HPLC fractionation and structural dynamics of humic acids

Different soil extracted humic acids as well as a commercial humic acid sodium salt were fractionated by HPLC. An almost complete recovery could be achieved for the dissolved material. All humic samples show a typical chromatogram of at least five fractions. The separation of humic substances is influenced by an altered hydrophobicity due to changes in the tertiary structure. Three fractions were further investigated by Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy and by HPLC/HPLC separation. DRIFT spectroscopy provided data on the primary structure, whereas HPLC/HPLC results gave insight into the secondary and tertiary structure and their changes. Received: 18 June 1996 / Revised: 9 October 1996 / Accepted: 12 October 1996     

Determination of heavy metal complexes with humic substances by HPLC/ICP-MS coupling using on-line isotope dilution technique

An isotope dilution mass spectrometric (IDMS) method has been developed for the simultaneous determination of the complexes of 11 heavy metals (Ag, Cd, Cu, Mo, Ni, Pb, Tl, U, W, Zn and Zr) with humic substances (HS) by coupling HPLC with ICP-MS and applying the on-line isotope dilution technique. The HPLC separation was carried out with size exclusion chromatography. This HPLC/ICP-IDMS method was applied to samples from a brown water, ground water, sewage and seepage water as well as for a sample containing isolated fulvic acids. The total contents of heavy metals and of their complexes were analyzed in these samples with detection limits in the range of 5–110 ng/L. The analysis of heavy metal/HS complexes from the different waters resulted in characteristic fingerprints of the distribution pattern of heavy metals in the separated HS fractions. A comparison between the total heavy metal concentrations and their portions bound to humic substances showed distinct differences for the various metals. Simultaneous ¹²C detection was used for the characterization of HS complexes not identified by UV detection and for the determination of relative DOC concentrations of chromatographic peaks. Received: 21 February 1997 / Revised: 27 May 1997 / Accepted: 28 May 1997     

Evaluation of gel permeation chromatography to Fourier transform infrared spectrometric detection using a modified thermospray for analysis of polystyrene samples

A heated gas flow modified thermospray was used to couple gel permeation chromatography (GPC) to Fourier transform infrared spectrometry (FTIR) for the analysis of the standard polystyrene samples. Effluents from the GPC column were evaporated and the solutes were deposited as a series of spots on the surface of a moving stainless steel belt (0.025 mm thickness × 13 mm width). The belt continuously transferred the spots into the diffuse reflectance (DRIFT) accessory of the FTIR spectrometer, enabling identification of the deposited solutes by measurement of the diffuse reflectance IR spectrum. The IR spectra of the separated components showed excellent agreement of the spectral features to those of standard FTIR spectra and no thermal degradation was observed. Received: 20 May 1996 / Revised: 17 October 1996 / Accepted: 28 November 1996     

HPLC of humic substances fractionated by Flow FFF

This communication reports a study of the effect of ionic strength and electrolyte composition on fractions, separately collected by flow FFF, of a mixture of humic substances. Reverse phase HPLC analysis of three early eluting fractions suggests that the components released by the column behave as organic acids. The baseline‐resolved peaks of the first two fractions, subject to higher retention in solutions of lower pH and/or higher polarity, substantiate this suggestion. The fraction with larger components, as measured by flow FFF, also appears to contain acidic species. Their retention level, however, may not be accurately modulated by varying the mobile phase properties as these species are either totally retained in acidic phases or released before the void peak at pH ≥ 4.2. Besides showing the effective separation achieved in the flow FFF channel, this study reveals the pronounced difference in the physicochemical properties of some components of a humic mixture even with very close particle size.     

Retention behavior of humic substances in reversed phase HPLC

Recovery as well as appearance and abundance (in percent) of different fractions of humic substances are found to depend on injected sample amounts in reversed phase HPLC. Sample amounts have been varied both by varying sample concentration and sample volume. In case of lowest amounts injected only two fractions were obtained for a commercial humic acid sodium salt, i.e. one for excluded molecules and one for hydrophobic components. The abundance of excluded molecules decreases upon increasing amounts injected. Another three fractions are obtained upon increasing amount injected: a hydrophilic fraction and two hydrophobic ones. This behavior is explained by auxiliary equilibria between excluded components and humic molecules previously adsorbed on the stationary phase.     

用于碱性物质分离的高效液相色谱键合相的制备及评价

采用２,４－戊二酮与正辛烷基二甲基氯硅烷反应制成活泼的中间体——硅醚型硅烷中间体,然后再与硅胶进行键合。经元素分析、漫反射红外光谱和高效液相色谱法对键合相进行了鉴定和评价。结果表明,键合反应按预定路线进行,硅胶表面被键合相基团覆盖的较均匀、完全。键合相具有较好的色谱性能,可有效地用于碱性化合物的分离分析中。     

Rhodium and palladium β-diketonate determination with on-line supercritical fluid extraction-high performance liquid chromatography via solid phase extraction

 An on-line system of supercritical fluid extraction (SFE) and high performance liquid chromatography (HPLC) via solid phase extraction (SPE) is described for the determination of palladium and rhodium 2,2,6,6-tetramethyl-3,5-heptanedione-(thd) as well as rhodium-acetylacetonate-(acac) and benzylacetonate-(bzac) chelates. The chelates were extracted with supercritical CO₂ from sand and humic acid, concentrated on SPE cartridges and analysed with HPLC. Two cartridge materials were tested and compared to off-line trapping. The percentage of the breakthrough and cartridge retained material were measured in liquid dichloromethane. The SFE conditions could be optimized to separate metal chelates during the extraction. The supercritical fluid (SF) behaviour of different ligands on rhodium were investigated. Received: 19 July 1996/Revised: 11 December 1996/Accepted: 14 December 1996     

Analysis of nitrophenols and other polar nitroaromatic compounds in ammunition wastewater by high-field proton nuclear magnetic resonance (1H-NMR) spectroscopy and chromatographic methods

 It is shown that by using high-field proton nuclear magnetic resonance (¹H-NMR) spectroscopy it is possible, without prior separation, to analyse nitrophenols and other acidic nitroaromatic compounds in the “pH 2 extract” of ammunition wastewater. The ¹H-NMR chemical shifts data of a variety of reference compounds are presented. Two groundwater samples from the former ammunition plant in Elsnig (Saxony) were analysed by ¹H-NMR and also by chromatographic methods (GC/MS, HPLC). The results are compared and discussed. Received: 17 January 1996/Revised: 15 May 1996/Accepted: 17 May 1996     

Analysis of nitrophenols and other polar nitroaromatic compounds in ammunition wastewater by high-field proton nuclear magnetic resonance (1H-NMR) spectroscopy and chromatographic methods

 It is shown that by using high-field proton nuclear magnetic resonance (¹H-NMR) spectroscopy it is possible, without prior separation, to analyse nitrophenols and other acidic nitroaromatic compounds in the “pH 2 extract” of ammunition wastewater. The ¹H-NMR chemical shifts data of a variety of reference compounds are presented. Two groundwater samples from the former ammunition plant in Elsnig (Saxony) were analysed by ¹H-NMR and also by chromatographic methods (GC/MS, HPLC). The results are compared and discussed. Received: 17 January 1996/Revised: 15 May 1996/Accepted: 17 May 1996     

Analytical fractionation of aquatic humic substances by metal affinity chromatography on iron(III)-coated cellulose

The chromatographic fractionation of aquatic humic substances (HS) onto iron(III)-coated cellulose (Cell-Fe(III)) as a metal-loaded adsorbent is described, analogously to the separation principle of the well-established metal affinity chromatography (MAC). For that purpose the sorption of HS from different aquatic origin on that collector was characterized by their kinetics and equilibrium distribution coefficients Kd. Based on Kd values of 10³ to 10⁴,mL/g, and fast sorption kinetics a preparative HPLC procedure, using stepwise increased pH-values (pH 8–12.5, borate buffer) as an eluent, was developed for the fractionation of dissolved HS (up to 7 fractions of different amount). The fractions obtained by this MAC procedure from selected aquatic HS samples were different in their Cu(II) complexation capacity, absorbance ratio E_{265 nm}/E_{365 nm} and Fourier transform infrared spectra. Received: 14 June 1999 / Revised: 6 August 1999 / Accepted: 10 August 1999     

Analytical fractionation of aquatic humic substances by metal affinity chromatography on iron(III)-coated cellulose

The chromatographic fractionation of aquatic humic substances (HS) onto iron(III)-coated cellulose (Cell-Fe(III)) as a metal-loaded adsorbent is described, analogously to the separation principle of the well-established metal affinity chromatography (MAC). For that purpose the sorption of HS from different aquatic origin on that collector was characterized by their kinetics and equilibrium distribution coefficients Kd. Based on Kd values of 10³ to 10⁴,mL/g, and fast sorption kinetics a preparative HPLC procedure, using stepwise increased pH-values (pH 8–12.5, borate buffer) as an eluent, was developed for the fractionation of dissolved HS (up to 7 fractions of different amount). The fractions obtained by this MAC procedure from selected aquatic HS samples were different in their Cu(II) complexation capacity, absorbance ratio E_{265 nm}/E_{365 nm} and Fourier transform infrared spectra. Received: 14 June 1999 / Revised: 6 August 1999 / Accepted: 10 August 1999     

Determination of caffeine in solid pharmaceutical samples by FTIR spectroscopy

Fourier transform infrared spectroscopy has been used for the quantitative determination of caffeine in several pharmaceutical products: acetyl salicylic acid, paracetamol and propyphenazone. The method is simple, rapid and selective, and allows the determination of caffeine without sample pretreatment and without separation from the matrix. Two techniques for measuring the infrared spectra of caffeine are described: transmission through pellets and diffuse reflectance from powder (DRIFT). In both methods, samples were diluted (1%) with KBr. Caffeine in pharmaceutical matrices was recovered within 5% error in pellets and 10% by DRIFT. Relative standard deviations were generally 1.5% for repeated measurements on a single pellet and 5% for measurements on different pellets. DRIFT in the vacuum mode gave rather large RSDs. The limit of detection of the pellet technique was about 0.5% caffeine in the original sample.     

Unambiguous detection of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana)

HPLC atmospheric pressure chemical ionization (APCI)/MS, GC MS, HPLC diode array detection (DAD), and NMR were used for the identification of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana). Matrix solid phase dispersion was applied for the extraction of the carotenoids. This gentle and expeditious extraction technique for solid and viscous samples leads to distinct higher enrichment rates than the conventional liquid-liquid extraction. The chromatographic separation was achieved employing a C30 RP column that allows the separation of shape-constrained geometrical isomers. A methanol/tert-butylmethyl ether/water gradient was applied. (all-E) Astaxanthin and the geometrical isomers were identified by HPLC APCI/MS, by coelution with isomerized authentical standard, by UV spectroscopy (DAD), and three isomers were unambiguously assigned by microcoil NMR spectroscopy. In this method, microcoils are transversally aligned to the magnetic field and have an increased sensitivity compared to the conventional double-saddle Helmholtz coils, thus enabling the measurement on small samples. The carotenol fatty acid esters were saponified enzymatically with Lipase type VII from Candida rugosa. The fatty acids were detected by GC MS after transesterification, but also without previous derivatization by HPLC APCI/MS. C14:0, C16:0, C16:1, C18:1, C20:0, C20:5, and C22:6 were found in astaxanthin monoesters and in astaxanthin diesters. (all-E) Astaxanthin was identified as the main isomer in six fatty acid ester fractions by NMR. Quantitation was carried out by the method of internal standard. (13-cis) Astaxanthin (70 microg/g), 542 microg/g (all-E) astaxanthin, 36 microg/g unidentified astaxanthin isomer, 62 microg/g (9-cis) astaxanthin, and 7842 microg/g astaxanthin fatty acid esters were found.     

Extraction of humic acids from a natural matrix by alkaline pyrophosphate. Evaluation of the molecular weight of fractions obtained by ultrafiltration

 Fractions of humic acids, resolved by ultrafiltration of extracts from a sample of peat treated with alkaline pyrophosphate solution, have been submitted to high-performance size-exclusion chromatography, with the aim of determining the molecular weight distributions. Anomalous peaks, located at retention volumes higher than those corresponding to the main signal, are present in the elution profiles relative to the lightest fractions. These peaks are more intense when using a refractive index detector rather than a UV detector. Elemental analysis data for the lightest fractions suggested that the spurious peaks are due to the presence of inorganic material. The hypothesis that pyrophosphate interacts with humic acids in the course of the extraction has been confirmed by colorimetric determination of the phosphorus content in the different fractions. As a consequence, the extraction procedure has been modified by using 0.1 mol/L NaOH as extractant. The average molecular weights of the various fractions, following NaOH extraction, result in substantial agreement with those obtained following pyrophosphate solution extraction (if the ‘pyrophosphate peaks’ present in the chromatographic profile are discarded in the calculations). Received: 18 November 1996 / Revised: 18 February 1997 / Accepted: 23 February 1997     

HPLC approach for revealing age-related changes of aquatic dissolved organic matter in sediment core

Analytical method based on HPLC has been used to characterize aquatic dissolved organic matter (DOM) from sediment core of Lake Võrtsjärv, South- Estonia. High-performance size exclusion chromatography (HPSEC) as separation method was coupled with diode-array detection (DAD) and separated molecular fractions of DOM were subject to qualitative and semi-quantitative analysis. Qualitative analysis based on UV- spectra revealed the presence of proteins and humic constituents in separated high molecular weight fraction and aromatic constituents in low molecular weight fraction. Statistical data treatment methods enabled clustering sediment layers into 4 periods according to sediment depth and age. The upper 0-30 cm sediment DOM had statistically relevant differences in comparison to other periods as revealed by lower total peak and humic substances (HS) fraction areas, and molecular weights. Samples from 80-120 cm depth differed from others by decreased low molecular weight (LMW) fraction content. The observed down-core trends suggest polymerization of LMW organic constituents and increasing humification. The statistical analyses revealed that some chromatographic and spectrometric parameters can be used to differentiate between sediment layers and to evaluate environmental changes.     

Determination of Triton X-100 in influenza vaccine by high-performance liquid chromatography and capillary electrophoresis

Triton X-100 is applied to influenza vaccines at different stages of the manufacturing process to prevent aggregation and precipitation of biomolecules. Furthermore it is used to disintegrate the virus particles in split vaccine and to guarantee the homogeneity during production and utilisation. The final concentration of Triton X-100 has to be determined because the concentration changes in manufacturing process. The determination of the total amount of Triton X-100 as well as the separation of its ethylene oxide oligomers was possible with high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). In HPLC a change of the column and eluent was necessary, in CE different electrolytes were used for the various separation effects. The HPLC method for the analysis of total Triton was preferred for the quantification of Triton X-100 in influenza vaccine because of better linearity, reproducibility and detection sensitivity compared to CE. In the end products an average concentration of 0.117 mg/mL was found. Received: 19 December 1996 / Revised: 27 February 1997 / Accepted: 6 March 1997     

Combination of TREF, high-temperature HPLC, FTIR and HPer DSC for the comprehensive analysis of complex polypropylene copolymers

A novel, powerful analytical technique, preparative temperature rising elution fractionation (prep TREF)/high-temperature (HT)-HPLC/Fourier transform infrared spectroscopy (FTIR)/high-performance differential scanning calorimetry (HPer DSC)), has been introduced to study the correlation between the polymer chain microstructure and the thermal behaviour of various components in a complex impact polypropylene copolymer (IPC). For the comprehensive analysis of this complex material, in a first step, prep TREF is used to produce less complex but still heterogeneous fractions. These chemically heterogeneous fractions are completely separated by using a highly selective chromatographic separation method—high-temperature solvent gradient HPLC. The detailed structural and thermal analysis of the HPLC fractions was conducted by offline coupling of HT-HPLC with FTIR spectroscopy and a novel DSC method—HPer DSC. Three chemically different components were identified in the mid-elution temperature TREF fractions. For the first component, identified as isotactic polypropylene homopolymer by FTIR, the macromolecular chain length is found to be an important factor affecting the melting and crystallisation behaviour. The second component relates to ethylene–propylene copolymer molecules with varying ethylene monomer distributions and propylene tacticity distributions. For the polyethylene component (last eluting component in all semi-crystalline TREF fractions), it was found that branching produced defects in the long crystallisable ethylene sequences that affected the thermal properties. The different species exhibit distinctively different melting and crystallisation behaviour, as documented by HPer DSC. Using this novel approach of hyphenated techniques, the chain structure and melting and crystallisation behaviour of different components in a complex copolymer were investigated systematically.

Utilisation of reversed-phase high-performance liquid chromatography as an alternative to silver-ion chromatography for the separation of cis- and trans-C18:1 fatty acid isomers

Silver ion-high-performance liquid chromatography (HPLC) has been commonly used for the separation and the analysis of trans-18:1 isomers in partially hydrogenated oils and milk fat. This paper describes an easy HPLC method using two reversed-phase columns. The cis- and trans-18:1 fatty acids isomers as methyl esters were eluted as two separate fractions. The collected fractions were analysed by gas chromatography (GC). The purity of the two fractions were tested by GC-MS and GC-Fourier transform IR.     

泥炭土连续碱抽提腐殖酸的分子结构特征

从Pahokee泥炭土中连续碱抽提分离出8个不同的腐殖酸级分，并对每一级分进行了元素分析、傅里叶变换红外光谱（FTIR）、固态^13C核磁共振（^13CNMR)、高效排阻色谱（HPSEC）等一系列定性、定量研究。结果表明：所分离出的8个腐殖酸级分存在明显的结构性质差异，随提取和蔼的增加，O／C原子比由0．52减少到0．36，H／C原子比由1．05增加到1．52，相应于结构中含氧基团的减少和脂族基的增加，表观分子量也由7．7K增加到22．1K。同时，^13C NMR显示长链脂肪碳结构由无定型向晶型转变。此工作表明在所研究的腐殖酸中可能存在分别具有芳香或脂肪特性的两类腐殖酸结构，每种类型都有不同的分子量分布、元素组成、基团结构和母质来源。在特定的环境因素下，不同类型的腐殖酸会共存于同一体系中，增加了腐殖物质的非均匀性和复杂性。     

Preparation and characterization of a new p-tert-butyl-calix[8]arene-bonded stationary phase for high-performance liquid chromatography.

A new p-tert-butyl-calix[8]arene-bonded silica gel stationary phase (CABS) was prepared via 3-glycidoxypropyltrimethoxysilane as a coupling reagent for HPLC. Its structure was characterized by diffuse reflectance infrared Fourier transform spectroscopy (DRIFT), elemental analysis and thermal analysis. The chromatographic performance of new packing was evaluated by using basic, acidic and neutral aromatic compounds as probes compared with conventional ODS. The results show that the new stationary phase has an excellent reversed-phase property and high selectivities for substituted aromatics compared with ODS, because CABS can provide various sites for the analytes, such as hydrogen-bonding interactions, pi-pi interactions, and inclusion complex, besides hydrophobic interactions.