After HPLC separation, fluorescence

Fluorescence and Ultraviolet Absorbance of Pesticides and Naturally Occurring Chemicals in Agricultural Products After HPLC Separation on a Bonded-CN Polar Phase... 

Detection of tocopherols and tocotrienols after HPLC separation is based on their ability to absorb ultraviolet light and create fluorescence. Tocopherols and tocotrienols show typical UV spectra with maximum absorption at 290-300 nm (Table 1.6). If the samples contain sufficient amounts of analytes, e.g., vegetable oils and supplemented products, a UV detector is sensitive enough. When higher sensitivity and better selectivity is needed, a fluorescence detector is the commonly used detector. With a fluorescence detector, it is possible to analyze tocopherols... 

Argauer, R.J. (1980), Fluorescence and ultraviolet absorbance of pesticides and naturally occurring chemicals in agricultural products after HPLC separation on a bonded-CN polar phase. Am. Chan. iV -, Symp. See. 1980-Vol. Pen. Anal. Methadai. 136, 103-126,... 

A selective and sensitive method for the determination of aliphatic anionic surfactants is reversed phase HPLC combined with postcolumn derivatization and fluorescence detection [62], After HPLC separation of the surfactants on a Q column, a UV-active cationic dye is added to the eluate in order to form fluorescent ion pairs. Then CHCI3 is added to the eluent stream as the extraction solvent for the ion pairs. The two phases are conducted through a sandwich-t) e phase separator where the major part of the organic phase is separated. Finally, the amount of ion pairs extracted into CHCI3 is determined by a fluorescence detector. 

In another study Bachas and coworkers have described two different postcolumn detection techniques for biotin and biocytin after HPLC separation based on fluorescence (116). One method involves avidin using 2,6-ANS (Fig. 10) as the fluorophore, while the other one relies on fiuoresceine covalently linked to streptavidin. The principle of these fluorescence detections has already been discussed. The HPLC separation was achieved on a C18 colunm using 0.1 M potassium phosphate buffer pH 7.0 with methanol (8 2) as mobile phase. The postcol-... 

Cells are typically concentrated by filtration and extracted into an organic solvent (usually acetone) after which, pigments are detected by fluorescence or absorption spectroscopy, sometimes after chromatographic separation (Bidigare and Trees, 2000). The application of HPLC to phytoplankton pigment analysis has lowered the uncertainty in the measurement of Chi a and accessory carotenoids, since compounds are physically separated and individually quantified. 

Histamine Extraction. Some secondary plant metabolites are very difficult to extract from their natural matrix and require lengthly soxhlet extractions. Complete histamine extraction was relatively simple. The HPLC separation was used to design a technique to confirm a complete extraction. The histamine peaks from three serial extractions on the same 3 grams of cotton plant leaves are shown in Figure 6. Post-column fluorescence detection of the fourth extraction showed only the slightest response even at the highest detector amplitude. Three extractions accounted for from 95% to 99% of the histamine content. The plant residues from these extractions yielded no additional histamine after standing at ambient conditions for two weeks. 

The basic methods to quantify biotin in food are bioassays, avidin-binding assays or fluorescent derivative assays. Although the avidin-binding assay of biotin and its metabolites after the separation by HPLC is considered as one of the best cnrrently available methods [587], it is still not largely diffnsed. 

A method for determination of various types of peroxides consists of RP-HPLC separation and applying post-column UV radiation to convert the peroxide to H2O2, which may be determined by CLD with bis(2,4,6-trichlorophenyl) oxalate (140) in the presence of 2,4,6,8-tetrathiomorpholinopyrimido[5,4-(J]pyrimidine (194) as fluorescence enhancer. Dibenzoyl peroxide and f-butyl perbenzoate have LOD (SNR 3) 6.8 and 7.5 p.M, respectively, and linearity range from 40 to 400 p.M. The method was applied for determination of dibenzoyl peroxide used as whitener of wheat flour, after extraction with ethanol . See a similar method in Section VLB.3. 

Figure 12.11—Comparison of UV andfluorescence detection after chromatographic separation. Aflatoxins, which are carcinogenic contaminants present in certain batches of grain cereals, are controlled by HPLC analysis. It can be seen that the peak intensities in UV detection vary with concentration whereas fluorescence detection is much more sensitive to aflatoxin G2 and B2. (Reproduced by permission of SUPELCO.)...

Determination of oxidized amino acids in urine is usually performed by isotope dilution gas chromatography-mass spectrometry (L9). DOPA is estimated by HPLC separation of acid protein hydrolysates with fluorescence detection (excitation 280 nm, emission at 320 nm) (A15). Other methods are based on borate-hydrochloric acid difference spectroscopy (this method suffers interference from tyrosine and tryptophan) (W2), derivatization of DOPA with nitrite and subsequent coulometric determination (W3), and fluorometric detection after derivatization with ethylenediamine (A15). 3-Hydroxylysine is quantitated by HPLC with 9-fluorenylmethyl chloroformate precolumn derivatization (M25) of amino acids obtained by gas-phase hydrolysis of proteins (F21). Other general methods to detect amino acid damage are mass spectometry methods applied to protein hydrolysates, such as tandem mass spectrometry (F6). 

Reversed-phase HPLC with fluorescence detection, after derivatization of plasma thiols with ammonium 7-fluorobenzo-2-oxa-l,3-diazole-4-sulphonate (SBD-F), is the most widely used method to determine total plasma amino thiols (cysteine, cysteinylglycine, and homocysteine). The time required for sample preparation (thiolic reduction, deproteinization, and precolumn derivatization g with SBD-F) and for thiol derivatives separation is nearly 2 h per sample. =... 

Determination of four tocopherols and four tocotrienols in vegetable oils and fats by the official American Oil Chemists Society method is based on separation by normal-phase HPLC and fluorescence detection (AOCS, 1990). Oil samples are dissolved in hexane, whereas margarines and other fats containing vitamer esters need a cold saponification step to liberate the vitamers. The American Association of Cereal Chemists has a method to analyze vitamin E in various foods. This method (AACC, 1997) is applicable to a vitamin E range of 1 x 10" - 100%, and it includes hot saponification and separation by reversed-phase HPLC. Results are calculated as a-tocopherol acetate. The Royal Society of Chemistry has approved a method to analyze vitamin E in animal feedstuffs by normal-phase HPLC after the vitamers have been liberated by hot saponification (Analytical Methods Committee, 1990). 

The hrst pTAS devices were based on GC. Soon after, HPLC-based pTAS platforms began to appear and then CE followed. In chip CE, an injection was performed and an electrophoretic separation of a sample mixture (different fluorescent dyes) and all liquid handling was achieved using electro-osmotic flow. Since then, electrophoresis and electrokinetic fluid handling have been the cornerstones of many miniaturised analytical devices. 

A logical step would be HPLC separation of the extracts and measurement of the native fluorescence after chromatography. Enhanced sensitivity in the detection of the individual phenols may be achieved by chromatography of dansyl derivatives (Cassidy et al., 1974) in which case ng quantities of phenolic compounds were estimated in physiological fluids. 

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