Effects of elevated temperature and mobile phase composition on a novel C18 silica column

A novel polydentate C18 silica column was evaluated at an elevated temperature under acidic, basic, and neutral mobile phase conditions using ACN and methanol as the mobile phase organic modifier. The temperature range was 40-200 degrees C. The mobile phase compositions were from 0 to 80% organic-aqueous v/v and the mobile phase pH levels were between 2 and 12. The maximum operating temperature of the column was affected by the amount and type of organic modifier used in the mobile phase. Under neutral conditions, the column showed good column thermal stability at temperatures ranging between 120 and 200 degrees C in methanol-water and ACN-water solvent systems. At pH 2 and 3, the column performed well up to about 160 degrees C at two fixed ACN-buffer compositions. Under basic conditions at elevated temperatures, the column material deteriorated more quickly, but still remained stable up to 100 degrees C at pH 9 and 60 degrees C at pH 10. The results of this study indicate that this novel C18 silica-based column represents a significant advancement in RPLC column technology with enhanced thermal and pH stability when compared to traditional bonded phase silica columns.     

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography has been investigated in the present study. Columns with inner diameters of 0.32, 0.53, 3.2 and 4.6 mm were compared with respect to retention and efficiency characteristics using temperature gradients from 30 to 90 degrees C with temperature ramps of 1, 5, 10 and 20 degrees C min(-1). The columns were all of 15 cm length and were packed with 3 microm Hypersil ODS particles. Alkylbenzenes served as model compounds, and the mobile phase consisted of acetonitrile-water (50:50, v/v). The study revealed that the column ID is not a critical limiting factor when performing temperature programming in LC, at least for columns narrower than 4.6 mm inner diameter in the temperature interval 30-90 degrees C. The retention times for all components on all columns were highly comparable, with similar peak profiles without any signs of peak splitting. The use of mobile phase pre-heating when using the larger bore columns was avoided by starting the temperature gradients close to ambient. However, the relative apparent efficiency was inversely proportional to column inner diameter, making the capillary columns generally more functional towards temperature gradients than the larger bore columns with respect to chromatographic efficiency. In addition, the capillary columns possessed higher robustness towards temperature programming than the conventional columns.     

Disengaging solutes in shale- and petroleum-derived jet fuels by altering GC programmed temperature rates

Overlapping chromatographic peaks of components from different hydrocarbon classes can be disengaged by exploiting their shifts in relative retention behavior with changes in linear rates of programmed temperature. Many co-eluting species in complex chromatograms of shale- and petroleum-derived jet fuels can be resolved without varying stationary phase, column length, or initial column temperature. Retention indices were simultaneously determined on two bonded-phase, fused silica capillary columns of slightly different polarities at three different linear programmed temperature rates. For certain hydrocarbon types, no change in index values was observed with an alteration in programming rate. However, the indices of other hydrocarbon classes shifted uniformly with programmed temperature rates on each of the two stationary phases. When applied, this phenomenon could help resolve coeluting members of different or even the same hydrocarbon type and elucidate their probable structure. The overall precision of the retention indices, i.e., the mean standard deviation at the 95% confidence levels, was less than ±0.13 for either column at any of the three programming rates. Since the above technique is automated, it could also be a useful screening tool to search for specific hydrocarbons in a myriad of unknown components of a complex hydrocarbon mixture.     

Effects of Blending Bare-Silica and Reversed-Phase on Retention of Neutral Compounds in Capillary Electrochromatography

Most commercially available instruments for capillary electrochromatography (CEC) have a fixed configuration and lack the flexibility to use shorter columns. Applying a blended stationary phase (a phase consisting of a given ratio of bare silica and reversed phase material) can simulate columns of different length in CEC. The goal of this work was to examine the effect of the degree of blending of reversed-phase columns (with bare silica) on the speed of the separation of neutral compounds in CEC. Optimum column packing mixture was determined from the variation of the solute retention factors as a function of the ratios of blending of reversed-phase and bare silica. By adjusting the column composition, solute retention factors and the analysis run time were halved when compared to a pure reversed-phase column of the same length. Stationary phase blending can be considered as an additional parameter to mobile phase variation, column temperature and applied electric field for the optimization of selectivity and analysis time. By adjusting the stationary phase composition, mobile phase composition, column temperature and applied electric field, the analysis run time of neutral components was decreased more than 75% when compared to a separation obtained on neat reversed-phase column of the same dimensions. The linear dependence of the retention factors as a function of the blend ratio (reversed phase/bare silica) offers a framework for designing a “blended” packed capillary column for CEC separations.     

Study of temperature-responsibility on the surfaces of a thermo-responsive polymer modified stationary phase

We investigated a thermo-sensitive polymer, poly(N-isopropylacrylamide) (PNIPAAm), which is the basis of an HPLC stationary phase. We prepared a PNIPAAm terminally-modified surface. In this study, we investigated the effect of PNIPAAm on the surface of a stationary phase on separation based on changes of the retention time with the temperature step gradient. As the temperature changed the surface property of the stationary phase switched from hydrophilic to hydrophobic. The retention on the polymer-modified stationary phase remarkably changed upon changing the temperature. Using a column packed with PNIPAAm-modified silica, the separation of steroids was carried out by changing the temperature. With increasing temperature, an increased interaction between solutes and PNIPAAm-grafted surfaces of the stationary phases was observed. A temperature-dependent resolution of steroids was achieved using only water as a mobile phase. The PNIPAAm-modified surface of the stationary phase exhibited temperature-controlled hydrophilic-hydrophobic changes. The drastic and reversible surface hydrophilic-hydrophobic property alteration for PNIPAAm terminally-grafted surfaces should be due to rapid changes in the polymer hydration state around the polymer's transition temperature. A solvent gradient elution-like effect could be achieved with a single mobile phase by programmed temperature changes during chromatographic runs. This system should be highly useful to control the function and property of the stationary phase for HPLC only by changing the temperature with an aqueous solvent.     

Comparison of hypercrosslinked polystyrene columns for the separation of nitrogen group-types in petroleum using High Performance Liquid Chromatography

High performance liquid chromatography in a quasi-normal phase mode (QNP) is used to separate the nitrogen group-types (pyrrole and pyridine) that are found in petroleum. A new type of stationary phase, hypercrosslinked polystyrene, is used to achieve this separation. Three different hypercrosslinked polystyrene stationary phases are compared under quasi-normal phase mode; a commercial 5-HGN packing, and two hypercrosslinked phases on silica particles. The utility of the columns for petroleum-based separations was explored with the use of 21 analytical standards. Partial elucidation of adsorption retention mechanisms for the columns are shown, as well as a comparison of retention characteristics for the three columns. The silica particle column derived with toluene (HC-Tol) was found to have the best selectivity for nitrogen group-types and polycyclic aromatic hydrocarbons (PAHs), attaining a separation under gradient conditions in less than 30 min.     

Combined effects of mobile phase composition and temperature on the retention of homologous and polar test compounds on polydentate C8 column

Combined effects of temperature and mobile phase on the reversed phase chromatographic behavior of alkylbenzenes and simple substituted benzenes were investigated on a Blaze C₈ polydentate silica-based column, showing improved resistance against hydrolytic breakdown at temperatures higher than 60 °C, in comparison to silica-based stationary phases with single attachment sites. For better insight into the retention mechanism on polydentate columns, we determined the enthalpy and entropy of the transfer of the test compounds from the mobile to the stationary phase. The enthalpic contribution dominated the retention at 80% or lower concentrations of methanol in the mobile phase. Entropic effects are more significant in 90% methanol and in acetonitrile–water mobile phases. Anomalies in the effects of mobile phase on the enthalpy of retention of benzene, methylbenzene and polar benzene derivatives were observed, in comparison to regular change in enthalpy and entropy of adsorption with changing concentration of organic solvent and the alkyl length for higher alkylbenzenes. The temperature and the mobile phase effects on the retention are practically independent of each other and – to first approximation – can be described by a simple model equation, which can be used for optimization of separation conditions.     

Temperature programmed retention indices: Calculation from isothermal data. Part 1: Theory

Direct conversion of isothermal to temperature programmed indices is not possible. In this work it is shown that linear temperature programmed retention indices can only be calculated from isothermal retention data if the temperature dependence of both the distribution coefficients and the column dead time are taken into account. Procedures are described which allow calculation of retention temperatures and from these, accurate programmed retention indices. Within certain limits the initial oven temperature and programming rate can be chosen freely. The prerequisite for this calculation is the availability of reliable isothermal retention data (retention times, retention factors, relative retention times, or retention indices) at two different temperatures for one column. The use of compiled isothermal retention indices at two different temperatures for the calculation of retention temperatures and thus temperature programmed indices is demonstrated. For the column for which programmed retention indices have to be determined, the isothermal retention times of the n-alkanes and the column dead time as a function of temperature have to be known in addition to the compiled data for a given stationary phase. Once the programmed retention indices have been calculated for a given column the concept allows the calculation of temperature programmed indices for columns with different specifications. The characteristics which can be varied are: column length, column inner diameter, phase-ratio, initial oven temperature, and programming rate.     

Studies on the long-term thermal stability of particulate and monolithic poly(styrene-divinylbenzene) capillaries in reversed-phase liquid chromatography

In the present study, the long-term high-temperature (>80 degrees C) and temperature programming stability of fused silica capillaries packed with 5 microm PLRP-S 300 A and monolithic PS-DVB capillaries (both 180 microm id x 6 cm) under reversed-phase conditions has been examined. In isothermal mode, the columns were defined as temperature-stable when a less than 10% change in apparent retention factors (k) and a less than 20% change in "retention time/peak width"-factors (n) of the probe solutes (proteins) were observed after passing 7,500 void volumes of effluent through the columns (about 100 h operation). According to these criteria, the PLRP-S and monolithic capillaries were defined temperature-stable at 100 and 130 degrees C, respectively. Furthermore, when continuously running temperature programs between 50 degrees C and the upper temperature limit determined in isothermal mode, virtually no change in k or n were observed on neither of the columns after running more than 35,000 void volumes or 1,600 temperature programs. Additionally, temperature-programmed reversed-phase separations of proteins on both types of capillaries are demonstrated and discussed.     

Separation of cannabinoids on three different mixed‐mode columns containing carbon/nanodiamond/amine‐polymer superficially porous particles

Three mixed‐mode high‐performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine‐polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C₁₈), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed‐mode column (C₁₈) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed‐mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C₁₈) mixed‐mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution.     

Non-trivial temperature effects on the cation exchange chromatography and chelation ion chromatography of metal ions

The effect of column temperature upon the retention of metal ions on sulfonated and mono-, di-, and amino-carboxylated cation exchange columns has been investigated. The retention of alkali, alkaline earth and transition metal ions on each of the above types of cation exchanger was studied over the temperature range 19-65 degrees C. A major difference between the behaviour of mono- and divalent metal ions was shown on each of the above stationary phases, with the monovalent alkali metals exhibiting clearly exothermic behaviour (a decrease in retention with increased temperature) under acidic eluent conditions and an apparent relationship between retention factor and the magnitude of the temperature effect. The effect of temperature upon alkaline earth metal ions was less defined, although strongly endothermic behaviour (increase in retention with temperature) could be seen on all stationary phases through correct choice of eluent. The transition metal ions studied showed endothermic behaviour on all four stationary phases, with the sulfonated column unexpectedly showing the largest increases in retention. The above behaviour can be partially explained through the dominance of the type of solute-stationary phase interaction governing retention. In several of the above columns, both ion-exchange and surface complexation interactions can occur, with the effects of temperature indicating which process dominates under specific eluent conditions.     

Separation of free sterols by high temperature liquid chromatography

Increasing the column temperature accelerates markedly elution in HPLC. The separation of five free sterols was studied on three packing materials that can withstand high temperatures. These stationary phases included graphitic carbon, a polymeric C18 silica, and a zirconia-based adsorbent. Measurements of retention data were made at up to 150 degrees C with mobile phases of different compositions. Since the columns tested afford different retention mechanisms, a variety of elution patterns were observed, with some being more advantageous than others for certain sterol separations. Effects observed include some selectivity improvements and some elution order reversals. The separation of free sterols in selected fruit juices is also presented. Albeit at the expense of a longer analysis time, the graphitic carbon column produced the best separation of the sterols in this study.     

Study of a monolithic silica capillary column coated with poly(octadecyl methacrylate) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds

The performance of a monolithic silica capillary column coated with poly(octadecyl methacrylate) (ODM column) for the reversed-phase liquid chromatographic separation of some polar and non-polar compounds was studied, and the results were compared to those obtained by using a monolithic silica capillary column modified with octadecylsilyl-(N,N-diethylamino)silane (ODS column). Benzene and naphthalene derivatives, polycyclic aromatic hydrocarbons (PAHs), steroids, alkyl phthalates, and tocopherol homologues were used as test samples. In general, compounds with aromatic character, rigid and planar structures, and lower length-to-breadth ratios (more compacted structures) seem to have more preference for the polymer coated stationary phase (ODM). Compounds with acidic character have also a higher retention on ODM columns because of the presence of ester groups in the stationary phase. The polymer coated column allowed the separation of some PAHs, alkyl phthalates, steroids, and of beta- and gamma-tocopherol isomers which cannot be separated under the same conditions on ODS columns, while keeping similar column efficiency. These results allowed to suggest ODM columns as a good alternative to conventional ODS columns for reversed-phase liquid chromatography.     

Evaluation of column bleed by using an ultraviolet and a charged aerosol detector coupled to a high-temperature liquid chromatographic system

In this study, five different HPLC columns were heated to 200 degrees C using a homemade heating system which can be operated in temperature programmed mode. The column bleed as an indicator of induced degradation of the stationary phase material was evaluated using a charged aerosol detector (CAD) and an ultraviolet diode array detector (UV-DAD) at different wavelengths. The silica based C-18 stationary phase gave the highest bleed, and the carbon clad titanium dioxide column the lowest bleed. This was independent of both the detection technique and the wavelength.     

Characterisation of stationary phases in subcritical fluid chromatography with the solvation parameter model IV. Aromatic stationary phases

The purpose of the present work was to systematically study the chromatographic behaviour of different aromatic stationary phases in a subcritical fluid mobile phase. We attempted to assess the chemical origin of the differences in retention characteristics between the different columns. Various types of aromatic stationary phases, all commercially available, were investigated. The effect of the nature of the aromatic bonding on interactions between solute and stationary phases and between solute and carbon dioxide-methanol mobile phase was studied by the use of a linear solvation energy relationship (LSER): the solvation parameter model. This study was performed to provide a greater knowledge of the properties of these phases in subcritical fluid chromatography, and to allow a more rapid and efficient choice of aromatic stationary phase in regard of the chemical nature of the solutes to be separated. Charge transfer interactions naturally contribute to the retention on all these stationary phases but are completed by various other types of interactions, depending on the nature of the aromatic group. The solvation vectors were used to compare the different phase properties. In particular, the similarities in the chromatographic behaviour of porous graphitic carbon (PGC), polystyrene-divinylbenzene (PS-DVB) and aromatic-bonded silica stationary phases are evidenced.     

High-speed gradient elution reversed-phase liquid chromatography of bases in buffered eluents. Part I. Retention repeatability and column re-equilibration

We studied the run-to-run repeatability of the retention times of both non-ionizable and basic compounds chromatographed using buffered eluents. The effect of flow rate, organic modifier and other additives, buffer type/concentration, stationary phase type, batch-to-batch preparation of the initial eluent, gradient time, sample type and intra-day changes on retention repeatability were examined. We also assessed the effect of column storage solvent conditions on the inter-day repeatability. Although retention repeatability is strongly influenced by many parameters (flow rate, solvent compressibility compensation, precision of temperature control, and buffer/stationary phase type), our primary finding is that with a reasonable size column (15cmx4.6mm (i.d.)) two column volumes of re-equilibration with initial eluent suffices to provide acceptable repeatability (no worse than 0.004min) for both non-ionizable and basic analytes under a wide variety of conditions. Under ideal conditions (e.g. the right buffer, flow rate, etc.) it is possible to obtain truly extraordinary repeatability often as good as 0.0004min. These absolute fluctuations in retention translate to worst case changes in resolution of 0.2 units and average changes of only 0.02 units.     

Separation of Polar and Basic Compounds in Hydrophilic Interaction Pressurized CEC Using Diethylenetriaminopropyl Silica Monolithic Columns

A novel silica-based monolithic column possessing diethylenetriaminopropyl ligands for hydrophilic interaction pressurized capillary electrochromatography is described. The preparation of monolithic stationary phase was based on the individual silica matrix forming and subsequent chemical bonding. The triamino groups on the surface of the novel stationary phase generated a sustainable anodic electroosmotic flow under acidic conditions. A variety of neutral and basic analytes were used to evaluate the column performance. The monolithic silica stationary phase exhibited hydrophilic interaction chromatographic behavior toward neutral solutes. For basic tetracycline antibiotics, hydrophilic interaction as well as electrophoretic migration process with the monoliths was observed and peak tailing was avoided.     

Repeatability and reproducibility of retention data and band profiles on reversed-phase liquid chromatography columns. V. Results obtained with Vydac 218TP C18 columns

The general goal of this work is to investigate the precision of chromatographic data and to determine which properties of chromatographic columns influence this factor. Chromatographic data were acquired under five different sets of experimental conditions for 30 neutral, acidic and basic test compounds on columns packed with Vydac 218TP C18, a polymeric, wide-pore silica-based stationary phase. Five columns packed with samples from the same batch of this packing material were used to measure the column-to-column reproducibility and six columns packed with material from six different batches to measure the batch-to-batch reproducibility. The parameters studied were the retention time, the retention and separation factors, the hydrophobic and the steric selectivities, the column efficiency, and the tailing factor.     

Development of a new C14 monolithic silica column containing embedded polar groups for pressurized capillary electrochromatography

Monolithic columns have been prepared with a novel bonded silica stationary phase, tetradecylamine bonded silica (TDAS), and used in pressurized capillary electrochromatography (pCEC). The monolithic silica column matrix was prepared by a sol-gel process and then chemically modified with the spacer (3-glycidoxypropyl)trimethoxysilane and tetradecylamine. The introduced embedded polar amine groups dominated the charge on the surface of the monolithic stationary phase and generated an EOF from cathode to anode under acidic conditions. The tetradecyl hydrophobic chains in TDAS provide chromatographic interactions. The chromatographic characteristics of the prepared monolithic column were studied. Some aromatic compounds including alkylbenzenes, aromatic hydrocarbons, phenols, and anilines were successfully separated on the TDAS monolithic column in pCEC mode. As expected, the TDAS monolithic stationary phases exhibit typical reversed-phase electrochromatographic behavior toward neutral solutes due to the introduced tetradecyl groups. Hydrophobic as well as electrophoretic migration processes within the monoliths were observed in the separation of basic anilines. Symmetrical peaks can be obtained for anilines because the embedded polar amine groups on the surface can effectively shield the adsorption of positively charged analytes onto the stationary phase.