Current Chromatography - Volume 4, Issue 2, 2017

Background: Cyanobacteria are a widely distributed group of photosynthetic prokaryotes and are considered relevant contributors for the formation of the Earth's atmosphere. Many of them produce toxins as secondary metabolites (cyanotoxins) that include a wide range of hepatotoxins (microcystins, nodularin, cilindrospermopsin), neurotoxins (anatoxins, saxitoxins and β- metilamino-L-alanine) and dermatotoxins (lyngbyatoxins, aplysyatoxin). In recent years, the occurrence of harmful cyanobacteria blooms (CyanoHABs) related to human-driven environmental factors has dramatically increased becoming a growing concern for the environment and public health. Despite the many applications used in routine investigations, cyanotoxins analysis remains challenging due to the number and diverse compounds involved. Several innovative methods based on biological and physico-chemical approaches are being developed to identify and quantify the toxins as well as the responsible harmful strains. Objective: Chromatographic methods with their separation capabilities are a powerful investigation tool, despite the huge number of molecules that need to be determined for many toxin types. HPLC is the most widely used separation method for cyanotoxins; it is highly sensitive, rapid and adaptable to a wide range of detectors based on UV absorbance, fluorescence or mass spectrometry. On the other hand, gas chromatography has been scarcely applied for toxin determination due to the requirement of high molecular weight molecules and the need for derivatization. Even more articles and applications are being published: the objective of this review is to give a synthetic and comprehensive view of the separation methods used in this field and their advances in the past years. Method: Chromatographic methods have been reviewed from 1990-2000 to 2017, with special attention to multitoxin applications, including additional techniques like MALDI-TOF mass spectrometry and capillary electrophoresis. Results: In this paper, a review of different chromatographic applications for cyanotoxins analysis is presented together with the sample preparation methods most widely used. Multitoxin applications are highlighted. Especial attention is given to the most recent advances in HPLC-MS, HPLC tandem-MS and high resolution MS techniques. Some capillary electrophoresis applications are described as well. Conclusion: Recent advances in chromatography include liquid chromatography and/or mass spectrometric investigations for characterization and determination in lake and river waters of different cyanobacterial toxins (microcystins, anatoxin-A, PSP toxins and related metabolites) simultaneously and new molecules like oligopeptides and novel secondary metabolites (anabaenopeptins, aeruginosins, microginins, microviridins and cyanopeptolins).

Background: Identification of monomeric and oligomeric organic compounds in Organosolv process wastewater is of growing importance in the context of isolating high value chemicals and producing biofuels. There were many studies devoted to identification of such compounds in precipitated Organosolv lignin, all of them using demanding, mostly hyphenated techniques such as HPLC-MS/MS and two dimensional NMR spectroscopy. However, the resulting Organosolv wastewater has not been studied as intensively in this respect, even though the application of these techniques to identify the components of dried/lyophilized Organosolv wastewater would be straightforward. Objective: The objective was to identify lignin-based breakdown products in Organosolv wastewater resulting from pulping of beech with aqueous ethanol solutions. Method: Low resolution GC/MS analysis in combination with derivatization reactions using nonlabeled and isotopically labeled derivatization agents and retention index information (where available) were used. Results: The application of isotopically labeled derivatization agents such as BSTFA-d9 (N,Obis( trimethylsilyl)trifluoracetamide-d9) turned out especially useful in structural identification work. In addition to monomers such as abundant syringaldehyde and vanillin, a multitude of dimeric products of the guaiacyl(syringyl) glycerol-β-guaiacyl(syringyl) ether-type, phenylcoumaran type and lignan-type could be detected, the most abundant surrogate being syringaresinol. Syringyl-type surrogates were more abundant compared to guaiacyl-type surrogates. Formation pathways of the monomeric breakdown products such as syringaldehyde and vanillin were proposed on the basis of the solvolytic cleavage of β-O-4 aryl ether linkages. Conclusion: Solvolytic cleavage of β(α)-O-4, β-5 and β-β linkages within the macromolecular lignin network under Organosolv process conditions results in dimeric products. Further activities should be focused on the use of high resolution MS and the synthesis of authentic standards.

Background: The disadvantages of the classical approach used to characterize column performance in liquid chromatography, based on the observation of Van Deemter plots, are the need of measuring the extra-column variance, the use of data from only one compound eluted with a single mobile phase, which limits the conclusions of the approach, and the uncertainty arising from the handling of position and variance of chromatographic peaks to obtain the theoretical plate height. Objective: In this work, a comprehensive study of the factors affecting the uncertainty of the chromatographic data used to build Van Deemter plots is carried out, and three complementary approaches are proposed to avoid the described disadvantages. Method: One of the approaches is based on the direct treatment of the peak variances of several probe compounds according to a linear relationship of the variance with the squared retention volume, which includes the extra-column variance as a fitting parameter. In a second approach, the peak variance is fitted, for each probe compound, against the flow rate, and the model parameters are further related to the retention factors for the set of compounds. The third approach is based on the combination of both above approaches, and allows the data treatment in a single step. Results: The three approaches were applied with good results to the characterization of three C18 columns (Zorbax Eclipse XDB, Spherisorb and Chromolith SpeedROD), using a set of five sulphonamides as probe compounds. In all cases, the prediction errors for the variance were highly satisfactory, being in the 2-4% range. Conclusion: The three approaches are complementary, and allow a complete visualization of the chromatographic performance, giving information on how the retention factor affects the A, B and C parameters in the Van Deemter equation.

Background: A common problem in HPLC is the inability to inject a sufficient volume of sample, particularly when the injection solvent (or diluent) is chromatographically strong. Experiments were conducted to evaluate the effectiveness of low-retention pre-columns in allowing larger injection volumes of chromatographically strong solvents. Method: Different combinations of pre-columns and analytical columns were evaluated with respect to their effectiveness in this regard. In addition, the effect of pre-column length, mobile phase conditions, and different injection solvents were evaluated with respect to the maximum injection volume that was possible. Lastly, experiments were conducted to evaluate the additional benefit of placing a mixer upstream of the pre-column. Results: It was demonstrated that significant increases in injection volume were possible by utilizing low-retention pre-columns, which allowed enhanced method sensitivity. Furthermore, it was found that solvents with very limited solubility in the mobile phase, such as ethyl acetate and MTBE, could be used as the sample solvent; and, in fact, the largest injection volumes were possible with these limited-solubility solvents when using the pre-column technique. Conclusion: The general approach was found to be effective in allowing larger injection volumes of chromatographically strong solvents. Furthermore, the focusing in this approach reduces band broadening due to all of the extra-column effects which act upstream of the analytical column. From these observations, it is clear that this technique could significantly reduce some of the difficulties associated with use of smaller diameter columns. Consequently, the use of pre-columns would also enable analytical laboratories to operate in a greener fashion by minimizing the quantity of waste that is generated.