Pseudolinear gradient ultrahigh-pressure liquid chromatography using an injection valve assembly

The use of ultrahigh pressures in liquid chromatography (UHPLC) imposes stringent requirements on hardware such as pumps, valves, injectors, connecting tubing, and columns. One of the most difficult components of the UHPLC system to develop has been the sample injector. Static-split injection, which can be performed at pressures up to 6900 bar (100,000 psi), consumes a large sample volume and is very irreproducible. A pressure-balanced injection valve provided better reproducibility, shorter injection time, reduced sample consumption, and greater ease of use; however, it could only withstand pressures up to approximately 1000 bar (15,000 psi). In this study, a new injection valve assembly that can operate at pressures as high as 2070 bar (30,000 psi) was evaluated for UHPLC. This assembly contains six miniature electronically controlled needle valves to provide accurate and precise volumes for introduction into the capillary LC column. It was found that sample volumes as small as several tenths of a nanoliter can be injected, which are comparable to the results obtained from the static-split injector. The reproducibilities of retention time, efficiency, and peak area were investigated, and the results showed that the relative standard deviations of these parameters were small enough for quantitative analyses. Separation experiments using the UHPLC system with this new injection valve assembly showed that this new injector is suitable for both isocratic and gradient operation modes. A newly designed capillary connector was used at a pressure as high as 2070 bar (30,000 psi).     

In-depth characterization of slurry packed capillary columns with 1.0-microm nonporous particles using reversed-phase isocratic ultrahigh-pressure liquid chromatography

Fused-silica capillary columns packed with 1.0-microm nonporous C18 bonded particles are evaluated with isocratic ultrahigh-pressure liquid chromatography (UHPLC). Improved UHPLC techniques have demonstrated column efficiencies as high as 730 000 plates/m and run pressures over 6800 bar (100 000 psi) for packed 10-microm-inner diameter (i.d.) columns. Columns as large as 150 microm have been tested with UHPLC and show no flow-induced heating effects on separation efficiencies. van Deemter plot analysis for column i.d.s ranging from 10 to 150 microm shows an increase in column efficiency with a decrease in column i.d. Reduced parameter analysis further illustrates a decrease in reduced parameter A term and C term values with decreasing i.d. However, reduced parameter C term values for columns evaluated with UHPLC are an order of magnitude larger than C term values for larger particles at conventional pressures. Retention factors for moderately retained compounds are observed to increase with column i.d., suggesting an increase in packing density. Highly ordered packing arrangement at the column wall is seen for packed beds extruded from large-diameter columns.     

Use of 1.5-microm porous ethyl-bridged hybrid particles as a stationary-phase support for reversed-phase ultrahigh-pressure liquid chromatography

A new ethyl-bridged hybrid packing material was evaluated in terms of its suitability for ultrahigh-pressure liquid chromatography (UHPLC). The 1.5-microm particles were obtained and packed into 30-microm-i.d. fused-silica capillary columns up to 50 cm in length. The particles were evaluated by isocratic reversed-phase UHPLC at pressures up to 4500 bar (65,000 psi). The chromatographic performance of these particles was found to be similar to the performance of 1.0-microm nonporous silica particles. The mechanical strength of the ethyl-bridged hybrid material was evaluated by running a 15-cm-long column at pressures up to 4500 bar. No breakdown of the particles in the packed bed was observed. The sample loading capacity of the hybrid material was evaluated and compared to 1.0-microm nonporous silica material by observing analyte peak width versus amount injected. The observed improvement in loading capacity for the hybrid material versus nonporous silica was consistent with the improvement predicted by comparing the phase ratios of the two materials.     

Injection valve for ultrahigh-pressure liquid chromatography

The increased interest in HPLC at elevated pressures, beyond the conventional 6000 psi (400 bar), has created a demand for injection systems capable of withstanding pressures beyond the 20,000 psi (1380 bar). To achieve high-resolution separations, an appropriate length of columns packed with sub 2-microm packing materials, a 30,000-40,000 psi (2070-2760 bar) pressure range is desirable. A new air-actuated needle valve injection system rated to withstand pressures of up to 40,000 psi (2760 bar) has been evaluated. Under isocratic chromatographic conditions, injecting 200 nL and operated at approximately 20,000 psi (1380 bar), the system showed a peak area reproducibility of approximately 2.5% RSD, contrasting the 5% RSD of a pressured-balanced injection system operated under similar conditions. Programmed for partial loop injections using injection times of 300-700 ms (injection volumes in the range of 1-2.5 microL) and operated at pressures close to 30,000 psi (2070 bar), the reproducibility in peak area for the amounts injected was approximately 1.5% RSD or lower, while an injection time of 100 ms resulted in a reproducibility of 3-4% RSD. The new injection system did not show any significant carryover, and after thousands of injections, the system has not shown sign of wear, loss of pressure during injection, or loss in chromatographic performance.     

Microchip HPLC of peptides and proteins

Rapid microchip reversed-phase HPLC of peptides and proteins at pressure gradients of 12 bar/cm (180 psi/cm) has been performed using a microdevice that integrates subnanoliter on-chip injection and separation with a miniaturized fluorescence detector. Proteins and peptides were separated on a C18 side-chain porous polymer monolith defined by contact lithography, and injection was achieved via a pressure-switchable fluoropolymer valve defined using projection lithography. Preliminary separations of peptide standards and protein mixtures were performed in 40-200 s, and switching between samples with no detectible sample carryover has been performed. The injections and separations were reproducible; the relative standard deviation (RSD) for retention time was 0.03%, and peak area RSD was 3.8%. Sample volumes ranging from 220 to 800 pL could be linearly metered by controlling the pressure injection pulse duration with conventional timing and valving. The current prototype system shows the potential for rapid and autonomous HPLC separations with varying modalities and the potential for direct connection to mass spectrometers at nanospray flow rates.     

Very high pressure gradient LC/MS/MS

A very high pressure liquid chromatography (VHPLC) system was constructed by modifying a commercially available pump in order to achieve pressures in excess of 1,200 bar (17,500 psi). A computer-controlled low-pressure mixer was used to generate solvent gradients. Protein digests were rapidly analyzed by reversed-phase VHPLC with linear solvent gradients coupled to either a tandem mass spectrometer using electrospray ionization or a UV/visible detector. The separations were performed at pressures ranging from 790 (11,500 psi) to 930 bar (13,500 psi) in 22-cm-long capillary columns packed with C18-modified 1.5-microm nonporous silica particles. A digest of bovine serum albumin (BSA) was analyzed by the VHPLC system connected to a mass spectrometer in MS mode. An analysis of 12.5 fmol of sample gave signal-to-noise ratios of tryptic peaks greater than 10:1 in the base peak plot mass chromatogram. This system was also used to analyze a proteolytic digest of a rat liver protein excised from a 2-D gel separation of a liver tissue lysate. For this analysis, the mass spectrometer was set up to perform data-dependent scanning (automated switching from MS mode to MS/MS mode when a peak was detected) for peptide sequencing and protein identification by database searching. The results of this analysis are compared to an analysis performed on the same sample using the nanoelectrospray-MS/MS technique. Though both techniques were able to identify the unknown protein, the VHPLC method gave twice as many sequenced peptides as nanoelectrospray and improved the signal-to-noise ratio of the spectra by at least a factor of 10. Direct comparisons with nanoelectrospray for MS and MS/MS data acquisition from a BSA digest were made. These comparisons show enhancements of greater than 20-fold for VHPLC over nanoelectrospray. In addition, the VHPLC/MS/MS data acquisition was accomplished in an automated manner.     

A CE-MALDI interface based on the use of prestructured sample supports

We have developed an off-line coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) based on CE fraction collection onto prestructured MALDI sample supports. Analyte carryover and detection sensitivity were investigated using a standard peptide mixture. Low femtomole amounts were detected, and no noticeable carryover was discovered. The performance of the method was evaluated with a mixture of tryptic digests of proteins from a human fetal brain cDNA expression library. The total number of identified peptides was increased from 47 to 211 when the CE-MALDI interface was used compared to direct MALDI-MS analysis. Sequence coverage with CE-MALDI was in the 25-60% range for the different proteins, corresponding to an increase of 1.3-4.9 times relative to that obtained with MALDI-MS of the crude mixture. Fractionation of sample components also facilitated protein identification by MALDI postsource decay analysis. Our initial results suggest this CE-MALDI interface can be used for the analysis of complex peptide mixtures isolated from biological tissues.     

Determination of Four Types of Hazardous Chemicals in Food Contact Materials by UHPLC‐MS/MS

A simple ultrahigh‐performance liquid chromatography (UHPLC) tandem mass spectrometric method for the identification and quantification of two photoinitiators 4‐methylbenzophenone and 2‐ethylhexyl‐4‐dimethylaminobenzoate; nine plasticizers including di(2‐ethylhexyl) adipate and diisobutyl adipate; three primary aromatic amines 4‐aminobiphenyl, 4‐amino‐2′,3‐dimethylazobenzene and bis‐(4‐aminophenyl) methane and six bisphenols bisphenol F diglycidyl ether, bisphenol A diglycidyl ether, bisphenol A (BPA), bisphenol B (BPB), bisphenol E (BPE) and bisphenol F (BPF) in food contact materials has been developed. The chromatographic conditions, pre‐treatment methods and matrix effects were studied and optimized. For the determination of the four bisphenols, BPA, BPB, BPE and BPF, the UHPLC method employed a mobile phase of aqueous ammonia and methanol in binary gradient mode, and measurement was based on a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in negative ion mode. The remaining chemicals were determined using the ESI source in positive ion mode and using the [M + NH₄]⁺ or [M + H]⁺ adducts as precursor ions for tandem mass spectrometry. The calibration graphs were linear with correlation coefficients of above 0.995. Detection limits for the method were in the range of 1–16 µg/kg. Analyte recovery values were in the range of 70–114%, and relative standard deviations were 1–14%. Under optimized conditions, the chromatographic separation was performed in 12 min. The validation data indicated that the method was effective for the determination of the four classes of hazardous chemicals in plastic packaging materials or in can lacquers. The optimized method was successfully applied to trace analysis of commercially available food contact materials. Copyright © 2014 John Wiley & Sons, Ltd.     

The use of ultra high-performance liquid chromatography for studying hydrolysis kinetics of CL-20 and related energetic compounds

Ultra high-performance liquid chromatography (UHPLC) utilizes columns packed with sub-2-mum stationary-phase particles and allows operation with pressures of up to 15,000 psi to yield increased resolution, speed, and sensitivity versus conventional HPLC. This promising new technology was used for the analysis of energetic compounds (RDX, HMX and CL-20) and a selective method was developed on an Acquity UPLC. A fast UHPLC method was applied to determine alkaline hydrolysis reaction kinetics of major energetic compounds. Activation energies of alkaline hydrolysis reaction for CL-20, RDX and HMX were comparable to those in literature, however they were determined in a shorter amount of time due to the speed of analysis of the chromatographic method. The use of liophilic salts (KPF(6)) as mobile-phase additives for the enhancement of separation selectivity of energetic compounds was demonstrated.     

聚丙烯对超高分子量聚乙烯加工流变性能的影响

PP能显著提高UHMWPE的加工流动性能，一定量PP的加入可使UHMWPE在通用加工设备上进行塑化加工．纯UHMWPE不存在常见的假塑性流动区，在低剪切速率下就无法正常挤出，挤出压力振荡不定．一定量PP的加入，UHMWPE呈现假塑性流动，未出现压力振荡现象。     

Automated 20 kpsi RPLC-MS and MS/MS with chromatographic peak capacities of 1000-1500 and capabilities in proteomics and metabolomics

Proteomics analysis based-on reversed-phase liquid chromatography (RPLC) is widely practiced; however, variations providing cutting-edge RPLC performance have generally not been adopted even though their benefits are well established. Here, we describe an automated format 20 kpsi RPLC system for proteomics and metabolomics that includes on-line coupling of micro-solid phase extraction for sample loading and allows electrospray ionization emitters to be readily replaced. The system uses 50 microm i.d. x 40-200 cm fused-silica capillaries packed with 1.4-3-microm porous C18-bonded silica particles to obtain chromatographic peak capacities of 1000-1500 for complex peptide and metabolite mixtures. This separation quality provided high-confidence identifications of >12 000 different tryptic peptides from >2000 distinct Shewanella oneidensis proteins (approximately 40% of the proteins predicted for the S. oneidensis proteome) in a single 12-h ion trap tandem mass spectrometry (MS/MS) analysis. The protein identification reproducibility approached 90% between replicate experiments. The average protein MS/MS identification rate exceeded 10 proteins/min, and 1207 proteins were identified in 120 min through assignment of 5944 different peptides. The proteomic analysis dynamic range of the 20 kpsi RPLC-ion trap MS/MS was approximately 10(6) based on analyses of a human blood plasma sample, for which 835 distinct proteins were identified with high confidence in a single 12-h run. A single run of the 20 kpsi RPLC-accurate mass MS detected >5000 different compounds from a metabolomics sample.     

Die Scheiteldruckprüfung korrosionsbeanspruchter Hoch- und Höchstdruckrohre zur Untersuchung des Bauteil-Rißwachstumsverhaltens unter Modus II-Schwingungsrißkorrosion

External Crown Fatigue Loading of High and Ultrahigh Pressure Tubes Subjected to Corrosion – a Highly Informative Test Predicting the Crack Growth Behaviour of Tubular Components under Mode II Corrosion Fatigue Conditions Stressing high and ultrahigh pressure tubes by external static or fatigue loads has been qualified as a convenient method to simulate the load case “internal pulsating pressure” by analysing the stress state of thick walled tubes when loaded by internal pressure and external crown loads, respectively. The results of different analytical calculations were compared with that of a Finite-Element-Computation demonstrating, for tubes with nominal pressures in the range of 325–3600 bar, an excellent correspondence. Tests with 86 tube cuttings of steel X 6 CrNiMoTi 17 12 2 (W.-No. 1.4571; ASTM UNS S 31635; BS 320531) showed the following results: In air, pulsating pressures of 325 bar (corresponding to the maximum allowable operating pressures) are sustained indefinitely. Under mode II-corrosion fatigue in 0,1 N H₂SO₄ (30°C) failure occures after 3,8 · 10⁷ mode cycles. A twentyfold H₂SO₄ concentration will lower the number of cycles to fracture to one tenth of this value without leaving mode II. Under mode II corrosion fatigue crack growth will propagate faster in radial direction than in air, so that leak-before-break under internal pressure will be likely. Crack growth rates in radial direction increased with increasing acid concentration so that the probability for leak-before-break will further increase. Highest priority for the surveillance strategy of components loaded in mode II CF has, however, the prove that crack initiation in this environment is commencing much earlier than in air, and definitely earlier than found for compact specimens tested in a mode II pulsating fatigue or rotating bending test.     

CMUTS with dual-electrode structure for improved transmit and receive performance

In this paper, we introduce capacitive micro-machined ultrasonic transducers (CMUTs) with electrically isolated multiple electrodes embedded in the same silicon nitride CMUT membrane. Some of the advantages of this structure are demonstrated using a dual-electrode CMUT with separate transmit and receive electrodes as an example. By locating the transmit electrodes near the edges of a rectangular CMUT membrane, the stable displacement range, hence the maximum pressure amplitude during transmit mode is increased without collapsing the membrane when operated within static collapse voltage range. In the receive mode, the center receive electrode is brought closer to the substrate by biasing the side electrodes, and a higher electromechanical transformer ratio is obtained at low direct current (DC) bias. Therefore, dual-electrode CMUT has an effectively larger gap as compared to conventional CMUT during transmit, and it has an effectively smaller gap during receive. Demonstrative experiments are performed on dual-electrode CMUTs with rectangular membranes with different side and center electrode sizes for transmit and receive measurements. By using the two 4-microm wide side electrodes and an 8-microm wide center electrode on a 20-microm wide membrane, a 6.8 dB increase in maximum output pressure is obtained with side electrode excitation as compared to conventional center electrode. Similarly, the receive performance improvement was demonstrated while reducing the DC bias requirements. Simple finite-element and equivalent circuit-based models were developed to successfully model the behavior of dual-electrode CMUTs. Simulations show that, with simple modifications, more than 10 dB overall sensitivity improvement is feasible with dual-electrode CMUTs with rectangular membranes.     

Giant Barocaloric Effect at the Spin Crossover Transition of a Molecular Crystal

The first experimental evidence for a giant, conventional barocaloric effect (BCE) associated with a pressure‐driven spin crossover transition near room temperature is provided. Magnetometry, neutron scattering, and calorimetry are used to explore the pressure dependence of the SCO phase transition in polycrystalline samples of protonated and partially deuterated [FeL₂][BF₄]₂ [L = 2,6‐di(pyrazol‐1‐yl)pyridine] at applied pressures of up to 120 MPa (1200 bar). The data indicate that, for a pressure change of only 0–300 bar (0–30 MPa), an adiabatic temperature change of 3 K is observed at 262 K or 257 K in the protonated and deuterated materials, respectively. This BCE is equivalent to the magnetocaloric effect (MCE) observed in gadolinium in a magnetic field change of 0–1 Tesla. The work confirms recent predictions that giant, conventional BCEs will be found in a wide range of SCO compounds.     

Pulsed ion beam characterization of CVD diamond surfaces under thin film deposition conditions

Diamond and diamond-like carbon have properties which in principle make them ideally suited to a wide variety of thin-film applications. The widespread use of diamond thin films, however, has been limited for a number of reasons related largely to the lack of understanding and control of the nucleation and growth processes. Real-time, in-situ studies of the surface of the growing diamond film are experimentally difficult because these films are normally grown under a relatively high pressure of hydrogen, and conventional surface analytical methods require an ultrahigh vacuum environment. Pulsed ion beam based analytical methods with differentially pumped ion sources and particle detectors are able to characterize the uppermost atomic layer of a film during growth at ambient pressures in the range 0.7–27 Pa (4–6 orders of magnitude higher than other surface-specific analytical methods). We describe here a system which has been developed for the purpose of determining the hydrogen concentration and bonding sites on diamond surfaces as a function of sample temperature and ambient hydrogen pressure under hot-filament chemical vapor deposition (CVD) growth conditions. It is demonstrated that as the hydrogen partial pressure increases the saturation hydrogen coverage of the surface of a CVD diamond film increases, but that the saturation level depends on the atomic hydrogen concentration and substrate temperature. At the highest temperatures studied (700 °C), it was found that the surface hydrogen concentration did not exceed 1/4 monolayer.     

Effects of pressure on the pore formation of carbon/carbon composites during carbonization

In this study, porosity and graphitizability of coal tar pitch with the treatment pressure were investigated. 4-directional carbon/carbon composites (4D C/C) were made from the matrix precursor of coal tar pitch through the process of impregnation and carbonization. Then the effects of applied pressure during the densification on the composites were observed. The matrix pitch which had 600 bar applied during the carbonization process had one and a half times less pore area ratio than that treated at 1 bar. When the pitch was heat treated up to 2300°C after the high pressure carbonization, the degree of graphitization was improved on a small scale and the crystal size tended to reduce. As the applied pressures to 4D C/C composites increased from 1 to 600 bar, the densification ratio was greatly improved. In the pore size distribution of the 4D C/C composites, the macropore portion was decreased while the mesopore portion increased, when high pressures were applied.     

Multistep ultrahigh performance liquid chromatography/tandem mass spectrometry analysis for untargeted quantification of glycating activity and identification of most relevant glycation products

The use of advanced glycation end-products (AGEs) as biomarkers for diagnosis and clinical studies is still hampered by insufficient knowledge on clinically relevant structures formed from precursors associated with defined disease states. The present study conducted untargeted analysis of the glycating activity of AGE-precursors by ultrahigh performance liquid chromatography/tandem mass spectrometry multiple reaction monitoring (UHPLC/MSMS-MRM), monitoring the loss of a nonapeptide as the glycation target. Thus, the glycating activities of seven important AGE-precursors were determined (glucose 13% and the reactive carbonyl compounds glucosone 39%, 3-deoxyglucosone 15%, 3-deoxygalactosone 26%, 3,4-dideoxyglucosone-3-ene 79%, methylglyoxal 94%, and glyoxal 97% peptide loss; 12 h/37 °C). Furthermore, UHPLC/MSMS with simultaneous precursor ion scan and information-dependent acquisition of enhanced resolution spectra and subsequent product ion scan was applied for untargeted analysis of the major AGE-structures derived from various AGE-precursors. The 20 most important modifications could be assigned to 8 AGE-structures previously reported in the literature. Seven loosely bound AGEs not yet covered by conventional methods were detected and assigned to hemiaminals. Five AGE structures did not match any known products. The method can be applied to analyze glycating activity and AGE-structures formed from various other precursors under defined reaction conditions, supporting the selection and evaluation of diagnostic AGE-markers for clinical studies.     

Mass recovery adsorption refrigeration cycle—improving cooling capacity

The study investigates the performance of two-bed, silica gel-water adsorption refrigeration cycle with mass recovery process. The cycle with mass recovery can be driven by the relatively low temperature heat source. In an adsorption refrigeration cycle, the pressures in adsorber and desorber are different. The chiller with mass recovery process utilizes the pressure difference to enhance the refrigerant mass circulation. Cooling capacity and coefficient of performance (COP) were calculated by cycle simulation computer program to analyze the influences of operating conditions. The mass recovery cycle was compared with conventional cycle such as the single stage adsorption cycle in terms of cooling capacity and COP. The results show that the cooling capacity of mass recovery cycle is superior to that of conventional cycle and the mass recovery process is more effective for low regenerating temperature.     

Thermodynamics of Refractory Nuclear Materials Studied by Mass Spectrometry of Laser-Produced Vapors

A new method of high-temperature mass spectrometry (MS) with laser-induced vaporization (LIV) has been developed. The initial problem of LIV MS, consisting of an inadequate correlation between the temperature of the surface and the MS signal, was successfully overcome.The method was developed on graphite, of which fast time-resolved MS measurements (ca. 20 ms) were performed over a large mass interval; the influence of geometrical parameters and of the laser pulse length on MS measurements was studied. Carbon sublimation relative partial pressures of C₁, C₂, C₃, and C₅ were measured up to 3810 K. This corresponds to a total pressure of about 0.8 bar estimated independently by the integral mass flux using the Hertz–Knudsen equation. The vaporization of UO₂ was studied at temperatures above ≈ 2500 K, where conventional Knudsen-cell mass spectrometry cannot be applied. The vaporization enthalpy obtained for the main species in UO₂ vapor was in good agreement with that of conventional mass spectrometry. Paper presented at the Seventh International Workshop on Subsecond Thermophysics, October 6–8, 2004, Orléans, France.     

Separation of peptides from myoglobin enzymatic digests by RPLC. Influence of the mobile-phase composition and the pressure on the retention and separation

The influence of the mobile-phase composition and the pressure on the chromatographic separation of the peptides from the enzymatic digest of myoglobin was studied under linear conditions. The retention behavior of these tryptic peptides was measured under isocratic conditions with different mobile-phase compositions, ranging from 9 to 28% (v/v) acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid. The effect of the pressure was studied by analyzing the separation of the tryptic peptides under different average column pressures between 14 and 220 bar, at 13, 20, and 26% (v/v) acetonitrile. The differences between the partial molar volumes of these peptides in the stationary and mobile phases were derived from these results. All the measurements were performed on a 10-cm-long C18-bonded, end-capped monolithic column. The results obtained illustrate the highly complicated behavior of the complex peptide mixtures afforded by tryptic digestion. The capacity factors of the analyzed peptides do not depend linearly on the acetonitrile concentration but follow exactly a quadratic relationship. The adsorption changes of partial molar volumes are in good agreement with other literature data. The consequences of the influence of the average column pressure (hence of the flow rate) on the column phase ratio and on the retention factors of the peptides are discussed. The retention pattern of the complex mixture is affected by both the mobile-phase composition and the pressure, and the resolution of certain peptide pairs is so much affected by the pressure that inversions in the elution order of some pairs are observed.