HPLC system. Type III systems are fully automated, dedicated systems used for cost-per-test, round-the-clock analysis of a variety of gradient and isocratic samples typical of clinical and environmental analysis laboratories. Type TV systems are fully automated gra-... 

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