High-temperature Liquid Chromatography Research Articles

Development of a superheated water chromatography method for the sustainable determination of preservatives in foods and cosmetics

The present research focused on the use of high temperatures to achieve fast separations in Liquid Chromatography (LC) and minimize the consumption of organic solvents. Particularly, superheated water was employed, by exploiting the decrease in dielectric constant of water when increasing the temperature. To realize this kind of application, the availability of LC columns resistant at high temperature is mandatory. Recently, porous graphitic carbon (PGC) columns have been proposed as stationary phases resistant up to 250 °C. Hardware modifications were also necessary to guarantee the efficient heating of the mobile phase prior of entering the column, as well as its rapid cooling before detection by photodiode array (PDA). Specifically, an LC system was interfaced to a GC oven, which hosted the LC column; to achieve a fast and efficient heating of the eluent coming from the autosampler prior to enter into the column, a pre-heating tube was interposed between the autosampler outlet and the column inlet. Instead, a cooling loop was connected between the column outlet and the PDA cell. A mixture of 1-nitroalkanes was used to evaluate the performance of the system in terms of dead volumes, band broadening and peak shape, as well as chromatographic efficiency and resolution. The use of higher temperatures allowed to perform analyses at higher flow rates in reduced analysis time and was also in terms of peak bandwidth resulting to increased efficiency for the most retained compound. Moreover, van't Hoff plots were obtained at different temperature, confirming that the most retained analyte greatly benefited of the temperature increase.The system was applied to the analysis of parabens in cosmetic and food products. The method combined eco-sustainable sample preparation with high-temperature LC separation on a PGC column and was fully validated in terms of linearity, precision, accuracy and detection and quantification limits. The developed method was critically evaluated by comparing the output of different metric tools implemented in the last decade for the quantitative assessment of the greenness.

Evaluation of analytical columns suitable for high-temperature liquid-chromatography-isotope-ratio-mass-spectrometry analysis of anabolic steroids

Isotope ratio mass spectrometry (IRMS) can be used to determine the carbon isotope ratio of anabolic steroids. For example, in sports doping and food safety control, it enables determining an endogenous or synthetic origin of anabolic steroids. Generally, the steroids of interest are purified by liquid chromatography (LC) and analysed by gas chromatography combustion IRMS. LC-IRMS is not used since only mobile phases without carbon atoms can be used. For analysing mid-to apolar compounds, heated water can be used as an eluent as it has a similar polarity to a weak polar organic solvent. The silica-based columns are not robust enough at elevated temperatures in aqueous conditions. However, modified silica particles, metal oxides coated with polymers, and porous graphitic carbon are promising column materials for high-temperature LC (HT-LC) applications. Here, the stability of the stationary phase is crucial, and their chromatographic performance needs to be evaluated under the conditions mentioned above for anabolic steroid separations. Six columns using temperatures up to 200 °C were assessed, and only two were found to be appropriate. The ZirChrom-PBD column can be used for HT-LC-IRMS research purposes but is not recommended for routine laboratory practice applications due to the substantial loss of retention and resolution over time at elevated temperatures. Sachtopore-RP columns are the only suitable option for routine HT-LC-IRMS applications, even though they suffer from peak broadening over time when operating at elevated temperatures

Separation of amorphous and semicrystalline parts of LLDPE with high‐temperature interactive liquid chromatography

AbstractLinear low‐density polyethylene (LLDPE) materials may contain components, which are soluble at room temperature. These may influence physical properties or raise concerns for end‐users and regulatory bodies because LLDPE is widely used for food packaging. Due to the nonpolar and amorphous nature of the room temperature soluble part of LLDPE, its molecular characterization with common methods for polyolefin analysis, such as Temperature Rising Elution Fractionation (TREF), Crystallization Fractionation (CRYSTAF), or Differential Scanning Calorimetry (DSC), has proven to be very challenging. Separation of the amorphous part of LLDPE, as well as its semicrystalline part, is, however, realizable with high‐temperature liquid chromatography (HT‐LC), which is demonstrated for the first time in this work. This technique enables to evaluate the chemical composition distribution of the room‐temperature solubles and non‐solubles in LLDPE samples. It was found that LLDPE synthesized with metallocene catalysts contained only a very small amount of amorphous material. In contrast, LLDPE prepared with Ziegler‐Natta catalysts contained up to 14 wt.% of the amorphous copolymer. The latter was soluble at room temperature, had a weight‐average molar mass ranging from 60 to 200 kg/mol, and contained about 10 mol% more 1‐olefin than the non‐soluble part of LLDPE.

Unraveling the comonomer distribution in ethylene – vinyl ester terpolymers through liquid chromatography with infrared detection

Polyolefins are the most commercially relevant polymers by volume. A readily available feedstock and their tailor-made microstructure allow to adapt polyolefins to many fields of application. Important molecular design features of olefin copolymers are the molar mass distribution (MMD) with the corresponding average values, comonomer type, chemical composition distribution (CCD) with the corresponding average and the tacticity distribution (TD). Advanced separation techniques i.e., high-temperature gel permeation chromatography (HT-GPC) as well as its hyphenation with high-temperature high performance liquid chromatography (HT-HPLC) in the form of high-temperature two-dimensional liquid chromatography (HT 2D-LC) have been successfully applied in this work. This allowed to deeply analyze the molecular heterogeneities of complex polyolefin terpolymers consisting of ethylene, vinyl acetate and branched vinyl ester monomers. By using filter-based infrared detection, the capabilities of HT-GPC are further extended so that the distribution of methyl- and carbonyl groups could be obtained along the molar mass axis. Using porous graphitic carbon (PGC) as a stationary phase for HT-HPLC separation provided information about the CCD of these complex polyolefins from experimental data as part of the hyphenated approach of HT 2D-LC. The latter revealed the full MMD x CCD distribution function, which is the key for a comprehensive analysis of the bivariate molecular structure of the polyolefin terpolymers.

Evaluation of PGC Stationary Phases Under High-Temperature LC Conditions for the Analysis of Parabens in Food Samples

Porous graphitic carbon (PGC) columns for liquid chromatography (LC) represent an alternative to octadecyl‑bonded silica columns for the separation of both polar and nonpolar molecules. This is accomplished by exploiting the polarizability of the stationary phase interacting with the functional groups of the analytes. However, the elution of nonpolar compounds requires a high percentage of organic solvent, losing the intrinsic advantage of reversed‑phase aqueous separations. In this article, we aimed to exploit an additional advantage of such columns, viz. the resistance at high temperatures. Superheated water was employed as the mobile phase, taking advantage of the decrease in water dielectric constant by increasing the temperature. In this context, our goal was to minimize the percentage of organic solvent utilizing high temperatures (up to 250 °C) to achieve fast and “green” separations. The new developed high-temperature LC instrument was applied to the analysis of parabens in food samples.

Reverse Engineering of Chemically Similar Bimodal High Density Polyethylenes: A Comprehensive Study Using Advanced Chromatographic Techniques

Back Cover: In article 2200149, Anthony Ndiripo, Albert Johannes van Reenen, and co-workers demonstrate the linking of mechanical properties of bimodal high-density polyethylene to molecular structure using tailored advanced separation techniques. Chemically similar resins with different end-use properties can be efficiently fractionated using tailored high-temperature twodimensional liquid chromatography.

Extra-thermodynamic study of the retention of anti-inflammatory 2-arylpropionic acid derivatives on a heat-resistive stationary phase: Application of HTLC approach for pharmaceutical and biological analysis

A study of the retention behavior of four frequently prescribed anti-inflammatory 2-arylpropionic acid derivatives on a reversed-phase thermostable stationary phase was conducted. Extra-thermodynamic investigations were carried out over the temperature range of 303–338 K. A modified van’t Hoff analysis was applied to assess the enthalpy, entropy, and Gibb’s free energy changes that take place during the mass transfer from the mobile phase to the stationary phase. It has been proven that enthalpy–entropy compensation exists as an extra-thermodynamic relationship. The effect of molecular structures of the investigated drugs on thermodynamic and retention behaviors was also explored. A validated high-temperature liquid chromatography method was established for the separation and analysis of ketoprofen, naproxen, diclofenac, and ibuprofen in pharmaceutical formulations and human urine with %recoveries within the range of 90.1–105.6 % and standard deviation not exceeding ±4.1% in all cases. The developed method greenness was positively assessed by the analytical eco-scale score tool.

Green method development approach of superheated water liquid chromatography for separation and trace determination of non-steroidal anti-inflammatory compounds in pharmaceutical and water samples and their extraction

Non-steroidal anti-inflammatory drugs (NSAIDs) are pharmaceutical compounds with anti-inflammatory, analgesic, and antipyretic effects. Herein, a simple and rapid high-temperature liquid chromatography and superheated water chromatography method was developed and validated for the trace determination of NSAID residues of ketoprofen, naproxen, sodium diclofenac, and ibuprofen in water samples. The NSAIDs were separated in less than five minutes using buffered distilled water as the mobile phase and ODS Zirconia RP-C18 column as the stationary phase. Linearity was observed in the van’t Hoff plots of the tested drugs by employing a low acetonitrile percentage (20% ACN) in the mobile phase, without any significant changes in their retention mechanisms. However, nonlinear van’t Hoff plots were obtained for the superheated water chromatography data of the tested drugs because of significant changes in their retention factors, transition stage of the stationary phase, or the mobile phase properties. The limits of detection for ketoprofen, naproxen, sodium diclofenac, and ibuprofen were 14, 2, 4.2, and 32 µg L −1 , respectively, and their limits of quantification were 44, 8, 12, and 98 µg L −1 , respectively. The accuracy and precision parameters were determined for selected drugs, where the relative standard deviations were in the range of ± 0.2179–2.6741%. In addition, these conditions were employed for the removal of NSAIDs from the water samples using carbon nanotubes. The proposed system was applied for the separation and analyses of drugs in water and pharmaceutical samples, and acceptable recoveries of 90.48–98.15% for the water samples and 99.9–100.08% for the pharmaceutical samples were obtained.

Investigation on the Solubility of the Antidepressant Drug Escitalopram in Subcritical Water

Escitalopram (ESC), the S-enantiomer of citalopram, is a selective serotonin reuptake inhibitor. Escitalopram is widely used to treat panic disorder, depression, obsessive compulsive disorder, and social anxiety disorder. The main goal of this present research is to determine the aqueous solubility of ESC at temperatures ranging from 298 to 473 K and a pressure of 5.0 MPa. ESC solubility increased significantly from 1.08 × 10–3 to 59.88 × 10–3 (mole fraction) when the temperature was raised from 298 to 473 K. This is a 55-fold enhancement. A mathematical model was developed based on our experimental solubility data. The model was used to successfully predict the solubility of ESC in subcritical water. Furthermore, experimental data were correlated using a modified Apelblat equation. The apparent thermodynamic analysis including apparent dissolution standard enthalpy (ΔsolH) and apparent dissolution standard entropy (ΔsolS) were calculated. Thermodynamic property calculations showed that the process is endothermic ΔsolH > 0, and the ΔsolS values were relatively high. Spectroscopic analyses revealed that ESC was stable in subcritical water at temperatures up to 473 K. The enhanced solubility of ESC in subcritical water enabled us to use high-temperature liquid chromatography to separate this antidepressant drug.

Aqueous solubility and chromatographic studies of antifungal drug-fluconazole at high temperature conditions

Fluconazole is a novel triazole antifungistatic drug, which can be administered both orally and intravenously and is currently used for the treatment of systemic and superficial fungal infections. In this study, the solubility of fluconazole in water at elevated temperature and pressure was investigated at temperatures in the range of 298 to 473 K under autogenous- 5.0 MPa pressure. The results showed that the solubility of fluconazole was increased 146-fold at the highest experimental temperature of 473 K. Based on the experimental data, a mathematical model was developed to predict the solubility of fluconazole in subcritical water. The model was validated successfully and the theoretical solubility values matched well with the experimental data. Furthermore, a modified Apelblat equation provided a good fit to the experimental values except at low temperature. The molar enthalpy and the molar entropy of dissolution of fluconazole in subcritical water at temperatures ranging from 298 to 473 K were calculated. The good solubility of fluconazole in subcritical water allowed us to perform high temperature liquid chromatography (HTLC) for the determination of this agent. Moreover, thermogravimetric (TG) and differential scanning calorimetry (DSC) analysis confirmed that fluconazole had excellent thermal stability under subcritical conditions.

High-temperature liquid chromatography for evaluation of the efficiency of multiwalled carbon nanotubes as nano extraction beds for removal of acidic drugs from wastewater. Greenness profiling and comprehensive kinetics and thermodynamics studies

The retention behavior of a series of acidic drugs, namely ketoprofen (KET), naproxen (NAP), diclofenac (DIC), and ibuprofen (IBU), on the heat-resisting ZORBAX 300SB-C18 column, was studied thermodynamically using high-temperature liquid chromatography (HTLC). A perfect correlation of the compounds' lipophilicity and the calculated thermodynamic indicators evidenced its contribution to the retention behavior. Besides, the steric fitting has a subsidiary effect on IBU retention. Isocratic HTLC separation of the four compounds was achieved using an aqueous mobile phase containing 30% acetonitrile-0.2% acetic acid-0.2% triethylamine at 60 °C. This method has been utilized to monitor the adsorption efficiency of multiwalled carbon nanotubes (MWCNTs) for the removal of the four NSAIDs from water. Different variables affecting the remediation process have been optimized such as the time of contact, pH, ionic strength, temperature, and the mass of MWCNTs. The kinetics and thermodynamics of the adsorption were investigated. The adsorption was evidenced to take place via pseudo-second-order kinetics and the intraparticle diffusion is the rate-controlling step. The thermodynamic investigation showed that the adsorption process is exothermic and enthalpy-driven, and the adsorption is more extensive at a lower temperature. The MWCNTs showed excellent adsorption efficiency of about 76.4 to 97.6% at the optimum conditions. The obtained results are promising and encouraging for the full-scale application of MWCNTs for remediation of NSAIDs-related water pollution. The green analytical chemistry metric “AGREE” and the analytical eco-scale score tool confirmed that the developed protocol is greener and more favorable to the environment and user than most of the reported literature.

Analyses of Short Chain Branches in Polyolefins with Improved 1H NMR Spectroscopy.

Polyolefin microstructures, for example, short chain branching (SCB) and short chain branch distribution (SCBD), have a direct impact on properties and thus ultimately influence end-use applications. The 1H NMR approach to analyze SCB and SCBD is particularly useful when only a limited amount of sample is available, for example, polyolefin film layers or the fractions from polyolefin separation techniques, such as gel permeation chromatography (GPC), crystallization elution fractionation (CEF), high temperature liquid chromatography (HTLC), and thermal gradient interaction chromatography (TGIC). In this paper, we discuss the best approach to find a good decoupling frequency and propose an improved 1H pulse sequence with homonuclear decoupling for better measuring SCB. With this new pulse it is possible to reach a S/N of 10 (level of quantification) for the methyl signal from SCB in an ethylene-hexene copolymer (EH, 3.6 mol % H) in 3.5 min with 0.5 μg of sample. We also show an easy method to calculate SCB/1000C and demonstrate the proper use of heteronuclear single quantum coherence (HSQC) to measure SCB in a complicated system. A very quick approach to examine the presence of a small amount of LDPE in a polyolefin sample is also suggested, which can reduce NMR acquisition time from a couple of days to a few minutes.

A Plug-and-Play High-Pressure ESI Source with an Emitter at Ground Potential and Its Application to High-Temperature Capillary LC-MS.

A new high-pressure ESI source that can be readily used for commercial API mass spectrometers in a plug-and-play manner without any modification on the ion sampling interface is introduced. The emitter can be operated at ground potential, and the positive mode electrospray is generated by applying a negative high potential to the counter electrode. A shielding electrode effectively shields the opposing electric field and improves the ion transmission. This feature facilitates the direct connection of the ESI emitter to the electrically grounded components. The application of the present ion source to the high-temperature (>100 °C) capillary liquid chromatography for high-speed separation of peptide and proteins is demonstrated using a monolithic polymeric column.

Heterophasic ethylene-propylene copolymers: New insights on complex microstructure by combined molar mass fractionation and high temperature liquid chromatography

The present study investigates the changes in the microstructure of heterophasic ethylene propylene copolymers (HEPCs) with increasing ethylene content, before and after visbreaking. Three samples obtained from commercial gas process reactors at Sasol Polymers at varying times after the addition of the ethylene comonomer are further visbroken to produce three more samples. Bulk sample analyses indicate that the ethylene-rich rubber phase which is essential in aiding peroxide mobility during visbreaking as observed in narrower dispersities and lower peak molar masses with increasing ethylene content. Further investigations of the microstructure via offline coupling of preparative molar mass fractionation (pMMF) to advanced analytical techniques such as solvent gradient interaction chromatography (SGIC) reveal that ethylene-rich copolymer chains are not significantly affected by the peroxide as the polypropylene homopolymer. Consequently, the polypropylene (PP) homopolymer and polypropylene-rich fractions with high molar mass diminish after visbreaking. Furthermore, the increase in ethylene content was observed to reduce the impact of the peroxide on the fraction quantities before and after visbreaking implying that visbreaking affects more the polyolefin chains with more PP segments and less those with more ethylene comonomer.

High-Temperature Liquid Chromatography and the Hyphenation with Mass Spectrometry Using High-Pressure Electrospray Ionization.

Increasing the operating temperature of the liquid chromatography (LC) column has the same effect as reducing the diameter of the packing particles on minimizing the contribution of C-term in the van Deemter equation, flattening the curve of plate height vs. linear velocity in the high-speed region, thus allowing a fast LC analysis without the loss of plate count. While the use of smaller particles requires a higher pumping pressure, operating the column at higher temperature reduces the pressure due to lower liquid viscosity. At present, the adoption of high-temperature LC lags behind the ultra-high-pressure LC. Nevertheless, the availability of thermally stable columns has steadily improved and new innovations in this area have continued to emerge. This paper gives a brief review and updates on the recent advances in high-temperature liquid chromatography (HTLC). Recent efforts of hyphenating the capillary HTLC with mass spectrometry via a super-atmospheric pressure electrospray ionization is also reported.

Recent Trends in Fast Liquid Chromatography for Pharmaceutical Analysis

Background: Liquid chromatography is the workhorse of analytical laboratories of pharmaceutical companies for analysis of bulk drug materials, intermediates, drug products, impurities and degradation products. This efficient technique is impeded by its long and tedious analysis procedures. Continuous efforts of scientists to reduce the analysis time resulted in the development of three different approaches namely, HTLC, chromatography using monolithic columns and UHPLC. Methods: Modern column technology and advances in chromatographic stationary phase including silica-based monolithic columns and reduction in particle and column size (UHPLC) have not only revolutionized the separation power of chromatographic analysis but also have remarkably reduced the analysis time. Automated ultra high-performance chromatographic systems equipped with state-ofthe- art software and detection systems have now spawned a new field of analysis, termed as Fast Liquid Chromatography (FLC). The chromatographic approaches that can be included in FLC are hightemperature liquid chromatography, chromatography using monolithic column, and ultrahigh performance liquid chromatography. Results: This review summarizes the progress of FLC in pharmaceutical analysis during the period from year 2008 to 2017 focusing on detecting pharmaceutical drugs in various matrices, characterizing active compounds of natural products, and drug metabolites. High temperature, change in the mobile phase, use of monolithic columns, new non-porous, semi-porous and fully porous reduced particle size of/less than 3μm packed columns technology with high-pressure pumps have been extensively studied and successively applied to real samples. These factors revolutionized the fast high-performance separations. Conclusion: Taking into account the recent development in fast liquid chromatography approaches, future trends can be clearly predicated. UHPLC must be the most popular approach followed by the use of monolithic columns. Use of high temperatures during analysis is not a feasible approach especially for pharmaceutical analysis due to thermosensitive nature of analytes.

Anti-Idiotype DNA Aptamer Affinity Purification⁻High-Temperature Reversed-Phase Liquid Chromatography: A Simple, Accurate, and Selective Bioanalysis of Bevacizumab.

This study presents a simple, accurate, and selective bioanalytical method of bevacizumab detection from plasma samples based on aptamer affinity purification–high-temperature reversed-phased liquid chromatography (HT-RPLC) with fluorescence detection. Bevacizumab in plasma samples was purified using magnetic beads immobilized with an anti-idiotype DNA aptamer for bevacizumab. The purified bevacizumab was separated with HT-RPLC and detected with its native fluorescence. Using aptamer affinity beads, bevacizumab was selectively purified and detected as a single peak in the chromatogram. HT-RPLC achieved good separation for bevacizumab with a sharp peak within 10 min. The calibration curves of the two monoclonal antibodies ranged from 1 to 50 μg/mL and showed good correlation coefficients (r2 > 0.999). The limit of detection (LOD) and lower limit of quantification (LLOQ) values for bevacizumab were 0.15 and 0.51 μg/mL, respectively. The proposed method was successfully applied to the bioanalysis of the plasma samples obtained from the patients with lung cancer and may be extended to plan optimal therapeutic programs and for the evaluation of biological equivalencies in the development of biosimilars.

Isomer separation of sialylated O- and N-linked glycopeptides using reversed-phase LC–MS/MS at high temperature

Analyses of intact glycopeptides using mass spectrometry is challenging due to the numerous types of isomers of glycan moieties attached to the peptide backbone. Here, we demonstrate that high-temperature reversed-phase liquid chromatography (RPLC) can be used to separate isomeric O- and N-linked glycopeptides. In general, high column temperatures enhanced the resolution for separation of sialylated O- and N-linked glycopeptide isomers with decreased retention times. Using the high-temperature RPLC method, α2–6-linked sialylated N-glycopeptides were eluted first, followed by α2–3-linked isomers. However, highly sialylated N-glycopeptides containing hydrophobic amino acids exhibited increased retention times at high temperature. The separation of sialylated O- and N-glycopeptides with different glycan isoforms using a high-temperature RPLC method was demonstrated. This study indicates that reversed-phase chromatographic separation at high column temperatures is suitable for the separation of glycopeptide structural isomers.

Simultaneous determination of citalopram, paroxetine, fluoxetine, and sertraline by high-temperature liquid chromatography

A lack of serotonin in the brain is associated with depression. Selective serotonin reuptake inhibitors (SSRIs) are widely used to help treat depression and associated symptoms. A method has been developed for the simultaneous determination of SSRIs by high-temperature liquid chromatography (HTLC). Citalopram, paroxetine, fluoxetine, and sertraline compounds, which are widely used as antidepressant active agents, have been chosen as SSRIs. The separation of the SSRIs have been carried out by using four different column types, including XTerra MS C18, Zorbax SB-Phenyl, Alltima C18 and Phenyl Hypersil columns, and their chromatographic performances have been evaluated. The best separation has been obtained on the Zorbax SB-Phenyl column (150 mm × 4.6 mm, 5 μm) among the four different columns studied. The separation temperature and the composition of mobile phase were examined for the optimization of chromatographic separation. Chromatographic separation of SSRIs has been carried out at temperatures ranging from 100 to 200 °C with variable flow rates (0.5-1.5 mL/min). Water:acetonitrile:acetic acid mixtures containing with 10 or 20% acetonitrile and 2% acetic acid have been used as mobile phase. The best separation was observed at volume ratio of 78:20:2 (water:acetonitrile:acetic acid) at elevated temperature on the Zorbax SB-Phenyl column. The wavelength of UV detector was set at 254 nm. All four analytes were eluted within 8 min at 200 °C. At the end of working, it was observed that the retention times of all four analytes decreased with increasing temperature and was stated that the temperature was an effective parameter for chromatographic separation. Furthermore, the relationship between retention factor and separation temperature was examined using Van’t Hoff plots and the results demonstrated with correlation coefficient greater than 0.91 on Zorbax SB Phenyl column. Consequently, the proposed HTLC method for separation and analysis of SSRIs may be used as a green alternative technique.

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.