Ultraviolet spectroscopy, HPLC

Ref. 277 unless otherwise noted gc = gas chromatography hplc = high pressure Hquid chromatography ir = infrared spectroscopy uv = ultraviolet spectroscopy glc = ga sliquid chromatography eia = enzyme immunoassay vis = visible spectroscopy. 

The predominant method of analyzing environmental samples for methyl parathion is by GC. The detection methods most used are FID, FPD, ECD, and mass spectroscopy (MS). HPLC coupled with ultraviolet spectroscopy (UV) or MS has also been used successfiilly. Sample extraction and cleanup varies widely depending on the sample matrix and method of detection. Several analytical methods used to analyze environmental samples for methyl parathion are summarized in Table 7-2. 

EC = electrical conductivity detector ECD = electron capture detector FPD = flame photometric detector GC = gas chromatography HPLC = high performance liquid chromatography NPD = nitrogen phosphorus detector TID = thermionic detector UV = ultraviolet spectroscopy... 

The methylxanthines can be determined in foods and biological systems by the chromatographic methods of TLC, GC, HPLC, or CE. Ultraviolet spectroscopy following a separation procedure can also be used. More recently, immunoassay methods have been developed. There is no single best method the analyst must balance the features of each assay with the final requirements for data precision and reproducibility. 

Similarly, organic liquids have a variety of applications. For example, hexane, which frequently contains impurities such as aromatic compounds, is used in a variety of applications for extracting non-polar chemicals from samples. The presence of impurities in the hexane may or may not be important for such applications. If, however, the hexane is to be used as a solvent for ultraviolet spectroscopy or for HPLC analysis with UV absorbance or fluorescence detection, the presence of aromatic impurities will render the hexane less transparent in the UV region. It is important to select the appropriate grade for the task you have. As an example, three different specifications for n-hexane ( Distol F , Certified HPLC and Certified AR ), available from Fisher Scientific UK, are shown in Figure 5.5 [10]. You will see that the suppliers provide extra, valuable information in their catalogue. 

An alternative method for fractionating and purifying petroleum hydrocarbons prior to GC or HPLC separation has been developed (Theobald 1988). The method uses small, prepacked, silica or Cjg columns that offer the advantage of rapid separation (approximately 15 minutes for a run) good recovery of hydrocarbons (85% for the Cjg column and 92% for the silica column) reusability of the columns and for the silica column in particular, good separation of hydrocarbon from non-hydrocarbon matrices as may occur with environmental samples. Infrared analysis and ultraviolet spectroscopy were used to analyze the aromatic content in diesel fuels these methods are relatively inexpensive and faster than other available methods, such as mass spectrometry, supercritical fluid chromotography, and nuclear magnetic resonance (Bailey and Kohl 1991). 

Numerous analyses in the quality control of most kinds of samples occurring in the flavour industry are done by different chromatographic procedures, for example gas chromatography (GC), high-pressure liquid chromatography (fiPLC) and capillary electrophoresis (CE). Besides the different IR methods mentioned already, further spectroscopic techniques are used, for example nuclear magnetic resonance, ultraviolet spectroscopy, mass spectroscopy (MS) and atomic absorption spectroscopy. In addition, also in quality control modern coupled techniques like GC-MS, GC-Fourier transform IR spectroscopy, HPLC-MS and CE-MS are gaining more and more importance. 

Bonazzi et al. used an HPLC method and a second derivative ultraviolet spectroscopy method for the analysis of benazepril and other angio-tensen-converting enzyme inhibitors [17]. For HPLC, 20 pL sample solutions containing the drug and an internal standard dissolved in 1 1 acetonitrile/20 mM sodium heptanesulfonate (pH 2.5) were used. HPLC was performed on a 5 pm Hypersil ODS column (25 cm x 4.5 mm) with a mobile phase mixture consisting of (A) 20 mM sodium heptanesulfonate (pH 2.5) and (B) 19 1 acetonitrile-tetrahydrofuran, eluted at a flow rate of 1 mL/min, and with detection at 215 nm. The A/B mixture used was 52 48 for benazepril. A low pH of 2.5 was essential to avoid peak splitting and band broadening. 

The elucidation and confirmation of structure should include physical and chemical information derived from applicable analyses, such as (a) elemental analysis (b) functional group analysis using spectroscopic methods (i.e., mass spectrometry, nuclear magnetic resonance) (c) molecular weight determinations (d) degradation studies (e) complex formation determinations (f) chromatographic studies methods using HPLC, GC, TLC, GLC (h) infrared spectroscopy (j) ultraviolet spectroscopy (k) stereochemistry and (1) others, such as optical rotatory dispersion (ORD) or X-ray diffraction. 

The first concern in the selection of the sample preparation solvent is to optimize recovery. However, a secondary consideration is the sample solvent s effect on the analysis. This is true whether the analytical technique is ultraviolet spectroscopy (UV), high-performance liquid chromatography (HPLC), or gas chromatography (GC). The method development sequence can be described as (a) development of the chromatographic separation, (b) development of the sample preparation method, and then (c) evaluation and optimization of the interaction of the sample preparation with the instrumental method. 

Initially, simple methods such as ultraviolet-visible (UV-Vis), fluorescence or infrared (IR) spectroscopy were proposed in order to estimate the total amount of antioxidants in various food samples. However, coupled methods such as gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography with ultraviolet-visible (HPLC-UV-Vis) or nuclear magnetic resonance detector (HPLC-NMR) and liquid chromatography-mass spectrometry (LC-MS) are employed more to quantify individual tocols or carotens from various corn-based food samples. In this chapter all these methods of analysis will be briefly described. 

These surfactants often contain a hindered phenol antioxidant to stabilize the polymer against degradation. Usually, a single antioxidant is present, and it may be determined by ultraviolet spectroscopy. The determination is also conventionally performed by reversed-phase HPLC or by gas chromatography, with or without prior derivatization. Positive identification of the stabilizer can usually be made by direct mass spectrometry. 

The most basic method for the determination of the methylxanthines is ultraviolet (UV) spectroscopy. In fact, many of the HPLC detectors that will be mentioned use spectroscopic methods of detection. The sample must be totally dissolved and particle-free prior to final analysis. Samples containing more than one component can necessitate the use of extensive clean-up procedures, ajudicious choice of wavelength, the use of derivative spectroscopy, or some other mathematical manipulation to arrive at a final analytical measurement. A recent book by Wilson has a chapter on the analysis of foods using UV spectroscopy and can be used as a suitable reference for those interested in learning more about this topic.1... 

A major consequence of using regulatory limits based on degradant formation, rather than absolute change of the API level in the drug product, is that it necessitates the application and routine use of very sensitive analytical techniques [ 10]. In addition, the need to resolve both structurally similar, as well as structurally diverse degradants of the API, mandates the use of analytical separation techniques, for example, HPLC, CE, often coupled with highly sensitive detection modes, for example, ultraviolet (UV) spectroscopy, fluorescence (F) spectroscopy, electrochemical detection (EC), mass spectroscopy (MS), tandem mass spectroscopy (MS-MS) and so forth. 

GC, gas chromatography HPLC, high-performance liquid chromatography MS, mass spectroscopy AA, atomic absorption GFAA, graphite furnace atomic absorption ICP, inductively coupled plasma UV-VIS, ultraviolet-visible molecular absorption spectroscopy IC, ion chromatography. 

Frequently industrial hygiene analyses require the identification of unknown sample components. One of the most widely employed methods for this purpose is coupled gas chromatography/ mass spectrometry (GC/MS). With respect to interface with mass spectrometry, HPLC presently suffers a disadvantage in comparison to GC because instrumentation for routine application of HPLC/MS techniques is not available in many analytical chemistry laboratories (3). It is, however, anticipated that HPLC/MS systems will be more readily available in the future ( 5, 6, 1, 8). HPLC will then become an even more powerful analytical tool for use in occupational health chemistry. It is also important to note that conventional HPLC is presently adaptable to effective compound identification procedures other than direct mass spectrometry interface. These include relatively simple procedures for the recovery of sample components from column eluate as well as stop-flow techniques. Following recovery, a separated sample component may be subjected to, for example, direct probe mass spectrometry infra-red (IR), ultraviolet (UV), and visible spectrophotometry and fluorescence spectroscopy. The stopped flow technique may be used to obtain a fluorescence or a UV absorbance spectrum of a particular component as it elutes from the column. Such spectra can frequently be used to determine specific properties of the component for assistance in compound identification (9). 

Specific Identity Tests. At least one specific identity test that is capable of distinguishing the drug substance from related compounds must be included. Spectrometric tests are usually used, such as ultraviolet, infrared, nuclear magnetic resonance, and mass spectroscopy. Retention times or factors derived from thin-layer, gas-liquid, and HPLC are also used as identification tests to verify a more specific spectral identity test. 

The main spectroscopic methods used for the structural characterization of isolated flavonoids are ultraviolet spectrophotometry (UV), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy (NMR). UV and NMR methods (both H NMR and C NMR) have been extensively covered in previous publications, and therefore will only be summarized in this chapter. MS, and particularly HPLC-MS/... 

The scope of ultraviolet and visible spectrophotometry can be further extended when combined with a chromatographic separation step such as HPLC. The development of rapid-scanning detectors based on the linear photodiode array permits spectra to be acquired during the elution of peaks. Computer-aided manipulation of these spectra has led to new strategies for the examination of chromatographic peak homogeneity, based on classical techniques in spectroscopy. The use of microcomputers enables the development of archive retrieval methods for spectral characterisation (A. F. Fell etal, J. Chromat., 1984, 316, 423-440). 

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