Reversed-phase HPLC with UV detection

A wide variety of methodologies have been employed for the analysis of antioxidants in polymers and some standard methods are available. For high-density polyethylene ASTM method D5524 (ASTM International) — Determination of phenolic antioxidants in high-density polyethylene, describes a method whereby the sample is ground to a small particle size and then extracted by refluxing with cyclohexane. The cyclohexane extract is then examined by reverse-phase HPLC with UV detection. 

Air is drawn through a midget impinger or a bubbler containing 0.1 ANaOH solution. Phenol and ciesols are trapped as phenolates. The pH of the solution is adjusted <4 by H2S04. The compounds are determined by reverse-phase HPLC with UV detection at 274 nm. An electrochemical or fluorescence detector may also be used. The solution may be analyzed by colorimetric or GC-FID technique. 

Aqueous samples buffered with citrate and pH adjusted to 3. Acidified sample derivatized with 2,4-dinitrophenylhydrazine (DNPH) derivative analyzed by GC-NPD or reverse phase HPLC with UV detection at 360 nm. 

Palaniswamy, U.R., Caporuscio, C. and Stuart, J.D. (2003) A chemical analysis of antioxidant vitamins in fresh curry leaf (Murraya koenigii) by reversed phase HPLC with UV detection. Acta Horticulturae 620, 475 178. 

Detection of ionic liquid [NB4MPy [BF4 ] was made by reversed-phase HPLC with UV detection in the following conditions sample injection volume 3 pi eluent 20 % acetonitrile, 80 % 0.02 M phosphate buffer pH 4.5 flow rate ... 

Nicotinic acid and nicotinamide These are sometimes known by the generic term niacin. Their importance is in combination with tryptophan, as the coenzyme forms nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NADP). HPLC is too insensitive to measure endogenous plasma levels, but the urinary metabolites N-methyl-2-pyridone-5-carboxylamide and N -methylnicotinamide can be measured to assess niacin status. Preliminary cleanup of urine by anion-exchange resins is followed by reversed-phase HPLC with UV detection. 

Trinitrobenzene derivatives of PE and PS from human red blood cells and rat brain were analysed by Hullin, Kim and Salem (1989). For quantification purposes, reversed-phase HPLC with UV detection was used. HPLC-TS (filament-on) was used for confirmation of molecular species separation. 

For reasons of space, it is not possible to abstract every relevant paper, and so at the end of some chapters an Annotated Bibliography lists other relevant papers. After the citation, a few features of the method that are not obvious from the title of the paper may be briefly mentioned to help the reader decide if this paper may be of use. For example, the limit of quantitation of the method maybe cited. Unless otherwise mentioned, it may be assumed that a method involves liquid-liquid extraction of a biological fluid from a human and uses reversed-phase HPLC with UV detection. Thus, if a method uses solid-phase extraction (SPE) or fluorescence detection, this will be mentioned. 

HPLC determination This technique has been used extensively for determining only one vitamer or both vitamers together. Simultaneous determination is difficult due to the differences in basicity and polarity of both vitamers and the interference problems. After water-phase extraction and sometimes cleanup steps as deproteinization, nicotinic acid and/or nicotinamide are analyzed by ion pair reversed-phase HPLC with UV detection. Most methods use different ion pair reagents for each vitamer, but a single ion pair reagent for nicotinamide and nicotinic acid can be also employed. 

The alternative technique for analyzing 1,4-dioxane is HPLC. Scalia proposed a method by solid-phase extraction using octadecyl-bonded silica cartridges and analyzed directly on a reverse phase column with UV detection at 200 nm and acetonitrile-water as eluent [328-330]. 

Methylene dithiocyanate (see Table 11) can be analysed in water samples and industrial materials using reverse-phase HPLC with UV detector at 254 nm. The detection limit is 2ppm. ... 

Samples were analyzed for free 3,3 -DCB using a modification of the technique described by Az, Dewald and Schnaitmann. Soxhlet toluene extractions were carried out under mild conditions to minimize pigment degradation and possible cleavage ofimide/amide DCB adducts during the course of the extraction. The samples were analyzed for 3,3 -DCB with CIS reverse phase HPLC using UV detection. Analysis results are presented in Table 2. The calculated limit of detection for the method was approximately 0.2 ppm. 

The test procedures described in this chapter are based on reversed-phase chromatography with UV detection and can be used for almost all HPLC systems, including those which are used with non-polar or more viscous mobile phases or other chromatographic methods. In such cases it is necessary to adjust the instrument parameters such as the suction speed of the autosampler or the compressibility factor of the pump. An important prerequisite is the thorough priming of the whole HPLC system with the mobile phases and flushing liquids in the case of non-miscibility, an intermediate solvent needs to be used. Do not forget the injection valve. 

For more specific analysis, chromatographic methods have been developed. Using reverse-phase columns and uv detection, hplc methods have been appHed to the analysis of nicotinic acid and nicotinamide in biological fluids such as blood and urine and in foods such as coffee and meat. Derivatization techniques have also been employed to improve sensitivity (55). For example, the reaction of nicotinic amide with DCCI (AT-dicyclohexyl-0-methoxycoumarin-4-yl)methyl isourea to yield the fluorescent coumarin ester has been reported (56). After separation on a reversed-phase column, detection limits of 10 pmol for nicotinic acid have been reported (57). 

Numerous high pressure Hquid chromatographic techniques have been reported for specific sample forms vegetable oHs (55,56), animal feeds (57,58), seta (59,60), plasma (61,62), foods (63,64), and tissues (63). Some of the methods requite a saponification step to remove fats, to release tocopherols from ceHs, and/or to free tocopherols from their esters. AH requite an extraction step to remove the tocopherols from the sample matrix. The methods include both normal and reverse-phase hplc with either uv absorbance or fluorescence detection. AppHcation of supercritical fluid (qv) chromatography has been reported for analysis of tocopherols in marine oHs (65). 

A method developed to analyze alkylbenzenesulfonates by reverse phase ion pair HPLC with UV detection has also been applied to the determination of alcohol ether sulfates [286]. 

Reverse-phase HPLC with ion pairing and UV detection at 227 and 210 nm was used to determine sweeteners (acesulfame-K, saccharin, and aspartame), preservatives (BA and SA), and... 

An HPLC method for chlorogenic acids with lactones in six different commercial brands of roasted coffee was developed by Schrader et al. (143). Hydroxycinnamic acid derivatives, including mono- and di-caffeoylquinic acids, corresponding lactones, and feruloylquinic acids were extracted from coffee with methanol at 80°C for 1 h under reflux. An HPLC method using step-gradient elution with 2% aqueous acetic acid (eluent A) and ACN (eluent B) for a 75-min run time was developed. Determination was carried out by HPLC with UV detection at 324 nm, and further confirmation was conducted by HPLC-thermospray (TSP)-MS and HPLC-diode array detection. Elution order for mono-caffeoylquinic acid (CQA) was 3-CQA, 5-CQA, followed by 4-CQA, which was different from the usual elution order of mono-CQA (Fig. 17). These results indicate that it is currently not possible to predict the elution order of different reversed-phase packings due to the different selectivity (143). 

Taylor EW, Qian MG, Dollinger GD. Simultaneous online characterization of small organic molecules derived from combinatorial libraries for identity, quantity, and purity by reversed-phase HPLC with chemiluminescent nitrogen, UV, and mass spectrometric detection. Anal Chem 1998 70 3339-3347. 

Figure 3.20. Analysis of carboxylic acids and alcohols by reversed phase HPLC, with indirect UV detection, (a) Carboxylic acids. Chromatography conditions mobile phase, 3 X 10 4 M l-phenethyl-2-picolinium in acetate buffer (pH 4.6) column, ju-Bondapak phenyl detection, indirect UV absorbance at 254 nm. Peaks 1, acetic acid 2, propionic acid 3, butyric acid 4, valeric acid 5, caproic acid S, system peak, (b) Aliphatic alcohols. Chromatography conditions mobile phase, 4 x 10 4 M nicotinamide in water column. Ultrasphere ODS detection, indirect UV absorbance at 268 nm. Peaks 1, methanol 2, propylene glycol 3, ethanol 4, 2-propanol 5, 1-propanol 6, system peak 7, 2-butanol 8, 2-methyl-l-propanol 9, 1-butanol. (Redrawn from Refs. 23 and 24 with permission.)...

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