Determination of phenolic compounds in river water with on-line coupling bisphenol A imprinted monolithic precolumn with high performance liquid chromatography

The bisphenol A (BPA) imprinted monolithic precolumn has been prepared by in situ polymerization using 4-vinylpyridine (4-VP) and ethylene dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. The column with good flow-through property was obtained by changing the molar ratio of the porogens (toluene and dodecanol). The selectivity and retention properties of the monolith for the BPA and other phenolic compounds were evaluated. The results show that the hydrophobic and hydrogen-bonding interaction plays important roles in the recognition process. The determination of BPA and other phenolic compounds with on-line solid-phase extraction (SPE) by monolithic precolumn coupled with conventional particulates packed and monolithic reversed-phase columns, respectively, was performed. The method was successfully applied to the analysis of phenolic compounds in river water.     

Determination of tetracyclines in food samples by molecularly imprinted monolithic column coupling with high performance liquid chromatography

A novel solid phase extraction (SPE) method for determination of tetracyclines (TCs) in milk and honey samples by molecularly imprinted monolithic column was developed. Using tetracycline (TC) as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, methanol as the solvent, cyclohexanol and dodecanol as the mixed porogenic solvents, a TC imprinted monolithic column was prepared by in situ molecular imprinting technique for the first time, and the optimal synthesis conditions and the selectivity of TC imprinted monolithic column were investigated. The interfering substances in food samples and TCs can be separated successfully on imprinted column. Molecularly imprinted solid phase extraction (MISPE) coupling with C18 column was used to determinate the TCs in milk and honey. The recoveries of this method for six tetracyclines antibiotics such as tetracycline (TC), oxytetracycline (OTC), minocycline (MINO), chlortetracycline (CTC), metacycline (MTC) and doxycycline (DTC) were investigated, and high recoveries of 73.3-90.6% from milk samples and 62.6-82.3% from honey samples were obtained. A method for determination of TCs at low concentration level in milk and honey samples was successfully developed by using the monolithic column as the precolumn for solid phase extraction of six TCs compounds.     

Determination of pituitary and recombinant human growth hormone molecular weights by modern high-performance liquid chromatography with low angle laser light scattering detection

The determination of molecular weight for pituitary and recombinant human growth hormone (p-hGH/Crescormon and r-hGH/Protropin) has been performed. This has involved on-line coupling of size-exclusion chromatography (SEC) and gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A 5-microns, 300 A, Delta-bond octyl column was used. Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for p-hGH and r-hGH. Known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data has been obtained for both proteins under conventional RP-HPLC gradient elution conditions. SEC data of both hGHs were found to be concentration, mobile phase, and column dependent for the particular analyses. Both medium- and high-resolution SEC-LALLS studies were performed, and all of these determinations further confirmed our RP-HPLC results. On-line LALLs provides certain advantages in identifying aggregates that may be present, even in medium-resolution SEC, where incomplete resolution occurs. The on-line coupling of modern RP-HPLC for biopolymers with LALLS detection represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus higher-order aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.     

Determination of biopolymer (protein) molecular weights by gradient elution, reversed-phase high-performance liquid chromatography with low-angle laser light scattering detection

The determination of molecular weights for certain proteins has been performed. This has involved the on-line coupling of gradient elution, reversed-phase high-performance liquid chromatography (RP-HPLC) with low-angle laser light scattering (LALLS) detection. A new 1.5-micron, non-porous, Monosphere RP-C8 column has been used in order to perform fast and conventional RP-HPLC gradients (5-45 min). Traditional specific refractive index increment (dn/dc) and refractive index (n) measurements have been performed in order to derive absolute weight-average molecular weight (Mw) information for ribonuclease A, lysozyme, and bovine serum albumin. Standard mixtures of known concentrations of each protein have been separated using reversed-phase gradients utilizing acetonitrile with on-line LALLS determination of excess Rayleigh scattering factors. Accurate Mw data have been obtained for all three proteins, but only under certain, conventional reversed-phase gradient elution conditions. Between 5-10 min of fast gradient elution, each protein appears to exhibit unusual Mw values, suggestive of aggregate formations. Methods have been developed to define the nature of such aggregates. The on-line coupling of modern RP-HPLC for biopolymers with LALLS represents a major step forward in the ability of bioanalytical chemists to determine the nature (monomer versus aggregate) of such materials. Other classes of biopolymers should prove suitable for studies with the same RP-HPLC-LALLS-UV approaches.     

整体柱离子对色谱-直接电导检测法快速测定微量碘酸根

建立了整体柱离子对色谱-直接电导检测快速测定微量碘酸根的方法.采用反相硅胶整体柱,以氢氧化四丁铵(TBA)-邻苯二甲酸-水-乙腈水溶液为淋洗液,分别讨论了淋洗液、流速及柱温对碘酸根保留的影响,并确定最佳色谱条件为:以0.25 mmol/L TBA-0.18 mmol/L邻苯二甲酸-3％乙腈(pH 5.5)水溶液为淋洗液...     

Molecularly imprinted monolith coupled on-line with high performance liquid chromatography for simultaneous quantitative determination of cyromazine and melamine

We report a novel method for simultaneous determination of cyromazine and melamine based on a molecularly imprinted monolith on-line coupled with high performance liquid chromatography (HPLC). The imprinted monolith was prepared by in situ polymerization using 2,4-diamino-6-undecyl-1,3,5-triazine (DAUTA) as a mimic template. Due to the better solubility of DAUTA in chloroform, hydrogen bonds were effectively developed between the template and the functional monomer and resulted in the formation of highly specific cavities in the obtained imprinted monolith. With methanol as the loading solvent, cyromazine and melamine were both selectively retained by the obtained imprinted monolith, while the nonspecific adsorption on the non-imprinted monolith was negligible. The imprinted monolithic column was on-line coupled with HPLC for purification and concentration of the two analytes from milk samples. To minimize the peak broadening during the on-line transfer of the analytes from the imprinted monolith to the following analytical column, a successive desorption program was developed for the elution step, which enabled on-line stacking of the target compounds before being analyzed by HPLC. Low detection limits of 0.12 μg mL(-1) for melamine and 0.05 μg mL(-1) for cyromazine were achieved with only 0.3 mL of milk sample and a low sensitivity HPLC-UVD instrument. The method may be further extended to detect other analytes of interest in a large variety of samples.     

Trace-level determination of polar phenolic compounds in aqueous samples by high-performance liquid chromatography and on-line preconcentration on porous graphitic carbon

The use of porous graphitic carbon (PGC) was investigated for the trace enrichment and the on-line liquid chromatographic separation of polar phenolic compounds (phenol, di- and trihydroxybenzenes, aminophenols, etc.) from aqueous samples. Comparison between retentions obtained with PGC and with the copolymer-based sorbent PRP-1 showed similar variations of the capacity factors with the mobile phase composition, but an inverse retention order. The capacity factor of a very polar analyte, such as 1,3,5-trihydroxybenzene (phloroglucinol), is 1000 in pure water, whereas this analyte is not retained by C₁₈-silica and is poorly retained by PRP-1 (k′ = 3 in water). A precolumn packed with PGC can be coupled to a PGC analytical column for simple separation in the reversed-phase mode. This methodology has been applied to the direct determination of pyrocatechol, resorcinol and phloroglucinol below the 0.1 μg/1 level in a 50-ml sample.     

Trace-level monitoring of chloroanilines in environmental waters using on-line trace-enrichment and liquid chromatography with UV and electrochemical detection

Summary An on-line procedure is described for the trace-level determination of mono-, di- and methyl-chloroanilines in aqueous samples using selective preconcentration with a cation-exchanger and liquid chromatography with UV and electrochemical detection. Because direct percolation through a cation-exchanger has to be avoided owing to the high content of inorganic anions present in natural waters, a two-step on-line preconcentration was carried out: chloroanilines were first trapped on a precolumn packed with an apolar polymeric sorbent (PRP-1) in their neutral form. Then the PRP-1 precolumn was coupled in series with a second precolumn containing cation exchange material. The chloroanilines were removed from the first precolumn with 3 mL of deionised water: acetonitrile (31) at pH 1 and retained by the cation exchange column. The contents of the cation exchange column were finally desorbed onto the analytical column and eluted with a water: acetonitrile gradient. The combination of selective trace enrichment and sensitive electrochemical detection allows the simultaneous determination of chloroanilines from 150 mL of river water samples with detection limits below 30 ng/l. Identification is confirmed by the selective preconcentration and the two detection modes.     

High-performance liquid chromatographic column-switching technique for the determination of intermediates of anaerobic degradation of toluene in ground water microcosm.

A reversed-phase liquid chromatographic column-switching system was used for the determination of phenol, benzoic acid and cresol (PBC) in the presence of toluene in ground water microcosm. A precolumn was connected in series with an analytical column via a column-switching valve. After the injection, as soon as PBC were eluted from the precolumn to the analytical column, the valve was switched so that the precolumn was between the analytical column and the UV detector. Toluene and other non-polar compounds were eluted from the precolumn in a very short time and detected along with the solvent front. Subsequently, PBC were separated on the analytical column and passed through the precolumn one more time before being detected by the UV detector. The total analysis time was 15 min. This technique facilitated the study of the basic mechanism and path way of anaerobic degradation of toluene in ground water aquifer.     

整体柱高效液相色谱法测定尿中核苷的方法研究

尿中修饰核苷已被研究用作可能的肿瘤标记物,目前最常用的分析方法为反相高效液相色谱法,其不足之处是分析周期太长。为此,建立了应用整体柱的高效液相色谱方法对尿中修饰核苷进行分析,所用缓冲液为25 mmol/L磷酸二氢钾溶液(pH 4.55)和60%(体积分数）的甲醇水溶液,线性梯度洗脱,检测波长为260 nm。12种目标核苷可被完全分离。所建立的方法在分离度、线性、重现性、灵敏度及回收率等指标上与普通反相液相色谱法相近,但分析周期大大缩短,仅为23 min,适合于大量临床样品的分析。     

Use of an on-line,precolumn photochemical reactor in high-performance liquid chromatography of naphthodianthrones in Hypericum perforatum preparations

A method has been developed for the determination of naphthodianthrones in Hypericum perforatum L. extracts and phytopharmaceutical preparations by high-performance liquid chromatography combined with on-line, precolumn photochemical conversion followed by photodiode-array detection. The chromatographic separation was performed on a C18 column under isocratic reversed-phase conditions. An on-line, precolumn photochemical reactor equipped with a knitted PTFE reaction coil around a visible light source was used in order to transform the light sensitive naphthodianthrones, protohypericin and protopseudohypericin, very easily into the non-protoforms, hypericin and pseudohypericin, respectively. Two UV chromatograms (photochemical reactor "on" and "off") were compared and were quite useful in characterizing the sample. Validation studies demonstrated that this HPLC method is simple, rapid, reliable and reproducible. The time-consumptive manual irradiation of the samples is omitted by this automated on-line irradiation step. The developed method was successfully applied to the quality control of Hypericum perforatum L. extracts and its phytopharmaceutical preparations.     

Large-bore particle-entrapped monolithic precolumns prepared by a sol-gel method for on-line peptides trapping and preconcentration in multidimensional liquid chromatography system for proteome analysis

The present report describes the preparation and characterization of large-bore particle-entrapped monolithic precolumns, which are suitable for incorporation into a two-dimensional liquid chromatography (2D-LC) system for proteome analysis. The fritless precolumns with different inner diameter (i.d.) (320 and 530 microm) were rapidly and successfully prepared by entrapping octadecylsilica (ODS) particles (5 microm, 300 A) prepacked into fused silica capillaries with a sol-gel network, which was formed by hydrolysis and polycondensation of methyltriethoxysilane (MTES). By optimizing the composition of the sol solution, the resulting large-bore monolithic precolumns of 5 mm length allow a flow rate of 20 microL/min loading buffer at a reasonable low back pressure of 25 bar or less and are capable of withstanding up to 300 bar inlet pressure. Scanning electron micrograms of the precolumns profile showed that the evolving sol-gel network joined particles to each other and onto the column wall, and no cracking or shrinkage of the column bed was observed even in 530 microm-i.d. capillary. The performance of the particle-entrapped monolithic precolumns used for preconcentration and desalting of proteolytic digest was evaluated by on-line coupling the large-bore precolumns with a capillary reversed-phase liquid chromatographic (RPLC) column followed by UV detection. The laboratory-made monolithic precolumns with 320 and 530 microm i.d. were characterized by using BSA tryptic digest or peptide standards as the analytes with respect to sample loading capacity, linearity, recovery and reproducibility, etc. The results indicate that the large-bore and short precolumns (5 mm x 320 microm i.d. or 5 mm x 530 microm i.d.) allow sample fast loading at a flow rate of 30 or 60 microL/min. The precolumns also have a mass loading capacity for BSA peptides of about 70 microg and for standard peptides of about 80 microg. Good linear calibration curves (R2 > 0.99) were obtained and the limits of detection (signal-to-noise ratio, S/N = 3) were improved by more than 60-fold and were between 0.53 and 1.32 ng/microL even with a UV absorbance detector. The total recovery was found to be approximately 90-100% for BSA digest and standard peptides. The day-to-day relative standard deviation (RSD) values for recoveries of BSA peptides on a single precolumn ranged from 4.66 to 7.56% and 2.68 to 3.05% for precolumn back pressure, while the column-to-column RSD values were 3.51-6.13% and 1.22-1.26% for recoveries of BSA peptides and precolumn back pressure, respectively. With good precolumn reproducibility, no significant degradation or decrease in precolumn performance was showed even after approximately 150 preconcentration/desorption cycles. The precolumns also proved to be resistant to salt buffer with high concentration and low-pH mobile phase. The large-bore particle-entrapped monolithic precolumns will be further used in a high-throughput 2D-LC array system coupled with tandem matrix assisted laser desorption/ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS) detection for proteome analysis.     

A molecularly imprinted organic–inorganic hybrid monolithic column for the selective extraction and HPLC determination of isoprocarb residues in rice

An IPC‐imprinted (IPC is isoprocarb) poly(methacrylic acid)/SiO₂ hybrid monolithic column was prepared and applied for the recognition of the template. The hybrid monolithic column was synthesized in a micropipette tip using methyltrimethoxysilane as the inorganic precursor, 3‐(methacryloxy)propyltrimethoxysilane as the coupling agent, and ethylene glycol dimethacrylate as the cross‐linker. The synthesis conditions, including the porogenic solvent, coupling agent, volume ratio of the inorganic alcoholysate and organic part, were optimized. The prepared monolithic column was characterized by SEM and FTIR spectroscopy. A simple, rapid, and sensitive method for the determination of IPC in rice using the imprinted monolithic column microextraction combined with HPLC was developed. Several parameters affecting the sample pretreatment were investigated, including the eluent, washing solution, and loading sample volume. The linearity of the calibration curve was observed in the range of 9.0–1000 μg/kg for IPC in rice with the correlation coefficient (r²) of 0.9983. The LOD was 3.0 μg/kg (S/N = 3). The assay gave recovery values ranging from 91 to 107%. The proposed method has been successfully applied for the selective extraction and sensitive determination of IPC in rice and a satisfactory result was obtained.     

Direct injection of plasma to determine pseudoephedrine by high performance liquid chromatography with column switching

An HPLC method has been developed for the determination of pseudoephedrine in plasma using column switching. Preparation of the sample was simple in that only 1000 microL of water was added to 200 microL of plasma before injection. A 900 microL aliquot was injected onto the precolumn. Double distilled water was used to elute and remove proteins and polar components in the sample. The components retained on the precolumn were flushed forward onto the analytical column by the mobile phase (acetonitrile-0.2 mol/L ammonium sulphate, 10:90 v/v) with automated column switching. The limit of determination of pseudoephedrine in plasma was 12 ng/mL. The relative standard deviations of intra- and inter-assay for the determination of pseudoephedrine in plasma were 1.2-9.8% over the concentration range 1020-21.8 ng/mL. The mean recovery by on-line solid phase extraction was 94.76% (RSD = 1.1%).     

Solid-phase extraction and high-performance liquid chromatographic determination of flumequine and oxolinic acid in salmon plasma

Two methods for determination of oxolinic acid and flumequine in salmon plasma are described. The first method applies sample pretreatment on C2 disposable solid-phase extraction columns. The second method is based on direct plasma injection and on-line sample clean-up on a polystyrene-divinylbenzene precolumn. After column-switching, the analytes are separated on a polystyrene-divinylbenzene analytical column and detected with a fluorescence detector. Validation of the methods showed good sensitivity, precision and reproducibility. Both methods are well suited for determination of plasma levels of the drugs in pharmacokinetic studies in Atlantic salmon.     

Selective and sensitive analysis of 1-nitropyrene in diesel exhaust particulate extract by multidimensional HPLC

Summary A multicolumn (MC) HPLC method for the determination of 1-nitropyrene (1-NP) at trace-levels via on-line reduction to 1-aminopyrene and fluorescence detection is presented. On the first column, packed with a pyrenebutyric acid amide stationary phase, the nitro-derivatives of PAHs are strongly retained and separated from other matrix components. The nitro-PAHs-containing fractions are transferred onto a RP₁₈-column via stepwise gradient elution and finally separated according to their various lipophilicities and sizes. To increase the overall selectivity and sensitivity of the multidimensional method (MD-HPLC system) post-column, on-line reduction of the nitro-PAHs to the respective amino-PAHs via a short catalysis column is performed thus enhancing the sensitivity significantly (to low pg levels). The applicability of this method for the determination of trace amounts of 1-NP in real samples (diesel particulate extracts) is demonstrated.     

Automated determination of disopyramide and N-monodealkyldisopyramide in plasma by reversed-phase liquid chromatography with a column switching system.

A rapid and simple column liquid chromatographic method involving a column switching system for the determination of disopyramide and its N-monodealkyl metabolite (NMD) in plasma is described. The deproteinized plasma is applied to an automated system. Purification and concentration were performed using a precolumn connected to a six-position valve; analytical separation was done on-line using a cyano reversed-phase column with a mobile phase consisting of 10 mmol/l trimethylamine (pH 2.5, adjusted with phosphoric acid)-acetonitrile-tetrahydrofuran (78:20:2, v/v/v). Absorbance was measured at 265 nm, with a minimum detectable amount of disopyramide and NMD of 0.1 micrograms/ml. The method can be applied to drug monitoring and pharmacokinetic studies.     

Measurement of the eddy diffusion term in chromatographic columns. I. Application to the first generation of 4.6mm I.D. monolithic columns

The corrected heights equivalent to a theoretical plate (HETP) of three 4.6mm I.D. monolithic Onyx-C(18) columns (Onyx, Phenomenex, Torrance, CA) of different lengths (2.5, 5, and 10 cm) are reported for retained (toluene, naphthalene) and non-retained (uracil, caffeine) small molecules. The moments of the peak profiles were measured according to the accurate numerical integration method. Correction for the extra-column contributions was systematically applied. The peak parking method was used in order to measure the bulk diffusion coefficients of the sample molecules, their longitudinal diffusion terms, and the eddy diffusion term of the three monolithic columns. The experimental results demonstrate that the maximum efficiency was 60,000 plates/m for retained compounds. The column length has a large impact on the plate height of non-retained species. These observations were unambiguously explained by a large trans-column eddy diffusion term in the van Deemter HETP equation. This large trans-rod eddy diffusion term is due to the combination of a large trans-rod velocity bias (?3%), a small radial dispersion coefficient in silica monolithic columns, and a poorly designed distribution and collection of the sample streamlets at the inlet and outlet of the monolithic rod. Improving the performance of large I.D. monolithic columns will require (1) a detailed knowledge of the actual flow distribution across and along these monolithic rod and (2) the design of appropriate inlet and outlet distributors designed to minimize the nefarious impact of the radial flow heterogeneity on band broadening.     

Reversed-phase high-performance liquid chromatographic determination of linear alkylbenzenesulphonates in river water at ppb levels by precolumn concentration.

A high-performance liquid chromatography method for the determination of linear alkylbenzenesulphonates (LASs) in river waters has been developed. The ppb levels of LASs can be determined by reversed-phase high-performance liquid chromatography with ultraviolet detection after on-line anion-exchange concentration and successive injection. LASs were quantitatively concentrated on the anion-exchange precolumn and easily cleaned up from river water matrix, because of its specific affinity, for the anion-exchange resin. A weak non-polar reversed-phase column was useful for the determination of LASs. The relationships between concentration and summation of peak areas were linear from 10 to 200 ppb for total LAS concentrated from 5 ml of standard solutions. Overall recovery for total LAS was found to be 99%. Total LAS in the Tama River waters was determined to be around 100 ppb by the proposed method.     

Molecularly Imprinted Monolithic Column for Selective On-Line Extraction of Enrofloxacin and Ciprofloxacin from Urine

A simple on-line molecularly imprinted solid-phase extraction coupled with liquid chromatographic separation was developed for the simultaneous determination of enrofloxacin and its active metabolite ciprofloxacin from urine samples. The molecularly imprinted monolithic columns were synthesized in water-containing systems using norflorxacin as dummy template and methanol–water (5:1, v/v) as porogenic solvent, which reveal high affinity to enrofloxacin and ciprofloxacin in the aqueous environment. Due to the unique porous structure and flow-through channels existing in the network skeleton of the imprinted monolith, urine samples could be injected directly into the imprinted column, proteins and other biological matrix were quickly washed out and the analytes were selectively retained and enriched. Good linearity was obtained from 0.05 to 200 mg L^?1 with relative standard deviations less than 3.1%. The recoveries were higher than 87% at three different concentrations and the limit of detection of the method was 0.01 mg L^?1. Moreover, by increasing the injection volume, the sensitivity of the method could be further improved.