Fluorescence HPLC separation

Popova and colleagues47 carried out TLC of oxidation products of 4,4 -dinitrodiphenyl sulphide (the sulphoxide and sulphone) on silica gel + a fluorescent indicator, using hexane-acetone-benzene-methanol(60 36 10 l) as solvent mixture. Morris130 performed GLC and TLC of dimethyl sulphoxide. For the latter, he applied a 6% solution of the sample in methanol to silica gel and developed with methanol-ammonia solution(200 3), visualizing with 2% aqueous Co11 thiocyanate-methanol(2 1). HPLC separations of chiral mixtures of sulphoxides have been carried out. Thus Pirkle and coworkers131-132 reported separations of alkyl 2,4-dinitrophenyl sulphoxides and some others on a silica-gel (Porosil)-bonded chiral fluoroalcoholic stationary phase, with the structure ... 

Another useful standard Is SRM 1647, priority pollutant polynuclear aromatic hydrocarbons (in acetonitrile). It can be used to calibrate liquid chromatographic Instruments (retention times. Instrument response), to determine percent recoveries, and to fortify aqueous samples with known PAH concentrations. Figure 8 Illustrates the HPLC separation and UV detection (fluorescence is also used extensively) for the 16 priority pollutants. 

Though we and others (27-29) have demonstrated the utility and the improved sensitivity of the peroxyoxalate chemiluminescence method for analyte detection in RP-HPLC separations for appropriate substrates, a substantial area for Improvement and refinement of the technique remains. We have shown that the reactions of hydrogen peroxide and oxalate esters yield a very complex array of reactive intermediates, some of which activate the fluorophor to its fluorescent state. The mechanism for the ester reaction as well as the process for conversion of the chemical potential energy into electronic (excited state) energy remain to be detailed. Finally, the refinement of the technique for routine application of this sensitive method, including the optimization of the effi-ciencies for each of the contributing factors, is currently a major effort in the Center for Bioanalytical Research. 

We inferred that these properties might be exploited in a series of unique derivatizing reagents designed specifically for trace analysis of organic compounds using HPLC separation and fluorescence detection. The use of these pyridones for the analytical purposes reported here is based on their acidic properties. Treatment of a lH-2-pyridone with a base converts the pyridone to its salt. 

Wetai Ion Analysis. We have reported a sensitive trace-metal analysis based upon HPLC separation of p-aminophenyl EDTA chelates and fluorescence detection by postcolumn reaction with fluorescamine (23). An application of the pyridone chemistry already discussed leads to a fluorescent-labeled EDTA (VIII). 

We have developed reverse-phase ion-pairing HPLC separations of substituted EDTA metal chelates of several transition metals (including Cd, Zn, Fb, and Hg) and several lanthanides (La, Ce, Eu, Dy, Er, Yb, Lu). Detection levels of these chelates are currently being assessed. A sensitive metal ion analysis employing an inherently fluorescent EDTA seems feasible. 

Plasma, Urine ( -aminolevuli nic acid) Derivatization of -aminolevulinic acid with formaldehyde and acetylacetone to form fluorescent compounds separation using HPLC HPLC/FL 3 pg/L No data Oishi et al. 1996... 

LEDs have already found a number of applications that demonstrate potential with respect to fluorescence measurements requiring routine operation, low cost, or compact implementations. A 50 mW LED (GaP) with a peak emission at 565 nm has been used in conjunction with HPLC separation and methylene blue fluorescence to detect... 

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. 

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 15 Representative RP-HPLC Separation of the Tryptic/Chymotryptic Peptides of Bovine Thyrotropin p-Subunit bTSHfS Labeled In Situ with the Fluorescent Reagent 5-[(Iodoacetamidoethyl)amino]naphthalene-l-sulfonic acid (5-l-AEDANS) MI a b...

Two analytical methods for priority pollutants specified by the USEPA (38) use HPLC separation and fluorescence or electrochemical detection. Method 605, 40 CFR Part 136, determines benzidine and 3,3-dichlorobenzidine by amperometric detection at +0.80 V, versus a silver/silver chloride reference electrode, at a glassy carbon electrode. Separation is achieved with a 1 1 (v/v) mixture of acetonitrile and a pH 4.7 acetate buffer (1 M) under isocratic conditions on an ethyl-bonded reversed-phase column. Lower limits of detection are reported to be 0.05 /xg/L for benzidine and 0.1 /xg/L for 3,3-dichlorobenzidine. Method 610, 40 CFR Part 136, determines 16 PAHs by either GC or HPLC. The HPLC method is required when all 16 PAHs need to be individually determined. The GC method, which uses a packed column, cannot adequately individually resolve all 16 PAHs. The method specifies gradient elution of the PAHs from a reversed-phase analytical column and fluorescence detection with an excitation wavelength of 280 nm and an emission wavelength of 389 nm for all but three PAHs naphthalene, acenaphthylene, and acenaphthene. As a result of weak fluorescence, these three PAHs are detected with greater sensitivity by UV-absorption detection at 254 nm. Thus, the method requires that fluores-... 

Because methylene-interrupted polyunsaturates do not have strongly absorbing chromo-phores in the UV region, detection by refractive index or far-UV detection (205-214 nm) has been utilized in RP-HPLC separations of free fatty acids and their aliphatic esters. Refractive index detection is less sensitive than UV detection. However, with far-UV detection, solvents absorbing UV below 210 cannot be used. The RP-HPLC separations reported to date have generally involved derivatization designed to incorporate aromatic chromophores allowing detection by fluorescence or UV detection. 

Data calculated from the peak areas of diphenylhexatriene fluorescence (340/460 nm) and UV absorption (205 nm) following HPLC separation of the phospholipids on a 5-/x,m Nucleosil 5 NH2 stationary phase. Egg PC was set as 1. 

Cyclamate can also be determined after its oxidation to cyclohexylamine. Cyclohexy-lamine can be quantified by HPLC with trinitrobenzenesulfonic acid precolumn derivatization and UV detection (72). Cyclohexylamine can also be converted into a fluorescent derivative, separated on a Cl8 reversed-phase column with acetonitrile Na2HP04 buffer (64 36, v/v), and quantified at 350 and 440-650 nm of excitation and emission, respectively (73). 

O-Phthaldialdehyde (OPA) is an amine detection reagent that reacts in the presence of 2-mercaptoethanol to generate a fluorescent product (for preparation, see Section 4.1, 2-mercaptoethanol) (Fig. 91). The resultant fluorophore has an excitation wavelength of 360 nm and an emission point at 455 nm. OPA can be used as a sensitive detection reagent for the HPLC separation of amino acids, peptides, and proteins (Fried et al., 1985). It is also possible to measure the amine content in proteins and other molecules using a test tube or microplate format assay with OPA. Detection limits are typically in the microgram per milliliter range for proteins. 

Mercapturates are proving to be very useful phase II reaction products for measuring exposure to xenobiotics, especially because of the sensitive determination of these substances by high-performance liquid chromatography (HPLC) separation, and fluorescence detection of their o-phthaldialdehyde derivatives. In addition to toluene, the xenobiotics for which mercapturates may be monitored include styrene, structurally similar to toluene acrylonitrile allyl chloride atrazine butadiene and epichlorohydrin. 

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

Figure 5. Fluorescence spectra in a nitrogen matrix at 15 K (excited by a mercury-xenon lamp) at two excitation wavelengths of the fifth fraction from the HPLC separation of Synthoil. Compounds BaA, benz[a]anthracene C, chrysene P, pyrene U. unknown...

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