Effect of urea on synchronous fluorescence spectra and electrochemical behaviour of cytochrome c

The changes of the synchronous fluorescence spectra and the electrochemical behaviour of cytochrome c with the urea concentration are studied. It has been found that with the increase of urea concentration, there occur sequentially the deaggregation of cytochrome c molecules, the increase of exposure extent of the heme group to the solvent, the disruption of Fe-S bond of the heme group and the change in the electrochemical behaviour of cytochrome c. It is suggested that the reason why the electrochemical reaction of cytochrome c is irreversible is that cytochrome c molecules exist in the concentrated solution as oligomers which are electrochemically inactive.

Fluorescence Characterization of Dissolved Organic Matter in East China Sea

Sea water samples were collected in the East China Sea in March and April, 2005, and three-dimensional fluorescence of dissolved organic matter was measured by fluorescence excitation-emission matrix spectroscopy. The position, number and intensity of fluorescence peak in the spectra and the relations of the peaks were analyzed to determine the type, distribution and origin of the fluorescence dissolved organic matter. Seven types of fluorescence peaks were detected from the samples. There are protein-like fluorescence peaks B with Ex(max)/Em(max) = 275/300 nm, D with Ex(max)/Em(max) = 225/295-305 nm, T with Ex(max)/Em(max) = 280/345 nm, and S with Ex(max)/Em(max) = 225-240/320-350 nm, two humic-like peaks A with Ex(max)/Em(max) = 250-255/410-455 nm and C 335-345/410-440 ran, and marine humic peak M with Ex(max)/Em(max) = 305 nm/400-420 nm. Peaks B, S and A appeared in all surveyed area. Peaks T and D appeared in the north of the surveyed area. Peaks M and C only appeared in a few stations. In the surface layer, the source of the fluorescence dissolved organic matter might be the fresh water outflow of the Yangtze River, while the fluorescence dissolved organic matter in the middle layer had double sources from the Yangtze River and the phytoplankton. The good correlationships of different fluorescence peaks showed the same source or some relationship between the protein-like and the humic-like fluorescence dissolved organic matter.

Three dimensional fluorescence excitation-emission on matrix spectrum of dissolved organic substance in marine microalgaes growth process

The three-dimensional fluorescence spectrum was used to detect the changes in dissolved organic substances from the cultured Skeletonema costatum, Alexandrium tamarense, Alexandrium mimutum, Scrippsiella trochodea, Prorocentrum donghaiense and Prorocentrum micans. The result indicates that all of the microalgaes can produce FDOM in the growth courses. Diatom such as Skeletonema costatum can produce humic-like FDOM. However dinoflagellate can produce protein-like FDOM at exponential growth phase. When the algae grows into decadency phase, the intensity of humic-like and protein-like fluorescence augments rapidly, which may be due to a mass of FDOM realeased by the old or dead cell fragmentation and the degradation of bacteria by using non-FDOM. The fluorescent intensity of Alexandrium tamarense, Alexandrium mimutum, Prorocentrum donghaiense and Prorocentrum micans can reduce at anaphase of decadency phase because of the degradation of bacteria and light. The same genus of algae can produce similar FDOM, for example: Alexandrium tamarense, and Alexandrium mimutum, Prorocentrum donghaiense and Prorocentrum micans, but the positions of the fluorescence peaks are different.

Application of 2-(11H-benzo[a] carbazol-11-yl) Ethyl Carbonochloridate as a Pro-column Derivatization Reagent of Amino Acid by High Performance Liquid Chromatography with Fluorescence Detection

On a reversed phase Hypersil BDS C-18 (200 mm x 4. 6 mm, 5 mu m) column, 20 amino acids, which were derivatized using 2-(11H-benzo [a] carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) as pre-column derivatization reagent, were separated in conjunction with a gradient elution. Optimum derivatization was obtained by reacting of amino acids with BCEC-Cl at room temperature for 5 min in the presence of sodium borate catalyst in acetonitrile solvent. The fluorescence excitation and emission wavelengths were 279 nm and 380 nm respectively. The identification of amino acid derivatives from hydrolyzed bovine serum albumin and bee pollen was carried out by post-column mass spectrometry with electrospray ion source in positive ion mode. Linear correlation coefficients of the amino acid derivatives were > 0.9990, and detection limits (at signal to noise of 3:1) were 1.49 - 19.74 fmol for the labeled amino acids.

Analysis of primary aromatic amines using precolumn derivatization by HPLC fluorescence detection and online MS identification

2-(2-Phenyl-1H-phenanthro-[9,10-d]imidazole-1-yl)-acetic acid (PPIA) and 2-(9-acridone)-acetic acid (AAA), two novel precolumn fluorescent derivatization reagents, have been developed and compared for analysis of primary aromatic amines by high performance liquid chromatographic fluorescence detection coupled with online mass spectrometric identification. PPIA and AAA react rapidly and smoothly with the aromatic amines on the basis of a condensation reaction using 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (EDC) as dehydrating catalyst to form stable derivatives with emission wavelengths at 380 and 440 nm, respectively. Taking six primary aromatic amines (aniline, 2-methylaniline, 2-methoxyaniline, 4-methylaniline, 4-chloroaniline, and 4-bromoaniline) as testing compounds, derivatization conditions such as coupling reagent, basic catalyst, reaction temperature and time, reaction solvent, and fluorescent labeling reagent concentration have also been investigated. With the better PPIA method, chromatographic separation of derivatized aromatic amines exhibited a good baseline resolution on an RP column. At the same time, by online mass spectrometric identification with atmospheric pressure chemical ionization (APCI) source in positive ion mode, the PPIA-labeled derivatives were characterized by easy-to-interpret mass spectra due to the prominent protonated molecular ion m/z [M + H](+) and specific fragment ions (MS/MS) m/z 335 and 295. The linear range is 24.41 fmol-200.0 pmol with correlation coefficients in the range of 0.9996-0.9999, and detection limits of PPIA-labeled aromatic amines are 0.12-0.21 nmol/L (S/N = 3). Method repeatability, precision, and recovery were evaluated and the results were excellent for the efficient HPLC analysis. The most important argument, however, was the high sensitivity and ease-of-handling of the PPIA method. Preliminary experiments with wastewater samples collected from the waterspout of a paper mill and its nearby soil where pollution with aromatic amines may be expected show that the method is highly validated with little interference in the chromatogram.

Extraction of Nitraria tangutorum seed oil by supercritical carbon dioxide and determination of free fatty acids by HPLC/APC/IMS with fluorescence detection

The seed oil from Nitraria tangutorum samples was obtained by supercritical carbon dioxide extraction methods. The extraction parameters for this methodology, including pressure, temperature, particle size and extraction time, were optimized. The free fatty acids in the seed oil were separated with a pre-column derivation method and 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-p-toluenesulfonate (BDETS) as a labeling regent, followed by high-performance liquid chromatography (HPLC) with fluorescence detection. The target compounds were identified by mass spectrometry with atmospheric pressure chemical ionization (APCI in positive-ion mode). HPLC analysis shows that the main compositions of the seed oil samples were free fatty acids (FFAs) in high to low concentrations as follows: linoleic acid, oleic acid, hexadecanoic acid and octadecanoic acid. The assay detection limits (at signal-to-noise of 3:1) were 3.378-6.572 nmol/L. Excellent linear responses were observed, with correlation coefficients greater than 0.999. The facile BDETS derivatization coupled with mass spectrometry detection allowed the development of a highly sensitive method for analyzing free fatty acids in seed oil by supercritical CO2 extraction. The established method is highly efficient for seed oil extraction and extremely sensitive for fatty acid profile determination. (C) 2007 Elsevier B.V. All rights reserved.

Determination of free fatty acids in soil and bryophyte plants by precolumn derivatization via HPLC with fluorescence detection and tandem mass spectrometry (HPLC-MS/MS)

A sensitive method for the determination of 30 kinds of free fatty acids (FFAs, C-1-C-30) with 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f] 9,10-phenan- threne (TSPP) as labeling reagent and using high performance liquid chromatography with fluorescence detection and identification by online postcolumn mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion mode (HPLC/MS/APCI) has been developed. TSPP could easily and quickly label FFAs in the presence of K2CO3 catalyst at 90 degrees C for 30 min in N,N-dimethylformamide (DMF) solvent, and maximal labeling yields close to 100% were observed with a 5-fold excess of molar reagent. Derivatives were stable enough to be efficiently analyzed by high performance liquid chromatography. TSPP was introduced into fatty acid molecules and effectively augmented MS ionization of fatty acid derivatives and led to regular MS and MS/MS information. The collision induced cleavage of protonated molecular ions formed specific fragment ions at m/z [MH](+)(molecular ion), m/z [M'+CH2CH2](+)(M' was molecular mass of the corresponding FFA) and m/z 295.0 (the, mass of protonated molecular core structure of TSPP). Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C-8 column (4.6 x 150 mm, 5 mu m, Agilent) with a good baseline resolution in combination with a gradient elution. Linear ranges of 30 FFAs are 2.441 x 10(-3) to 20 mu mol/L, detection limits are 3.24 similar to 36.97 fmol (injection volume 10 mu L, at a signal-to-noise ratio of 3, S/N 3:1). The mean interday precision ranged from 93.4 to 106.2% with the largest mean coefficients of variation (R.S.D.) < 7,5%. The mean intraday precision for all standards was < 6.4% of the expected concentration. Excellent linear responses were observed with correlation coefficients of > 0.9991. Good compositional data could be obtained from the analysis of extracted fatty acids from as little as 200 mg of bryophyte plant samples.Therefore, the facile TSPP derivatization coupled with HPLC/MS/APCI analysis allowed the development of a highly sensitive method for the quantitation of trace levels of short and long chain fatty acids from biological and natural environmental samples.

Determination of 30 free fatty acids in two famous Tibetan medicines by HPLC with fluorescence detection and mass spectrometric identification

A simple and sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection and mass spectrometric identification has been developed for analysis of 30 long-chain and short-chain free Fatty acids (FFAs). The fatty acids were derivatized to their esters with 1-[2-(p-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) in N,N-dimethylformamide (DMF) at 90 degrees C with anhydrous K2CO3 as catalyst. A mixture Of C-1-C-30 fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C-8 column. Qualitative identification of the acids was performed by atmospheric-pressure chemical ionization mass spectrometry (APCI-MS) in positive-ion mode. The fluorescence excitation and emission wavelengths were 260 and 380 nm, respectively. Quantitative determination of the 30 acids in two Tibetan medicines Gentiana straminea and G. dahurica was performed. The results indicated that the medicines contained many FFAs. Linear correlation coefficients for the FFA derivatives were > 0.9991. Relative standard deviations (RSDs, n = 6) for the fatty acid derivatives were < 3%. Detection limits (at a signal-to-noise ratio of 3:1) were 3.1-38 fmol. When the fatty acid derivatives were determined in the two real samples results were satisfactory and the sensitivity and reproducibility of the method were good.

Analysis of saturated free fatty acids from pollen by HPLC with fluorescence detection

A sensitive method for the determination of free fatty acids using 2-(2-(anthracen-10-yl)-1H-naphtho[2,3-dimidazol-1-yl) ethyl-p-toluenesuIfonate (ANITS) as tagging reagent with fluorescence detection has been developed. ANITS could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90 degrees C for 40 min in N,N-dimethylformamide solvent. From the extracts of rape bee pollen samples, 20 free fatty acids were sensitively determined. Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C8 column by HPLC in conjunction with gradient elution. The corresponding derivatives were identified by post-column APCI/MS in positive-ion detection mode. ANITS-fatty acid derivatives gave an intense molecular ion peak at mlz [M+H](+); with MS/MS analysis, the collision-induced dissociation spectra of m/z [M+H](+) produced the specific fragment ions at mlz [M-345](+) and mlz 345.0 (here, m/z 345 is the core structural moiety of the ANITS molecule). The fluorescence excitation and emission wavelengths of the derivatives were lambda(ex) = 250 nm and lambda(em) = 512 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are > 0.9999. Detection limits, at a signal-to-noise ratio of 3 : 1, are 24.76-98.79 fmol for the labeled fatty acids.

HPLC-APCI-MS determination of free fatty acids in Tibet folk medicine Lomatogonium rotatum with fluorescence detection and mass spectrometric identification

A simple and sensitive method for the determination of free fatty acids (FFAs) using acridone-9-ethyl-p-toluenesulfonate (AETS) as a fluorescence derivatization reagent by high performance liquid chromatography (HPLC) has been developed. Free fatty acid derivatives were separated on an Eclipse XDB-C-8 column with a good baseline resolution and detected with the fluorescence of which excitation and emission wavelengths of derivatives were set at lambda(ex) 404 and lambda(em) 440 nm, respectively. Identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with an atmospheric pressure chemical ionization (APCI) source under positive-ion detection mode. Nineteen FFAs from the extract of Lomatogonium rotatum are sensitively determined. The results indicate that the plant Lomatogonium rotatum is enriched with an abundance of FFAs and FFAs of higher contents, which mainly focus on even carbon atoms, C-14, C-16, and C-18. The validation of the method including linearity, repeatability, and detection limits was examined. Most linear correlation coefficients for fatty acid derivatives are > 0.9989, and detection limits (at signal-to-noise of 3: 1) are 12.3-43.7 fmol. The relative standard deviations (RSDs) of the peak areas and retention times for 19 FFAs standards are < 2.24% and 0.45%, respectively. The established method is rapid and reproducible for the separation determination of FFAs from the extract of Lomatogonium rotatum with satisfactory results.

Coke formation during the methanol conversion to olefins in zeolites studied by UV Raman spectroscopy

Coke formation on/in ZSM-5, USY and SAPO-34 zeolites was investigated during the methanol conversion to olefins at temperatures from 298 to 773 K using ultraviolet (UV) Raman spectroscopy. The fluorescence interference that usually obscures the Raman spectra of zeolites in the conventional Raman spectroscopy, particularly for coked catalysts, can be successfully avoided in the UV Raman spectroscopy. Raman spectra are almost the same for adsorbed methanol on the three zeolites at room temperature. However, the Raman spectra of the surface species formed at elevated temperatures are quite different for the three zeolites. Coke species formed in/on SAPO-34 are mainly polyolefinic species, and in/on ZSM-5 are some aromatic species, but polyaromatic or substituted aromatic species are predominant in USY at high temperatures. Most of the coke species can be removed after a treatment with O-2 at 773 K, while some small amount of coke species always remains in these zeolites, particularly for USY. The main reason for the different behavior of coke formation in the three zeolites could be attributed to the different pore structures of the zeolites. (C) 2000 Elsevier Science B.V. All rights reserved.

Characterization of aluminosilicate zeolites by UV Raman spectroscopy

A series of aluminosilicate zeolites are characterized by UV Raman spectroscopy for the first time, and UV Raman spectra of various zeolites give strong and clear bands with high resolution, while conventional Raman spectra of these zeolites are difficult to obtain because of a strong background fluorescence. Additionally, these zeolites show several new bands in UV Raman spectroscopy. A summary of these UV Raman spectra over various zeolites suggests that the bands at 470-530, 370-430, 290-410, and 220-280 cm(-1) can be assigned to the bending modes of 4-, 5-, 6-, and 8-membered rings of aluminosilicate zeolites, respectively. Furthermore, it is found that the band intensity of zeolites in UV Raman spectroscopy is dependent on the Si/Al ratio. Moreover, the UV Raman spectra of crystallization, for zeolite X at various times show that, in the initial stage of crystallization, the 4-membered rings (510 cm(-1)) interconnect each other to form beta -cages with 6-membered rings (390 cm(-1)), which further crystallize to zeolite X. (C) 2001 Elsevier Science B.V. All rights reserved.