Eluent Temperature Research Articles

Studies on the Effects of Process Conditions on Separation of B1, B2 and B3 Vitamin Mixture Using HILIC and RPLC Chromatography

In this work, a series of experiments on the retention of B1 (riboflavin), B2 (thiamine) and B3 (nicotinic acid) vitamins in the HILIC and RPLC conditions have been performed involving the effects of organic modifier type and content, pH of the eluent and buffering salt (ammonium acetate) concentration in the mobile phase as well as temperature of the system. For the needs of this study, three columns of different features have been chosen: Acclaim™ Mixed–Mode HILIC–1 (Dionex, Sunnyvale, CA, USA), Eurospher II 100–5 HILIC (Knauer, Berlin, Germany) and the RPLC column Nucleodur® C18 Gravity-SB (Macherey-Nagel, Duren, Germany). The influence of acetonitrile and methanol content in the eluent and process temperature have been tested and, based on that, the most promising systems have been selected regarding the possible separation of the vitamin mixture. Both the pH and buffering salt concentrations in the eluent have been adjusted in order to indicate the most effective system, which turned out to be the one involving the Nucleodur column and the eluent with 90% methanol, at pH 6 and Cbuff = 20 mmol/dm3, which enables separation of the mixture within a time as short as 2.5 min at a 1.0 mL/s flowrate in isocratic conditions.

DEVELOPMENT OF THE TECHNOLOGY FOR THE SEPARATION OF THE COMPONENTS FROM GREEN TEA USING THE PREPARATIVE LIQUID CHROMATOGRAPHY BY DESIGN OF PERIODICAL PROCESS

Leaves of the green tea plant camellis sinensis contain up to 36 % polyphenols. Epigallocatechin gallate (EGCG) is the most interesting polyphenols because it exhibits a strong antioxidant effect. Furthermore, it has been demonstrated that EGCG has an antimutagenic and an anticancer effects, an antibacterial effect and a beneficial effect on cholesterol level in blood. Therefore, is needed to isolate EGCG in a pure form in high yield by simple and commercial process. This method for isolation of EGCG contents the following steps: a) selection of macroporous polar resin for the preparative liquid chromatography; b) selection of polar elution solvent; c) determination the temperature and pressure for adsorbtion and desorbtion of the polyphenols of the green tea. Therefore, it was searched the interconnection between yield of EGCG and the range of temperature and amount of eluent. Finally, the process for the separation of Epigallocatechin gallate from green tea extract was developed in the laboratory and EGCG was isolated with the concentration 92% by the yield 43%. Accordingly, the technology of EGCG production based on the preparative liquid chromatography was launched and introduced on the market.

Gel-permeation chromatography as a method for monitoring transformations of heavy petroleum fractions during secondary oil refining

A rapid and easy to use method for monitoring transformations of heavy oil fractions during secondary processes of oil refining is proposed. The method is based on the study of changes in the molecular weight characteristics by gel-permeation chromatography (GPC) using a refractometric detector. Optimal conditions for registration of the chromatograms (temperature, eluent consumption and its composition) were specified. Heptane, toluene, and toluene added with 1% and 5% of methanol were studied as eluents, the polarity indices being arranged in a series 0.2; 2.4; 2.43; 2.54. The effect of the nature of the solvent on the type of the product chromatogram is determined. It is shown that addition of a polar solvent to a nonpolar one increases the polarity index, reduces both the degree of association of petroleum molecules and adsorption on the gel. The most available toluene which meets the GPC requirements is chosen as an eluent. Addition of 1% CH3OH to the eluent promotes suppression of polyelectrolyte effects and adsorption. The revealed absence of the dependence of the retained volume and the type of chromatograms on the eluent flow rate and temperature indicates that separation is carried out under conditions of exclusion and completely obeys the theory of exclusive liquid chromatography. The transformation of raw materials during a technological process of oil production at the JSC «ANKhK» was studied using the GPC method under above-mentioned chromatographic conditions. We have studied vacuum distillate of the primary oil refining unit ELOU+AVT-6 (VD) and the product of hydrocracking of the vacuum distillate of the 123PM unit (HVD). It is shown that determination of the molecular weight characteristics provides an important information about the behavior of technological processes. The HVD meets the requirements for hydrocracking products. A decrease in the content of aromatic hydrocarbons in comparison with the raw feedstock is proved by the data of thin layer chromatography (TLC).

Enhanced Anaerobic Mixed Culture Fermentation with Anion-Exchange Resin for Caproate Production

The bioproduction of caproate from organic waste by anaerobic mixed culture is a very attractive technology for upgrading low-grade biomass to a high-value resource. However, the caproate production process is markedly restricted by the feedback inhibition of caproate. In this study, four types of anion-exchange resin were investigated for their enhancing capability in caproate fermentation of anaerobic mixed culture. The strong base anion-exchange resin D201 showed the highest adsorption capacity (62 mg/g), selectivity (7.50), and desorption efficiency (88.2%) for caproate among the test resins. Subsequently, the optimal desorption temperature and NaOH concentration of eluent for D201 were determined. The adsorption and desorption efficiency of D201 remained stable during eight rounds of the adsorption–desorption cycle, indicating a satisfactory reusability of D201. Finally, performances of caproate fermentation with and without resin adsorption for carboxylate were evaluated. The results demonstrated that the final concentration of caproate was improved from 12.43 ± 0.29 g/L (without adsorption) to 17.30 ± 0.13 g/L (with adsorption) and the maximum caproate production rate was improved from 0.60 ± 0.01 g/L/d to 2.03 ± 0.02 g/L/d. In the group with adsorption, the cumulative caproate production was increased to 29.10 ± 0.33 g/L broth, which was 134% higher than that of the control group. Therefore, this study provides effective approaches to enhance caproate production.

Solvation characteristics of porous graphitic carbon Hypercarb in subcritical water chromatography

Retention of 17 organic compounds was studied in high-temperature liquid chromatography mode on Hypercarb porous graphitic carbon with subcritical water as an eluent. Three different eluent temperatures were studied: 150, 175 and 200 °C. Abraham solvation parameters model was used for quantitative evaluation of retention data. It was found that increase of eluent temperature from 150 to 200 °C results in decrease of cavity formation system parameter v from 2.9 to 1.7. No clear tendency can be observed for temperature dependency of other system parameters.

Sorption technology of indium extraction from zinc production solutions

The article studies the dynamic sorption of indium ions from polycomponent solutions with pre-reduced iron (III) containing, g/dm3: 0,084 In3+, 6,2 Fe2+, 67,0 Zn2+ and 19,6 H2SO4, on the Metosol reagent. The latter is a montmorillonite with the following composition (Na,Ca)0,3(Al,Mg)2Si4O10(OH)2(H2O)n, modified by di(2-ethylhexyl) phosphoric acid. Its absorption dynamics of metal ions was studied by the method of frontal chromatography. The article determines the values of working (DEC) and total (TDEC) dynamic exchange capacities of the mineral sorbent depending on the specific throughput rate and the eluent temperature. The article justifies and calculates the basic parameters of the sorption technology of selective indium extraction from process solutions used for zinc production operations on the Metosol reagent in columns with subsequent desorption of metal by hydrochloric acid solution (1 : 1).

Repeatability of gradient ultrahigh pressure liquid chromatography–tandem mass spectrometry methods in instrument-controlled thermal environments

The impact of viscous friction on eluent temperature and column efficiency in liquid chromatography is of renewed interest as the need for pressures exceeding 1000bar to use with columns packed with sub-2μm particles has grown. One way the development of axial and radial temperature gradients that arise due to viscous friction can be affected is by the thermal environment the column is placed in. In this study, a new column oven integrated into an ultrahigh pressure liquid chromatograph that enables both still-air and forced-air operating modes is investigated to find the magnitude of the effect of the axial thermal gradient that forms in 2.1×100mm columns packed with sub-2μm particles in these modes. Temperature increases of nearly 30K were observed when the generated power of the column exceeded 25W/m. The impact of the heating due to viscous friction on the repeatability of peak capacity, elution time, and peak area ratio to an internal standard for a gradient UHPLC-MS/MS method to analyze neurotransmitters was found to be limited. This result indicates that high speed UHPLC-MS/MS gradient methods under conditions of high viscous friction may be possible without the negative effects typically observed with isocratic separations under similar conditions.

Application of modified nano-γ-alumina as an efficient adsorbent for removing malachite green (MG) from aqueous solution

This study describes a simple, inexpensive and sensitive extraction procedure using γ-Al2O3 nanoparticles modified with sodiumdodecylsulfate, as an efficient solid phase, for removal, preconcentration and spectrophotometric determination of trace amounts of malachite green (MG) by fiber optic-linear array detection spectrophotometry. The properties of the produced modified nanoparticles were determined by FESEM and TEM. Various effective parameters on the preconcentration of MG, such as pH, sample volume, amount of sorbent, type, concentration, contact time, temperature, and volume of eluent were investigated. Under the optimized operating conditions, the calibration graph was linear over the range of 1.2–250 μg L−1 and relative standard deviation of 1.50% at 100 μg L−1 were obtained (n = 5). The limit of detection (S/N = 3) was obtained 0.34 μg L−1. The Langmuir adsorption capacity (qmax) was found to be 72.4 mg g−1 of the adsorbent. The method has been successfully used to determine of MG in fish farming water with satisfactory results.

Effects of eluent temperature and elution bandwidth on detection response for aerosol-based detectors

Aerosol detectors provide generally uniform response for most analytes, independent of their optical properties, and have the advantage of being compatible with elevated temperature mobile phases. Therefore, aerosol detectors present an attractive detection alternative for high temperature liquid chromatography (HTLC) separations. The present study has investigated the effects of HTLC conditions using aqueous mobile phases on the detection response of an evaporative light-scattering detector (ELSD) and a corona-charged aerosol detector (C-CAD). The response of the ELSD was increased up to 5-fold by increasing the separation temperature from 30°C to 180°C. The C-CAD showed much smaller increases in response under the same conditions. This increase in response was found not to result from the increased temperature for the mobile phase but rather from compression of the elution bandwidth at elevated temperature. The effect of bandwidth on detector response was confirmed using flow-injection studies in which the same amount of analyte was introduced into the detector at varying bandwidths. Furthermore, it is shown that a temperature gradient can be used to counteract the effects of varying bandwidths associated with isocratic–isothermal separations, with relatively constant bandwidth and detector response being observed with appropriate temperature gradients. This study demonstrates the necessity to consider the elution bandwidth in HTLC-aerosol detector analysis.

Realization and potential advantages of gradient separations performed under steady state temperature regime

This paper describes a new method designed to perform gradient separations while keeping constant the distribution of temperature throughout the column during the three phases of a gradient run (the ramp up of the strong eluent concentration, the column wash, and the re-equilibration steps) in high pressure liquid chromatography (vHPLC). The method is based on the solution of the integrated heat balance equation along the column knowing the variations of the eluent specific heat, its thermal expansion coefficient, and its viscosity with the concentration of the strong eluent. This new method is called gradient under constant wall heat (cWH). Its value is demonstrated by the stability of the eluent temperature at the column outlet during a series of consecutive gradient runs. The potential advantages of cWH gradients over classical gradients execution mode at constant flow rate (cF) are illustrated in the example of two such gradients run in the same time. They include (1) a diminution by a factor 8 of the largest temperature variation during the sequence of injections (0.4°C versus 3.2°C); (2) a reduction of the baseline noise by a factor two (0.05mAU versus 0.10mAU); and (3) the achievement of an excellent injection-to-injection repeatability of the vHPLC system used to measure the first (average RSDs, 0.04%) and the second central (average RSDs≃0.75%) moments of the peaks of an eight-compounds mix, baseline separated, from the first to the last injection.

Gradient chromatography under constant frictional heat: Realization and application

A new mode of gradient elution in liquid chromatography is proposed. It is derived from simple theoretical considerations and is particularly suitable for applications to fast, very-high pressure gradients. It is designed to improve the injection-to-injection repeatability of chromatographic runs at either constant flow (cF, cooling case scenario) or constant pressure (cP, heating case scenario). The purpose of this original gradient mode is to minimize the variations of the temperatures and the pressures across and along the column during the gradient time. These variations are caused by the heat generated in the column due to the friction of the eluent percolating the bed. Any temperature fluctuation affects to some extent the precision of the measurements of retention times and bandwidths of eluted compounds. The minimization of this effect was achieved by maintaining constant the frictional heat power (i.e., the product of the flow rate by the column pressure drop) generated during the gradient run, the washing step, and the re-equilibration time. The eluent temperature was recorded at the column outlet. One useful application of gradient chromatography at constant frictional heat (cFH) is illustrated for a 50–100% volume gradient of acetonitrile in water using a 4.6mm × 150mm column packed with 3.5μm BEH-C18 particles and operated with an Agilent 1290 Infinity liquid chromatograph. The reproducibility (eleven consecutive injections) of the retention times and peak variances of nine small molecules (RPLC check-out sample mixture) using cF, cP, and cFH gradients were compared for the same amount of heat produced (403J) during each run time. The RSDs of the retention times and the peak variances for four consecutive injections were systematically below 0.035 and 0.50% in constant frictional heat gradient chromatography, after three initial injections. These RSDs were markedly higher for cF (0.050 and 0.90%) and cP (0.070 and 1.80%) gradients.

Chromatographic Separation and Characterization of Capsaicinoids and Their Analogues

An attempt was made to establish a chromatographic separation and analysis method for capsaicinoids and their analogues. A series of factors,such as choice of column and eluents, eluent composition,flow rate of eluent and operational temperature were correlated, countereacted and optimized to attain appropriate separation efficiency and finalize the experimentally oprational procedures as a whole, permiting a mixture comprising 8 capsaicinoids including analogues to be separated and Characterized with an allowed deviations and within a period of 36 minutes via 1 test as well.

Heat Exchanges in Fast, High-Performance Liquid Chromatography. A Complete Thermodynamic Study

The successive physical transformations of the mobile phase that take place in very high pressure liquid chromatography were studied based on the formalism of classical thermodynamics. The eluent is initially under atmospheric pressure ( P (0)) and at ambient temperature ( T ext). In a first step, it is compressed to a high pressure ( P max of the order of 1 kbar) in the pump heads of the chromatograph. In a second step, the pressurized eluent is transferred to the inlet of the chromatographic column, along which, in a third step, it is decompressed to atmospheric pressure. Both the compression and the decompression of the fluid were considered to take place under conditions that can be either adiabatic or nonadiabatic and either reversible or irreversible. Applications of the first and second principles of thermodynamics allow the determination of the heat and energy exchanged between the eluent and the external surroundings during each transformation. Experimental data were acquired using acetonitrile as the mobile phase. The true state equation, rho( P, T), of liquid acetonitrile was used in the theoretical calculations. A series of four different flow rates (0.55, 0.85, 1.15, and 1.45 mL/min, corresponding to inlet pressures of 357.2, 559.5, 765.1, and 972.9 bar, respectively), were applied to a 2.1 x 100 mm column packed with 1.7-mum bridged ethane-silicon hybrid particles. Thermocouples were used to measure the eluent temperature before and after its passage through the column. These data provide estimates of the variation of the internal energy of the eluent. The heat lost through the external wall of the column during the eluent decompression was estimated by measuring the surface temperature of the column tube under steady state. Both the compression and the decompression of acetonitrile were found to be nonadiabatic and irreversible transformations. The results showed that, during the eluent decompression, the heat released by the friction forces serves four different purposes: (1) it increases the eluent entropy at constant temperature (for approximately 35%); (2) it increases the temperature of the eluent (for approximately 5%); (3) it provides heat to the laboratory atmosphere (for approximately 5%); and (4) it provides some work inside the column (for approximately 5%). This quantitative heat balance description accounts well for the actual performance of the new, very high pressure liquid chromatographic technique.

Consequences of the radial heterogeneity of the column temperature at high mobile phase velocity

When a high velocity stream of mobile phase percolates through a chromatographic column, the bed cannot remain isothermal. Due to the mobile phase decompression, heat is generated along the column. Longitudinal and radial temperature gradients take place along and across its bed. The various consequences of this thermal heterogeneity are calculated and their effects on the column efficiency investigated for a 0.46cm×25cm stainless steel column packed with 5μm particles. The maximum pressure drop applied was varied from 0.1 to 2kbar. The amplitude of the longitudinal temperature gradient can be estimated on the basis of the integral heat balance equation applied to the whole column and of measurements of the eluent temperature at the column exit. Assuming that the radial gradient is parabolic and the longitudinal gradient linear, the amplitude of the radial gradient can be determined on the basis of the energy balance across the column and of direct measurements of the radial gradient at high inlet pressures. A radial temperature gradient causes a radial distribution of the eluent viscosity, hence of its local velocity. The result is that bands move faster in their center than along the wall, become warped, hence a radial concentration gradient, similar in origin to the one observed in open cylindrical tubes. Diffusion relaxes this gradient. If there is only a longitudinal temperature gradient, the column efficiency would be 30% smaller for a 2kbar pressure drop than if there is no longitudinal temperature gradient. However, when both a longitudinal and a radial temperature gradient coexist, there is a large loss of efficiency. If the influence of the diffusive relaxation of the radial concentration gradient is neglected, the peak shape would be broad and exhibit a marked shoulder.

Optimization strategies for separation of sulfadiazines using Box-Behnken design by liquid chromatography and capillary electrophoresis

Development of effective chromatographic or electrophoretic separation involves judicious deciding of selection of optimal experimental conditions that can provide an adequate resolution at a reasonable run time for the separation of interested components. Box-Behnken factorial design was effectively applied for the separation optimization of eight structurally related sulfonamides using capillary zone electrophorosis and reverse high performance liquid chromatography. Optimum values for volume ratio of THF to H2O in eluent, column temperature and flow rate of eluent are found as 12 to 88, 35 °C and 1.0 mL/min, respectively. Box-Behnken modified optimization model is extended to separation by capillary electrophoresis (CE). While using CE, a satisfactory separation is achieved with a minimum resolution larger than 1.0 for a separation time less than 10 min.

Development and application of a specially designed heating system for temperature-programmed high-performance liquid chromatography using subcritical water as the mobile phase

A specially designed heating system for temperature-programmed HPLC was developed based on experimental measurements of eluent temperature inside a stainless steel capillary using a very thin thermocouple. The heating system can be operated at temperatures up to 225 °C and consists of a preheating, a column heating and a cooling unit. Fast cycle times after a temperature gradient can be realized by an internal silicone oil bath which cools down the preheating and column heating unit. Long-term thermal stability of a polybutadiene-coated zirconium dioxide column has been evaluated using a tubular oven in which the column was placed. The packing material was stable after 50 h of operation at 185 °C. A mixture containing four steroids was separated at ambient conditions using a mobile phase of 25% acetonitrile:75% deionized water and a mobile phase of pure deionized water at 185 °C using the specially designed heating system and the PBD column. Analysis time could be drastically reduced from 17 min at ambient conditions and a flow rate of 1 mL/min to only 1.2 min at 185 °C and a flow rate of 5 mL/min. At these extreme conditions, no thermal mismatch was observed and peaks were not distorted, thus underlining the performance of the developed heating system. Temperature programming was performed by separating cytostatic and antibiotic drugs with a temperature gradient using only water as the mobile phase. In contrast to an isocratic elution of this mixture at room temperature, overall analysis time could be reduced two-fold from 20 to 10 min.

Investigations into the HPLC Retention Behavior of Simvastatin and Structurally Related Compounds

The HPLC retention behavior of simvastatin and its β,δ‐dihydroxy acid and methylester forms were investigated. The retention of the lactone and the methylester was found to be highly dependent on the eluent composition and mildly dependent on temperature at low temperatures. The retention of the β,δ‐dihydroxy acid was found to be highly dependent on eluent pH and temperature as well as the composition. Atypical dependence of the retention of the β,δ‐dihydroxy acid on temperature prompted further investigation. It was determined that the observed temperature–retention relationship for the β,δ‐dihydroxy acid was caused by a shift in the w s pK of the acid under the elution conditions. As the elution temperature increases, the w s pK of the acid changes resulting in an increase in retention at low temperatures. Thermodynamic analysis shows that retention of simvastatin and the methyl ester is predominantly enthalpy driven. Similar analysis for the β,δ‐dihydroxy acid shows that retention is entropy driven at low temperatures at all pH's, but it is enthalpy driven at higher temperatures.

High Temperature Liquid Chromatography of Tocol-Derivatives on Polybutadiene-Coated Zirconia Stationary Phases

High temperature reversed-phase high performance liquid chromatography is evaluated as a new approach for the separation of vitamin E isomers (α-, γ-, δ-tocopherol, and α-, γ-, δ-tocotrienol) and α-tocopherol acetate. The separations of each analyte were examined by varying the eluent composition and column temperature. Using a polybutadiene-coated zirconia column at temperatures of 80 °C–140 °C, complete separations were achieved within 10–20 min using organic modifier (acetonitrile) in the range of 40–50%. Irregularity of the Van’t Hoff analysis was noted over the temperature studied. The plot tends to deviate from linearity at higher operating temperatures (140 °C and 150 °C) with linear deviation errors of 12.7% and 41.6%, respectively, for the mobile phase examined.

Liquid Chromatography of Macromolecules Under Limiting Conditions of Solubility (LC LCS): A Mechanistic Study

A coupled liquid chromatographic technique combining an enthalpic contribution, precipitation, and the entropic size exclusion effect is reported. Liquid chromatography under limiting conditions of solubility (LC LCS) results in the elution of the polymer solute on the front shoulder of the injection zone. In the experiments carried out herein 70/30–65/35 wt.% THF/n-hexane mixtures were found to provide a retention independent of molar masses for poly(methyl methacrylate)s up to molar mass of approximately 1000 K daltons. This far exceeds the feasible range of liquid chromatography at the critical adsorption point. The effects of the mobile phase, composition and further flow rate, injected concentration, and temperature, were investigated. The polymer retention volumes were found to be dependent on the eluent composition and temperature and to a lesser extent also on injected polymer concentration and eluent flow rate. At high molar masses and high injected concentrations, the system can be overloaded, shifting the balance in favor of precipitation, while at low molar masses and flow rates the macromolecules have time to equilibrate and the balance shifts in favor of solubility. In LC LCS the enthalpic effects leading to adsorption and partition may be sufficiently strong to result in large retention of high molar mass compounds on the stationary phase, implying that the choice of the column packing is critical.

Preparative Chiral Chromatography of trans‐Stilbene Oxide Using Cellulose tris(Phenylcarbamate), Chiralcel® OC, as Stationary Phase

Two approaches were studied to improve the productivity of preparative chiral separation. For a model case, trans‐stilbene oxide as a racemate and cellulose tris(phenylcarbamate), “CHIRALCEL® OC,” as a stationary phase were chosen. The productivity per unit CSP was improved as much as 52% by connecting two different sizes of columns (10 cm ID × 50 cm L and 5 cm ID × 50 cm L) in series. Further improvement may be possible by adjusting the eluent temperature several degrees lower than the jacket temperature of the columns. Preparative separation conditions regarding composition of mobile phase, separation temperature, loading amount of racemic substance, positions of fractions to be taken, and so on were also studied.