Comparative study on separation of diastereomers by HPLC

Summary Reversed (RP-HPLC) and normal phase chromatographic (NP-HPLC) separations have been developed for diastereomers ofN-acyl-1-methyl-1,2,3,4-tetrahydo-β-carbolines which are acylated derivatives of simple natural β-carboline alkaloids. Separations of derivatives having different acyl moieties in theO,O-diacyl-tartaric acid ester subtituent differed remarkably. Little or no resolution in either NP-HPLC or RP-HPLC could be achieved with the diacetyl-tartrate derivative. Base-line separation by RP-HPLC but no separation by NP-HPLC was possible with the bulkier and more apolar dipivaloyl derivative. Retention order of the bis-benzoylated diastereomers was reversed and separation time increased dramatically by RP-HPLC. Good separation of the medium polarity, but rigid,N-camphanyl derivative by NP-HPLC has been achieved, whereas RP-HPLC could not be used for separation of these diastereomers. Separability of different diastereomers was highly dependent on polarity and rigidity of the derivatizingN-acyl moieties. Conformational analysis by molecular mechanics and comparison of the lowest energy conformational states of the diastereomers was applied to rationalise separation-retention behaviour of stereoisomers by RP-HPLC. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Analysis of cyanobacterial toxins (anatoxin-a, cylindrospermopsin, microcystin-LR) by capillary electrophoresis

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) were applied to the simultaneous separation of cyanobacterial toxins (anatoxin-a, microcystin-LR, cylindrospermopsin). The analytical performance data of both methods, optimized for the three toxins, were similar with a precision of migration times smaller than 0.8 RSD% and a detection limit in the range of 1-4 microg/mL, using spectrophotometric detection at 230 nm. Both methods were applied to an analysis of cyanotoxins in water bloom samples and crude cyanobacterial extracts. The results obtained indicate that, for complex matrices, the sequential application of CZE and MEKC is necessary. It is recommended to use both CE techniques for the analysis of the same sample in order to confirm the results by an orthogonal approach.     

Compression of long-cavity Ti:sapphire oscillator pulses with large-mode-area photonic crystal fibers

Motivated by the pulse compression challenge of novel long-cavity, high-pulse-energy Ti:sapphire laser oscillators, we report on ~280 nm supercontinuum generation and 4.5-times compression of close to transform limited, high-energy oscillator pulses using different large-mode-area photonic crystal fibers and standard chirped mirrors. As input, we used pulses of a long-cavity Ti:sapphire oscillator with 190 nJ pulse energy, 70 fs pulse length and 3.6 MHz repetition rate. Compressed pulses at the fiber/compressor output had a duration of 15–18 fs with up to 100 nJ pulse energy representing as much as 53 % throughput for the fiber/chirped mirror system. Using transform-limited input pulses, we could use short fiber pieces and thus a simple, low-dispersion chirped mirror compressor comprised of one pair of mirrors.     

A New Oxygen Containing Pyclen-Type Ligand as a Manganese(II) Binder for MRI and 52Mn PET Applications: Equilibrium,Kinetic, Relaxometric,Structural and Radiochemical Studies

A new pyclen-3,9-diacetate derivative ligand (H₂3,9-OPC2A) was synthesized possessing an etheric O-atom opposite to the pyridine ring, to improve the dissociation kinetics of its Mn(II) complex (pyclen = 3,6,9,15-tetraazabicyclo(9.3.1)pentadeca-1(15),11,13-triene). The new ligand is less basic than the N-containing analogue (H₂3,9-PC2A) due to the non-protonable O-atom. In spite of its lower basicity, the conditional stability of the [Mn(3,9-OPC2A)] (pMn = −log(Mn(II)), c_L = c_Mn(II) = 0.01 mM. pH = 7.4) remains unaffected (pMn = 8.69), compared to the [Mn(3,9-PC2A)] (pMn = 8.64). The [Mn(3,9-OPC2A)] possesses one water molecule, having a lower exchange rate with bulk solvents (k_ex²⁹⁸ = 5.3 ± 0.4 × 10⁷ s⁻¹) than [Mn(3,9-PC2A)] (k_ex²⁹⁸ = 1.26 × 10⁸ s⁻¹). These mild differences are rationalized by density-functional theory (DFT) calculations. The acid assisted dissociation of [Mn(3,9-OPC2A)] is considerably slower (k₁ = 2.81 ± 0.07 M⁻¹ s⁻¹) than that of the complexes of diacetates or bisamides of various 12-membered macrocycles and the parent H₂3,9-PC2A. The [Mn(3,9-OPC2A)] is inert in rat/human serum as confirmed by ⁵²Mn labeling (nM range), as well as by relaxometry (mM range). However, a 600-fold excess of EDTA (pH = 7.4) or a mixture of essential metal ions, propagated some transchelation/transmetalation in 7 days. The H₂3,9-OPC2A is labeled efficiently with ⁵²Mn at elevated temperatures, yet at 37 °C the parent H₂3,9-PC2A performs slightly better. Ultimately, the H₂3,9-OPC2A shows advantageous features for further ligand designs for bifunctional chelators.     

Enzyme-purification and catalytic transformations in a microstructured PASSflow reactor using a new tyrosine-based Ni-NTA linker system attached to a polyvinylpyrrolidinone-based matrix

The synthesis of a Ni-nitrilotriacetic acid (Ni-NTA) attached via a new tyrosine-based linker matrix on monolithic crosslinked poly(vinyl benzyl chloride)/poly(vinylpyrrolidinone) is described. This matrix is incorporated inside a microstructured PASSflow reactor which was used for automatic purification and immobilisation of His(6)-tagged proteins. These could be used as stable and highly active biocatalysts for the synthesis of (R)-benzoin (6), (R)-2-hydroxy-1-phenylpropan-1-one (7) and 6-O-acetyl-D-glucal (17) in a flow-through mode.     

Pinoresinol from Ipomoea cairica cell cultures

Ipomoea cairica cell cultures produced a tetrahydrofuran lignan, (+)-pinoresinol, identified by UV, IR, MS and NMR methods, not yet found in the intact plant, and new in the Convolvulaceae family. Pinoresinol was found to have antioxidant and Ca2+ antagonist properties. As it could be requested for its biological activity, we examined the possibility to raise the pinoresinol yield of I. cairica cultures, as well as we continued investigations on lignans' response to optimization.     

Origin Identification of Hungarian Honey Using Melissopalynology,Physicochemical Analysis,and Near Infrared Spectroscopy

The objective of the study was to check the authenticity of Hungarian honey using physicochemical analysis, near infrared spectroscopy, and melissopalynology. In the study, 87 samples from different botanical origins such as acacia, bastard indigo, rape, sunflower, linden, honeydew, milkweed, and sweet chestnut were collected. The samples were analyzed by physicochemical methods (pH, electrical conductivity, and moisture), melissopalynology (300 pollen grains counted), and near infrared spectroscopy (NIRS:740–1700 nm). During the evaluation of the data PCA-LDA models were built for the classification of different botanical and geographical origins, using the methods separately, and in combination (low-level data fusion). PC number optimization and external validation were applied for all the models. Botanical origin classification models were >90% and >55% accurate in the case of the pollen and NIR methods. Improved results were obtained with the combination of the physicochemical, melissopalynology, and NIRS techniques, which provided >99% and >81% accuracy for botanical and geographical origin classification models, respectively. The combination of these methods could be a promising tool for origin identification of honey.     

Comparative study of recent wide-pore materials of different stationary phase morphology, applied for the reversed-phase analysis of recombinant monoclonal antibodies

Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ～250 Å and macropores of ～?1.1 μm, a column packed with 3.6 μm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 μm particles having pore size of ～300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene–divinyl benzene) organic monolithic column, with a macropore size of approximately 1 μm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 μm and fully porous 1.7 μm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 μm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 μm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.     

Comparison of various silica‐based monoliths for the analysis of large biomolecules†

In the present study, three types of silica‐based monoliths, i.e. the first and second generations of commercial silica monolithic columns and a wide‐pore prototype monolith were compared for the analysis of large biomolecules. These molecules possess molecular weights between 1 and 66 kDa. The gradient kinetic performance of the first‐generation monolith was lower than that of the second generation, for large biomolecules (>14 kDa) but very close with smaller ones (1.3–5.8 kDa). In contrast, the wide‐pore prototype column was particularly attractive with proteins larger than 19 kDa (higher peak capacity). Among these three columns, the selectivity and retention remained quite similar but a possible larger number of accessible and charged residual silanols was noticed on the wide‐pore prototype material, which led to unpredicted small changes in selectivity and slightly broader peaks than expected. The peak shapes attained with the addition of 0.1% formic acid in the mobile phase remained acceptable for MS coupling, particularly for biomolecules of less than 6 kDa. It was found that one of the major issues with all of these silica‐based monoliths is the possible poor recovery of large biomolecules (principally with monoclonal antibody fragments of more than 25 kDa).     

Thionation of a cyanoxime derivative to form the sulphur-containing derivative,a novel ligand for complexation with transitional metal ions

Structural Chemistry - 2-Cyano-2-(hydroxyimino)dithioacetic acid was prepared starting from cyanoacetic acid methylester via 2-cyano-2-(hydroxyimino)acetic acid methylester. Before thionation, the...