Simulating Capillary Gas Chromatographic Separations including Thermal Gradient Conditions.

This article presents a method of simulating molecular transport in capillary gas chromatography (GC) applicable to isothermal, temperature-programmed, and thermal gradient conditions. The approach accounts for parameter differences that can occur across an analyte band including pressure, mobile phase velocity, temperature, and retention factor. The model was validated experimentally using a GC column comprised of microchannels in a stainless-steel plate capable of isothermal, temperature-programmed, and thermal gradient GC separations. The parameters governing retention and dispersion in the transport model were fitted with 12 experimental isothermal separations. The transport model was validated with experimental data for three analytes using four temperature-programmed and three thermal gradient GC separations. The simulated peaks (elution time and dispersion) give reasonable predictions of observed separations. The magnitudes of the maximum error between simulated peak elution time and experiment were 2.6 and 4.2% for temperature-programmed and thermal gradient GC, respectively. The magnitudes of the maximum error between the simulated peak width and experiment were 15.4 and 5.8% for temperature-programmed and thermal gradient GC, respectively. These relatively low errors give confidence that the model reflects the behavior of the transport processes and provides meaningful predictions for GC separations. This transport model allows for an evaluation of analyte separation characteristics of the analyte band at any position along the length of the GC column in addition to peak characteristics at the column exit. The transport model enables investigation of column conditions that influence separation behavior and opens exploration of optimal column design and heating conditions.

Comparison of the Dynamic Thermal Gradient to Temperature-Programmed Conditions in Gas Chromatography Using a Stochastic Transport Model.

This paper compares dynamic (i.e., temporally changing) thermal gradient gas chromatography (GC) to temperature-programmed GC using a previously published stochastic transport model to simulate peak characteristics for the separation of C12-C40 hydrocarbons. All comparisons are made using chromatographic conditions that give approximately equal analyte retention times (tR). As shown previously, a static thermal gradient does not improve resolution (Rs) equally for all analytes, which highlights the need for a dynamic thermal gradient. An optimal dynamic thermal gradient should result in constant analyte velocities at any instant in time for those analytes that are actively being separated (i.e., analytes that have low retention factors). The average separation temperature for each analyte is used to determine the thermal gradient profile at different times in the temperature ramp. Because many of the analytes require a similar thermal gradient profile when actively being separated, the thermal gradient profile in this study was held fixed; however, the temperature of the entire thermal gradient was raised over time. From the simulations performed in this study, optimized dynamic thermal gradient conditions are shown to improve Rs by up to 13% over comparative temperature-programmed conditions, even with a perfect injection (i.e., zero injection bandwidth). In the dynamic thermal gradient simulations, all analytes showed improvements in Rs along with slightly shorter tR values compared to simulations for traditional temperature-programmed conditions.

Comparison of Static Thermal Gradient to Isothermal Conditions in Gas Chromatography Using a Stochastic Transport Model.

This paper compares static (i.e., temporally unchanging) thermal gradient gas chromatography (GC) to isothermal GC using a stochastic transport model to simulate peak characteristics for the separation of C12-C14 hydrocarbons resulting from variations in injection bandwidth. All comparisons are made using chromatographic conditions that give approximately equal analyte retention times so that the resolution and number of theoretical plates can be clearly compared between simulations. Simulations show that resolution can be significantly improved using a linear thermal gradient along the entire column length. This is mainly achieved by partially compensating for loss in resolution from the increase in mobile phase velocity, which approximates an ideal, basic separation. The slope of the linear thermal gradient required to maximize resolution is a function of the retention parameters, which are specific to each analyte pair; a single static, thermal gradient will not affect all analytes equally. A static, non-linear thermal gradient that creates constant analyte velocities at all column locations provides the largest observed gains in resolution. From the simulations performed in this study, optimized linear thermal gradient conditions are shown to improve the resolution by as much as 8.8% over comparative isothermal conditions, even with a perfect injection (i.e., zero initial bandwidth).

Portable capillary liquid chromatography for pharmaceutical and illicit drug analysis.

A newly developed portable capillary liquid chromatograph was investigated for the separation of various pharmaceutical and illicit drug compounds. The system consists of two high-pressure syringe pumps capable of delivering capillary-scale flow rates at pressures up to 10 000 psi. Capillary liquid chromatography columns packed with sub-2 µm particles are housed in cartridges that can be inserted into the system and easily connected through high-pressure fluidic contact points by simply applying a specific, predetermined torque rather than using standard fittings and less precise sealing protocols. Several over-the-counter analgesic drug separations are demonstrated, along with a simple online measurement of tablet dissolution. Twenty illicit drug compounds were also separated across six targeted drug panels. The results described in this study demonstrate the capability of this compact liquid chromatography instrument to address several important drug-related applications while simplifying system operation, and greatly reducing solvent usage and waste generation essential for onsite analysis.

Stainless-Steel Column for Robust, High-Temperature Microchip Gas Chromatography.

This paper reports the first results of a robust, high-performance, stainless-steel microchip gas-chromatography (GC) column that is capable of analyzing complex real-world mixtures as well as operating at very high temperatures. Using a serpentine design, a 10 m column with an approximately semicircular cross-section with a 52 µm hydraulic diameter ( Dh) was produced in a 17 × 6.3 × 0.1 cm rectangular steel chip. The channels were produced using a multilayer-chemical-etch and diffusion-bonding process, and metal nuts were brazed onto the inlet and outlet ports allowing for column interfacing with ferrules and fused silica capillary tubing. After deactivating the metal surface, channels were statically coated with a ≈0.1 µm layer of 5% phenyl-1% vinyl-methylpolysiloxane (SE-54) stationary phase and cross-linked with dicumyl peroxide. By using n-tridecane ( n-C13) as a test analyte with a retention factor ( k) of 5, a total of 44 500 plates (≈4500 plates per meter) was obtained isothermally at 120 °C. The column was thermally stable to at least 350 °C, and rapid temperature programming (35 °C/min) was demonstrated for the boiling-point range from n-C5 to n-C44 (ASTM D2887 simulated-distillation standard). The column was also tested for separation of two complex mixtures: gasoline headspace and kerosene. These initial experiments demonstrate that the planar stainless-steel column with proper interfacing can be a viable alternative platform for portable, robust microchip GC that is capable of high-temperature operation for low-volatility-compound analysis.

Compact Ultrahigh-Pressure Nanoflow Capillary Liquid Chromatograph.

A compact ultrahigh-pressure nanoflow liquid chromatograph (LC) was developed with the purpose in mind of creating a portable system that could be easily moved to various testing locations or placed in close proximity to other instruments for optimal coupling, such as with mass spectrometry (MS). The system utilized innovative nanoflow pumps integrated with a very low volume stop-flow injector and mixing tee. The system weighed only 5.9 kg (13 lbs) or 4.5 kg (10 lbs) without a controller and could hold up to 1100 bar (16000 psi) of pressure. The total volume pump capacity was 60 µL. In this study, the sample injection volume was determined by either a 60 nL internal sample groove machined in a high-pressure valve rotor or by a 1 µL external sample loop, although other sample grooves or loops could be selected. The gradient dwell volume was approximately 640 nL, which allowed significant reduction in sample analysis time. Gradient performance was evaluated by determining the gradient step accuracy. A low RSD (0.6%, n = 4) was obtained for day-to-day experiments. Linear gradient reproducibility was evaluated by separating a three-component polycyclic aromatic hydrocarbon mixture on a commercial 150 µm inner diameter capillary column packed with 1.7 µm particles. Good retention-time reproducibility (RSD < 0.17%) demonstrated that the pumping system could successfully generate ultrahigh pressures for use in capillary LC. The system was successfully coupled to an LTQ Orbitrap MS in a simple and efficient way; LC-MS of a trypsin-digested bovine serum albumin (BSA) sample provided narrow peaks, short dwell time, and good peptide coverage.

Detection and confirmation of serum lipid biomarkers for preeclampsia using direct infusion mass spectrometry.

Despite substantial research, the early diagnosis of preeclampsia remains elusive. Lipids are now recognized to be involved in regulation and pathophysiology of some disease. Shotgun lipidomic studies were undertaken to determine whether serum lipid biomarkers exist that predict preeclampsia later in the same in pregnancy. A discovery study was performed using sera collected at 12-14 weeks pregnancy from 27 controls with uncomplicated pregnancies and 29 cases that later developed preeclampsia. Lipids were extracted and analyzed by direct infusion into a TOF mass spectrometer. MS signals, demonstrating apparent differences were selected, their abundances determined, and statistical differences tested. Statistically significant lipid markers were reevaluated in a second confirmatory study having 43 controls and 37 preeclampsia cases. Multi-marker combinations were developed using those lipid biomarkers confirmed in the second study. The initial study detected 45 potential preeclampsia markers. Of these, 23 markers continued to be statistically significant in the second confirmatory set. Most of these markers, representing several lipid classes, were chemically characterized, typically providing lipid class and potential molecular components using MS(2) Several multi-marker panels with areas under the curve >0.85 and high predictive values were developed. Developed panels of serum lipidomic biomarkers appear to be able to identify most women at risk for preeclampsia in a given pregnancy at 12-14 weeks gestation.

Global "omics" evaluation of human placental responses to preeclamptic conditions.

BACKGROUND: Preeclampsia (PE) is a leading cause of maternal death. Its cause is still debated but there is general agreement that the placenta plays a central role. Perhaps the most commonly proposed contributors to PE include placental hypoxia, oxidative stress, and increased proinflammatory cytokines. How the placenta responds to these abnormalities has been considered but not as part of a comprehensive analysis of low-molecular-weight biomolecules and their responses to these accepted PE conditions. OBJECTIVE: Using a peptidomic approach, we sought to identify a set of molecules exhibiting differential expression in consequence of provocative agents/chemical mediators of PE applied to healthy human placental tissue. STUDY DESIGN: Known PE conditions were imposed on normal placental tissue from 13 uncomplicated pregnancies and changes in the low-molecular-weight peptidome were evaluated. A t test was used to identify potential markers for each imposed stress. These markers were then submitted to a least absolute shrinkage and selection operator multinomial logistic regression model to identify signatures specific to each stressor. Estimates of model performance on external data were obtained through internal validation. RESULTS: A total of 146 markers were increased/decreased as a consequence of exposure to proposed mediators of PE. Of these 75 changed with hypoxia; 23 with hypoxia-reoxygenation/oxidative stress and 48 from exposure to tumor necrosis factor-α. These markers were chemically characterized using tandem mass spectrometry. Identification rates were: hypoxia, 34%; hypoxia-reoxygenation, 60%; and tumor necrosis factor-α, 50%. Least absolute shrinkage and selection operator modeling specified 16 markers that effectively distinguished all groups, ie, the 3 abnormal conditions and control. Bootstrap estimates of misclassification rates, multiclass area under the curve, and Brier score were 0.108, 0.944, and 0.160, respectively. CONCLUSION: Using this approach we found previously unknown molecular changes in response to individual PE conditions that allowed development biomolecular signatures for exposure to each accepted pathogenic condition.

LED-based UV absorption detector with low detection limits for capillary liquid chromatography.

A 260 nm deep UV LED-based absorption detector with low detection limits was developed and integrated with a small nanoflow pumping system. The detector is small in size (5.2 × 3.0 cm) and weighs only 85 g (without electronics). This detector was specifically designed and optimized for on-column detection to minimize extra-column band broadening. No optical reference was included due to the low drift in the signal. Two ball lenses, one of which was integrated with the LED, were used to increase light throughput through the capillary column. Stray light was minimized by the use of a band-pass filter and an adjustable slit. Signals down to the parts per billion level (nanomolar) were easily detected with a short-term noise level of 4.4 µAU, confirming a low limit of detection and low noise. The detection limit for adenosine-5'-monophosphate was 230 times lower than any previously reported values. Good linearities (3 orders of magnitude) were obtained using sodium anthraquinone-2-sulfonate, adenosine-5'-monophosphate, dl-tryptophan, and phenol. The LC system was demonstrated by performing isocratic separation of phenolic compounds using a monolithic capillary column (16.5 cm × 150 µm i.d.) synthesized from poly(ethylene glycol) diacrylate.

Hand-Portable Gradient Capillary Liquid Chromatography Pumping System.

In this work, a novel splitless nanoflow gradient generator integrated with a stop-flow injector was developed and evaluated using an on-column UV-absorption detector. The gradient pumping system consisted of two nanoflow pumps controlled by micro stepper motors, a mixer connected to a serpentine tube, and a high-pressure valve. The gradient system weighed only 4 kg (9 lbs) and could generate up to 55 MPa (8000 psi) pressure. The system could operate using a 24 V DC battery and required 1.2 A for operation. The total volume capacity of the pump was 74 µL, and a sample volume of 60 nL could be injected. The system provided accurate nanoflow rates as low as 10 nL/min without employing a splitter, making it ideal for capillary column use. The gradient dwell volume was calculated to be 1.3 µL, which created a delay of approximately 4 min with a typical flow rate of 350 nL/min. Gradient performance was evaluated for gradient step accuracy, and excellent reproducibility was obtained in day-to-day experiments (RSD < 1.2%, n = 4). Linear gradient reproducibility was tested by separating a three-component pesticide mixture on a poly(ethylene glycol) diacrylate (PEGDA) monolithic column. The retention time reproducibility was very good in run-to-run experiments (RSD < 1.42%, n = 4). Finally, excellent separation of five phenols was demonstrated using the nanoflow gradient system.

Modeling transthyretin (TTR) amyloid diseases, from monomer to amyloid fibrils.

ATTR amyloidosis is caused by deposition of large, insoluble aggregates (amyloid fibrils) of cross-ß-sheet TTR protein molecules on the intercellular surfaces of tissues. The process of amyloid formation from monomeric TTR protein molecules to amyloid deposits has not been fully characterized and is therefore modeled in this paper. Two models are considered: 1) TTR monomers in the blood spontaneously fold into a ß-sheet conformation, aggregate into short proto-fibrils that then circulate in the blood until they find a complementary tissue where the proto-fibrils accumulate to form the large, insoluble amyloid fibrils found in affected tissues. 2) TTR monomers in the native or ß-sheet conformation circulate in the blood until they find a tissue binding site and deposit in the tissue or tissues forming amyloid deposits in situ. These models only differ on where the selection for ß-sheet complementarity occurs, in the blood where wt-wt, wt-v, and v-v interactions determine selectivity, or on the tissue surface where tissue-wt and tissure-v interactions also determine selectivity. Statistical modeling in both cases thus involves selectivity in fibril aggregation and tissue binding. Because binding of protein molecules into fibrils and binding of fibrils to tissues occurs through multiple weak non-covalent bonds, strong complementarity between ß-sheet molecules and between fibrils and tissues is required to explain the insolubility and tissue selectivity of ATTR amyloidosis. Observation of differing tissue selectivity and thence disease phenotypes from either pure wildtype TTR protein or a mix of wildtype and variant molecules in amyloid fibrils evidences the requirement for fibril-tissue complementarity. Understanding the process that forms fibrils and binds fibrils to tissues may lead to new possibilities for interrupting the process and preventing or curing ATTR amyloidosis.

Organic monoliths for high-performance reversed-phase liquid chromatography.

RPLC is the most common mode of LC. It is widely used for separations of both small and large molecules. Monolithic columns, which are currently under intensive study by many groups, have the potential of becoming attractive alternatives to particle-packed columns. They are generally easier and faster to fabricate, and they demonstrate a lower pressure drop, less nonspecific adsorption, and richer chemistry (in the case of organic polymer monoliths) for providing broad selectivity. Silica monoliths, as is also true for columns packed with silica particles, are best applied to small-molecule separations. Organic polymer monoliths, on the other hand, have shown advantages for large-molecule separations. Recently, improvements in organic monoliths have led to efficiencies for small molecules that are approaching and even surpassing 100,000 plates/m. While this performance is still far short of what is currently available using modern small particles and silica monoliths in RPLC, steady progress is being made. This review describes recent developments in the synthesis and performance of organic polymer RPLC monoliths, and identifies areas where additional work is needed to significantly improve their performance for both small- and large-molecule separations.

Characterizing organic monolithic columns using capillary flow porometry and scanning electron microscopy.

Polyethylene glycol diacrylate monoliths prepared using different amounts of monomer, porogen ratio, and capillary dimensions were characterized using capillary flow porometry (CFP) and scanning electron microscopy (SEM). Our results reveal good agreement between SEM and CFP measurements for through-pore size distribution. The CFP measurements for monoliths prepared by the same procedure in capillaries with different diameters (i.e., 75, 150, and 250 µm) clearly confirmed a change in through-pore size distribution with capillary diameter, thus, certifying the need for in-column measurement techniques over bulk measurements (e.g., mercury intrusion porosimetry). The mean through-pore size varied from 3.52 to 1.50 µm with a change in capillary diameter from 75 to 250 µm. Consistent mean through-pore size distribution for capillary columns with the same internal diameter but with different lengths (1.5, 2, and 3 cm) confirms the high interconnectivity of the pores and independence of CFP measurements with respect to capillary length. CFP and SEM measurements not only allow pore structure analysis but also prediction of relative column performance. Monoliths with narrow through-pore size distribution (0.8-1.2 µm), small mean through-pore size, and thin skeletal size (0.55 µm) gave the best performance in terms of efficiency for polyethylene glycol diacrylate monoliths.

GC/MS method for positive detection of Bacillus anthracis endospores.

A simple method was developed for detection of Bacillus anthracis (BA) endospores and for differentiation of them from other species in the Bacillus cereus group. Chemical profiles that include lipids (i.e., fatty acids), carbohydrates (i.e., sugars), and the spore-specific biomarker, dipicolinic acid, were generated by one-step thermochemolysis (TCM) at 140 °C in 5 min to provide specific biomarker signatures. Anthrose, which is a biomarker characteristic of the B. cereus group of bacteria, was determined from a fragment produced by TCM. Surprisingly, several virulent BA strains contained very low levels of anthrose, which confounded their detection. A statistical discrimination algorithm was constructed using a combination of biomarkers, which was robust against different growth conditions (medium and temperature). Fifteen endospore-forming Bacillus species were confirmed in a statistically designed test (~90%) using the algorithm, including six BA strains (four virulent isolates), five B. thuringiensis (BT) isolates, and one isolate each for B. cereus (BC), B. mycoides (BM), B. atrophaeus (BG), and B. subtilis (BS). The detection limit for B. anthracis was found to be 50,000 endospores, on the basis of the GC/MS detection limits for 3-methyl-2-butenoic acid methyl ester, which is the biomarker derived from TCM of anthrose.

Wood used by Stradivari and Guarneri.

Whether or not the great Italian violin-makers used wood that had been chemically processed in order to preserve it and enhance the instrument's sound quality has long been a contentious issue. Here we use nuclear magnetic resonance and infrared spectroscopy to analyse organic matter in wood taken from antique instruments made by Stradivari and Guarneri del Gesu. Our results indicate that the wood used by the masters could indeed have been chemically treated, a technique that may inspire an approach to violin making that is more chemistry-based.

Long-term economic growth stimulus of human capital preservation in the elderly.

Health care is a crucial factor in US economic growth, because growing health care costs have made US corporations less competitive than their counterparts in countries where central governments assume most of those costs. In this paper we illustrate a second, possibly more powerful, effect of health care expenditures on the long term pace of US economic growth, i.e., that such investments in aging populations helps preserve human capital to later ages. In addition, as current investment in health care improves health and functional status, the future demand for health care as well as future health care costs will be constrained. These are crucial factors in countries experiencing rapid population aging. US labor force projections do not directly represent the effects of health care investment on the health of the future labor force, and federal health cost projections do not reflect the trajectory of health changes. Health dynamic projections suggest the effects of health care investment are large and growth stimulating. Projections done for the time period used by the Congressional Budget Office in budget mark-ups (2010-2020) are presented in the supporting information.

NIH funding trajectories and their correlations with US health dynamics from 1950 to 2004.

To determine optimal future National Institutes of Health (NIH) funding levels, the longitudinal correlation of the level of investment in NIH research with population changes in the risk of specific diseases should be analyzed. This is because NIH research is the primary source of new therapies and treatments for major chronic diseases, many of which were viewed as relatively untreatable in the 1950s. NIH research is also important in developing preventative and screening strategies to support public health interventions. These correlations are examined 1938 to 2004 for 4 major chronic diseases [cardiovascular disease (CVD), stroke, cancer, and diabetes] and the NIH institutes responsible for research for those diseases. This analysis shows consistent non-linear temporal correlations of funding to mortality rates across diseases. The economic implications of this are discussed assuming that improved health at later ages will allow projected declines in the rate of growth of the US labor force to be partly offset by a higher rate of labor force participation in the US elderly population due to reduced chronic disease risks and functional impairment.

Preparation of zwitterionic polymeric monolithic columns for hydrophilic interaction capillary liquid chromatography.

Porous zwitterionic monolithic columns based on photo-initiated copolymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betaine and poly(ethylene glycol) diacrylate in a binary porogen system comprising isopropanol and decanol were prepared in 75-µm-id fused silica capillaries. The resulting monolith was evaluated by hydrophilic interaction chromatography. Inverse size-exclusion chromatography was used to characterize the pore structure of the resulting monolith. A typical hydrophilic interaction chromatography mechanism was observed when the organic content in the mobile phase was higher than 60%. Good separations of amides, phenols, and benzoic acids were achieved. An efficiency of 75,000 plates/m was obtained. The effects of mobile phase pH, salt concentration, and organic modifier content on retention were investigated. For polar-charged analytes, both hydrophilic interactions and electrostatic interactions contributed to the selectivity.

Weak cation-exchange monolithic column for capillary liquid chromatography of peptides and proteins.

A stable poly(2-carboxyethyl acrylate-co-poly(ethylene glycol) diacrylate) monolith was synthesized inside a 75-µm id capillary by direct in situ photo-initiated polymerization in a binary porogenic solvent consisting of methanol and ethyl ether. The resulting monolith was evaluated for weak cation-exchange capillary liquid chromatography of peptides and proteins. A high dynamic binding capacity of 72.7 mg lysozyme per cm3 column volume was measured. Fast mass transfer was demonstrated by steep breakthrough curves. The resulting monolith exhibited negligible hydrophobicity, leading to good separation of peptides and proteins. Peak capacities of 11 for peptides with a 10-min salt gradient and 39 for proteins with a 20-min salt gradient were measured. An efficiency of 37,000 plates/m for proteins was obtained under isocratic conditions. The effects of functional group concentration, porogenic solvent composition, mobile phase pH, salt gradient rate, and hydrophobicity on the retention of analytes were investigated. Good run-to-run relative standard deviation (RSD) <1.93% and column-to-column RSD <4.63% were achieved.

Transformation of matter in living organisms during growth and evolution.

Growth of an organism involves transformations of the state of matter from unstructured food or photosynthate into the highly organized matter in the living organism. Biological evolution involves random changes in the structure of DNA that lead to changes in the organization of the matter in an organism. Thermodynamic data show the organized biomass in living organisms has the same thermodynamic properties as a random mixture of the same elemental composition and is not in an energetically metastable, low entropy state. Therefore, the central thesis of this work is that building biological structures and organization from foodstuffs incurs no direct thermodynamic cost. The implication is that growth and evolution occur with little or no thermodynamic cost. In consequence, the fundamental difference between living biomass and lifeless organic sludge is in the information constraints that direct and govern the organization of the system. These constraints within a living organism override random processes to produce an organized distribution of biomass within the organism. Similarly, the information in DNA constrains the outcome of biological evolution across organisms within a population of a species in a predictable way that leads to convergent evolution. Although individuals and molecules act or are acted upon in a random manner, the outcome in a constrained system is predictable within an organism and across organisms. As a consequence evolution will produce similar outcomes at the macro level in similar environments. Stochastic determinism is proposed as a method that could be used to model convergent evolution.