Theory of Multiple‐Detection Size‐Exclusion Chromatography of Complex Branched Polymers

SEC separates complex branched polymers by hydrodynamic volume, rather than by molecular weight or branching characteristics. Equations relating the response of different types of detectors are derived including band broadening, by defining a distribution function N′(M,V_h), the number of chains with molecular weight M and hydrodynamic volume V_h. While the true molecular weight distribution of complex polymers cannot be determined by SEC, irrespective of the detector used, the formalism enables multiple detection SEC data to be processed to both analyze the polymer sample and reveal mechanistic information about polymer synthesis. The formalism also shows how the true weight‐ and number‐average molecular weight, and , can be obtained from correct processing of the hydrodynamic volume distributions.

     

The Influence of Stop-Flow on Band Broadening of Peptides in Micro-Liquid Chromatography

Stop-flow techniques are occasionally needed in combinations of LC-NMR and LC-MS. During the interval when there is no flow on the column, axial diffusion of components not yet eluted can be expected to take place. In this paper the size of the band broadening which is caused by diffusion during stop-flow has been determined for two peptides on reversed-phase packed micro columns. Within a temperature range of 20–40 °C, stop-flow could be extended to 30 minutes for peptides having k values in the range of 0.7–5.1 with little increase in band width on 1.0 mm i.d. columns at isocratic conditions. Stop-flow for 6 h at 20 °C increased the peak width of bradykinin and leucine-enkephalin by 25% and 60%, respectively, depending on the secondary interactions of the peptides. The peak broadening increased with increasing temperature (from 20 to 40 °C), as expected, and the impact was significant at stop-flow times larger than 2 h. Stop-flow during gradient elution resulted in less increase in peak width than isocratic elution due to the peak compression obtained when re-entering the gradient. At 20 °C the effective diffusion coefficients of leucine-enkephalin and bradykinin were determined to 6.5 × 10⁻⁷cm ²/s and 5.5 × 10⁻⁷ cm²/s, respectively, on the packed micro column.     

Large Volume Injection in Fast Gas Chromatography with On‐Column Injector

In this work a fast gas chromatography set‐up with on‐column injection was optimized and evaluated with a model mixture of C₈–C₂₈ n‐alkanes. Usual injection volumes when using narrow‐bore (e. g., 0.1 mm i.d.) analytical columns are ca. 0.1 μL. The presented configuration allows introduction of 10–30‐fold larger sample volumes without any distortion of peak shapes. In the set‐up a normal‐bore retention gap (1 m×0.32 mm i. d.) was coupled to a narrow‐bore (4.8 m×0.1 mm i. d.×0.4 μm film thickness) analytical column using a low dead volume column connector. The effects of the experimental conditions such as inlet pressure, sample volume, initial injection temperature, and oven temperature on a peak focusing are discussed. H‐u curves for helium and hydrogen are used to compare their suitability for high speed gas chromatography and to show the dependence of separation efficiency on the carrier gas velocity at high inlet pressures. In the fast gas chromatography system a baseline separation of C₁₀–C₂₈ n‐alkanes was achieved in less than 3 minutes.     

分子印迹柱-高效液相色谱法测定猪肉中磺胺嘧啶(英文)

建立了分子印迹柱-高效液相色谱法检测猪肉中磺胺嘧啶残留方法。制备了磺胺嘧啶的分子印迹柱,并优化了分子印迹柱的萃取条件。研究结果表明,在最佳萃取条件下,分子印迹柱-高效液相色谱法的加标回收率≥75.6%,相对标准偏差≤6.1%。分子印迹柱为固相萃取柱可以预浓缩与纯化猪肉样品中磺胺嘧啶。与氧化铝萃取柱比,分子印迹柱具有较好的重复性和萃取效率。本方法已成功用于实际猪肉样品中磺胺嘧啶含量的检测,结果满意。     

Determination of Reserpine in Plasma Using High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

A high-performance liquid chromatographic method for determining reserpine in plasma has been developed. The procedure involves extraction of reserpine from buffered plasma into benzene, oxidation of reserpine to a fluorophor by treatment with vanadium pentoxide in phosphoric acid, and chromatographic separation of the reserpine fluorophor on an octadecylsilane column by ion-pairing with heptanesulfonate ions. Fluorescence monitoring of the column effluent provides high sensitivity of detection and increases the specificity of the procedure. A detection limit of approximately 100 pg of reserpine per ml of plasma was obtained following analysis of 2 ml samples. Analysis of a number of samples demonstrated the applicability of this method in confirming the presence of reserpine in equine plasma specimens collected at various horse shows and in evaluating the pharmacokinetic behavior of reserpine following intramuscular administration to horses.     

Commercial Screen‐Printed Gold Electrodes for the Detection and Quantification of Aminothiols in Human Plasma by Liquid Chromatography with Electrochemical Detection

In order to show the possibilities of screen‐printed electrodes in HPLC detection, a method is presented for routine quantification of aminothiols in human plasma. It uses commercial gold screen‐printed electrodes and provides a rapid, simple and economical procedure with a disposable detector and without any additional electrode. By working at 98 : 2 (0.05 % trifluoroacetic acid: methanol) and 0.9 V, it allows us to determine cysteine (Cys), cysteinyl‐glycine (Cys‐Gly), and homocysteine (HCys) at μmol/L levels. The method was validated, and linearity range, detection and quantification limits, precision and accuracy were evaluated by external calibration and by using N‐acetylcysteine as internal standard, which exhibited better precision.     

Rapid and Sensitive Method for Citrinin Determination Using High-Performance Liquid Chromatography with Fluorescence Detection

Citrinin is a toxic product of secondary metabolism of fungi, such as certain Aspergillus, Penicillium, and Monascus species that are usually contaminating cereals. A new sensitive liquid chromatographic method with fluorescence detection was developed, validated, and applied for citrinin determination. The method is based on reversed-phase separation at pH 2.5, where citrinin exhibits the highest fluorescence quantum yield. In this setup, no derivatization step is needed. The method shows linearity in the range between 0.2 μg/mL and 0.1 mg/mL. The detection limit reached is 90 ng/mL (3.6 × 10^?7 M). Validated method was successfully applied on analysis of spiked and real cereal samples.     

Separation of intact proteins on γ‐ray‐induced polymethacrylate monolithic columns: A highly permeable stationary phase with high peak capacity for capillary high‐performance liquid chromatography with high‐resolution mass spectrometry

Polymethacrylate‐based monolithic capillary columns, prepared by γ‐radiation‐induced polymerization, were used to optimize the experimental conditions (nature of the organic modifiers, the content of trifluoroacetic acid and the column temperature) in the separation of nine standard proteins with different hydrophobicities and a wide range of molecular weights. Because of the excellent permeability of the monolithic columns, an ion‐pair reversed‐phase capillary liquid chromatography with high‐resolution mass spectrometry method has been developed by coupling the column directly to the mass spectrometer without a flow‐split and using a standard electrospray interface. Additionally, the high working flow and concomitant high efficiency of these columns allowed us to employ a longer column (up to 50 cm) and achieve a peak capacity value superior to 1000. This work is motivated by the need to develop new materials for high‐resolution chromatographic separation that combine chemical stability at elevated temperatures (up to 75°C) and a broad pH range, with a high peak capacity value. The advantage of the γ‐ray‐induced monolithic column lies in the batch‐to‐batch reproducibility and long‐term high‐temperature stability. Their proven high loading capacity, recovery, good selectivity and high permeability, moreover, compared well with that of a commercially available poly(styrene‐divinylbenzene) monolithic column, which confirms that such monolithic supports might facilitate analysis in proteomics.     

Selective Analysis of Secondary Amines Using Liquid Chromatography with Electrochemical Detection (LC‐EC)

In a mixture of primary and secondary aliphatic amines, the primary amines were derivatized (masked) with o‐phthalaldehyde (OPA) followed by derivatization of the remaining secondary amines with ferrocenecarboxylic acid chloride (FAC). The “tagged” amines were analyzed by LC‐EC (liquid chromatography with electrochemical detection) using in‐series dual electrode detection. Chemically‐reversible oxidation of the FAC tagged secondary amines and their subsequent complementary oxidation and reduction signals coupled with chemically‐irreversible oxidation of OPA tagged primary amines provided the selectivity for quantitative secondary amine analysis. The procedure was also applied for the selective identification of fragment 4–11 (N‐terminus‐proline) of Substance P in the presence of other Substance P fragments with primary amino acids as their N‐termini.     

Analysis of Phenylpropanolamine by Micellar Electroknetic Chromatography with Laser‐induced Fluorescence Detection

A new analytical method for phenylpropanolamine based on micellar electrokinetic chromatographic separation and laser‐induced fluorescence detection has been developed. Naphthalene‐2,3‐dicarboxaldehyde was used for precolumn derivatization of the nonfluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) containing 15 mM sodium dodecyl sulfate and a He‐Cd laser (Δ_ex: 442 nm, Δ_em: 500 nm). Linearity (r ≥ 0.99) of two orders of magnitude was generally obtained and the concentration limit of detection was in the ng/mL level. Coupled with a simple cleanup procedure, the method can be applied to the analysis of phenylpropanolamine in human plasma, with a limit of detection at 15 ng/mL. Recovery of phenylpropanolamine from plasma samples was about 90%.     

过氧化氢柱前荧光衍生化液相色谱法测定麻痹性贝毒的研究

液相色谱-荧光检测法(LC-FLD)测定贝类样品中石房蛤毒素(STX)和decarbamoylsaxitoxin(dcSTX)。样品经30 mmol/L HAc超声提取,C18固相萃取柱净化,2%碱性H2O2荧光衍生,C18色谱柱(4.6 mm×250 mm,5μm)分离,以乙腈-0.1 mol/L甲酸铵溶液(5∶95,V/V)作流动相,流速1.0 mL/m in。结果表明,STX和dcSTX衍生物在7m in内获得完全分离。在空白样品中添加标准品使浓度0.01~2.0μg/g,得到峰面积与浓度呈良好线性,线性相关系数>0.998。添加浓度在0.1、0.8和1.6μg/g的回收率为87%~97%(n=8);相对标准偏差为8%~13%。方法检出限(S/N=3)分别为STX 1.0 ng/g和dcSTX 0.3 ng/g。另外,采用四极杆-飞行时间质谱(Q-TOF-MS)对STX和dcSTX衍生物进行了结构解析。     

The Combination of Laser-Induced Fluorescence and Intensified Linear Diode Array Detection in Column Liquid Chromatography

Abstract

The frequency-doubled 514-nm argon-ion laser line providing excitation at 257 nm is combined with an intensified linear diode array (ILDA) detector to enable on-the-fly identification by laser-induced fluorescence (LIF) in conventional-size column liquid chromatography (LC). The potential of this detection system is demonstrated for the analysis of a standard mixture of polynuclear aromatic hydrocarbons (PAHs). Detection limits are at the 1.5 μg 1^?1 level (15 pg injected); the identitication limits are about one order of magnitude higher.     

Comparative Study of the Determination of Parabens in Shampoos by Liquid Chromatography with Amperometric and Coulometric Detection

The simultaneous determination of four para‐hydroxybenzoic acid esters (parabens) in shampoos was studied by liquid chromatography (LC) with amperometric (LC‐AD) and coulometric (LC‐CD) detection. The parabens were separated on an ODS C18 reversed column by isocratic elution with a mobile phase based on methanol‐0.1 M acetic acid (60 : 40%, v/v) with 0.02 M NaClO₄ at a flow rate of 0.8 mL min^?1. The limit of detection (S/N>3) for the analytes was in the 15–25 pg (injected mass) range at an applied potential of 1.20 V vs. Ag/AgCl using the LC‐AD and in the 2–3 pg range at a potential of 0.790 V vs. Pd using the LC‐CD. The peak ratio of the internal standard peak (IS: 4‐hydroxybenzoic acid sec‐butyl ester) versus the analyte peak was found to be related to the amount injected from 0.1 ng to 100ng (r=0.996–0.999) with the LC‐AD and from 0.050 ng to 100 ng range (r=0.999–1.000) with the LC‐CD. The relative standard deviation (RSD, n=10) was comprised between 1.8 to 3.5% by LC‐AD ( 5 ng injected) and between 2.0 to 2.4% by LC‐CD (0.5 ng injected). The determination of four most used parabens in ten different shampoos was successfully realized.     

Determination of Homologue Imidazolium Ionic Liquid Cations by Ion Chromatography Using a Carboxyl Acid Cation-Exchange Column with Direct Conductivity Detection

An investigation into the determination of four imidazolium ionic liquid cations by ion chromatography using a carboxyl acid cation exchange column and direct conductivity detection was carried out. This research has developed a simple, selective, and accurate ion chromatographic method for separation of imidazolium ionic liquid cations. Detection limits (S/N = 3) for the cations were 3.2–24.3 mg/L. Relative standard deviations (RSD, n = 5) for peak areas were less than 1.6%. The method has been successfully applied to the determination of two ionic liquids synthesized by organic chemistry lab.     

凝胶柱前处理-气相色谱法测定火腿中多种有机膦农药残留量

用乙腈萃取火腿试样中的残留农药,所得萃取液经凝胶渗透色谱柱净化后用气相色谱法(带火焰光度法检测器)测定了9种农药的残留量,用外标法定量。HP-1701型毛细管色谱柱(30 m×0.53 mm,1.0μm)用于气相色谱分离,采用柱温250℃和检测器温度230℃,载气为氮气,流速为10 mL.min-1,测得三唑膦农药的测定限为2.8μg.kg-1,其它8种有机膦农药的测定限均为5μg.kg-1。用标准加入法测定回收率,9种农药的回收率均在70%以上。     

Simultaneous Determination of Palladium and Platinum by On‐Line Enrichment Followed by Rapid Column High Performance Liquid Chromatography

In this paper, a new method for the simultaneous determination of palladium and platinum ions was developed using a rapid column high performance liquid chromatograph equipped with an on‐line enrichment technique. The palladium and platinum ions were pre‐column derivatized with 5‐(p‐aminobenzylidene)‐thiorhodanine (ABTR) to form colored chelates. The Pd‐ABTR, Pt‐ABTR chelates can be absorbed onto the front of an enrichment column when they were injected into the injector and sent to the enrichment column [ZORBAX Stable Bound, 4.6 × 10 mm, 1.8 μm] with a buffer solution of 0.05 mol/L sodium acetate‐acetic acid buffer solution (pH 3.5) as mobile phase. After the enrichment had finished, by switching the six‐ports switching valve, the retained chelates were back‐flushed by mobile phase and traveled towards the analytical column. These chelates separation on the analytical column [ZORBAX Stable Bound, 4.6 × 50 mm, 1.8 μm] was satisfactory with 65% methanol (containing 0.05 mol/L of pH 3.5 sodium acetate‐acetic acid buffer salt and 0.01 mol/L of tritonX‐100) as mobile phase. The palladium and platinum were separated completely within 2 min. The detection limits (S/N = 3) of palladium and platinum are 1.4 ng/L and 1.6 ng/L, respectively. This method was applied to the determination of palladium and platinum in water and urine samples with good results.     

Peak purity assessment in a triple‐active fixed‐dose combination drug product related substances method using a commercial two‐dimensional liquid chromatography system

Pharmaceutical formulations containing multiple active components challenge the development of analytical methods, especially as the individual active ingredients diverge in their physicochemical properties. Establishing specificity, especially peak purity, is one of the major evaluation criteria when developing a related substances method for drug substances or products. Fixed‐dose combination products may not be amenable to common strategies for assessing peak purity, such as performing orthogonal separations, due to the complexity of the separation and/or diversity of the active ingredients. An alternate approach to evaluating peak purity is demonstrated for a triple‐active component fixed‐dose combination product under development. A commercially available automated two‐dimensional liquid chromatography system was used to perform a selective comprehensive multidimensional separation of an active ingredient peak. The first dimension performed the drug product impurity/degradant profiling method; the second dimension assayed these fractions using the drug substance profiling method, which was pseudo‐orthogonal to the first dimension. A total of 14 targeted fractions were sampled across the first dimension main peak, with 11 containing detectable analytes and the remaining fractions bracketing the main peak. This degree of sampling allowed profiling of a coeluting degradant present at a 0.2% w/w level throughout the main peak.     

Determination of Nitrophenols in Water Using Dynamic Liquid‐Liquid‐Liquid Microextraction under Non‐Equilibrium Consideration

To pursue optimum condition in liquid‐liquid‐liquid microextraction (LLLME), extraction parameters dominating extraction efficiency were investigated by theoretical considerations. The theoretical considerations discussed equilibrium model for equilibrium LLLME and non‐equilibrium model for dynamic LLLME. A method described here is a dynamic LLLME technique combined with high‐performance liquid‐chromatography ultraviolet absorbance detection (HPLC/UV) to determine traces of nitrophenols in water. Analytical parameters such as organic phase, acceptor phase volume, sample agitation, extraction time, acceptor phase NaOH concentration, donor phase HCl concentration, salt addition, and absorption wavelength were identified as variable settings. Relative standard deviation (RSD, 1.8‐4.4%), coefficient of estimation (R², 0.9994‐0.9999), and detection limit (0.032‐0.065 ng mL^?1) were achieved under the variable settings. The proposed method was successfully applied to the analysis of a lake water sample, and the relative recoveries of nitrophenols from spiked water sample were up to 92.5%. The variable settings of LLLME close to optimization was responsible for an acceptable extraction efficiency.     

Determination of Uric Acid in Human Urine by Ion‐exclusion Chromatography with UV Detection Using Pure Water as Mobile Phase

A simple, rapid and accurate ion‐exclusion chromatographic method coupled with a UV detector for the determination of uric acid in human urine samples has been developed. The separation was carried out on an ion‐exclusion column using only pure water as mobile phase. The detection wavelength was 254 nm and urine sample was injected directly without any pretreatment. Furthermore, the retention behavior of uric acid on the ion‐exclusion column was researched when pure water and 1 mmol·L^?1 HCl were used as mobile phase, respectively. The stability of uric acid was also further investigated within 28 days. In this method, the linear range of the calibration curve for uric acid was 0.25–100 mg·L^?1, and the detection limit calculated at S/N=3 was 0.02 mg·L^?1. The proposed ion‐exclusion chromatographic method has been used for the determination of uric acid in human urine.     

Determination of Acrolein‐Derived 3‐Hydroxypropylmercapturic Acid in Human Urine Using Solid‐phase Extraction Combined with Molecularly Imprinted Mesoporous Silica and LC‐MS/MS Detection

A novel method for the analysis of (3‐hydroxypropyl)mercapturic acid (HPMA), a major acrolein metabolite in human urine incorporating a molecularly imprinted solid‐phase extraction (MISPE) process using N‐acetylcysteine ‐imprinted mesoporous silica particles coupled with LC‐MS/MS detection was developed. The molecularly imprinted mesoporous silica particles were synthesized based on the supported material of ordered mesoporous silica SBA‐15 with N‐acetylcysteine (NAC) as template using surface molecular imprinting technology. The condition of MISPE procedures was optimized. The use of MISPE improved the accuracy and precision of the LC‐MS method and lowered the limit of detection (0.23 ng/mL). The recoveries at three spiked levels ranged between 88.5% to 108.6%. The developed MISPE method enabled the selective extraction of HPMA successfully in human urine and could be used as an effective approach for the determination of ultra‐trace HPMA in complex biological matrices. The results in real samples showed that median levels of HPMA were significantly higher (1922.0 ng/mg of creatinine, N = 75) in smokers than in nonsmokers (759.1 ng/mg of creatinine, N = 5), demonstrating the higher acrolein uptake in smokers than in nonsmokers.