Controlling the kinetic chain length of the crosslinks in photo-polymerized biodegradable networks.

Biodegradable polymer networks were prepared by photo-initiated radical polymerization of methacrylate functionalized poly(D,L-lactide) oligomers. The kinetic chains formed in this radical polymerization are the multifunctional crosslinks of the networks. These chains are carbon-carbon chains that remain after degradation. If their molecular weight is too high these poly(methacrylic acid) chains can not be excreted by the kidneys. The effect of the photo-initiator concentration and the addition of 2-mercaptoethanol as a chain transfer agent on the molecular weight of the kinetic chains was investigated. It was found that both increasing the initiator concentration and adding 2-mercaptoethanol decrease the kinetic chain length. However, the effect of adding 2-mercaptoethanol was much larger. Some network properties such as the glass transition temperature and the swelling ratio in acetone are affected when the kinetic chain length is decreased.

Column Selection and Optimization for Comprehensive Two-Dimensional Gas Chromatography: A Review.

Compared to one-dimensional gas chromatography, comprehensive two-dimensional gas chromatography (GC × GC) method development is significantly more complex because more method development choices need to be made and because of the complex interplay of the primary and secondary parameters; the individual dimensions cannot be optimized separately. Also, optimization is restricted by requirements such as the modulation criterion and upper temperature limits of the individual columns. In general, the internal diameter of the primary column is larger than the internal diameter of the secondary column which complicates the optimization and leads to sub-optimal flow settings, column loadability issues and indirectly a reduction of the overall separation efficiency. In this review, papers concerning method development for comprehensive two-dimensional gas chromatography (GC × GC) are discussed and general guidelines are proposed with the focus on selecting the GC × GC instrumental set-up and column-set and optimization of the GC × GC settings.

Flow injection analysis electrospray ionization mass spectrometry for high-throughput screening of a gene library for amidase activity.

In high-throughput screening of gene and mutant libraries, high analysis speeds and short method development times are important factors. Mass spectrometry (MS) is considered to be a generic analytical technique with a relatively short development time. Furthermore, when applying flow injection analysis (FIA) for sample introduction, the requirements for high throughput are met. In this work, the use of a single quadrupole electrospray MS instrument for assaying amidase activity in a gene library is demonstrated. The desired selectivity for measuring the amino acid, the reaction product of the amidase reaction, in the presence of high concentrations of the corresponding amino acid amide substrate was obtained by selective ionization of the amino acid in negative ion mode electrospray. The only sample preparation required was a 200-fold dilution of the reaction mixture. For obtaining quantitative results, a complementary calibration procedure was set up to correct for the change in ionization suppression as a function of conversion. This approach was used to screen a Mycobacterium neoaurum gene library consisting of 11,520 clones with alpha-methylleucine amide as substrate within 24h. Conversion was measured on the [M-H]- species of the corresponding alpha-methylleucine (m/z 144). Five positive clones were detected with a conversion ranging from 0.2% to 3.4%.

Two metrics for measuring orthogonality for two-dimensional chromatography.

Orthogonality can be used as a selection parameter for two-dimensional chromatography column selection (e.g. in GC × GC or LC × LC) or for method optimization purposes, both aiming for maximal orthogonality for a particular analytical application. In order to improve the concurrence of two-dimensional chromatography expert's orthogonality grading, two orthogonality metrics, %FIT and %BIN, were developed, evaluated and compared with the Asterisks orthogonality metric. The %BIN is a bin counting approach where the number of bins is fixed at 25 and deviations from the expected average number of peaks per bin is used as the basis for the orthogonality calculation. The %FIT is based on fitting polynomials of degree two, through the xy and the yx data and calculating the average minimal distance and standard deviation of all data points above and below the fitted polynomials. The orthogonality metrics were evaluated by using 14 different types of computer generated xy datasets and two measured LC × LC datasets. Both %FIT and %BIN, were shown to have a larger discriminative power than the Asterisks equations, and are in good agreement with the orthogonality scores for 2D-chromatograms provided by nine experts.

Enhancing detectability of anabolic-steroid residues in bovine urine by actively modulated online comprehensive two-dimensional liquid chromatography - high-resolution mass spectrometry.

In this study we describe an approach to enhance the sensitivity of an online comprehensive two-dimensional liquid chromatography (LC × LC) high-resolution mass spectrometry method for the separation and detection of trace levels of anabolic-steroid residues in complex urine matrices. Compared to one-dimensional liquid chromatography (1D-LC), LC × LC methods offer higher separation power, thanks to the combined effect of two different selectivities and a higher peak capacity. However, when using state-of-the-art LC × LC instrumentation, the price paid for the increase in separation power is a decrease in sensitivity and detectability of trace-level analytes. This can be ascribed to the sample dilution that takes place during each of the two chromatographic steps. The way in which fractions are collected and transferred from the first to the second column is also of paramount importance, especially the volume and the solvent composition of the fractions injected in the second column. To overcome the detection limitation, we present an active-modulation strategy, based on concentrating the fractions of the first-dimension effluent using a modulation interface that employs trap columns. We obtained a signal enhancement for anabolic-steroid compounds in a bovine-urine sample by a factor of 2.4-7.6 and an increase in the signal-to-noise ratio up to a factor of 7 in comparison with a standard loop-based modulation interface. In addition, thanks to the increased sensitivity of our method, a substantially larger number of peaks were detected (76 vs. 36). Moreover, we could reduce the solvent consumption by a factor of three (160 mL vs. 500 mL per run).

Size-exclusion chromatography using core-shell particles.

Size-exclusion chromatography (SEC) is an indispensable technique for the separation of high-molecular-weight analytes and for determining molar-mass distributions. The potential application of SEC as second-dimension separation in comprehensive two-dimensional liquid chromatography demands very short analysis times. Liquid chromatography benefits from the advent of highly efficient core-shell packing materials, but because of the reduced total pore volume these materials have so far not been explored in SEC. The feasibility of using core-shell particles in SEC has been investigated and contemporary core-shell materials were compared with conventional packing materials for SEC. Columns packed with very small core-shell particles showed excellent resolution in specific molar-mass ranges, depending on the pore size. The analysis times were about an order of magnitude shorter than what could be achieved using conventional SEC columns.

Comprehensive lipidomic analysis of human plasma using multidimensional liquid- and gas-phase separations: Two-dimensional liquid chromatography-mass spectrometry vs. liquid chromatography-trapped-ion-mobility-mass spectrometry.

Recent advancements in separation science have resulted in the commercialization of multidimensional separation systems that provide higher peak capacities and, hence, enable a more-detailed characterization of complex mixtures. In particular, two powerful analytical tools are increasingly used by analytical scientists, namely online comprehensive two-dimensional liquid chromatography (LC×LC, having a second-dimension separation in the liquid phase) and liquid chromatography-ion mobility-spectrometry (LC-IMS, second dimension separation in the gas phase). The goal of the current study was a general assessment of the liquid-chromatography-trapped-ion-mobility-mass spectrometry (LC-TIMS-MS) and comprehensive two-dimensional liquid chromatography-mass spectrometry (LC×LC-MS) platforms for untargeted lipid mapping in human plasma. For the first time trapped-ion-mobility spectrometry (TIMS) was employed for the separation of the major lipid classes and ion-mobility-derived collision-cross-section values were determined for a number of lipid standards. The general effects of a number of influencing parameters have been inspected and possible directions for improvements are discussed. We aimed to provide a general indication and practical guidelines for the analyst to choose an efficient multidimensional separation platform according to the particular requirements of the application. Analysis time, orthogonality, peak capacity, and an indicative measure for the resolving power are discussed as main characteristics for multidimensional separation systems.

A procedure for comprehensive two-dimensional gas chromatography retention time locked dual detection.

In this paper, a novel, and easy to perform, retention time locking procedure for locking primary and secondary retention times of detector signals in comprehensive two-dimensional gas chromatography (GCxGC) dual-detection is proposed and its advantages are demonstrated and discussed. The dual detection retention time locking procedure is a 2-step process for a GCxGC system in which the effluent of the primary column is split, by using a pressure regulated splitter, towards the GCxGC modulator using two identical secondary GC columns of which one is installed in the main GC oven and the other is installed in a secondary GC oven. The first step of the locking procedure is to minimize the secondary retention time difference between both detectors of a compound, which has a retention factor (k) close to 0. This is done by stepwise altering the effective secondary column length, simply by sliding the secondary column, which is installed in the main oven, forwards or backwards through the modulator. The second step is to minimize the secondary retention time difference of a compound which has a significant retention in both dimensions. This is done by stepwise altering the secondary oven temperature rate. This locking procedure was successfully demonstrated for the analysis of a diesel sample by GCxGC coupled to a time of flight mass spectrometer (TOFMS) and a nitrogen chemiluminescence detector (NCD) and by GCxGC coupled to a TOFMS and a flame ionization detector (FID). For all compounds the average absolute secondary retention time differences between the NCD or the FID and the TOFMS detectors were 0.03, and 0.07s, respectively, which are significantly less than the average peak widths at half heights, which was 0.2s.

Tunable secondary dimension selectivity in comprehensive two-dimensional gas chromatography.

In this paper two tunable two-dimensional gas chromatography setups are compared and described in which the secondary dimension consists of two different capillary columns coupled in series. In the first setup the selectivity of the second dimension can be tuned by adjusting the effective column length of the first secondary dimension column, simply by sliding it stepwise back or forward through the GC×GC modulator. In the second setup, in which the first secondary dimension column is installed in a separate GC-oven (oven-2), the overall selectivity of the second dimension can be tuned by adjusting the oven-2 temperature offset with respect to the main oven. The contribution of the first secondary dimension column to the overall secondary dimension separation can be decreased by applying a higher temperature offset. A real-life sample, the headspace of a coffee powder, was used to demonstrate the added value of tunable GC×GC by solving coelutions of some specific aroma compounds. Besides optimizing the overall GC×GC separation, by altering the second dimension column selectivity, these set-ups also offer enhanced possibilities for qualitative analysis. By stepwise altering the selectivity of the second dimension, classes of compounds showing similar retention behavior could be discriminated.

Quantitative analysis of morphine in dried blood spots by using morphine-d3 pre-impregnated dried blood spot cards.

Two different internal standard dried blood spot (DBS) pre-impregnation procedures (prior to blood spotting) were investigated. In the first procedure DBS pre-impregnation is performed by immersing the DBS card fully into an internal standard solution. In the second procedure pre-impregnation is performed by pipetting a certain volume of an internal standard solution onto the DBS card. Morphine-d3 was used as the model compound for all experiments. The pre-impregnation procedure by immersing was further investigated with respect to homogeneity of impregnation, influence of different blood spotting techniques and the influence of spotting different blood volumes on the internal standard distribution, calibration and stability of pre-impregnated cards. Finally, the immersing procedure was used for the analysis of morphine in dried blood spots and the results were compared to the conventional procedure in which the internal standard morphine-d3 was added to the extraction solvent. The new pre-impregnated cards couple simplicity of operation and convenient use in the field to results equivalent to the conventional procedure.

Temperature-tunable selectivity in comprehensive two-dimensional gas chromatography.

A temperature-tunable two-dimensional gas chromatography setup, consisting of three capillary columns with different selectivity, is described. In this setup the selectivity of the primary dimension can be tuned by adjusting the temperature offset of two in series-coupled capillary columns, both columns being part of the primary dimension and positioned in two separate GC ovens. The overall GC×GC separation can be optimized by altering the selectivity of the primary dimension. Besides tuning selectivity, in order to achieve optimal separation, this 2D-GC setup also offers enhanced opportunities for qualitative analysis. Sequentially altering the selectivity of the primary dimension enables one to identify groups of compounds which show similar chromatographic retention behavior.

Alignment and clustering strategies for GC×GC-MS features using a cylindrical mapping.

Comprehensive two-dimensional gas chromatography coupled to mass spectrometry is a powerful tool to analyze complex samples. For application of the technique in studies like biomarker discovery in which large sets of complex samples have to be analyzed, extensive preprocessing is needed to align the data obtained in several injections (analyses). We developed new alignment and clustering algorithms for this type of data. New in the current procedures is the consistent way in which the phenomenon referred to as wrap-around is treated. The data analysis problems associated with this phenomenon are solved by treating the 2D display as the surface of a three-dimensional cylinder. Based on this transformation we developed a new similarity metric for features as a function of both the cylindrical distance (reflecting similarity in chromatographic behavior) and of the mass spectral correlation (reflecting similarity in chemical structure). The concepts are used in warping and clustering, and include a protection against greedy warping. The methods were applied - for the purpose of an example - to the analysis of 11 replicates of a human urine sample concentrated by solid phase extraction. It is shown that the alignment is well protected against greedy warping which is important with respect to analytical qualities as robustness and repeatability. It is also demonstrated that chemically similar features are clustered together. The paper is organized as follows. First a brief introduction is provided addressing the background of the GC×GC-MS data structure followed by a theoretical section with a conceptual description of the procedures and details of the algorithms. Finally an example is given in the experimental section, illustrating the application of the procedures.

Fast and efficient size-based separations of polymers using ultra-high-pressure liquid chromatography.

Ultra-high-pressure liquid chromatography (UHPLC) has great potential for the separations of both small molecules and polymers. However, the implementation of UHPLC for the analysis of macromolecules invokes several problems. First, to provide information on the molecular-weight distribution of a polymer, size-exclusion (SEC) columns with specific pore sizes are needed. Development of packing materials with large pore diameters and pore volumes which are mechanically stable at ultra-high-pressures is a technological challenge. Additionally, narrow-bore columns are typically used in UHPLC to minimize the problem of heat dissipation. Such columns pose stringent requirements on the extra-column dispersion, especially for large (slowly diffusing) molecules. Finally, UHPLC conditions generate high shear rates, which may affect polymer chains. The possibilities and limitations of UHPLC for size-based separations of polymers are addressed in the present study. We demonstrate the feasibility of conducting efficient and very fast size-based separations of polymers using conventional and wide-bore (4.6 mm I.D.) UHPLC columns. The wider columns allow minimization of the extra-column contribution to the observed peak widths down to an insignificant level. Reliable SEC separations of polymers with molecular weights up to ca. 50 kDa are achieved within less than 1 min at pressures of about 66 MPa. Due to the small particles used in UHPLC it is possible to separate high-molecular-weight polymers (50 kDa ≤ M(r) ≤ 1-3 MDa, upper limit depends on the flow rate) in the hydrodynamic-chromatography (HDC) mode. Very fast and efficient HDC separations are presented. For very large polymer molecules (typically larger than several MDa, depending on the flow rate) two chromatographic peaks are observed. This is attributed to the onset of molecular deformation at high shear rates and the simultaneous actions of hydrodynamic and slalom chromatography.

Deformation and degradation of polymers in ultra-high-pressure liquid chromatography.

Ultra-high-pressure liquid chromatography (UHPLC) using columns packed with sub-2 µm particles has great potential for separations of many types of complex samples, including polymers. However, the application of UHPLC for the analysis of polymers meets some fundamental obstacles. Small particles and narrow bore tubing in combination with high pressures generate significant shear and extensional forces in UHPLC systems, which may affect polymer chains. At high stress conditions flexible macromolecules may become extended and eventually the chemical bonds in the molecules can break. Deformation and degradation of macromolecules will affect the peak retention and the peak shape in the chromatogram, which may cause errors in the obtained results (e.g. the calculated molecular-weight distributions). In the present work we explored the limitations of UHPLC for the analysis of polymers. Degradation and deformation of macromolecules were studied by collecting and re-injecting polymer peaks and by off-line two-dimensional liquid chromatography. Polystyrene standards with molecular weight of 4 MDa and larger were found to degrade at UHPLC conditions. However, for most polymers degradation could be avoided by using low linear velocities. No degradation of 3-MDa PS (and smaller) was observed at linear velocities up to 7 mm/s. The column frits were implicated as the main sources of polymer degradation. The extent of degradation was found to depend on the type of the column and on the column history. At high flow rates degradation was observed without a column being installed. We demonstrated that polymer deformation preceded degradation. Stretched polymers eluted from the column in slalom chromatography mode (elution order opposite to that in SEC or HDC). Under certain conditions we observed co-elution of large and small PS molecules though a convolution of slalom chromatography and hydrodynamic chromatography.

Retention time locking procedure for comprehensive two-dimensional gas chromatography.

In gas chromatography (GC) reproducible retention times are in many cases highly favorable or in some cases even required. In one-dimensional GC, retention time shifts can be eliminated or minimized using a procedure called retention time locking (RTL). This procedure is based on adjusting the (constant) column head pressure. Unfortunately, this RTL procedure cannot be used in comprehensive two-dimensional gas chromatography (GC×GC) given the fact that peaks will shift in both dimensions. Adjusting the column head pressure in GC×GC will only minimize or eliminate the primary retention time shifts. In this paper, a fast and easy to perform, two-step retention time locking procedure for two-dimensional gas chromatography (2D-RTL) is proposed and its feasibility is demonstrated. This 2D-RTL procedure involves adjustment of the column head pressure or constant column flow, followed by the adjustment of the so-called effective secondary column length. The secondary column length is increased or decreased, simply by moving it stepwise through the modulator. It is demonstrated that retention time shifts in both the primary- and secondary-dimension, which may occur after e.g. replacing the column set, can be minimized to less than half peak base width. The proposed 2D-RTL procedure is used successfully for approximately 1 year in our laboratory.