Mobile phase viscosity and velocity dependence on protein retention using nonequilibrium chromatographic techniques

Nonequilibrium chromatography (NEC) is an alternative chromatographic procedure for the separation of macromolecules. The retardation of a protein series is studied using a phosphate buffer as a mobile phase with various concentrations of glycerol fraction (used as a viscosity modifier) at different mobile phase velocities and a C1 column with a very low packing particle diameter as a stationary phase. It is shown that the two factors (viscosity and velocity) of the mobile phase constituted important parameters in the retention mechanism of the proteins in NEC. The retardation velocity domain is divided into two regions. For low velocity regions, the protein retention decreased with a mobile phase velocity increase. This retention is enhanced above a critical value of the mobile phase velocity. The transition between the two well-known NEC methods, slalom chromatography and hydrodynamic chromatography, is clearly visualized for the first time for the protein retention of particular values of the mobile phase velocity.     

Supercoiled circular DNA retention in nonequilibrium chromatography: viscosity and velocity dependence--behavior difference with proteins

This study demonstrates that the retention behavior of various circular double-stranded DNA molecules (3, 5, and 10 kb) increases over the entire flow-rate range (0.02-1.8 mL/min) at all the mobile phase viscosities (h). The transition between the two well-known nonequilibrium chromatography methods (slalom and hydrodynamic chromatography) is clearly visualized for proteins and does not appear for plasmids because of their strong compact structure. Also, the optimal conditions for F and h are determined to obtain the most efficient separation of these three plasmids in a minimum analysis time.     

Superheated water as eluent in high-temperature high-performance liquid chromatographic separations of steroids on a polymer-coated zirconia column

High-temperature liquid chromatography (HTLC), with a superheated water mobile phase, has been shown to be a feasible replacement for medium-polarity acetonitrile-water mixtures as an eluent in reversed-phase HPLC. Instrumental parameters of flow-rate, injection volume and mobile phase preheating were shown to have significant effects on the quality of the chromatographic peaks. The selectivity and retention patterns of testosterone and several related compounds were investigated on a porous zirconia, polybutadiene-coated column at temperatures up to 200 degrees C and compared with that of a porous silica, octadecylsilane-coated column and the zirconia column under traditional reversed-phase conditions of an acetonitrile-water mobile phase at 40 degrees C. The selectivity differences observed for testosterone and related compounds show that the separation mechanisms are complementary and unique selectivity is obtained with the zirconia column under HTLC conditions.     

Temperature effects in Microcolumn Size Exclusion Chromatography

Abstract

Effects of column temperature on the column efficiency, retention time and stability of analytes were studied in microcolumn size-exclusion chromatography. Larger theoretical plates were achieved at column temperatures betwen 70 and 100 °C. In the constant-flow mode the retention time of analytes decreased with increasing column temperature, which was due mainly to thermal expansion of the mobile phase. When the column temperature was around or higher than the critical temperature of the mobile phase, the retention times of the analytes observed under supercritical pressure conditions still indicated dominancy of the size-exclusion mechanism, while another retention mechanism was involved under subcritical pressure conditions. In the case of the analysis of saccharides, the column temperature should be lower than 100 °C because oligosaccharides were decomposed at higher temperatures.     

Preparation and characterization of a novel dual‐retention mechanism mixed‐mode stationary phase with PEG 400 and succinic anhydride as ligand for protein separation in WCX and HIC modes

A novel dual‐retention mechanism mixed‐mode stationary phase based on silica gel functionalized with PEG 400 and succinic anhydride as the ligand was prepared and characterized by infrared spectra and elemental analysis. Because of the ligand containing PEG 400 and carboxyl function groups, it displayed hydrophobic interaction chromatography (HIC) characteristic in a high‐salt‐concentration mobile phase, and weak cation exchange chromatography (WCX) characteristic in a low‐salt‐concentration mobile phase. As a result, it can be employed to separate proteins with both WCX and HIC modes. The resolution and selectivity of the stationary phase was evaluated under both HIC and WCX modes with protein standards, and its performance was comparable to that of conventional ion‐exchange chromatography and HIC columns. The results indicated that the novel dual‐retention mechanism column, in many cases, could replace two individual WCX and HIC columns as a ‘2D column’. In addition, the mixed retention mechanism of proteins on this ‘2D column’ was investigated with stoichiometric displacement theory for retention of solute in liquid chromatography in detail in order to understand why the dual‐retention mechanism column has high resolution and selectivity for protein separation under WCX and HIC modes, respectively. Based on this ‘2D column’, a new 2DLC technology with a single column was developed. It is very important in proteome research and recombinant protein drug production to save column expense and simplify the processes in biotechnology. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of Column Temperature on the Retention of Inorganic Anions and Organic Acids in Non-Suppressed Anion-Exchange IC

An investigation has been conducted into the effect of column temperature on the retention of inorganic anions and organic acids in non-suppressed ion chromatography on an anion-exchange column. Potassium biphthalate and p-hydroxybenzoic acid–tris–boric acid were used as mobile phases. The column temperature was from 25 to 50 °C. Endothermic and exothermic retention of inorganic anions were both observed when potassium biphthalate was used as mobile phase. When p-hydroxybenzoic acid–tris–boric acid was used as mobile phase, however, endothermic behavior only was observed. Moreover, for the two mobile phases, variation of the retention time of the system peaks with changing temperature was reversed. For retention of the organic acids, only endothermic behavior was observed with the two mobile phases. Variation of retention time was greater when p-hydroxybenzoic acid–tris–boric acid was used as mobile phase than when potassium biphthalate was used. These results indicated the exchange reaction in anion-exchange chromatography could be either endothermic or exothermic, depending on the solute and mobile phase ions involved. Different relative changes of retention time were observed for individual inorganic anions and organic acids with increasing column temperature. In general, variation of retention time with increasing temperature was greater for strongly retained inorganic anions and organic acids than for weakly retained species. Van’t Hoff plots for inorganic anions, organic acids, and system peaks were linear. Selectivity variation of the retention of inorganic anions and organic acids was achieved by changing the temperature. In achieving optimum separation of inorganic anions and organic acids, temperature was a valuable tool. To reduce the retention times of the ions and avoid interference from system peaks in non-suppressed anion-exchange ion chromatography with the two mobile phases, a low column temperature, for example, 35 °C, was best.     

Prediction of the plate height of capillary columns operated at any inlet pressure of the carrier gas by using few retention data measured under isobaric conditions

Programming inlet pressure in gas chromatography permits to decrease the analysis time without changing the elution order of compounds of different polarity whose relative retention changes with changing temperature. The choice of the best values of the inlet pressure and flow-rate of the carrier gas often requires many preliminary analyses with different parameters to be carried out. A method for the prediction of the separation by starting from few experimental data measured in isothermal and isobaric conditions decreases the time required for the optimisation of the analysis. The efficiency of the separation depends on the change of the theoretical plate height at various pressures and temperatures, due to pressure drop along the column. By calculation of the diffusion coefficients of the analysed compounds into the mobile and stationary phase it is possible to evaluate the column efficiency and predict the number of theoretical plates at any inlet pressure. A procedure for the prediction of the plate height of a capillary column at any inlet pressure of the carrier gas and column temperature by using retention data of polar and non-polar compounds (1-alcohols and linear alkanes) obtained in few isobaric runs is described.     

Chromatographic behavior of small organic compounds in low‐temperature high‐performance liquid chromatography using liquid carbon dioxide as the mobile phase

Low‐temperature high‐performance liquid chromatography, in which a loop injector, column, and detection cell were refrigerated at –35ºC, using liquid carbon dioxide as the mobile phase was developed. Small organic compounds (polyaromatic hydrocarbons, alkylbenzenes, and quinones) were separated by low‐temperature high‐performance liquid chromatography at temperatures from –35 to –5ºC. The combination of liquid carbon dioxide mobile phase with an octadecyl‐silica (C₁₈) column provided reversed phase mode separation, and a bare silica‐gel column resulted in normal phase mode separation. In both the cases, nonlinear behavior at approximately –15ºC was found in the relationship between the temperature and the retention factors of the analytes (van't Hoff plots). In contrast to general trends in high‐performance liquid chromatography, the decrease in temperature enhanced the separation efficiency of both the columns.     

Stationary phase retention and preliminary application of a spiral disk assembly designed for high-speed counter-current chromatography

A spiral disk assembly composed of five single-channel units was designed for high-speed counter-current chromatography (HSCCC). The retention of different solvent systems ranging from moderately polar to polar organic-aqueous systems to aqueous two-phase systems (ATPs) was investigated under different elution modes. The results indicated that the spiral disk assembly can produce excellent retention of stationary phase for moderately polar organic-aqueous solvent systems, such as chloroform-methanol-water (4:3:2) and hexane-ethyl acetate-methanol-water (1:1:1:1) by pumping lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at a high flow-rate (8 mL/min, Sf>70%), regardless of whether the inlet is at the inner or outer terminal of the channel. This makes it possible for fast analysis of some small molecular compounds. This has been proved in the separation of mixtures of three flavones, including isorhamnetin, kaempferol, and quercetin. The spiral disk assembly can also provide satisfactory retention for polar to ATPS such as 1-butanol-acetic acid-water (4:1:5) (<3 mL/min, Sf>70%), 12.5% poly(ethylene glycol) (PEG) 1000-12.5% K2HPO4-75% water (< or =1 mL/min, Sf>70%) and 4% PEG 8000-5% Dextran T500-91% water (< or =0.5 mL/min, Sf>50%) by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a low flow-rate, while this is not possible with the multilayer coil column. Acceptable resolutions were achieved when it was used for the separation of peptides such as Leu-Tyr and Val-Tyr, and proteins including cytochrome c and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins.     

Analysis of proanthocyanidins by high-performance gel permeation chromatography

A high-performance gel permeation chromatography method was developed for the analysis of proanthocyanidins. The isocratic method consisted of two porous polystyrene-divinylbenzene columns (300 x 7.5 mm each, 5 microm, 100 and 500 A individual pore size) and a mobile phase consisting of N,N-dimethylformamide containing 1% (v/v) acetic acid, 5% (v/v) water and 0.15 M lithium chloride. The flow-rate was maintained at 1 ml/min, with a column temperature of 60 degrees C and with detection at 280 nm. The method was used to analyze proanthocyanidin fractions of increasing molecular mass and from different plant tissues. The average molecular mass of proanthocyanidin fractions as determined by acid catalysis in the presence of phloroglucinol, related well with their gel permeation chromatography column retention, yet significant differences in the retention properties between individual plant tissue isolates existed. Proanthocyanidin compositional differences between isolates may explain these differences. A second-order calibration curve was generated from fractionated grape seed proanthocyanidins and this curve was used to analyze grape seed proanthocyanidins isolated from grapes harvested at extremes of maturity.     

Vacancy ion-exclusion chromatography of aromatic carboxylic acids on a weakly acidic cation-exchange resin

Determination of aromatic carboxylic acids by conventional ion-exclusion chromatography is relatively difficult and methods generally rely on hydrophobic interaction between the solute and the resin. To overcome the difficulties in determining aromatic carboxylic acids a new approach is presented, termed vacancy ion-exclusion chromatography, which is based on use of the sample as mobile phase and an injection of aqueous 10% methanol onto a weakly acidic cation-exchange column (TSKgel OApak-A). Highly sensitive conductivity detection occurred with sharp and well-shaped peaks, leading to very efficient separations. The effects of sulfuric acid concentration added to the mobile phase, flow-rate, and column temperature on the retention volume of tested aromatic carboxylic acids was investigated. Retention times were found to be affected by the concentration of the analytes in the mobile phase and to some extent also by the addition of an organic modifier such as methanol to the injected water sample. Separation of sulfuric acid (SA), naphthalenetetracarboxylic acid (NTCA), phthalic acid (PA) and benzoic acid (BA) was satisfactory using this new approach. Detection limits were 0.66, 0.67, 0.42 and 0.86 microM and detector responses were linear in the range 1-100, 1-80, 2.5-100 and 10-40 microM, for SA, NTCA, PA and BA, respectively. Precision for retention times was 0.36% and for peak areas was 1.5%.     

Molecular mass distribution analysis of ethyl(hydroxyethyl)cellulose by size-exclusion chromatography with dual light-scattering and refractometric detection

Dual low-angle light scattering and refractometric detection coupled to size-exclusion chromatography provided proof for the presence of a low amount of stable aggregates/particles in ethyl(hydroxyethyl)cellulose. Unlike the correct size-exclusion chromatographic behavior of the parent polysaccharide itself, the aggregates exhibit variable size-dependent weak retention as a function of flow-rate and of ionic strength of the aqueous mobile phase. Therefore, determination of the molecular mass of non-aggregated polymer is possible in aqueous mobile phase containing 0.1 M NaCl under conditions at which aggregates are completely adsorbed on the column packing irrespective of the flow-rate used. Flow-rate and ionic strength-dependent variations of aggregate behavior as well as model size-exclusion experiments with latex particles indicate that they partly carry a minute charge and have a compact structure. Their weak retention under the separation conditions used suggests a difference in their surface chemistry when compared with the dissolved polymer coils which exhibit a correct size-exclusion behavior.     

Investigation of the low temperature effect in reversed phase micro high performance liquid chromatography

The low temperature effect has been investigated in reversed phase micro high performance liquid chromatography with various aqueous mobile phase systems by an approach based on enthalpy-entropy compensation. The compensation temperatures, Tc , were determined for these systems, and the results show that the retention mechanism in a lower temperature column is similar to that in a normal temperature column in the reversed phase mode at mobile phase water contents above 9.8%. At lower water contents, the separation mechanism is different from that in the reversed phase mode, but otherwise very similar to that in the normal phase mode.     

Liquid chromatographic separation of the enantiomers of metoprolol and its alpha-hydroxy metabolite on Chiralcel OD for determination in plasma and urine.

The two enantiomers of metoprolol and the four enantiomeric forms of alpha-hydroxymetoprolol were separated by liquid chromatography on a Chiralcel OD column containing a cellulose tris(3,5-dimethyl-phenylcarbamate) chiral stationary phase. The column efficiency was strongly dependent on the flow-rate and the enantioselectivity was influenced by temperature. Of utmost importance for the chiral separation was the water content of the mobile organic phase. The separation system was used for the separation and determination of the enantiomers in plasma and urine samples. The metoprolol enantiomers could be determined by fluorescence down to 10 nmol/l of each in plasma with a relative standard deviation of less than 15%.     

Analysis of polymer additives in high-temperature liquid chromatography

A high-temperature liquid chromatographic technique is employed for the separation of commercially available polymer additives to enhance the resolution and speed. Separation efficiencies and elution behaviors for seven phthalate plasticizers and five antioxidants are evaluated at elevated column temperatures and with a thermal gradient. Diamondbond C18 (octadecylsilica), Zirchrom PS (zirconia-based polystyrene), and Zirchrom PBD (zirconia-based polybutadiene) columns are selected for the study because of their thermal stability. The temperature programming is controlled with a column oven in conjunction with an independent mobile phase preheater and a post-column effluent cooling assembly. Van't Hoff plots show that the reverse-phase liquid chromatography mechanism is maintained over a wide range of column temperatures. A 1% increase of acetonitrile in the mobile phase is estimated to have a comparable effect as a 7-7.5 degrees C column temperature increase on the retention time changes.     

Temperature-dependent separation of bryostatin 18 and 10 by high performance liquid chromatography

Summary The temperature-dependent separation of bryostatins by HPLC was examined on an octadecyl bonded stationary phase, using column temperatures between 0 and 40°C and mobile phase temperatures from 0 to 25°C. The retention time and resolution of bryostatins changed drastically and separation improved with decreasing temperature. A column temperature of less than 5°C and a mobile phase temperature of less than 15°C is recommended for a good resolution of bryostatins for routine work.     

Rapid analysis of a plasmid by hydrophobic-interaction chromatography with a non-porous resin

A HPLC technique has been developed, based on hydrophobic-interaction Chromatography with a non-porous packing (TSKgel Butyl-NPR, Tosoh Biosep LLC), that allows separation of the open circular (nicked) and supercoiled forms of five DNA plasmids, ranging in size from 4 to 30 kilo base pairs (kb). The identity of the bands was determined through light scattering and gel electrophoresis. Several buffers, gradients, flow-rates and temperatures were evaluated in determining the optimum operating conditions for the separation. For all plasmids a reversed ammonium sulfate in phosphate buffer (pH 7.1) gradient was established. The chromatographic resolution between the supercoiled and nicked peaks was found to be a function of flow-rate and temperature. The resolution and the elution order did not vary with plasmid size, with the open-circular form always being eluted before the supercoiled form. Hydrophobic-interaction chromatography is a useful alternative to ion exchange or size exclusion for the chromatography of large plasmids, up to 30 kb.     

Effect of high-temperature on high-performance liquid chromatography column stability and performance under temperature-programmed conditions

Six commercially available analytical (4.1 or 4.6 mm i.d.) columns were evaluated under temperature-programmed high-temperature liquid chromatography (HTLC) conditions to access their stability and performance at extreme temperatures. Seven components consisting of acidic, basic and neutral compounds were analyzed under temperature-programmed conditions and solvent gradient conditions using three different mobile phase compositions (acidic, basic and neutral). Each column was checked with a two-component test mix at various stages of the evaluation to look for signs of stationary phase collapse. Three zirconia based stationary phases studied exhibited column bleed under temperature-programmed conditions. The other three columns, a polydentate silica column, a polystyrene-divinylbenzene (PS-DVB) polymeric column, and a graphitic carbon column performed well with no evidence of stationary phase degradation. The R.S.D. for the retention times and efficiencies were less than 10% for most conditions, and not more than 15% during the course of the evaluation for each column. The polydentate silica stationary phase was temperature programmed to 100 degrees C, the PS-DVB stationary phase was temperature programmed up to 150 degrees C, and the graphitic carbon column was used with temperature programming up to 200 degrees C. Comparable peak capacities and similar retention behaviors were observed under solvent gradient and temperature-programmed conditions. Temperature programming with dynamic mobile phase preheating can replace solvent gradient analysis without a loss of peak capacity when used with 4.1 or 4.6 mm columns.     

Separation of oligogalacturonic acids by high-performance gel filtration chromatography on silica gel with diol radical.

Oligogalacturonic acids (OLGAs) ranging from two to nineteen residues in length were separated using high-performance gel filtration chromatography on a silica gel with diol radical. The optimum conditions (eluent, column temperature) for separation of OLGAs by high-performance gel filtration chromatography were investigated. The column used in this experiment allowed a high pressure of 4900 p.s.i. and a flow-rate of 2 ml/min. The stationary phase of silica gel stabilized the separation of OLGAs. The peaks of OLGAs separated using this column were assigned by comparing retention times with standards, and the molecular weights of the corresponding OLGAs were determined by fast atom bombardment mass spectrometry.     

Advantages of pentafluorophenylpropyl stationary phase over conventional C18 stationary phase--application to analysis of triamcinolone acetonide

A pentafluorophenylpropyl (PFPP) stationary phase was for the first time tested for the simultaneous determination of triamcinolone acetonide, its degradation product triamcinolone and two preservatives, methylparaben, and propylparaben. A new simple isocratic reversed phase HPLC method with UV detection, using estradiol hemihydrate as an internal standard, has been developed and validated. Chromatography was performed on a Discovery HS F5 column (150 mm x 4.6 mm, 5 microm) using a binary mobile phase composed of acetonitrile and water 45:55 (v:v). The flow-rate was 0.6 mL/min, the column temperature 25 degrees C and the UV detection was accomplished at 240 nm. The chromatography results using PFPP stationary phase were compared with those obtained using conventional C18 columns.