Analytical and Bioanalytical Chemistry (v.371, #1)

Online First publication by Peter W. Enders (1-1).

Tribute to J.F.K. Huber by Ernst Kenndler; Franz Dickert (2-3).

Internet-assisted exercises in structural analysis by H. Thomas; S. Paasch; S. Machill; S. Thiele; K. Herzog; M. Hemmer; J. Gasteiger; R. Salzer (4-10).
In this report we describe our experience in the analytical chemistry curriculum of teaching spectrometer principles and preparing spectroscopy laboratory exercises by means of virtual instruments. The benefits of the intensified preparation of laboratory exercises by virtual instruments will be evaluated with respect to the subsequent handling of real instruments. The utilization of in-house electronic media with Internet resources for elucidation and verification of a structural assignment will also be considered.

Highly selective and robust polymer coatings for the detection of polycyclic aromatic hydrocarbons (PAHs) in liquid media have been generated by use of an innovative method of molecular imprinting. By imprinting with two different templates, the selectivity of the polyurethanes used was increased by creating diffusion pathways and molecular cavities. Analyte inclusion was detected both by fluorescence and by use of mass-sensitive transducers. It is possible to optimize layers in respect of the extraction of two different analytes or to achieve extremely high selectivity for a distinct analyte. In this way coatings can be tuned to the lean chrysene, e.g., and it is enriched by a factor of approximately fifty compared with the more quadratic pyrene with the same number of aromatic rings. Measurements of PAHs in water were also performed with a quartz crystal microbalance, which shows that humic acids are not incorporated by the layers and thus do not influence the fluorescence properties of the layers.

A novel, multidimensional SPE sample-processing platform for complex fluids, which relies on the combination of small LC columns packed with restricted access materials (RAM) and molecular imprinted polymers (MIP) is described. It is called the Six-S ProcEdure (Six-SPE). Six-SPE involves a size-selective sample-separation step followed by a solvent-switch. Six-SPE efficiently removes interfering matrix components of complex aqueous samples and creates optimal conditions for selective recognition, i.e. binding of the imprinted target analyte(s). A Six-SPE analysis cycle consists of four distinct steps: 1. separation of a given sample (e.g. plasma, urine, saliva, milk, etc.) by adsorptive extraction (e.g. reversed-phase partitioning) of low molecular weight components on to the stationary phase of a RAM column and simultaneous size-exclusion, i.e. quantitative disposal of macromolecular matrix constituents to waste; 2. desorption and transfer of the extract from the RAM column on to a series-connected MIP column using a pure organic mobile phase (e.g. acetonitrile) [solvent switch]; 3. molecular recognition, i.e. selective binding of the target analyte(s) by a tailor-made MIP column; and 4. desorption and transfer of the analyte fraction on to a series-connected separation (e.g. HPLC) and/or detection system (e.g. UV, FD, MS).As a first application we coupled the Six-SPE platform to a conventional HPLC system for on-line analysis of the analgesic drug Tramadol in human plasma using LiChrospher ADS RP-18 as a RAM precolumn for the fractionation step in the first and second chromatographic dimension and a Tramadol imprinted polymer for the molecular recognition step, i.e. third chromatographic dimension.

Methods have been developed which enable attachment amino and epoxy groups to the surface of integrated optical wave-guide sensors for immunosensor applications. The SiO2–TiO2 surfaces were modified by use of the trifunctional silane reagents γ-aminopropyltriethoxysilane (APTS) and γ-glycidoxypropyltrimethoxysilane (GOPS) in organic and/or in inorganic phases. Silanization methods were optimized taking into consideration the concentration of silane reagent used and the temperature and time of reaction. To evaluate the layers formed, immobilization experiments were undertaken on the modified surfaces using the bovine serum albumin (BSA)–anti-BSA IgG antibody model molecule pair. The regenerability of the sensitized surfaces was also studied.

Selected agrochemicals (s-triazines and phenoxy acids) have been investigated with partial-filling micellar electrokinetic chromatography (PFMEKC) and non-aqueous capillary electrophoresis (NACE). Because these two techniques are compatible for coupling of capillary electrophoresis with mass spectrometry, different conditions affecting the separation efficiency (reproducibility, method linearity) were systematically tested, and the results were compared with those from classical MEKC. The conditions tested included buffer molarity, pH, the concentrations of the organic modifier and surfactant, the applied voltage, the injection time of the sample, and the length of the partial-filling plug. The respective limits of detection (LOD) using UV-detection were determined. Reduction of the electrophoretic raw data using the mobility scale transformation (µ-scale) improved qualitative comparison of the electropherograms and the reproducibility of quantitative data (integrated peak area) thus extending this data treatment from CZE to other endoosmotic flow-driven CE-techniques such as PFMEKC and NACE.

Interference of sulfate(VI) with poly(pyrrole) electrochemistry by A. Michalska; U. Nadrzycka; K. Maksymiuk (35-38).
The effect of the SO4 2– anion on both potentiometry and voltammetry with poly(pyrrole) films doped with chloride or hexacyanoferrate(II/III) ions has been studied and compared with the responses obtained in KCl solutions. Strong interference was observed when the SO4 2– to Cl molar ratio was 1:1000, leading to a decrease in polymer film electroactivity at higher sulfate concentrations. The effect is attributed to incorporation of the divalent anion into the polymer film structure, a hypothesis supported by analysis of the composition of the elemental film.

Comparison of denuder and impinger sampling for determination of gaseous toluene diisocyanate (TDI) by Y. Nordqvist; J. Melin; U. Nilsson; R. Johansson; A. Colmsjo (39-43).
An air-sampling method employing denuders coated inside with a chemisorptive stationary phase has been evaluated for analysis of the hazardous gaseous 2,4 and 2,6 isomers of toluene diisocyanate (TDI). The denuder stationary phase consisted of polydimethylsiloxane (SE-30) to which dibutylamine (DBA) was added as a reagent for derivatization of TDI. The accuracy and precision of sampling by means of denuders were shown to differ only slightly from those of the established impinger method. The denuder method was, however, also shown to be suitable for long-term measurements (up to 8 h). The limit of determination (LOD) of the method, including LC–APCI–MS–MS analysis, was found to be 1.9 µg m–3 and 1.2 µg m–3 for 2,4- and 2,6-TDI, respectively, for short-term measurements (15 min). Significant lower LOD was obtained for long-term measurements.This is well below the Occupational Safety and Health Administration (OSHA) 8-h TWA (time-weighted average) exposure limit, which is 40 µg m–3 for the sum of the TDI isomers. The denuder method was also found to be robust and easy to handle. The samplers can be prepared several days before sampling with no loss in performance. The contents of denuders should, on the other hand, be extracted immediately after sampling to prevent degradation of the isocyanate derivatives formed.

Lucifer yellow CH is shown to be a highly selective fluorescent reagent for the determination of Cu(II) in the µg L–1 concentration range. The fluorophore is statically quenched by Cu(II); the carbohydrazide group was assigned as the complexing part of the dye molecule. A total range of Cu(II) determination from 0.06 mg L–1 (1 µmol L–1) to 6.3 mg L–1 (100 µmol L–1) with a limit of detection of 0.019 mg L–1 (0.3 µmol L–1) was obtained, along with surprisingly high selectivity. There was no interference from alkaline and earth alkaline metal ions. The cross sensitivity to heavy metal ions was evaluated by the separate solution method and by competitive binding experiments. Calibration plots are shown for Cu(II) determination at different pH and the dissociation constant was determined. The application of the reagent was demonstrated by the determination of the Cu(II) content of tap water samples.

A simple, rapid, cost-efficient, and robust method for separation of 237Np with an extraction chromatographic column (TOA: tri-n-octylamine on Teflon powder) is outlined in detail and further improved for direct ICP–MS analysis. The column efficiently retained 237Np in 2 mol L–1 HNO3 medium and all of the 237Np was easily eluted with 0.02 mol L–1 oxalic acid in 0.16 mol L–1 HNO3 at 95 °C. The separated solutions were free from most matrix elements and were aspirated into the ICP–MS directly. The decontamination factor for 238U is more than 104. The instrumental detection limit for 237Np was 0.46 pg mL–1, which corresponds to 1.2×10–5 Bq mL–1. The method is more rapid than traditional radiometric techniques. It is also considered to be more suitable for environmental monitoring than existing methods based on TOA.

A chemical solution-deposited multilayer system of SrTiO3 ("STO")/La0.5Sr0.5CoO3 ("LSCO") on a platinized wafer with a layer sequence Pt/TiO2/SiO2/Si(bulk) has been investigated by dynamic SIMS (secondary ion mass spectroscopy) and TEM (transmission electron microscopy); element determination was performed with EELS (electron energy-loss spectroscopy). The STO layer is intended to serve as a dielectric layer for a microelectronic capacitor; the conducting LSCO layer is a buffer layer intended to eliminate fatigue effects which usually occur at the STO/Pt interface. The SIMS depth profiles obtained for the main components revealed intense diffusion processes which must have occurred during the deposition/crystallization processes. Ti is found to diffuse from the (insulating) STO layer into the conductive LSCO layer where a region of constant concentration is observable. TEM–EELS experiments showed that these Ti plateaus are caused by precipitates approximately 20–80 nm in diameter.

The enantiomeric composition of the chiral flavoring agent limonene was analyzed by means of a quartz-crystal microbalance (QCM) sensor. As chiral selectors three different modified β-cyclodextrins were investigated. The selector molecules were applied as mixtures in different polysiloxane matrices. The chiral separation factors α for limonene obtained at 30°C by gas chromatography and by use of the QCM sensor were comparable. Evaluation of sensor data was performed by use of an artificial neuronal network (ANN); this enabled prediction of the enantiomeric composition of the gas mixtures.

A high-performance liquid-chromatographic method has been developed for the determination of five penicillin compounds (benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, and dicloxacillin) at trace levels in commercially available milk samples. This method comprises extraction of the lipids with ethyl acetate, clean-up and concentration on a C-18 solid-phase extraction column, and derivatization with 1,2,4-triazole and mercury(II) chloride solution, pH 8, at 65 °C for 10 min. The derivatized compounds are eluted from a C-2 column with a mobile phase containing acetonitrile and phosphate buffer loaded with sodium thiosulfate and tetrabutylammonium hydrogen sulfate as ion-pairing reagent. The limit of determination was found to be 4 µg L–1 milk for benzylpenicillin and 10 µg L–1 for the others. This meets EU criteria according to decision No. 93/256/EEC.

Improvement of the HPLC analysis of anthocyanins in red wines by use of recently developed columns by Bálint Berente; Manfred Reichenbächer; Klaus Danzer (68-72).
Comparison of the separation performance of five newly developed pH-stable HPLC columns is described for the analysis of anthocyanins in red wines. Separation of twenty anthocyanins in a single run is described using the most appropriate method.

Determination of flavonoids and stilbenes in red wine and related biological products by HPLC and HPLC–ESI–MS–MS by Günther Stecher; Christian W. Huck; Michael Popp; Günther K. Bonn (73-80).
To investigate probable health benefits of flavonoids and stilbenes in red wine a new reversed-phase (RP) high-performance liquid-chromatographic (HPLC) method with enhanced separation efficiency and improved selectivity, sensitivity, and speed has been established for determination of the flavonoids quercetin, myricetin and kaempferol and the stilbenes cis- and trans-resveratrol, in a single run . UV-absorbance, fluorescence (FLD), and mass-spectrometric (MS) detection were also evaluated. UV-absorbance detection at 320 nm for stilbenes and 377 nm for flavonoids enables their determination up to the nanogram range with a linearity of R2>0.9999 (linear range 50 ng mL–1–50 µg mL–1). Calculated values of average recoveries were between 95 and 105% for all analytes. For resveratrol, fluorescence detection was highly selective and twice as sensitive as UV detection, and linearity was satisfactory (R2>0.9996; linear range see UV detection). For the detection of the hydrophilic glycosidic compounds piceid and rutin, which are coeluted with other hydrophilic ingredients, the validated RP HPLC system was coupled to a quadrupole ion-trap mass-spectrometer (MS) via an electrospray interface (ESI) with 25% ammonia solution as sheath liquid. MS detection was, highly linear (R2>0.9878; linear range 50 ng mL–1–50 µg mL–1) for all investigated analytes and the limits of detection were in the low nanogram range. Compared with UV detection MS detection resulted in a 200% increase in signal intensity for myricetin and 400% increases for quercetin and kaempferol, but equal signal intensity for resveratrol. Calculated values of average recoveries were 102% for myricetin and 79% for piceid. Collision induced dissociation (CID) was also used to obtain characteristic fragmentation fingerprints to facilitate qualitative and quantitative analysis even in complex matrices. Finally, this hyphenated HPLC–ESI–MS method was highly suitable and an essential improvement compared with UV- and fluorescence detection.

A method is described which enables the group-separation of proteinaceous binding media from vegetable glues (carbohydrates), and simultaneous hydrolysis of the proteins in mixtures of both. The mixtures of the binders are suspended in aqueous–ethanolic solvent with the H+ form of a strong cation exchanger and treated at elevated temperature in sealed vials. The polypeptides are cleaved by H+-catalysed hydrolysis. On abstraction the amino acids are transformed into the ammonium ions by the protons, and the cations are adsorbed by the exchanger resin. The amino acids are removed from solution in this way, thus suppressing interfering reactions with other binders, e.g. humin formation with carbohydrates. Clear and colourless solutions were obtained with all mixtures of vegetable and animal glues. Two fractions can be obtained after separation of the solid resin from the liquid supernatant – the resin fraction with the adsorbed amino acids, and the aqueous–ethanolic solution with the carbohydrates. In each of these fractions the two classes of binder can be identified separately by GC–MS; this avoids the occurrence of unresolved GC peaks and superimposed mass spectra.The method has been used to identify the binder found between fabric layers of a Burgundian liturgical vestment of the Order of the Golden Fleece from the first half of the 15th century, the Cope of the Virgin Mary. With the aid of the GC pattern obtained, and the mass spectra of the main peaks, which were identified as glucopyranose anomers, the binding medium was identified as starch.