Current Chromatography - Volume 3, Issue 1, 2016

Background: Sulfobetaine zwitterionic stationary phases are offering various retention modes, but the influence of the capacity has never been investigated systematically. Objective: The capacity dependence of the retention and selectivity in zwitterionic ion chromatography mode has been investigated for common UV absorbing anions. Methods: Stationary phases with variable distances between the charged groups (one to four methylen groups) were prepared with capacities from 296 to 790 (one and two methylene groups as charge separator) or from 212 to 298 μeq g-1 (three and four methylene groups) by grafting polymerization on highly porous divinylbenzene polymers. The different spacer lengths and capacities are used as an investigative tool for the retention behavior of the anions nitrate, nitrite, bromide, iodide and thiocyanate. The separation mechanism is explored by varying eluent ionic strength and pH. Results: Remarkably, an increasing capacity leads depending on the charge distance to an increase for two and three methylen groups, decrease for four or no significant change in the retention times of inorganic anions for one methylene group charge distance. No significant change of selectivity occurred in investigated range of capacities. It should be noted that the higher capacity of the exchanger with two methylene groups between the oppositely charged groups exhibit a different separation behavior for inorganic anions in comparison to a previous study. Conclusions: The capacity plays in zwitterionic ion chromatography an important role for the retention behavior. The changes in retention behavior are depending on the spacer length between the charged groups.

Background: The use of a silica hydride-based stationary phase is investigated for the analysis of capsaicinoids in various hot sauces. These compounds are important in a variety of food products. Aqueous normal phase chromatography is a technique involving the use of silica hydridebased stationary phases that can be used to retain both polar and nonpolar compounds. Methods: Five of these compounds were investigated in three types of sauces. The use of time-offlight mass spectrometry with accurate mass measurement is used as a means analyzing for these closely related compounds in the shortest time possible. Additional improvement in resolution through higher efficiency is investigated by using 2 μm particles under near UPLC conditions. Results: A simple experimental protocol for the analysis of capsaicinoids in peppers and hot sauces has been developed. With exact mass measurements, all of the components do not need to be chromatographically separated since they all have different m/z and can be developed into a validated method if needed. Conclusion: The use of silica hydride-based columns results in a fast analysis time, excellent run-to-run reproducibility, MS compatible mobile phase conditions and fast column re-equilibration.

Background: Linear alkanes, between nC5 and nC9, and aromatic hydrocarbons, also called BTX (benzene, toluene, xylenes are important constituents of raw materials in the petroleum and chemical industries. Their separation is relevant for both industrial and analytical point of views. Industrial separation and purification involve the use of appropriate molecular sieve materials. Methods: The separation of linear alkanes (nC5-nC9), and benzene, toluene and o-xylene, also called BTX, by using two inorganic polymers based on tetraethoxysilane (TEOS) as stationary phase was evaluated from inverse gas chromatography data. Both materials were able to separate the mixtures of those organic volatile hydrocarbons. The nature of such behavior is discussed according to the possible guest-host interactions of the molecules adsorption and retention in the chromatographic column. Results: The retention times were obtained from the recorded chromatographic profiles and then used to obtain differential adsorption heats and separation coefficients. The adsorption heats were estimated for each probe molecule from the slopes of the straight lines at four temperatures. The effects of these surface differences on the molecules adsorption and separation are discussed. Conclusion: The main difference between the two studied materials is the percentage of water on their surface due to the synthesis conditions. This fact modulates the pore accessibility and the adsorption interactions for BTX probe molecules but not for linear alkanes adsorption.

Background: Vitamin K deficiency of the newborn is preventable by routine administration of vitamin K1 post-partum. Although this practice is essentially successful, little is known about the efficacy of the dosage, the preferred route of administration and variations required for safe treatment of term and preterm neonates. Methods: We describe a method which has been successfully employed for the determination of the 5C- and 7C-metabolites of vitamin K in neonatal faecal matter by HPLC with redox-mode electrochemical detection. Results: Peaks attributable to the 5C- and 7C-metabolites of vitamin K were detected in neonatal faecal material post prophylaxis. Metabolite identity was confirmed by chromatographic retention time compared to pure standards and by LCMS. Vitamin K metabolite elimination curves were in line with that expected post vitamin K1 administration. Conclusion: The method described is suitable for investigation of vitamin K metabolism post vitamin K prophylaxis in term and preterm neonates.

Background: Current liquid chromatography instruments may yield two-way signals with full spectra collected as a function of time. These signals can be processed to recover chromatographic peaks in case of low resolution in the time order without any peak shape assumption. In Part I of this work, a two-way chromatographic objective function (COF) that incorporates both time and spectral information using diode array detection, based on the multivariate selectivity concept (figure of merit derived from the net analyte signal), was reported. The approach was applied to improve the chromatographic analysis of a mixture of 25 phenolic compounds with appreciable spectral differences, which remained unresolved in the time order. Objective: Part 2 of this work is aimed to illustrate the usefulness of the two-way COF to reduce the analysis time for compounds exhibiting small spectral differences. Method: The COF locates experimental conditions where the spectrochromatograms can be recovered with low error, using the combination of the orthogonal projection approach and alternating least squares. Results: The probe compounds (19 o-phthalaldehyde/N-acetylcysteine amino acid derivatives) were satisfactorily resolved using multi-linear and multi-isocratic gradients of acetonitrile-water, but at too long analysis times. By increasing the elution strength, the analysis time decreased, but producing overlapped peaks. The approach found the experimental conditions where the information for these peaks could be accurately recovered based on their spectral information. Conclusion: This demonstrates that the developed two-way COF succeeds to find convenient separation conditions for mixtures containing overlapped peaks for compounds exhibiting small spectral differences.

Background: Expanded Bed Adsorption Chromatography (EBAC) has emerged as a powerful technique in downstream processing mainly for avoiding the need for a clarification step such as filtration or centrifugation, during the initial capture of the target molecule, thus reducing both the step numbers and overall costs as well as increasing productivity. The use of models to represent the EBAC systems is crucial during the process scale-up. Among the many models used to model chromatographic systems it can be cited the phenomenological general rate model (white-box) as the blackbox models as for instance Artificial Neural Network (ANN). Methods: The goal of this work is to use the general rate model (porous diffusion model) to predict both flow-through as well as washing steps and to use the ANN to predict the chromatogram profile during chitosanase purification protocol using EBAC. Results: Using the general rate model it was observed that superficial velocity has a major influence on the breakthrough curve compared to axial dispersion as well as the initial enzymatic concentration. Also, change on the effective diffusivity and axial solid coefficient did not change the breakthrough curve profile. A backpropagation-ANN with twelve at hidden layer adjusted the three steps quite well. Conclusion: Phenomenological models such as the porous diffusion here used depend on parameter estimation. This can be a quite challenge for EBAC since a lot of parameters are involved therefore learning network such as ANN can stand out.

Background: Chiral selectors play a vital role in the separation efficiency for chiral separation techniques. Recently much attention has been devoted to chiral ionic liquids (CILs) derived from natural sources, which could be used as chiral selectors in chiral discrimination. Objective: In this study, chiral ionic liquids with L-phenylalanine ester as cations (L-phenylalanine ethyl ester bis (trifluoromethanesulphonyl) imide) were synthesized and applied as novel chiral ligand for chiral separation. Method: Ligand exchange chromatography with L-phenylalanine ethyl ester bis (trifluoromethanesulphonyl) imide as chiral selector was used to separate ofloxacin enantiomers. The effects of copper ion concentration, Lphenylalanine ethyl ester bis(trifluoromethanesulphonyl)imide concentration, organic modifier ratio, pH of the mobile phase as well as temperature on efficiency of chiral separation were investigated and evaluated. Results: Optimal separation conditions were obtained, where 8 mmol/L this chiral ionic liquids and 4 mmol/L Cu2 were dissolved in methanol/water (12:88, v/v) with pH 4.8. Under the optimum conditions, well enantioseparation of ofloxacin enantiomers could be observed with the resolution of 1.84. Conclusion: The results demonstrated the good applicability of the chiral ionic liquids with amino acid ester as cations to separate ofloxacin enantiomers in ligand exchange chromatography. It also exhibited the potential for the separation and determination of other chiral medicines by an ionic liquid-assisted ligand-exchange method, and would extend the application of chiral ionic liquids in separation science.

Background: Presence of toxic chemicals in daily diet is one of the major challenges before mankind today, especially a diet like milk, which not only is the main food for infants and children, but also is amongst the most consumed foods of animal origin by adults. The presence of various persistent and toxic pollutants like polychlorinated biphenyls (PCBs) in milk is a well-known fact. However, the analysis of milk for monitoring of its quality is a tedious job due to the current complex methods of analysis. The current methods are time consuming multistep methods hence chances of introduction of error in analysing trace level pollutants in the environmental matrices are more. To overcome this difficulty, a new methodology was developed. Methods: This new methodology is a simple and rapid method based on the solvent-less headspace-solid phase micro extraction (HS-SPME) technique followed by gas chromatography with electron-capture detection (GC-ECD) and was developed for the simultaneous determination of 14 congeners of PCBs in packaged milk samples. Several experiments were performed to assess the influence of various factors on SPME procedure and to determine the optimal extraction conditions. The final conditions were optimized with PDMS/DVB/Car fibre in headspace mode SPME with vigorous stirring at 100°C and exposure time of 50 minutes. The analysis was carried out in a GC-ECD while the confirmation of sample peaks was done in GC-MS. Results: During standardisation of the method, the fiber with coating of PDMS/DVB/Car was found to give maximum response, hence was used for all further analyses. Limits of detection reaching pg/ml were obtained using this method. When compared with the general LLE method of extraction, for spiked and non-spiked samples, the results obtained were in good agreement with each other. Additionally, the method was found to be reproducible and very efficient for trace level detection of PCBs contamination in Milk. Conclusion: As compared to the conventional methods, this method is fast, accurate, reproducible, easy, nearly interference free, hence does not require additional clean up and evaporative pre-concentration steps thus reducing possibilities of error and generation of large amounts of waste. This method can be used for quick and efficient ultra-trace level analysis of chlorinated pollutants like the PCBs.