Flow Field Thermal Gradient Gas Chromatography

Negative temperature gradients along the gas chromatographic separation column can maximize the separation capabilities for gas chromatography by peak focusing and also lead to lower elution temperatures. Unfortunately, so far a smooth thermal gradient over a several meters long separation column could only be realized by costly and complicated manual setups. Here we describe a simple, yet flexible method for the generation of negative thermal gradients using standard and easily exchangeable separation columns. The measurements made with a first prototype reveal promising new properties of the optimized separation process. The negative thermal gradient and the superposition of temperature programming result in a quasi-parallel separation of components each moving simultaneously near their lowered specific equilibrium temperatures through the column. Therefore, this gradient separation process is better suited for thermally labile molecules such as explosives and natural or aroma components. High-temperature GC methods also benefit from reduced elution temperatures. Even for short columns very high peak capacities can be obtained. In addition, the gradient separation is particularly beneficial for very fast separations below 1 min overall retention time. Very fast measurements of explosives prove the benefits of using negative thermal gradients. The new concept can greatly reduce the cycle time of high-resolution gas chromatography and can be integrated into hyphenated or comprehensive gas chromatography setups

Comprehensive Theory of the Deans’ Switch As a Variable Flow Splitter: Fluid Mechanics, Mass Balance, and System Behavior

The Deans’ switch is an effluent switching device based on controlling flows of carrier gas instead of mechanical valves in the analytical flow path. This technique offers high inertness and a wear-free operation. Recently new monolithic microfluidic devices have become available. In these devices the whole flow system is integrated into a small metal device with low thermal mass and leak-tight connections. In contrast to a mechanical valve-based system, a flow-controlled system is more difficult to calculate. Usually the Deans’ switch is used to switch one inlet to one of two outlets, by means of two auxiliary flows. However, the Deans’ switch can also be used to deliver the GC effluent with a specific split ratio to both outlets. The calculation of the split ratio of the inlet flow to the two outlets is challenging because of the asymmetries of the flow resistances. This is especially the case, if one of the outlets is a vacuum device, such as a mass spectrometer, and the other an atmospheric detector, e.g. a flame ionization detector (FID) or an olfactory (sniffing) port. The capillary flows in gas chromatography are calculated with the Hagen–Poiseuille equation of the laminar, isothermal and compressible flow in circular tubes. The flow resistances in the new microfluidic devices have to be calculated with the corresponding equation for rectangular cross-section microchannels. The Hagen–Poiseuille equation underestimates the flow to a vacuum outlet. A corrected equation originating from the theory of rarefied flows is presented. The calculation of pressures and flows of a Deans’ switch based chromatographic system is done by the solution of mass balances. A specific challenge is the consideration of the antidiffusion resistor between the two auxiliary gas lines of the Deans’ switch. A full solution for the calculation of the Deans’ switch including this restrictor is presented. Results from validation measurements are in good accordance with the developed theories. A spreadsheet-based flow calculator is part of the Supporting Information

Hyperfast Flow-Field Thermal Gradient GC/MS of Explosives with Reduced Elution Temperatures

Hyperfast GC/MS below 60 s of measurement time has been used for the measurement of explosives. The new flow-field thermal gradient GC (FF-TG-GC) utilizes a modified transport process of the explosives at lowered temperatures. In combination with the focusing effect of the gradient, high-resolution chromatograms are obtained even in very short time intervals. The reduction of the elution temperature by applying a thermal gradient along the chromatographic column is demonstrated by the simulation of the migration of analytes through the column. The simulation shows an interesting effect of the difference between maximum temperature and elution temperature of analytes during their separation with the spatial gradient. The results show the benefit of the gradient elution both from a modeling perspective and by measurements of explosives with low limits of detection (LOD) in the range from 0.1 to 20 μg/mL (0.5 to 150 pg of analyte mass on column). Results were compared to state-of-the-art vacuum outlet GC/MS as a reference method. A correlation between the reduction of elution temperatures and lower LODs are found for thermal labile nitrate ester explosives (EGDN, NG, ETN, and PETN), while no significant influence of the reduced elution temperature on LODs of more stable explosives, like DNT and TNT, was found

Comprehensive Theory of the Deans’ Switch As a Variable Flow Splitter: Fluid Mechanics, Mass Balance, and System Behavior

The Deans’ switch is an effluent switching device based on controlling flows of carrier gas instead of mechanical valves in the analytical flow path. This technique offers high inertness and a wear-free operation. Recently new monolithic microfluidic devices have become available. In these devices the whole flow system is integrated into a small metal device with low thermal mass and leak-tight connections. In contrast to a mechanical valve-based system, a flow-controlled system is more difficult to calculate. Usually the Deans’ switch is used to switch one inlet to one of two outlets, by means of two auxiliary flows. However, the Deans’ switch can also be used to deliver the GC effluent with a specific split ratio to both outlets. The calculation of the split ratio of the inlet flow to the two outlets is challenging because of the asymmetries of the flow resistances. This is especially the case, if one of the outlets is a vacuum device, such as a mass spectrometer, and the other an atmospheric detector, e.g. a flame ionization detector (FID) or an olfactory (sniffing) port. The capillary flows in gas chromatography are calculated with the Hagen–Poiseuille equation of the laminar, isothermal and compressible flow in circular tubes. The flow resistances in the new microfluidic devices have to be calculated with the corresponding equation for rectangular cross-section microchannels. The Hagen–Poiseuille equation underestimates the flow to a vacuum outlet. A corrected equation originating from the theory of rarefied flows is presented. The calculation of pressures and flows of a Deans’ switch based chromatographic system is done by the solution of mass balances. A specific challenge is the consideration of the antidiffusion resistor between the two auxiliary gas lines of the Deans’ switch. A full solution for the calculation of the Deans’ switch including this restrictor is presented. Results from validation measurements are in good accordance with the developed theories. A spreadsheet-based flow calculator is part of the Supporting Information

Expression of proteases and Pearson correlation.

(A) Comparison of the relative amounts of ADAM (a disintegrin and metalloprotease domain) proteases as well as BACE (beta-secretase) and MMP2 (matrix metallopeptidase 2) in the SLGC lines indicated (data from proteome array). Based on the assumption that the antibodies spotted on the filters of the proteome array possessed similar KDs, the relative expression of the proteases was calculated. The sum of the signals of all proteases was arbitrary set at 100%. The bars indicate to what percentage the individual proteases contribute to this expression. (B) Similar assay as in (A). The relative Cathepsin D levels were compared to the relative expression of all other proteases, depicted in panel (A). (C) Similar analysis as in (A) comparing the relative expression of the protease inhibitors TIMP (tissue inhibitor of metalloproteinases) 1, 2, 3 and 4.–Parts D and E graphically summarize some of the correlation data shown in Figs 8 and 9. In (B) the coefficients are indicated on the y-axis, highlighting expression levels with a positive correlation to the Sox2 (blue) or CD133 (violet) dots. In (C) the Pearson correlation coefficients were calculated relative to the levels of the neural proteins Tau and GFAP, as well as the hyaluronan receptor CD44, the integrin αv, and the N- and E-cadherin, respectively. In all cases, the expression levels that entered the calculations were determined with the same whole cell extracts. The calculations were confirmed with biological replicates. (TIF)</p

Growth factor requirements and growth curves.

(A) Expression (RT-PCR analysis) of mRNAs coding for EGF (epidermal growth factor), TGFα (transforming growth factor α), HB-EGF (Heparin-binding EGF-like growth factor), and bFGF (basic fibroblast growth factor). Bars depict the mean of a minimum of three replicates, whiskers the standard deviation. Significant differences relative to the expression in the T1338 cell line are indicated (**, pB) Effects of growth factor depletion on proliferation (BrdU ELISA). The combination of growth factors added into the medium is indicated by distinct levels of gray(white: EGF/bFGF; light grey: EGF; dark grey: bFGF; black: no growth factor added). The bars depict mean values and standard deviations. Significant reduction of BrdU incorporation is indicated by p-values (**, pC) Growth curves of the SLGC lines indicated were performed in the presence of EGF (red line), bFGF (black line) or both (blue line), or in the absence of growth factors (violet line). After six days (d6) cells were re-plated for studies with extended incubation times. Values for d9 were determined from both the original and the replated cultures. Significant differences between the growth curves at specific time points are indicated by p-values (*, pD) Growth factor ELISA. The amounts of EGF and bFGF present in T1371 and T1447 cultures were determined at days d2 and d9 after plating. The assays were performed in DMEM/Ham’s F12 containing fetal calf serum (10% FCS) or the serum supplements BIT (bovine albumin, insulin, and transferrin) and B27, respectively. The presence of exogenous growth factors is indicated by EGF/bFGF. Each assay encompassed a minimum of four replicates. Significant differences are indicated (**, p (TIF)</p

Pearson correlation coefficients (k): SLGC markers.

In (A) cell lines were ranked according to their Sox2 expression at the time of analysis. The Actin-normalized relative expression of the respective proteins is indicated in the plots. Group 1 displayed significantly higher Sox2 levels than Group 2. Group E comprises the established cell lines U87 and CaCo2, both of which lack Sox2 expression. The ranking was as follows: [T1452, T1371, T1495-SC, T1586, T1440, T1587, T1447-SC, T1495, T1447, T1338], [T1522, T1389, T1467, T1442, T1464, T1454, T1600, T1439], [CaCo2, U87]. In (B) cell lines were ranked according to their CD133 expression at the time of analysis. The Actin-normalized relative expression of the respective proteins is indicated in the plots. The CD133 levels were highest in the CaCo2 cell line, followed by the SLGCs assigned to groups with decreasing CD133-levels. The non-SLGC line CaCo2, which encompasses >90% of CD133 positive cells, is indicated on the very left. The ranking was as follows: [CaCo2], [T1452, T1333, T1495-Sc], [T1447-SC], [T1586, T1442, T1587, U87, T1600, T1464, T1447, T1495, T1440], [T1522, T1371, T1389, T1467, T1439, T1454].–For abbreviations, see the legends to Figs 5 and 6.</p

GBM; glioblastoma multiforme; GS, gliosarcoma; GS*, recurrent gliosarcoma; suffix “SC”, indicates that the cell lines was established from an orthotopic tumor grown in a SCID mouse (SC2, was derived from xenotransplanted T1495-SC); PTEN, <i>Phosphatase and tensin homolog;</i> +/loss*, PTEN status in T1440 subpopulations is +/+, +/- or -/-; T1389 mutant*, subpopulations with mixed Tp53 status in exons 5 and 6; T1338 WT*, a subpopulation of T1338 cells is heterozygote for Tp53 mutation; RTK, receptor tyrosine kinase, IDH, isocitrate dehydrogenase; E, [EGFR] epidermal growth factor receptor; E^§, amplification of truncated EGFR; Pα, Pβ, platelet-derived growth factor [PDGF] receptors α and β; MERTK, tyrosine protein kinase Mer—n.t., not tested.

GBM; glioblastoma multiforme; GS, gliosarcoma; GS*, recurrent gliosarcoma; suffix “SC”, indicates that the cell lines was established from an orthotopic tumor grown in a SCID mouse (SC2, was derived from xenotransplanted T1495-SC); PTEN, Phosphatase and tensin homolog; +/loss*, PTEN status in T1440 subpopulations is +/+, +/- or -/-; T1389 mutant*, subpopulations with mixed Tp53 status in exons 5 and 6; T1338 WT*, a subpopulation of T1338 cells is heterozygote for Tp53 mutation; RTK, receptor tyrosine kinase, IDH, isocitrate dehydrogenase; E, [EGFR] epidermal growth factor receptor; E§, amplification of truncated EGFR; Pα, Pβ, platelet-derived growth factor [PDGF] receptors α and β; MERTK, tyrosine protein kinase Mer—n.t., not tested.</p

Expression of cell surface proteins.

(A, B, C) Flow cytometry analyses of SLGCs expanded in serum-free (N) and serum-containing (F) medium. Fluorochrome-coupled antibodies directed against CD133/Prominin-1, CD44, integrin α6/CD49f, and integrin αv/CD51, as well as the endothelial CAM (cell adhesion molecule) PECAM/CD31 or the endothelial cadherin VE-cadherin/CD144 were used. A minimum of 10,000 cells was analysed. Assays with corresponding N- and F cultures were done in parallel. Bars represent mean values, whiskers represent the variation between technical replicates. (D) Box plots summarizing the real-time PCR (qRT-PCR) analyses using primers directed against integrins (Int) or cadherins (Cdh). The plots indicate the mean values calculated from the qRT-PCR data of 12 distinct SLGC lines, in which each SLGC line was analyzed in three individual reactions. The standard deviations are indicated by the upper and lower borders of the boxes, and the median is symbolized by the central line. The range of minimal and maximal values is represented by the whiskers. The qRT-PCR values were normalized against the reference genes gapdh, ubiquitin ligase, and 18 s r-rna prior to calculation of the Box plots.</p

Expression of SLGC and GBM subtype markers.

(A-C) Western blot analyses. The loading controls Actin (42 kDa) or GAPDH (glyceraldehyde-3 phosphate dehydrogenase; 36 kDa) are shown below the corresponding blots. Brackets indicate that the same nitrocellulose filter was used for the respective detections. (D) Immunofluorescence analysis of the expression of the platelet-derived growth factor receptors-1 and -2 (PDGFR-α, -β). The primary antibodies were revealed with goat anti-mouse DyLight®488 (green) and goat anti-rabbit Cy3 (red), respectively. K58 served as a marker for the Golgi apparatus.—Bars, 50 μm; DAPI, 4′,6-diamidino-2-phenylindole.–Akt, protein kinase B; CD133, Prominin-1; CD44, cell surface receptor that engages extracellular matrix components such as hyaluronan; CDK6, cyclin-dependent kinase 6; EGFR, epidermal growth factor receptor; FABP7, fatty acid binding protein 7; MERTK, tyrosine-protein kinase Mer; Musashi, RNA binding protein; Nanog, DNA binding homeobox transcription factor; PDGFR-α, -β, platelet-derived growth factor receptor α and β; PTEN, phosphatase and Tensin homolog; p53, tumor suppressor p53; Sox2, SRY-box transcription factor 2; SC, derived from orthotopic tumor.</p