Erwin Kaal

Extending the molecular application range of gas chromatography

Gas chromatography is an important analytical technique for qualitative and quantitative analysis in a wide range of application areas. It is fast, provides a high peak capacity, is sensitive and allows combination with a wide range of selective detection methods including mass spectrometry. However, the application area of GC is limited because the molecules to be analysed have to be thermally stable and sufficiently volatile. Numerous molecules do not meet these requirements and hence are not amenable to direct GC analysis. Recent research has resulted in better chromatographic columns and methods for sample preparation that enable a significant expansion of the molecular application range of GC. The strategies exploited include conversion of (macro)molecules into smaller species and approaches to reduce the polarity of molecules. In this review we identify four generic routes for extending the applicability of GC. These include high-temperature GC, derivatisation, pyrolysis and thermochemolysis. The principles, recent developments and future perspectives of these routes are discussed and examples of applications using the different options will be shown. Life sciences, metabonomics and profiling strategies for sample characterization are identified as important future drivers for the continued development of GC.

A fast method for the identification of Mycobacterium tuberculosis in sputum and cultures based on thermally assisted hydrolysis and methylation followed by gas chromatography–mass spectrometry

A fast gas chromatography-mass spectrometry (GC-MS) method with minimum sample preparation is described for early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are then methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM). The THM-GC-MS procedure was optimized for the injection of sputum samples. For the identification of Mycobacterium tuberculosis the known marker tuberculostearic acid (TBSA) and other potential markers were evaluated. Hexacosanoic acid in combination with TBSA was found to be specific for the presence of M. tuberculosis. For validation of the method several sputum samples with different viscosities spiked with bacterial cultures were analyzed. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed in Amsterdam by microscopy after decontamination/concentration and using the new THM-GC-MS method. No false positives were found by THM-GC-MS and all patients who were diagnosed with TB were also found positive using our newly developed THM-GC-MS method. These results show that the new fast and sensitive THM-GC-MS method holds great potential for the diagnosis of TB.

Fully automated system for the gas chromatographic characterization of polar biopolymers based on thermally assisted hydrolysis and methylation

Pyrolysis-gas chromatography (Py-GC) is a powerful tool for the detailed compositional analysis of polymers. A major problem of Py-GC is that polar (bio)polymers yield polar pyrolyzates which are not easily accessible to further GC characterization. In the present work, a newly developed fully automated procedure for thermally assisted hydrolysis and methylation (THM) of biopolymers is described. Drying of the sample, addition of the reagent, incubation and pyrolysis are performed inside the liner of a programmable temperature vaporizer injector. The new system not only allows efficient analysis of large series of samples, but also allows automated optimization of the experimental parameters based on an experimental design approach. The performance of the automated THM-procedure was evaluated by performing THM-GC of a poly(acrylic acid)-poly(maleic anhydride) copolymer (PAA/PMAH) and several polysaccharides. The optimized THM-procedure was applied for the structural characterization and differentiation of several lignins and hydroxypropylmethyl-celluloses. It was also applied to proteins. Here myoglobin and cytochrome c were used as the model compounds. Both conventional GC-mass spectrometry (MS) and comprehensive two-dimensional gas chromatography (GCxGC)-time-of-flight (TOF) MS were used for separation and identification of the species formed. The information obtained can aid in structure elucidation of polar biopolymers as well as in providing detailed compositional information which can be used to differentiate structurally similar biopolymers.

Aqueous size‐exclusion chromatographic separations of intact proteins under native conditions: Effect of pressure on selectivity and efficiency

The selectivity and separation efficiency of aqueous size-exclusion chromatographic separations of intact proteins were assessed for different flow rates, using columns packed with 3 and 5 μm silica particles containing 150 and 290 Å stagnant pores. A mixture of intact proteins with molecular weights ranging between 17 000 and 670 000 Da was used to construct the calibration curves. Both the model fit and the predictive properties, using a leave-one-out strategy, of different polynomial models (up to fifth order) were evaluated for different flow rates. The best compromise between model fit and predictive properties was obtained using a third-order polynomial model. The accuracy of the predictive properties decreased with 10% with an eightfold increase in the flow rate. No changes in retention factors (hence selectivity) were observed in the flow-rate range applied. A strong correlation between molecular weight and plate height was observed. Exclusion of large-molecular-weight proteins led to a significant reduction in the stationary-phase mass-transfer contribution to the total plate-height value, and this effect was also independent of the flow rate applied. The kinetic-performance limits, in terms of plate number and time, and optimal column-length particle-size combinations were determined at the maximum recommended operating pressure of the size-exclusion chromatography columns (20 MPa). Finally, the possibilities of method speed-up using ultra-high-pressure size-exclusion chromatography in combination with columns packed with sub-2 μm particles are discussed.

Determination of cholesterol and triglycerides in serum lipoproteins using flow field-flow fractionation coupled to gas chromatography-mass spectrometry

Asymmetric flow field flow fractionation (AsFlFFF) was combined with pyrolysis-gas chromatography mass spectrometry for a sized based fractionation and a detailed compositional study of the triglycerides and cholesterol associated with the various lipoprotein subclasses present in human serum. Serum samples were injected in the AsFlFFF instrument and fractionated with a time-delayed exponential decay cross flow program. The fractions collected after AsFlFFF elution were injected into a programmable temperature vaporizer (PTV) GC-injector, containing a fritted liner. A temperature and split-flow program for the PTV injector was optimized for the thermally assisted hydrolysis and methylation of the compounds of interest. The resulting fatty acid and cholesterol methyl esters were separated by GC and characteristic fragment ions were detected by MS. The system was optimized and calibrated with triglyceride and cholesterol standards for quantitative analysis. The possible interference by phospholipids with the quantitative results was investigated and found to be of minor importance. The concentrations and lipoprotein profiles of triglycerides and cholesterol were determined in a pooled serum sample of healthy volunteers and a serum sample of a sepsis patient. The results obtained with the GC-MS approach were compared with those of a previously developed method based on AsFlFFF with a dual enzymatic reaction detection system. A good agreement of the profiles was found, for cholesterol as well as for the triglycerides, even when the GC-MS method quantifies the fatty acids while with the enzymatic reaction method the glycerol concentrations are determined. Total cholesterol and triglyceride concentration values for the serum samples showed good agreement with the results of the standard enzymatic method as used in practice in the university hospital.

Comprehensive multidimensional systems incorporating GC×GC

Publisher Summary The chapter discusses the principles, difficulties, and applications of multidimensional systems incorporating a 2D gas chromatography (GCxGC) separation procedure. The chapter assesses the possibilities of incorporating GCxGC in a truly 3D comprehensive setup, with either GC or liquid chromatography (LC) in the first dimension, abbreviated as XCxGCxGC. The chapter discusses the practical aspects of GCxGCxGC, LCxGCxGC, and LC–GCxGC. Mass spectrometry (MS) can be used as a specific detector providing an additional dimension of information. Comprehensive 3D chromatography is very slow, so care should be taken not to transfer more fractions than necessary. Comprehensive 3D systems can provide the strongly desired match between sample dimensionality and system dimensionality. However, more work in different aspects of XC–GCxGC or XCxGCxGC is needed to fully reap the benefits of the higher-dimension comprehensive systems.

How can we improve ion-exchange separations in LC?

Ion-exchange chromatography (IEX) emerged as an analytical technique for the separation of ions and ionizable analytes in 1975, when Small et al. described a novel approach to suppress counter ions in the mobile phase prior to conductivity detection [1]. Nowadays, dedicated instrumentation is available and compatible with normal bore (4.6 mm i.d.) and capillary (400 μm i.d.) column formats. Whereas the technology was initially applied only for the analysis of small inorganic anions and cations, its scope has expanded and IC is now commonly applied to analyze a wide range of species, including organic acids and aliphatic amines, and a wide range of biomolecules including antibodies, intact proteins, peptides and carbohydrates, among others. To analyze these analytes in contemporary sample mixtures with a large dynamic range as encountered in emerging fields, such as biotechnology research and clinical diagnostics, the demands on separation efficiency have increased significantly [2,3]. The objective of this editorial is to provide a short overview of the IEX performance limits that can be achieved with current instrumentation and column technology, and to raise discussion on how to realize the next leap in performance optimization in IC. The resolution (R s ) of two Gaussian peaks separated in a chromatographic experiment depends on the plate number (N ), the retention factor of the most retained compound (k), and the selectivity factor (α), according to: To improve IEX separations, considerable effort has been spent on tuning α via optimization of the stationary-phase chemistry and architecture [4]. By tuning the stationary-phase surface of the resins and eluent conditions different IC mechanisms, that is, Coulomb interactions, ion exclusion and ion pairing, can be utilized to realize the desired critical-pair separation. Both silicaand polymer-based resins have been developed, the latter being stable at high pH mobile phases. An additional advantage of polymerbased resins is the better biocompatibility (e.g., no undesired interactions with residual silanol groups), and hence reduced carry over [5]. Outstanding overviews describing the latest stationary-phase developments to tune the selectivity can be found in the literature [4,6–8]. Whereas major improvements in N and analysis time have been realized in HPLC by the introduction of ultra-high-pressure systems in combination with columns packed with sub-2-μm particles, the developments in the field of IEX separations have lagged behind. Conventional columns are packed with particle diameters between 6 and 10 μm, and the column length varies typically between 150 and 250 mm, yielding approximately 13,000 plates in isocratic mode. Only recently columns packed with 4-μm particle technology have become commercially available. The separation efficiency in terms of How can we improve ion-exchange separations in LC?

Novel approaches in diagnosing tuberculosis

The WHO declared tuberculosis (TB) a global emergency. An estimated 8-9 million new cases occur each year with 2-3 million deaths. Currently, TB is diagnosed mostly by chest-X ray and staining of the mycobacteria in sputum with a detection limit of 1x104 bacteria /ml. There is an urgent need for better diagnostic tools for TB especially for developing countries. We have validated the electronic nose from TD Technology for the detection of Mycobacterium tuberculosis by headspace analysis of 284 sputum samples from TB patients. We used linear discriminant function analysis resulting in a sensitivity of 75% a specificity of 67% and an accuracy of 69%. Further research is still required to improve the results by choosing more selective sensors and sampling techniques. We used a fast gas chromatography- mass spectrometry method (GC-MS). The automated procedure is based on the injection of sputum samples which are methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM-GC-MS). Hexacosanoic acid in combination with tuberculostearic acid was found to be specific for the presence of M. tuberculosis. The detection limit was similar to microscopy. We found no false positives, all microscopy and culture positive samples were also found positive with the THM-GC-MS method. The detection of ribosomal RNA from the infecting organism offers great potential since rRNA molecules outnumber chromosomal DNA by a factor 1000. It thus may possible to detect the organism without amplification of the nucleic acids (NA). We used a capture and a tagged detector probe for the direct detection of M. tuberculosis in sputum. So far the detection limit is 1x106 bacteria / ml. Currently we are testing a Lab-On-A-Chip Interferometer detection system.