HPLC systems components

For high sensitivity measurements special care must be taken with respect to some HPLC system components. 

A block diagram of an HPLC system, illustrating its major components, is shown in Figure 2.1. These components are discussed in detail below. 

In this chapter, an HPLC system has been described in terms of its component parts and the effect of each of these on the use of a mass spectrometer as a detector... 

FTIR in multiply hyphenated systems may be either off-line (with on-line collection of peaks) [666,667] or directly on-line [668,669]. Off-line techniques may be essential for minor components in a mixture, where long analysis times are required for FT-based techniques (NMR, IR), or where careful optimisation of the response is needed. In an early study a prototype configuration comprised SEC, a triple quadrupole mass spectrometer, off-line evaporative FTIR with splitting after UV detection see Scheme 7.12c [667]. Off-line IR spectroscopy (LC Transform ) provides good-quality spectra with no interferences from the mobile phase and the potential for very high sensitivity. Advanced approaches consist of an HPLC system incorporating a UV diode array, FTIR (using an ATR flow-cell to obtain on-flow IR spectra), NMR and ToF-MS. 

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... 

Males were anesthetised and mental glands (N 200) were surgically removed. Secreted components were extracted into 0.8 mM acetylcholine chloride in l/2x PBS (cf Rollmann, Houck and Feldhoff 1999). Gland extracts were centrifuged for 10 min at 14,000 g and the supernatant was removed. The supernatant was filtered (0.2 pm) and loaded as aliquots onto a Sephadex Superfine G-75 gel filtration column (1.6 cm x 15.5 cm Pharmacia, Piscataway, NJ) on a Waters HPLC system (Mil-lipore, Milford, MA). The column had previously been equilibrated with one-half strength Dulbecco s phosphate buffered saline (l/2x PBS). The column was eluted... 

Again, there s a lot of variety among HPLC systems, so what I say won t necessarily apply to your system in every respect. But it should help. I ve based my observations on a Glencoe HPLC unit. It is simple and rugged, performs very well, and uses very common components carried by almost every HPLC supplier. Parts are easy to get. 

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... 

Common HPLC solvents with adequate purity are commercially available. Halogenated solvents may contain traces of acidic impurities that can react with stainless steel components of the HPLC system. Mixtures of halogenated solvents with water should not be stored for long periods, as they are likely to decompose. Mixtures of halogenated solvents with various ethers, e.g., diethyl ether, react to form products that are particularly corrosive to stainless steel. Halogenated solvents such as methylene chloride react with other organic solvents such as acetonitrile and, on standing, form crystalline products. 

The mass spectrometer is also incompatible with the HPLC system, but for a different reason. The ordinary mass spectrometer operates under very low pressure (a high vacuum see Chapter 10), and thus the liquid detection path must rapidly convert from a very high pressure and large liquid volume to a very low pressure and a gaseous state. Several approaches to this problem have been used, but probably the most popular is the thermospray (TS) technique. In this technique, the column effluent is converted to a fine mist (spray) as it passes through a small-diameter heated nozzle. The analyte molecules, which must be thermally stable, are preionized with the presence of a dissolved salt. A portion of the spray is introduced into the mass spectrometer. The analyte and mobile phase must be polar if the TS technique is used because the mobile phase must dissolve the required salt and the components must interact with the analyte molecule. See Workplace Scene 13.5. 

Detail the path of the mobile phase through the HPLC system from the solvent reservoir to the waste receptacle, giving brief explanatory descriptions of instrument components along the way. 

A guard column is a short, less-expensive liquid chromatography column that is placed ahead of the analytical column in an HPLC system. The purpose of a guard column is to adsorb and retain mixture components that would contaminate the more expensive analytical column. In-line filters are relatively coarse filters (compared to prefilters) placed in the mobile phase line to filter out particulates that maybe introduced on-line, such as from sample injection. 

Complete listing and identification of components to be installed on the system, to include system components and peripheral device identification (HPLC, UV, etc.). 

Unfortunately, mixtures of natural pigments generally contain one or more unidentified pigment components making impossible the prediction of their retention behaviour in any RP-HPLC system. 

HPTLC is a very fast and convenient assay to separate samples components and is often used in Organic Chemistry and in Synthetic approach. Unknown substances, after different display assay, were generally scraped off from the TLC/HPTLC plate, diluted into a tube and transferred into the MS system for structural elucidation and characterization. Now, a TLC-MS interface was developed by CAMAG, which can semi-automatically extract zones of interest and on-line direct them into any brand of a HPLC-MS system. The TLC-MS interface is connected by two fittings to any HPLC instrument coupled with mass spectrometer, without other system configuration adjustments or mass spectrometer modifications. By this way, the unknown substances can be directly extracted from a TLC/HPTLC plate, eluted and resolved by HPLC system and sensitive and selective mass spectrometric signals are obtained within a minute per substance zone [33],... 

The nitrobenzene oxidation mixture was analyzed using the HPLC method. 0.2 mL of the stock solution was pipetted into a 25 mL volumetric flask and acetonitril-water (1 2 vA ) was added to it. About 20 gL of the sample solution was next injected into the HPLC system (Shimatzu) equipped with a Hypersil bond C,g coluitm (particle size 5 gL, 25 x 4.6 mm i.d.) to quantitatively determine the vanillin component while another component was determined qualitatively. Acetonitril-water (1 8) containing 1% acetic acid was used as an eluent with a flow rate of 2 tuL/min. The eluent was then monitored with an UV (ultraviolet) detector at 280 ran [6]. 

Figure 5 represents a correlogram of this analysis obtained with the correlator and a modified HPLC system. The concentration of each component is only 0.2 ppm, an enhancement of 30. In Figure 6 the response trace leading to the correlogram of Figure 5 is shown. 

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. 

A system is typically comprised of multiple instrument components. Therefore, there is usually an individual IQ for each of these instruments and for any corresponding instrument control/data-handling software. The typical instrument components making up an HPLC system include a binary or quaternary HPLC pump, an autosampler supporting multiple vials or microtiter plates (autosamplers often include cooled Peltier trays for sample stability), a column oven, and a UV-Vis or photodiode array (PDA) detector. 

Operational testing is the main body of an OQ. It should include those tests and corresponding instructions that will help validate that each instrument component in an HPLC system is operating properly according to the vendor s functional specifications. 

For each instrument component that is tested as part of the overall HPLC system, the PQ should contain a corresponding section to document the results. This section should include the actual test results, calculated performance parameters, pass/fail status for each test, as well as any comments. 

Following pre-installation qualification and the actual installation of an HPLC system, both the IQ and the OQ protocols should be implemented, back to back, soon after the installation. Again, the IQ is used to verify that the installation of the system was successful, with all instrument components powering-up properly. The OQ follows, verifying that the system components perform as they were functionally specified by the vendor. Finally, the PQ protocol serves to verify that the system as a whole performs to the URS established by the user and within the functional limitations of the system as a whole. As part of the PQ, it is recommended to test the system as a whole, called holistic validation. This... 

A second approach to on-line SPE is to use an SPE extraction column that can be used for hundreds of samples. In the simplest of systems, two pumps (either HPLC systems or stand-alone pumps) are connected to an extraction column and an analytical column via 6 or 10 ports, and these are further linked to an MS system. The pump that is connected in-line with the autosampler loads the sample under high flow rate (3 to 5mL/min). The large molecules from the matrix are not retained by the SPE sorbent and are diverted to waste. The analytes of interest are retained by the sorbent. The valve then switches so that the second pump with the elution solvent is now in-line with the SPE column and elutes the analytes onto the analytical column for HPLC/MS analysis. This type of system has proved useful for the analysis of small molecules in a variety of sample matrices such as plasma and urine. While it is relatively straightforward to plumb this type of system with components already in the laboratory, commercial systems are available from such companies... 

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