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.

Identification of phenolics and nucleoside derivatives in Gastrodia elata by HPLC-UV-MS

An HPLC-UV-MS method for simultaneous identification of predominant phenolics and minor nucleoside derivatives in Gastrodia elata was developed, which was based on their UV and MS characteristics summarized through a series of homemade reference standard experiments. Phenolics showed characteristic UV lambda(max) at 267 nm, [M + NH4](+) base peak in positive mode and [M - H](-) base peak in negative mode while nucleosides exhibited UV lambda(max) at 255 nm, [M + H](+), [M - H + 2H(2)O](-) or [M - H + CH3COOH](-). Phenolics conjugates mainly underwent the consecutive loss of gastrodin residue (- 268 U) and the combined loss of H2O and CO2 from the citric acid unit under negative MS/MS conditions whereas nucleosides simply lost the ribose (- 132 U) under positive MS/MS conditions. According to these characteristics, a special pattern under MS/MS conditions and reported compound data for G. elata in the literature, not only 15 phenolics were identified but also 6 nucleoside derivatives were identified. Among these compounds, seven phenolics and three nucleoside derivatives have not been reported yet from G. elata.

Enhancement of atmospheric pressure chemical ionization for the determination of free and glycine-conjugated bile acids in human serum

A highly sensitive and accurate method based on the precolumn derivatization of bile acids (BA) with a high ionization efficiency labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-benzenesulfonate (BDEBS) coupled with LC/MS has been developed. After derivatization, BA molecules introduced a weak basic nitrogen atom into the molecular core structure that was readily ionized in commonly used acidic HPLC mobile phases. Derivatives were sufficiently stable to be efficiently analyzed by atmospheric pressure chemical ionization (APCI)-MS/MS in positive-ion mode. The MS/MS spectra of BA derivatives showed an intense protonated molecular ion at m/z [M + H](+). The collision-induced dissociation of the molecular ion produced fragment ions at [MH - H2O](+), [MH - 2H(2)O](+), [MH - 3H(2)O](+). The characteristic fragment ions were at m/z 320.8, 262.8, and 243.7 corresponding to a cleavage of N - CO, O - CO, and C - OCC, respectively, and bonds of derivatized molecules. The selected reaction monitoring, based on the m/z [M + H]+ -> [MH - H2O](+), [MH - H2O](+), [MH - 2H(2)O](+), [MH-3H(2)O](+), 320.8, 262.8, and 243.7 transitions, was highly specific for the BA derivatives. The LODs for APCI in a positive-ion mode, at an S/N of 5, were 44.36-153.6 fmol. The validation results showed high accuracy in the range of 93-107% and the mean interday precision for all standards was < 15% at broad linear dynamic ranges (0.0244-25nmol/mL). Good linear responses were observed with coefficients of > 0.9935 in APCI/MS detection. Therefore, the facile BDEBS derivatization coupled with mass spectrometric analysis allowed the development of a highly sensitive and specific method for the quantitation of trace levels of the free and glycine-conjugated BA from human serum samples.

Total analytical method for Radix astragali extract using two-binary multi-segment gradient elution liquid chromatography

There is an urgent need for thorough analysis of Radix astragali, a widely used Chinese herb, for quality control purposes. This paper describes the development of a total analytical method for Radix astragali extract, a multi-component complex mixture. Twenty-four components were separated step by step from the extract using a series of isocratic isopropanol-methanol elutions, and then 42 components were separated similarly using methanol-water elutions. Based on the log k(w) and -S of the 66 components obtained from the above procedure and the optimization software developed in our laboratory, an optimum elution program consisting of seven methanol-water segments and four isopropanol-methanol segments was developed to finish the task of analyzing the total components in a single run. Under optimized gradient conditions, the sample of Radix astragali extract was analyzed. As expected, most of the components were well separated and the experimental chromatogram was in a good agreement with the predicted one.

Modification of a poly(methyl methacrylate) injection-molded microchip and its use for high performance analysis of DNA

Inexpensive and permanently modified poly(methyl methacrylate)(PMMA) microchips were fabricated by an injection-molding process. A novel sealing method for plastic microchips at room temperature was introduced. Run-to-run and chip-to-chip reproducibility was good, with relative standard deviation values between 1-3% for the run-to-run and less than 2.1% for the chip-to-chip comparisons. Acrylonitrile-butadiene-styrene (ABS) was used as an additive in PMMA substrates. The proportions of PMMA and ABS were optimized. ABS may be considered as a modifier, which obviously improved some characteristics of the microchip, such as the hydrophilicity and the electro-osmotic flow (EOF). The detection limit of Rhodamine 6G dye for the modified microchip on the home-made microchip analyzer showed a dramatic 100-fold improvement over that for the unmodified PMMA chip. A detection limit of the order of 10(-20) mole has been achieved for each injected phiX-174/HaeIII DNA fragment with the baseline separation between 271 and 281 bp, and fast separation of 11 DNA restriction fragments within 180 seconds. Analysis of a PCR product from the tobacco ACT gene was performed on the modified microchip as an application example.

Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteins

A new set-up was constructed for capillary isoelectric focusing (CIEF) involving a sampling capillary as a bypass fixed to the separation capillary. Sample solutions were subjected to a previously established pH gradient from the sample capillary. Besides performing conventional CIEF, the separation of ampholytic compounds with isoelectric points (p/s) beyond the pH gradient was carried out on this system. This method was termed as pH gradient driven electrophoresis (PGDE) and the basic mathematical expressions were derived to express the dynamic fundamentals. Proteins such as lysozyme, cytochrome C, and pepsin with p/s higher than 10 or below 3 were separated in a pH gradient provided by Pharmalyte (pH 3-10). Finally, this protocol convincingly exhibited its potential in the separation of a solution of chicken egg white.

Preparation of monolithic silica column with strong cation-exchange stationary phase for capillary electrochromatography

A monolithic silica based strong cation-exchange stationary phase was successfully prepared for capillary electrochromatography. The monolithic silica matrix from a sol-gel process was chemically modified by treatment with 3-mercaptopropyltrimethoxysilane followed by a chemical oxidation procedure to produce the desired function. The strong cation-exchange stationary phase was characterized by its substantial and stable electroosmotic flow (EOF), and it was observed that the EOF value of the prepared column remained almost unchanged at different buffer pH values and slowly decreased with increasing phosphate concentration in the mobile phase. The monolithic silica column with strong cation-exchange stationary phase has been successfully employed in the electrochromatographic separation of beta-blockers and alkaloids extracted from traditional Chinese medicines (TCMs). The column efficiencies for the tested beta-blockers varied from 210,000 to 340,000 plates/m. A peak compression effect was observed for atenolol with the mobile phase having a low phosphate concentration.

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.

Capillary array electrophoresis for the research of asymmetric transformation reaction of L-proline to D-proline

One asymmetric transformation reaction Of L-proline (L-Pro) to D-proline was studied by a home-made capillary array electrophoresis (CAE) for the first time. The aldehyde catalysts and the organic acid solvents for the asymmetric transformation reaction were rapidly screened and the enantiomeric excess values of the asymmetric product Of L-Pro were directly obtained from the electrophoretogram of CAE.

Synthesis and characterisation of novel superficially porous silica based stationary phases for use in liquid chromatography

The concept of pellicular particles was suggested by Horváth and Lipsky over fifty years ago. The reasoning behind the idea of these particles was to improve column efficiency by shortening the pathways analyte molecules can travel, therefore reducing the effect of the A and C terms. Several types of shell particles were successfully marketed around this time, however with the introduction of high quality fully porous silica under 10 μm, shell particles faded into the background. In recent years a new generation of core shell particles have become popular within the separation science community. These particles allow fast and efficient separations that can be carried out on conventional HPLC systems. Chapter 1 of this thesis introduces the chemistry of chromatographic stationary phases, with an emphasis on silica bonded phases, particularly focusing on the current state of technology in this area. The main focus is on superficially porous silica particles as a support material for liquid chromatography. A summary of the history and development of these particles over the past few decades is explored, along with current methods of synthesis of shell particles. While commercial shell particles have a rough outer surface, Chapter 2 focuses on the novel approach to growth of smooth surface superficially porous particles in a step-by-step manner. From the Stöber methodology to the seeded growth technique, and finally to the layer-bylayer growth of the porous shell. The superficially porous particles generated in this work have an overall diameter of 2.6 μm with a 350 nm porous shell; these silica particles were characterised using SEM, TEM and BET analysis. The uniform spherical nature of the particles along with their surface area, pore size and particle size distribution are examined in this chapter. I discovered that these smooth surface shell particles can be synthesised to give comparable surface area and pore size in comparison to commercial brands. Chapter 3 deals with the bonding of the particles prepared in Chapter 2 with C18 functionality; one with a narrow and one with a wide particle size distribution. This chapter examines the chromatographic and kinetic performance of these silica stationary phases, and compares them to a commercial superficially porous silica phase with a rough outer surface. I found that the particle size distribution does not seem to be the major contributor to the improvement in efficiency. The surface morphology of the particles appears to play an important role in the packing process of these particles and influences the Van Deemter effects. Chapter 4 focuses on the functionalisation of 2.6 μm smooth surface superficially porous particles with a variety of fluorinated and phenyl silanes. The same processes were carried out on 3.0 μm fully porous silica particles to provide a comparison. All phases were accessed using elemental analysis, thermogravimetric analysis, nitrogen sorption analysis and chromatographically evaluated using the Neue test. I observed comparable results for the 2.6 μm shell pentaflurophenyl propyl silica when compared to 3.0 μm fully porous silica. Chapter 5 moves towards nano-particles, with the synthesis of sub-1 μm superficially porous particles, their characterisation and use in chromatography. The particles prepared are 750 nm in total with a 100 nm shell. All reactions and testing carried out on these 750 nm core shell particles are also carried out on 1.5 μm fully porous particles in order to give a comparative result. The 750 nm core shell particles can be synthesised quickly and are very uniform. The main drawback in their use for HPLC is the system itself due to the backpressure experienced using sub – 1 μm particles. The synthesis of modified Stöber particles is also examined in this chapter with a range of non-porous silica and shell silica from 70 nm – 750 nm being tested for use on a Langmuir – Blodgett system. These smooth surface shell particles have only been in existence since 2009. The results displayed in this thesis demonstrate how much potential smooth surface shell particles have provided more in-depth optimisation is carried out. The results on packing studies reported in this thesis aims to be a starting point for a more sophisticated methodology, which in turn can lead to greater chromatographic improvements.

Preparation and evaluation of Fuller's Earth Beads for removal of Cesium from waste streams

This work deals with removal of Cesium (radioactive waste) in dilute aqueous phase by adsorption. Fullers earth was used as an adsorbent. The adsorption capacity of Fullers earth with respect to Cesium was found to be high, 26.3 mg / g of adsorbent.