A Comparative Study of Diamine-Modified and Amine-Modified Silica Nanoparticles as Pseudo-Stationary Phases for CE Separation

The use of silica nanoparticles with surface modification in different applications is a topic of growing interest. This article provides a systematic comparison of diamine-modified and amine-modified silica nanoparticles as pseudo-stationary phases (PSPs) in capillary electrophoresis. The different electrophoretic behavior of these PSPs was evaluated by analysis of four auxins, indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (dCPAA), and 2-(1-naphthyl)acetic acid (NAA). As expected, fast separation of the auxins was achieved, because they migrate in the same direction as the electro-osmotic flow under the optimum experimental conditions. However, the two nanoparticles had different selectivity and efficiency in the separation of the auxins, which could be related to the primary and secondary amines in their structures. The results indicated that even slight structural changes of the functional groups of the nanoparticles have pronounced effect on the interactions between the pseudo-stationary phase and the analytes.     

Polydopamine-based permanent coating capillary electrochromatography for auxin determination

A novel, simple, and economical method for the preparation of open-tubular capillary column using polydopamine coating was reported for the first time. After the capillary was filled with dopamine solution for 20h, polydopamine was formed and deposited on the inner wall of capillary as permanent coating via the oxidation of dopamine by the oxygen dissolved in the solution. Moreover, the electroosmotic flow of the coated capillaries was measured to be dependent on the repetitive coating times. The performance of the polydopamine-coated capillary electrochromatography was validated by the analysis of four auxins, indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (dCPAA), indole-3-acetic acid (IAA), and phenoxyacetic acid (PAA). The precisions (RSD, n=5) were in the range of 1.6-2.4% for migration time, 4.0-6.5% for peak area response, and 3.6-4.7% for peak height response for the four auxins at 1microgmL(-1) level. The detection limits were 0.185, 0.172, 0.177, and 0.259microg/mL for IBA, dCPAA, IAA, and PAA, respectively. The method was successfully used to the determination of IAA in the culture media of IAA-producing bacteria.     

Dual-cloud point extraction and tertiary amine labeling for selective and sensitive capillary electrophoresis-electrochemiluminescent detection of auxins

The low concentrations of the auxins in samples of plant tissue necessitate the use of selective and sensitive techniques for their quantification. Herein a selective and sensitive method based on dual-cloud point extraction (dCPE) and tertiary amine labeling for the quantification of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) by capillary electrophoresis-electrochemiluminescence (CE-ECL) is proposed. The procedure for dCPE included two cloud point processes with Triton X-114 as the extractant. The two auxins became hydrophobic in an acidic solution and were extracted into surfactant-rich phase after the first cloud point procedure. They were then back-extracted into the alkaline aqueous phase during the second cloud point step. The extracted auxins were reacted with 2-(2-aminoethyl)-1-methylpyrrolidine (AEMP) in acetonitrile that contained N,N′-dicyclohexylcarbodiimide and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine to produce their AEMP-derivatives. The two auxin-AEMP-derivatives were subjected into CE and detected by Ru(bpy)₃²⁺-based ECL. The preconcentration factors for IAA and IBA with dCPE were 40.5 and 43.4, respectively. The on-capillary detection limits (S/N = 3) were 2.5 and 2.8 nM for IAA and IBA. This protocol presents a clear advantage in that it reduces the interference from the matrixes extensively and gives a high sensitivity for the detection of auxins. The proposed method was applied successfully to the detection of the two auxins in acacia tender leaves, buds, and bean sprout.     

Preparation of chitosan‐modified silica nanoparticles and their applications in the separation of auxins by capillary electrophoresis

In recent years, nanoparticles have gained more attention when used in separation science. In this study, chitosan‐modified silica nanoparticles were successfully synthesized and characterized by transmission electron microscopy, elemental analysis and zeta potential measurements, etc. When added into the running buffer solution as pseudo‐stationary phase in capillary electrophoresis, the separation of four representative auxins, i.e., indole‐3‐acetic acid, indole butyric acid, 2,4‐dichlorophenoxyacetic acid, 1‐naphthaleneacetic acid, was carried out. Some important factors, such as the nanoparticles concentration, the pH and concentration of the running buffer solution, were also investigated on the separation. Under optimized experimental conditions, all the auxins investigated can be baseline separated within 5 min with higher column performance. The method established can also be used for quantitative analysis. The relative standard deviations obtained for indole‐3‐acetic acid, indole butyric acid, 2,4‐dichlorophenoxyacetic acid, 1‐naphthaleneacetic acid were in the range of 1.6–5.7% for peak area and 0.53–1.60% for migration time. The calibration curves obtained from the peaks areas for auxins were linear in the range of 0.1–80 mg/L with the correlation coefficients of 0.994–0.999. The limit of detection (S/N = 3) was 11–75 μg/L. The developed method was also successfully used for the determination of auxins in fruits and vegetables samples with good recoveries.     

The application of functional silica nanoparticles to fulfill the rapid and improved enantioselective capillary electrophoresis separation of amino acid derivatives

In this study, diamino moiety functionalized silica nanoparticles with the size of 118 ± 12 nm were successfully synthesized and directly introduced into a chiral capillary electrophoresis system to improve the enantioseparation of 9‐fluorenyl methoxycarbonyl derivatized amino acids using norvancomycin as chiral selector. Under acidic background electrolyte conditions, functional silica nanoparticles can be readily adsorbed onto the inner surface of bare silica capillary column through electrostatic interaction to form a dynamic coating, resulting in a reversed anodic electro‐osmotic flow (i.e. from cathode to anode). As expected, chiral amino acid derivatives (usually negatively charged) can be rapidly separated under co‐electro‐osmotic flow conditions in the current separation system. Furthermore, the column performance and detection sensitivity for the enantioseparation were also obviously improved because the adsorption of chiral selector of norvancomycin to the capillary wall was greatly suppressed. Some important factors influencing the separation, such as the coating thickness, background electrolyte concentration, functional silica nanoparticles concentration, and the organic modifier were also investigated and the optimized separation conditions were obtained.     

Simultaneous Determination of Phytohormones in Plant Extracts using SPME and HPLC

Solid-phase microextraction followed by HPLC was used for the determination of indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) in plant samples. Parameters influencing performance, including pH, salinity, extraction time, fiber coating and temperature, were optimized. A Carbowax-coated fiber was chosen for determination due to much higher extraction efficiency compared to polyacrylate fibers. The dynamic ranges spanned over three orders of magnitude. The LOD/(LOQ) values of the target compounds in pure water were 0.149(0.497), 0.442(1.472), 0.121(0.403), 0.058(0.193) μg L⁻¹ for IAA, ABA, IBA and NAA respectively. The method was successfully applied to the analysis of xylem fluid from Musa basjoo stem obtaining recoveries of 98.85% (IAA), 94% (IBA) and 94.30% (NAA). The method was also successfully applied to the analysis of these four target compounds in the hyperaccumulating plant, Viola baoshanensis. The results matched quite well with ones obtained by solid phase extraction followed by HPLC. The method developed was superior when applied to liquid samples because matrix effects could be eliminated.     

Preparation of magnetic indole-3-acetic acid imprinted polymer beads with 4-vinylpyridine and β-cyclodextrin as binary monomer via microwave heating initiated polymerization and their application to trace analysis of auxins in plant tissues

Auxin is a crucial phytohormone for precise control of growth and development of plants. Due to its low concentration in plant tissues which are rich in interfering substances, the accurate determination of auxins remains a challenge. In this paper, a new strategy for isolation and enrichment of auxins from plant tissues was obtained by the magnetic molecularly imprinted polymer (mag-MIP) beads, which were prepared by microwave heating initiated suspension polymerization using indole-3-acetic acid (IAA) as template. In order to obtain higher selective recognition cavities, an enhanced imprinting method based on binary functional monomers, 4-vinylpyridine (4-VP) and β-cyclodextrin (β-CD), was adopted for IAA imprinting. The morphological and magnetic characteristics of the mag-MIP beads were characterized by scanning electron microscopy, Fourier-transform infrared spectroscopy and vibrating sample magnetometry. A majority of resultant beads were within the size range of 80-150μm. Porous surface morphology and good magnetic property were observed. Furthermore, the mag-MIP beads fabricated with 4-VP and β-CD as binary functional monomers exhibited improved recognition ability to IAA, as compared with the mag-MIP beads prepared with the individual monomer separately. Competitive rebinding experiment results revealed that the mag-MIP beads exhibited a higher specific recognition for the template than the non-imprinted polymer (mag-NIP) beads. An extraction method by mag-MIP beads coupled with high performance liquid chromatography (HPLC) was developed for determination of IAA and indole-3-butyric acid (IBA) in plant tissues. Linear ranges for IAA and IBA were in the range of 7.00-100.0μgL(-1) and 10.0-100.0μgL(-1), and the detection limits were 3.9 and 7.4μgL(-1), respectively. The analytical performance was also estimated by seedlings or immature embryos samples from three different plant tissues, pea, rice and wheat. Recoveries were in the range of 70.1-93.5%. The results show that the present imprinting method is a promising approach for preparation of selective adsorbents for sample preparation of auxin analysis in plant tissues.     

Multi-walled carbon nanotube as a solid phase extraction adsorbent for analysis of indole-3-butyric acid and 1-naphthylacetic acid in plant samples

In this work,a new sample pretreatment method prior to HPLC separations was developed for the determination of auxins in plant samples.Owing to its large surface area and high adsorption capacity, multi-walled carbon nanotube(MWCNT) was chosen as the adsorbent for the extraction of auxins from plant samples.In this study,two important auxins were selected as model analytes,namely indole-3-butyric acid(IRA) and 1-naphthylacetic acid(NAA).They could be extracted and concentrated due to theirπ-πstacking interactions with MWCNT.Then HPLC-UV was introduced to detect IBA and NAA after sample pretreatment.Factors that may affect the enrichment efficiency were investigated and optimized.Comparative studies showed that MWCNT was superior to C18 for the extraction of the two analytes.Validation experiments showed that the optimized method had good linearity(0.9998 and 0.9960),high recovery(81.4%-85.4%),and low detection limits(0.0030 mg/L and 0.0012 mg/L).The results indicated that the novel method had advantages of convenience,good sensitivity,high efficiency, and it was feasible for the determination of auxins in plant samples.     

植物生长激素的毛细管胶束电动色谱法分离

以高效毛细管胶束电动色谱法对赤霉素（ＧＡ）、脱落酸（ＡＢＡ）、吲哚丁酸（ＩＢＡ）、吲哚乙酸（ＩＡＡ）、萘乙酸（ＮＡＡ）等植物生长激素的分离和测定进行了研究。考察了各种操作参数及有机添加剂对分离的影响,得到良好的分离结果。对各组分进行了定量测定研究,ＡＢＡ、ＧＡ、ＩＢＡ、ＩＡＡ及ＮＡＡ的最低检测浓度依次为５．０,３．０,０．５８,０．１５,０．１４ｍｇ／Ｌ。     

Development of sample preparation method for auxin analysis in plants by vacuum microwave-assisted extraction combined with molecularly imprinted clean-up procedure

A novel sample preparation method for auxin analysis in plant samples was developed by vacuum microwave-assisted extraction (VMAE) followed by molecularly imprinted clean-up procedure. The method was based on two steps. In the first one, conventional solvent extraction was replaced by VMAE for extraction of auxins from plant tissues. This step provided efficient extraction of 3-indole acetic acid (IAA) from plant with dramatically decreased extraction time, furthermore prevented auxins from degradation by creating a reduced oxygen environment under vacuum condition. In the second step, the raw extract of VMAE was further subjected to a clean-up procedure by magnetic molecularly imprinted polymer (MIP) beads. Owing to the high molecular recognition ability of the magnetic MIP beads for IAA and 3-indole-butyric acid (IBA), the two target auxins in plants can be selectively enriched and the interfering substance can be eliminated by dealing with a magnetic separation procedure. Both the VMAE and the molecularly imprinted clean-up conditions were investigated. The proposed sample preparation method was coupled with high-performance liquid chromatogram and fluorescence detection for determination of IAA and IBA in peas and rice. The detection limits obtained for IAA and IBA were 0.47 and 1.6 ng/mL and the relative standard deviation were 2.3% and 2.1%, respectively. The IAA contents in pea seeds, pea embryo, pea roots and rice seeds were determined. The recoveries were ranged from 70.0% to 85.6%. The proposed method was also applied to investigate the developmental profiles of IAA concentration in pea seeds and rice seeds during seed germination.     

Separation of plant hormones from biofertilizer by capillary electrophoresis using a capillary coated dynamically with polycationic polymers.

A new, simple and rapid capillary electrophoresis (CE) method, using hexadimethrine bromide (HDB) as electroosmotic flow (EOF) modifier, was developed for the identification and quantitative determination of four plant hormones, including gibberellin A3 (GA3), indole-3-acetic acid (IAA), alpha-naphthaleneacetic acid (NAA) and 4-chlorophenoxyacetic acid (4-CA). The optimum separation was achieved with 20 mM borate buffer at pH 10.00 containing 0.005% (w/v) of HDB. The applied voltage was -25 kV and the capillary temperature was kept constant at 25 degrees C. Salicylic acid was used as internal standard for quantification. The calibration dependencies exhibited good linearity within the ratios of the concentrations of standard samples and internal standard and the ratios of the peak areas of samples and internal standard. The correlation coefficients were from 0.9952 to 0.9997. The relative standard deviations of migration times and peak areas were < 1.93 and 6.84%, respectively. The effects of buffer pH, the concentration of HDB and the voltage on the resolution were studied systematically. By this method, the contents of plant hormone in biofertilizer were successfully determined within 7 min, with satisfactory repeatability and recovery.     

Effect of auxins and cytokinins on efficient plant regeneration and multiple-shoot formation from cotyledons and cotyledonary-node expiants of groundnut (Arachis hypogaea L.) by in vitro culture technology

Tissue cultures were established from cotyledon and cotyledonarynode segments ofArachis hypogaea L. on Murashige and Skoog (MS) medium supplemented with different concentrations of auxins (IAA, NAA, IBA, and 2, 4-D) and cytokinins (KIN and BAP). For callus initiation, high concentration of auxins and low concentration of cytokinins were used, whereas high concentration of cytokinins and low concentration of auxins were used for shoot-bud differentiation. Callus induction and shoot-bud regeneration frequency, however, varied with genotype, expiant, and the different plant-growth regulators combination in the medium. The shoot-bud multiplication was also influenced by genotype, explant type, and growth regulators. The combination of BAP and NAA produced more shoots than other combinations. The maximum number of shoots was obtained from cotyledonary-node segments on a medium containing BAP (5.0 mg/L) and IBA (1.0 mg/L). Rooting of regenerated shoots was achieved on a medium augmented with NAA or IBA (2.0 mg/L) in combination with KIN (0.5 mg/L). Rooted plantlets were successfully established in the soil, where 95% of them survived. Tissue-culture studies of these expiants suggests the shoots to be ofde nova origin, which would make the system suitable for gene-transfer technology.     

Polyamidoamine‐grafted silica nanoparticles as pseudostationary phases for capillary electrochromatographic separation of proteins

Polyamidoamine‐grafted silica nanoparticles were synthesized, characterized and investigated for the feasibility as pseudostationary phases in alkaline buffer for separation of cationic and anionic proteins, viz., lysozyme, cytochrome C, gamma globulin, and myoglobin. Neither bare silica nanoparticles nor polyamidoamines nor their mixtures as pseudostationary phases could lead to simultaneous separation of the four proteins. However, polyamidoamine‐grafted silica nanoparticles not only suppressed the irreversible wall adsorption of the cationic lysozyme and cytochrome C, but also provided selectivity toward all the proteins. We found that polyamidoamine generation two‐modified silica nanoparticles were the optimum pseudostationary phases with respect to detection sensitivity and separation efficiency; presence of the nanoparticles at 0.01% in the running buffer of 12.5 mM tetraborate/phosphate at pH 9.1 resulted in baseline resolution of the four proteins.     

Simultaneous determination of four plant hormones in bananas by molecularly imprinted solid-phase extraction coupled with high performance liquid chromatography

A highly selective molecularly imprinted solid-phase extraction (MISPE) combined with liquid chromatography-ultraviolet detection was developed for the simultaneous isolation and determination of four plant hormones including indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA) in banana samples. The new molecularly imprinted microspheres (MIMs) prepared by aqueous suspension polymerization using 3-hydroxy-2-naphthoic acid and 1-methylpiperazine as mimic templates performed with high selectivity and affinity for the four plant hormones, and applied as selective sorbents of solid-phase extraction could effectively eliminate the interferences of the banana matrix. Good linearity was obtained in a range of 0.04-4.00 μg g(-1) and the recoveries of the four plant hormones at three spiked levels ranged from 78.5 to 107.7% with the relative standard deviations (RSD) of less than 4.6%. The developed MISPE-HPLC protocol obviously improved the selectivity and eliminated the effect of template leakage on quantitative analysis, and could be applied for the determination of plant hormones in complicated biological samples.     

Determination of phytohormones in plant samples based on the precolumn fluorescent derivatization with 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene by HPLC for routine use

Six phytohormones including indole butyric acid (IBA), naphthalene acetic acid (NAA), 2,4-dichloro-phenoxy acetic acid (2,4-D), indole-3-acetic acid (IAA), abscisic acid (ABA), and salicylic acid (SA) in crude plant extractions have been quantitated by means of high-performance liquid chromatography (HPLC) with fluorescence detection based on the precolumn derivatization using 1,3,5,7-tetramethyl-8-aminozide-difluoroboradiaza-s-indacene (BODIPY-aminozide), a fluorescent reagent synthesized in our lab recently. The optimization of derivatization conditions was carefully studied by an L(25) (5(6)) orthogonal array design (OAD) with five factors at five levels that are important influence parameters in the improvement of derivatization efficiency. The separation conditions were also studied in detail. Under the optimal conditions, the detection limits (S/N=3) of the six phytohormones were found from 0.12 to 0.75 nM. The proposed method was the first investigation of aminozide for the analysis of phytohormones and has been successfully applied to the determination of phytohormones in plant samples such as cucumber and tomato with recoveries of 94-105%.     

Enhanced separation of seven quinolones by capillary electrophoresis with silica nanoparticles as additive

This paper describes the enhanced separation of lomefloxacin, sparfloxacin, fleroxacin, norfloxacin, ofloxacin, gatifloxacin and pazufloxacin by capillary zone electrophoresis (CZE) using silica nanoparticles (SiNPs) as running buffer additive. The impact of SiNPs concentration on the resolution and selectivity of separation was investigated and a given value of SiNPs was finally chosen under the optimum conditions. The addition of the SiNPs to the running buffer enabled electroosmotic flow (EOF) decrease and permitted full interaction between SiNPs and analytes. The influence of separation voltage, pH and buffer concentration on the separation in the presence of SiNPs was examined. Interactions between drugs and nanoparticles during the separation are discussed; the determination of interaction constants is also achieved. A good resolution of seven quinolones was obtained within 15 min in a 50 cm effective length fused-silica capillary at a separation voltage of +10 kV in a 12 mM disodium tetraborate-phosphate buffer (pH 9.08) containing 5.2 μg mL⁻¹ SiNPs.     

气相色谱法直接测定植物生长素

 建立了一种采用 5 3 0 μm大口径毛细管色谱柱、不经衍生化处理而直接测定吲哚乙酸 (IAA)、吲哚丁酸(IBA)和萘乙酸 (NAA)等植物生长素的气相色谱分析方法。以邻苯二甲酸二丁酯为内标物 ,用 FID检测 ,IAA,IBA和 NAA的相对标准偏差分别为 1 .1 4% ,0 .61 %和 0 .78%。方法简便、快速、准确、重现性好 ,可用于生长素类单组分和混合制剂的质量检测。     

Identification and quantitation of auxins in plants by liquid chromatography/electrospray ionization ion trap mass spectrometry

Auxin is an important phylohormone, which regulates specific physiological responses such as division, elongation and differentiation of cells. A new method using liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) has been developed for identification and quantitation of four auxins. Under the optimum conditions, four auxins (indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and 1-naphthylacetic acid) were completely separated and quantitated within 7 min with a minimum detection limit of 8.0 ng mL(-1) with relative standard deviations lower than 5.0%. This method also has been applied to analysis of auxins in Chinese cabbage where, even with a complicated serious background perturbation due to the natural biological matrix, the mean recoveries ranged from 77.5% to 99.8%. Finally, we discuss the MS-relevant properties of the identified auxins in detail.     

Capillary enzyme immunoassay with electrochemical detection for determining indole-3-acetic acid in tomato embryos

An amperometric immunoassay for indole-3-acetic acid (IAA) in tomato embryos was developed. The antibody against IAA was covalently bound to the chemically modified inner surface of a 22 μL silica capillary. The competitive binding of methylated IAA and an IAA alkaline phosphatase conjugate for a limited amount of antibody was accomplished in the silica capillary in 20 min. The enzymatic product p-aminophenol, produced from p-aminophenyl phosphate, was determined by flow injection analysis with amperometric detection. A detection limit for IAA of 3 pg/μL was obtained. The average concentration of IAA in mature embryos measured by this method is 0.37 ± 0.03 μg/g. Although isotope dilution GC-SIM-MS can achieve the same detection limit, the sample analysis is not feasible in such a complicated matrix and further purification and separation are required.     

Fourier transform infrared spectroscopy with a sample deposition interface as a quantitative detector in size-exclusion chromatography

Micellar electrokinetic capillary chromatography was developed to analyze plant hormones including gibberellic acid, abscisic acid, indole-3-acetic acid, alpha-naphthaleneacetic acid, 2,4-dichlorophenoxyacetic acid, kinetin-6-furfurylaminopurine and N6-benzyladenine. The influences of some crucial parameters including buffer concentration, pH value, micelle concentration and applied voltage on electrophoretic separation were investigated. Under optimum conditions (50 mM borate as the running buffer containing 50 mM sodium dodecylsulfate, pH 8.0; separation voltage: -15 kV; injection: hydrodynamic injection, 5 s at 50 mbar; temperature: 25 degrees C), a complete separation of seven plant hormones was accomplished within 30 min. Emphasis was placed on improving detection sensitivity in order to detect small amounts of hormones in plant tissue. Multiple wavelength detection and expanded bubble cell capillary were used with enrichment factors of 2 and 3, respectively. In addition, an on-line concentration method of large volume sample stacking was designed. Enrichment factors of up to approximately 10-600 were achieved for these hormones with detection limits down to 0.306 ng/ml. The method was successfully applied to analyzing abscisic acid in flowers of transgenic tobacco.