HPLC instrumentation photodiode array detectors

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. 

The photodiode-array detector is a powerful analytical instrument that has provided enhanced detection capabilities with the addition of detailed spectral information via its multisignal detection technology. Its applications are HPLC based and can be found in basic research, automated analysis, pharmaceutical product development, and the clinical laboratory environment. Through spectral acquisition and analysis, a wealth of information can be obtained about the identity and purity of a compound. Combined with high selectivity and sensitivity, this mode of de-... 

A computer associated with an HPLC equipment may control the instrument or part of it, e.g., the photodiode array detector, or acquire data generated by the instrument, such as the peak area of analytes. 

The method of Thurnham etal. (1988) was modified for the quantification of plasma j3C and retinol. Plasma extracts were dissolved in 40 /il of dimeth-ylforamide and vortexed and then 210 pil of acetonitrile/methanol/chloro-form (47/47/6, v/v/v) was added. Reconstituted samples were vortexed and sonicated for 40 sec prior to being transferred to autosampler vials and sealed under nitrogen. The HPLC system consisted of a photodiode array detector (Waters 9%, Miliipore Corp., Milford, MA) with Millennium software, a Waters 717 plus autosampler, and a Hewlett-Packard Model 1050 pump. Analytes of interest were separated using acetonitrile/methanol/ chloroform (47/47/6, v/v/v), with 0.05 M of ammonium acetate and 1% triethylamine at a flow rate of 1.2 ml/min and a 4.6 X 15-cm Spherisorb ODS-2 column (LKB Instruments Ltd., Surrey, UK) maintained at 26°C using a column heater (Timberline Instruments Ltd., Boulder, CO). This analysis does not discriminate between C-enriched and nonenriched analytes, but rather measures the total concentration of each isotopomer. The retention times of retinol, retinyl acetate (internal standard), and /3-carotene were 2.1, 2.6, and 16.9 min, respectively. Plasma concentrations of retinol and /3C were calculated using a standard curve for each analyte and an internal standard to correct for volume recovery. 

RP HPLC has proved to be the method of choice for the separation of a variety of flavonoids in different samples. The phenolic nature of these compounds requires the use of acidic mobile phases for satisfactory separation and peak shapes, whereas the detection is usually carried out with photodiode array detectors which are also very helpful for their identification of the characteristic absorption spectra of the flavonoids. In the last decade, mass spectrometers connected to HPLC systems introduced a greater selectivity and sensitivity in flavonoid analysis. Improving the characteristics of the stationary phases and developing more sophisticated instruments as well as devices for more efficient and faster sample preparation are the challenges for all modem analysts. Discovering... 

HPLC analyses of vanillin were performed using a HP1090M instrument equipped with a photodiode array detector. Vanillin was monitored by reversed-phase using a Capcell Pak C18 SGI20 (S-5 pm) column (Shishedo, 4.6 mm x 250 mm) with a constant elution gradient from 10% (V/V) acetonitrile in water to pure acetonitrile. The flow rate was 1 mL/min and the detection wavelength was 280 nm. 

High-performance liquid chromatography (HPLC) analysis is carried out on an XL ODS Cjg, 3-fim (70 x 4.6 mm) column (Beckman Instruments Inc., Altex Division, San Ramon, CA). HPLC purification is carried out on a Nucleosil Cjg, 5- im (250 x 10 mm) column (SFCC/ Shandon, Eragny, France) with an increasing proportion of acetonitrile (5-15%) in 0.05M triethylammonium acetate, pH 7, as eluant. A Waters U6K injector. Model 510 pumps, an M 720 gradient controller, and a Waters 990 photodiode array detector are employed. 

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. 

After optimization of the correct capillary parameters (ID, OD, Lj), detection at the microscale level became the next major challenge for the survival of CE. Despite the challenges, many of the common HPLC detectors have a CE complement, e.g., absorbance, fluorescence, conductivity, photodiode array, and mass spectroscopy. Small dimensions mean universal detectors such as refractive index cannot be used. A sample of detectors will be discussed. The technical aspects of each detector will not be covered except in relation to the CE instrument. Readers are advised to consult an instrumentation textbook for more details on theory of operation. 

A Varian Unity Inova 600-MHz NMR instrument (Palo Alto, CA) equipped with a H C/ N pulse field gradient triple resonance microliow NMR probe (flow cell 60pL 3mm O.D.) was used. Reversed-phase HPLC of the samples was carried out on a Varian modular HPLC system (a 9012 pump and a 9065 photodiode array UV detector). The Varian HPLC software was also equipped with the capability for programmable stop-flow experiments based on UV peak detection. An LCQ classic MS instrument, mentioned in the previous section, was connected on-line to the HPLC-UV system of the LC-NMR by contact closure. The H resonance of the D2O was used for field-frequency lock, and the spectra were centered on the ACN methyl resonance. Suppression of resonances from HOD and methyl of ACN and its two C satellites was accomplished using a train of four selective WET pulses, each followed by a Bo gradient pulse and a composite 90-degree read pulse [41]. 

A common feature of modem HPLC instruments is a diode array detector, as described in Chapter 16. The instantaneous recording of absorption spectra provides a powerful qualitative tool. The focused radiation source passes through the detector flow cell and is dispersed by a grating to a photodiode array for detection. The ability to mathematically resolve overlapping spectra can provide additional separating ability when a chromatographic peak may consist of two or more analytes. 

A Varian Unity Inova 600 MHz NMR instrument (Palo Alto, CA), equipped with a H C/ N pulse field gradient triple resonance microflow NMR probe (flow cell 60 pL 3 mm O.D.), was used. Reverse-phase HPLC of the samples was carried out with a Varian modular HPLC system (a 9012 pump and a 9065 photodiode array UV detector). The Varian HPLC software was also... 

FIGURE 7 Diagram of a photodiode array instrument. For HPLC, the cuvette is replaced by a flow cell similar to that used for the standard UV-VIS detector. [From Siouffi, A-M.. Chapter 1, in Food Analysis by HPLC. (L. M. L. Nollet, ed.), Marcel Dekker, New York. 

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