Reprint of: Application of micro-thin-layer chromatography as a simple fractionation tool for fast screening of raw extracts derived from complex biological,pharmaceutical and environmental samples

The main goal of present paper is to demonstrate the separation and detection capability of micro-TLC technique involving simple one step liquid extraction protocols of complex materials without multi-steps sample pre-purification. In the present studies target components (cyanobacteria pigments, lipids and fullerenes) were isolated from heavy loading complex matrices including spirulina dried cells, birds’ feathers and fatty oils as well as soot samples derived from biomass fuel and fossils-fired home heating systems. In each case isocratic separation protocol involving less that 1 mL of one component or binary mixture mobile phases can be completed within time of 5–8 min. Sensitive detection of components of interest was performed via fluorescence or staining techniques using iodine or phosphomolybdic acid. Described methodology can be applied for fast fractionation or screening of whole range of target substances as well as chemo-taxonomic studies and fingerprinting of complex mixtures, which are present in raw biological or environmental samples.     

Micro-TLC Approach for Fast Screening of Environmental Samples Derived from Surface and Sewage Waters

In this work we demonstrated analytical capability of micro-planar (micro-TLC) technique comprising one and two-dimensional (2D) separation modes to generate fingerprints of environmental samples originated from sewage and ecosystems waters. We showed that elaborated separation and detection protocols are complementary to previously invented HPLC method based on temperature-dependent inclusion chromatography and UV-DAD detection. Presented 1D and 2D micro-TLC chromatograms of SPE (solid-phase extraction) extracts were optimized for fast and low-cost screening of water samples collected from lakes and rivers located in the area of Middle Pomerania in northern part of Poland. Moreover, we studied highly organic compounds loaded in the treated and untreated sewage waters obtained from municipal wastewater treatment plant “Jamno” near Koszalin City (Poland). Analyzed environmental samples contained number of substances characterized by polarity range from estetrol to progesterone as well as chlorophyll-related dyes previously isolated and pre-purified by simple SPE protocol involving C18 cartridges. Optimization of micro-TLC separation and quantification protocols of such samples were discussed from the practical point of view using simple separation efficiency criteria including total peaks number, log(product ΔhR _F), signal intensity and peak asymmetry. Outcomes of the presented analytical approach, especially using detection involving direct fluorescence (UV366/Vis) and phosphomolybdic acid (PMA) visualization are compared with UV-DAD HPLC-generated data reported previously. Chemometric investigation based on principal components analysis revealed that SPE extracts separated by micro-TLC and detected under fluorescence and PMA visualization modes can be used for robust sample fingerprinting even after long-term storage of the extracts (up to 4 years) at subambient temperature (?20 °C). Such approach allows characterization of wide range of sample components that are present in given extract in high and middle concentration range. Due to protocol simplicity and low cost of analysis this method can be useful for preliminary sample screening.     

Low-parachor solvents extraction and thermostated micro-thin-layer chromatography separation for fast screening and classification of spirulina from pharmaceutical formulations and food samples

The goal of this paper is to demonstrate the separation and detection capability of eco-friendly micro-TLC technique for the classification of spirulina and selected herbs from pharmaceutical and food products. Target compounds were extracted using relatively low-parachor liquids. A number of the spirulina samples which originated from pharmaceutical formulations and food products, were isolated using a simple one step extraction with small volume of methanol, acetone or tetrahydrofuran. Herb samples rich in chlorophyll dyes were analyzed as reference materials. Quantitative data derived from micro-plates under visible light conditions and after iodine staining were explored using chemometrics tools including cluster analysis and principal components analysis. Using this method we could easily distinguish genuine spirulina and non-spirulina samples as well as fresh from expired commercial products and furthermore, we could identify some biodegradation peaks appearing on micro-TLC profiles. This methodology can be applied as a fast screening or fingerprinting tool for the classification of genuine spirulina and herb samples and in particular may be used commercially for the rapid quality control screening of products. Furthermore, this approach allows low-cost fractionation of target substances including cyanobacteria pigments in raw biological or environmental samples for preliminary chemotaxonomic investigations. Due to the low consumption of the mobile phase (usually less than 1 mL per run), this method can be considered as environmentally friendly analytical tool, which may be an alternative for fingerprinting protocols based on HPLC machines and simple separation systems involving planar micro-fluidic or micro-chip devices.     

Separation and characterization of nanoparticles in complex food and environmental samples by field-flow fractionation

The thorough analysis of natural nanoparticles (NPs) and engineered NPs involves the sequence of detection, identification, quantification and, if possible, detailed characterization. In a complex or heterogeneous sample, each step of this sequence is an individual challenge, and, given suitable sample preparation, field-flow fractionation (FFF) is one of the most promising techniques to achieve relevant characterization.The objective of this review is to present the current status of FFF as an analytical separation technique for the study of NPs in complex food and environmental samples. FFF has been applied for separation of various types of NP (e.g., organic macromolecules, and carbonaceous or inorganic NPs) in different types of media (e.g., natural waters, soil extracts or food samples).FFF can be coupled to different types of detectors that offer additional information and specificity, and the determination of size-dependent properties typically inaccessible to other techniques. The separation conditions need to be carefully adapted to account for specific particle properties, so quantitative analysis of heterogeneous or complex samples is difficult as soon as matrix constituents in the samples require contradictory separation conditions. The potential of FFF analysis should always be evaluated bearing in mind the impact of the necessary sample preparation, the information that can be retrieved from the chosen detection systems and the influence of the chosen separation conditions on all types of NP in the sample. A holistic methodological approach is preferable to a technique-focused one.     

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time III. Improving the accuracy of computer simulation.

Previous studies have shown that four experimental runs, where both temperature T and gradient time tG are varied, can be used for the reliable prediction of separation as a function of these two variables (two-dimensional optimization). Computer simulation (e.g., DryLab) can then be used to predict "optimized" conditions for maximum sample resolution using either isocratic or gradient elution. Samples that contain a large number of components (e.g., n>15-20) present a greater challenge. Resolution for these more complex samples is often quite sensitive to small changes in T or tG in turn requiring greater accuracy in predictions that result from computer simulation. In the present study of several samples, we have examined computer simulation errors that can arise from inexact expressions for retention time as a function of T, tG or isocratic %B. Resulting conclusions are applicable to both complex and simpler samples, in either one- or two-dimensional optimization. Means to anticipate and minimize the impact of these predictive errors are examined.     

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time II. Two-run assay procedures.

By optimizing column temperature T and gradient time tG, complex samples can often be separated by means of reversed-phase high-performance liquid chromatography (RP-LC). Conclusions reached in Part I suggest that the complete separation of such samples will be difficult, however, when more than 15-20 components are present in the sample. An alternative approach is to carry out two separations with different conditions (T, tG) in each run. The combination of results from these two runs then allows the total analysis of the sample, providing that every sample component is adequately resolved in one run or the other. Examples of this approach, carried out by means of computer simulation, are shown here for several samples of varying complexity. Also considered is the ability of a single separation where T and tG are optimized to enable the separation and analysis of one or more individual sample components from complex mixtures (e.g., drugs in animal plasma), including the resolution of isomeric compounds from each other.     

Evaluation of signal and noise and identification of a suitable target function in the tuning of an ESI ion trap mass spectrometer by multivariate pattern recognition tools

When mass spectrometry is not combined to separation techniques, the evaluation of signal and noise in a complex mass spectrum is not trivial. The tuning of the spectrometer based only on the increase of the signal of a selected number of m/z values does not ensure the achievement of the best experimental conditions: signal could improve and noise could increase as well. The scope of this work is the development of a function separating signal and noise (for evaluating the S/N) from complex mass spectra for potential use as target function for the automatic tuning of the instrument. Two different methods were applied: the first is based on the separation of a pool of m/z values attributable to the signal from the m/z values due to the noise, while the second is based on the application of principal component analysis to separate the signal (present in the significant components) from the noise (present in the residuals). The comparison of the two methods was carried out by the evaluation of the stability of the signal and the target functions obtained, and the evaluation of the variation of the target functions as a function of concentration.     

Analysis of octopamine in human doping control samples

The biogenic amine octopamine [4‐(2‐amino‐1‐hydroxyethyl)phenol] is prohibited in sports owing to its stimulating and performance‐enhancing properties. Adverse analytical findings in athletes' doping control samples commonly result from surreptitious applications; however, the occurrence of octopamine in nutritional supplements and in selected invertebrates as well as the assumption that its N‐methylated analog synephrine [4‐(1‐hydroxyethyl‐2‐methylamino)phenol, not banned by anti‐doping authorities but currently monitored in prevalence studies) might be converted in‐vivo into octopamine have necessitated a study to investigate the elimination of synephrine and octopamine present in over‐the‐counter products. Urine samples collected after administration of nutritional supplements containing octopamine and/or synephrine as well as urine samples collected after therapeutic application of octopamine‐ or synephrine‐containing drugs were analyzed using a validated solid‐phase extraction‐based procedure employing a weak cation exchange resin and liquid chromatographic/tandem mass spectrometric detection with electrospray ionization and multiple reaction monitoring. In the case of therapeutic octopamine application, the urinary concentration of the target compound increased from baseline levels below the lower limit of detection to 142 µg/mL, while urine samples collected after synephrine as well as dietary supplement administration did not yield any evidence for elevated renal excretion of octopamine. Copyright © 2011 John Wiley & Sons, Ltd.     

Fractionation of bacteria by electrophoresis as pre-separation method before MALDI-MS detection

The present study attempted to apply the capillary electrophoresis technique for the fractionation and separation of S. Staphylococcus hominis and Escherichia coli bacteria isolated from urine samples and the detection of migrated fraction with spectrometric method. This involved the selection of suitable conditions for separation as well as the identification of pathogens. The result of the research was the separation of Gram-negative and Gram-positive bacteria, as well as their subsequent identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using two different approaches (culture of fractions on an agar plate and direct analysis of the collected fractions). The preliminary results provide a solid basis for further research on the use of electromigration techniques with LDI detection to identify pathogens such as bacteria and viruses in biological samples.     

Highly sensitive determination of haloperidol in human serum by capillary gas chromatography using a surface ionization detector

A method has been developed for highly sensitive determination of haloperidol in human serum involving a simple extraction procedure followed by gas chromatographic separation. Target components were separated from the extracting solvents with a Van den Berg type solventless sample injector before introduction Into a DB-1 capillary separation column. A surface ionization detector (SID), which has highly selective sensitivity for Substituted amines, was employed for quantitation using bromperidol as an internal standard. Chloroform proved to be the best extracting solvent, yielding a quantitative detection limit of 5 ng/ml (S/N = 2). Comparison of the response to target compounds obtained by the SID, FTD (flame thermionic detector), and FID (flame ionization detector) showed the SID to be superior.     

Enhancement in MS-based peptide detection by microfluidic free-flow zone electrophoresis

Matrix components are known to significantly alter the ionization of a target analyte in ESI-based measurements particularly when working with complex biological samples. This issue however may be alleviated by extracting the analyte of interest from the original sample into a relatively simple matrix compatible with ESI mass-spectrometric analysis. In this article, we report a microfluidic device that enables such extraction of small peptide molecules into an ESI-compatible solvent stream significantly improving both the sensitivity and reproducibility of the measurements. The reported device realizes this analyte extraction capability based on the free-flow zone electrophoretic fractionation process using a set of internal electrodes placed across the width of the analysis channel. Employing lateral electric fields and separation distances of 75 V/cm and 600 µm, respectively, efficient extraction of the model peptide human angiotensin II was demonstrated allowing a reduction in its detection limit by one to three orders of magnitude using the ESI-MS method. The noted result was obtained in our experiments both for a relatively simple specimen comprising DNA strands and angiotensin II as well as for human serum samples spiked with the same model peptide.     

CE separation of various analytes of biological origin using polyether ether ketone capillaries and contactless conductivity detection

The development of efficient and sensitive analytical methods for the separation, identification and quantification of complex biological samples is continuously a topic of high interest in biological science. In the present study, the possibility of using a polyether ether ketone (PEEK) capillary for the CE separation of peptides, proteins and other biological samples was examined. The performance of the tubing was compared with that of traditional silica capillaries. The CE analysis was performed using contactless conductivity detection (C⁴D), which eliminated any need for the detection window and was suitable for the detection of optically inactive compounds. In the PEEK capillary the cathodic EOF was low and of excellent stability even at extremes pH. In view of this fast biological anions were analyzed using an opposite end injection technique without compromising separation. A comparison of the performances of fused‐silica and polymer capillaries during the separation of model sample mixtures demonstrated the efficiency and separation resolution of the latter to be higher and the reproducibility of the migration times and peak areas is better. Furthermore, PEEK capillaries allowed using simple experimental conditions without any complicated modification of the capillary surface or use of an intricate buffer composition. The PEEK capillaries are considered as an attractive alternative to the traditional fused‐silica capillaries and may be used for the analysis of complex biological mixtures as well as for developing portable devices.     

Determination of unconjugated aromatic acids in urine by capillary electrophoresis with dual electrochemical detection – Potential application in fast diagnosis of phenylketonuria

A novel method of CE coupled with dual electrochemical detection has been developed for the determination of pathological metabolites of phenylalanine in urine samples. Factors influencing the separation and detection were examined and optimized. Five aromatic acid metabolites and a major coexisting interfering compound uric acid could be well separated within 23 min at a separation voltage of 16 kV using a 35 mmol/L SDS/60 mmol/L H₃BO₃‐Na₂B₄O₇ running buffer (pH 8.2). Highly linear response was obtained for these five biomarker compounds over three orders of magnitude with detection limits ranging from 6.6 to 0.064 μg/mL (S/N=3). The average recovery and RSD were within the range of 92.6–121.0 and 1.0–12.0%, respectively. The proposed method has been used to detect the unconjugated aromatic acids simultaneously in urine samples with the advantages of obtaining more information about target analytes and avoiding redundant measurements and high assay cost, thus could find potential applications involving assays of biomarker compounds for the purpose of fast diagnose of some metabolic diseases including phenylketonuria.     

Inertial focusing of microparticles,bacteria, and blood in serpentine glass channels

Early detection of pathogenic microorganisms is pivotal to diagnosis and prevention of health and safety crises. Standard methods for pathogen detection often rely on lengthy culturing procedures, confirmed by biochemical assays, leading to >24 h for a diagnosis. The main challenge for pathogen detection is their low concentration within complex matrices. Detection of blood-borne pathogens via techniques such as PCR requires an initial positive blood culture and removal of inhibitory blood components, reducing its potential as a diagnostic tool. Among different label-free microfluidic techniques, inertial focusing on microscale channels holds great promise for automation, parallelization, and passive continuous separation of particles and cells. This work presents inertial microfluidic manipulation of small particles and cells (1–10 μm) in curved serpentine glass channels etched at different depths (deep and shallow designs) that can be exploited for (1) bacteria preconcentration from biological samples and (2) bacteria-blood cell separation. In our shallow device, the ability to focus Escherichia coli into the channel side streams with high recovery (89% at 2.2× preconcentration factor) could be applied for bacteria preconcentration in urine for diagnosis of urinary tract infections. Relying on differential equilibrium positions of red blood cells and E. coli inside the deep device, 97% red blood cells were depleted from 1:50 diluted blood with 54% E. coli recovered at a throughput of 0.7 mL/min. Parallelization of such devices could process relevant volumes of 7 mL whole blood in 10 min, allowing faster sample preparation for downstream molecular diagnostics of bacteria present in bloodstream.     

Ultrasensitive chemiluminescent immunochemical identification and localization of protein components in painting cross-sections by microscope low-light imaging

The characterization and localization of proteins and other organic components in the complex stratigraphy of paintings is crucial for authentication and studies of painting techniques. With this aim we have developed a new ultrasensitive immunochemical procedure for the detection of the protein ovalbumin (chicken egg white albumin), present in binding media or varnishes, in painting cross-sections. The technique is based on chemiluminescence imaging detection combined with optical microscopy, and allowed the sensitive localization of the target protein in cross-sections with high spatial resolution. In order to evaluate its performance, the method was first applied to standard samples (also containing different common pigments), then used for the localization of ovalbumin in samples obtained from a Renaissance wood painting.     

Comparing chemical fingerprints of herbal medicines using modified window target-testing factor analysis

A chromatographic fingerprint of a herbal medicine is essentially its chromatographic spectrum: a characteristic representation of its chemical components, some of which are pharmacologically active. Since a wide variety of factors, such as the geographical location, the harvest season, and the part used can influence the chemical constituents (and therefore the pharmacological activity) of any particular herbal medicine and its products, these fingerprints provide a way to compare and contrast the compositions of different variants of the same herbal medicine. In particular, it is possible to ascertain whether particular components present in one herbal fingerprint are also present in another fingerprint. In this work we use a novel method—modified window target-testing factor analysis (MWTTFA), based on the use of target factor analysis (TFA), fixed-size moving window evolving factor analysis (FSMWEFA) and a Gaussian shape correction to the chromatographic profiles—to achieve this end. To demostrate the strategy, the fingerprints of samples from garlics produced in different geographical locations were compared, as well as the fingerprints of samples taken from above-ground and below-ground parts of Houttuynia cordata Thunb. The results from these comparisons clearly show that four chemical components present in Hunan common edible garlic are absent in Xingping base garlic, while seven components are present in Xingping base garlic but absent in Hunan common edible garlic. Also, eleven components are present in the sample from the above-ground part of Houttuynia cordata Thunb but not in the sample from the below-ground part, while seven components are present in the sample from the below-ground part of Houttuynia cordata Thunb that are not present in the sample from the above-ground part. These interesting conclusions should be very useful for future pharmacological and clinical research into these herbal medicines, and the novel MWTTFA technique can also be used for quality control purposes.     

Classification of highly similar crude oils using data sets from comprehensive two-dimensional gas chromatography and multivariate techniques

Comprehensive two-dimensional gas chromatography (GCxGC) has proven to be an extremely powerful separation technique for the analysis of complex volatile mixtures. This separation power can be used to discriminate between highly similar samples. In this article we will describe the use of GCxGC for the discrimination of crude oils from different reservoirs within one oil field. These highly complex chromatograms contain about 6000 individual, quantified components. Unfortunately, small differences in most of these 6000 components characterize the difference between these reservoirs. For this reason, multivariate-analysis (MVA) techniques are required for finding chemical profiles describing the differences between the reservoirs. Unfortunately, such methods cannot discern between 'informative variables', or peaks describing differences between samples, and 'uninformative variables', or peaks not describing relevant differences. For this reason, variable selection techniques are required. A selection based on information between duplicate measurements was used. With this information, 292 peaks were used for building a discrimination model. Validation was performed using the ratio of the sum of distances between groups and the sum of distances within groups. This step resulted in the detection of an outlier, which could be traced to a production problem, which could be explained retrospectively.     

Targeted three-dimensional liquid chromatography: a versatile tool for quantitative trace analysis in complex matrices

Targeted multidimensional liquid chromatography (MDLC), commonly referred to as 'coupled-column' or 'heartcutting', has been used extensively since the 1970s for analysis of low concentration constituents in complex biological and environmental samples. A primary benefit of adding additional dimensions of separation to conventional HPLC separations is that the additional resolving power provided by the added dimensions can greatly simplify method development for complex samples. Despite the long history of targeted MDLC, nearly all published reports involve two-dimensional methods, and very few have explored the benefits of adding a third dimension of separation. In this work we capitalize on recent advances in reversed-phase HPLC to construct a three-dimensional HPLC system for targeted analysis built on three very different reversed-phase columns. Using statistical peak overlap theory and one of the most recent models of reversed-phase selectivity we use simulations to show the potential benefit of adding a third dimension to a MDLC system. We then demonstrate this advantage experimentally by developing targeted methods for the analysis of a variety of broadly relevant molecules in different sample matrices including urban wastewater treatment effluent, human urine, and river water. We find in each case that excellent separations of the target compounds from the sample matrix are obtained using one set of very similar separation conditions for all of the target compound/sample matrix combinations, thereby significantly reducing the normally tedious method development process. A rigorous quantitative comparison of this approach to conventional 1DLC-MS/MS also shows that targeted 3DLC with UV detection is quantitatively accurate for the target compounds studied, with method detection limits in the low parts-per-trillion range of concentrations. We believe this work represents a first step toward the development of a targeted 3D analysis system that will be more effective than previous 2D separations as a tool for the rapid development of robust methods for quantitation of low concentration constituents in complex mixtures.     

LC-amperometric determination of tert-butylcatechol in N-methyl-2-pyrrolidone stream containing polar and nonpolar impurities

Amperometric detection is shown to be highly selective and sensitive for the determination of tert-butylcatechol (TBC) added as an antioxidant in the circulating N-methyl-2-pyrrolidone (NMP) used for the purification of 1,3-butadiene by extractive distillation. The separation of TBC from numerous polar and nonpolar species present in the analyte is achieved on a 5-cm reversed-phase C-18 column using acetonitrile-water gradient conditions, both solvent components containing tetrabutyl ammonium dihydrogen phosphate as the amphiphilic modifier. The separation process is optimized for the resolution of TBC from nonpolar hydrocarbons as well as polar compounds, and a complete elution of all species from the column. An applied potential of 0.6 V relative to the Ag/AgCI electrode is found to provide an optimum sensitivity by the amperometric detector. It is also shown that the hydrocarbon content in NMP can be monitored simultaneously by coupling a spectrophotometric detector in series.     

Purification of lignans from Fructus Arctii using off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography

As a common traditional Chinese medicine, Fructus Arctii has important clinical medical values. Its main components are lignans, which are difficult to separate and analyze because of the complex composition, similar chemical structures, and close properties. In this study, an off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography method, as well as an effective sample pretreatment method based on hydrophilic interaction chromatography material, was developed to enrich the minor lignan fractions and obtain high‐purity compounds. In total, 12 high‐purity compounds were isolated from Fructus Arctii . Their structures were identified by using high‐resolution mass spectrometry and nuclear magnetic resonance spectroscopy, which showed that all were lignans and that most of them were isomers. The results demonstrated the effective off‐line two‐dimensional supercritical fluid chromatography/reversed‐phase liquid chromatography method for the purification of lignans from Fructus Arctii . The separation protocol established here will be beneficial for the separation of complex samples from other kinds of natural products.