Chromatographic Mobile Phase Research Articles

Hydrophilic interaction liquid chromatography method for the determination of ascorbic acid

Hydrophilic interaction liquid chromatography (HILIC) method using internal standard for the determination and stability study of ascorbic acid was developed. HILIC method was very fast and simple using the following analytical conditions: ZIC HILIC (150 x 2.1 mm, 3.5 microm) chromatographic column and mobile phase composed of ACN and 50 mM ammonium acetate buffer pH 6.8 (78:22 v/v). Diode array detection was performed and chromatograms were processed at 268 nm, the maximum wavelength of absorbance of ascorbic acid. An extensive stability study of ascorbic acid as a function of various factors including temperature, stabilizing agents, oxygen presence and its concentration in solution was performed in order to gain information about the quantitative influence of individual stability factors. Low temperature and stabilizing agents (o-phosphoric acid and oxalic acid) were found to be key factors enabling substantial enhancement of the stability of ascorbic acid.

Mixture design optimization of extraction and mobile phase media for fingerprint analysis of Bauhinia variegata L.

Two statistical mixture designs were used to optimize the proportions of solvents used in both the extraction medium and the reversed liquid chromatographic mobile phase to improve the quality of chromatographic fingerprints of Bauhinia variegata L extracts. For modeling, the number of peaks was used as a measure of fingerprint information. Three mobile phases, each with a chromatographic strength of two, gave good results. A methanol/water (77:23 v/v) mixture resulted in 17 peaks in the chromatographic fingerprint whereas acetonitrile/water (64.5:35.5 v/v) and methanol/acetonitrile/water (35:35:30 v/v/v) mixtures resulted in 18 and 20 peaks, respectively. The corresponding optimum solvent compositions to extract chemical substances for these three mobile phases were ethanol/acetone (25:75 v/v/v) and dichloromethane/acetone (70:30 v/v) mixtures, and pure dichloromethane, respectively. The mixture designs are useful for understanding the influence of different solvents on the strengths of the extraction medium and the mobile phase.

Influence of the presence of methyl cyclodextrins in high-performance liquid chromatography mobile phases on the separation of β-carboline alkaloids

The presence of cyclodextrins (CDs) in the mobile phase alters the chromatographic equilibria and induces a secondary chemical equilibrium associated to the chromatographic separation by HPLC. In this study the influence of the presence of CDs in the mobile phase as chemically modified β-CDs, i.e. 2,3-di- O-methyl-β-cyclodextrin (DMβ-CD) and 2,3,6-tri- O-methyl-β-cyclodextrin (TMβ-CD) on the separation of the alkaloids norharmane, harmane and harmine is described. These β-carboline alkaloids are chemically and structurally related and their quantitation by RP-HPLC is of interest due to their biological and pharmacological properties. Two stationary phases (methyl-, C 1 and octadecyl-, C 18) were employed, with methanol:buffered aqueous solution and ethanol:buffered aqueous solution as mobile phases. The role of tert-butyl alcohol as a mobile phase modifier and also as an inclusion complex stabiliser was also considered. The concentrations of DMβ-CD and TMβ-CD vary from 0 to 17 mM. The presence of increasing amounts of CDs in the mobile phase reduces the retention factor. The changes observed in the retention factor allow the determination of the alkaloid/CD apparent association constants, whose magnitude is influenced by the chemical and structural properties of the guest molecules but also by the composition of the mobile phase. Assuming a 1:1 stoichiometry for the inclusion complexes, the apparent association constants obtained were higher for norharmane and for both DMβ-CD and TMβ-CD. The strength of the complexes is higher for DMβ-CD than for TMβ-CD and this behaviour can be explained considering the steric problems associated to the permethylated-β-CDs. Besides significant differences in the magnitude of the apparent association constants were observed for the two stationary phases employed and thus can be related to the adsorption of CDs on the stationary phase. A significant reduction in the proportion of organic solvent in the mobile phase (50%) without a decrease in the selectivity or resolution of the separation is a favourable consequence of the presence of the CDs in the mobile phase.

Dynamic ultrasound-assisted extraction coupled on-line with solid support derivatization and high-performance liquid chromatography for the determination of formaldehyde in textiles

An on-line method was developed for the extraction, derivatization and determination of formaldehyde in textile samples. Formaldehyde was first extracted with water by ultrasound assisted, and directly introduced into a derivatization column which was packed with a moderately sulfonated cation-exchange resin. The resin used as solid support for the derivatization was charged with 2,4-dinitrophenylhydrazine (DNPH) previously. The formaldehyde DNPH derivative was eluted with the chromatographic mobile phase into an analytical column for the separation, and then monitored by UV detector. The maximum extraction yield was achieved when the extraction vessel was located at 10 mm from the ultrasonic source and 10 mg textile sample was extracted with 5 mL pure water at a flow rate of 1.0 mL min −1 at 50 °C. The detection limit of the proposed method was 0.06 mg kg −1. This method was applied to the determination of formaldehyde in different textile samples, and compared with the state standard method (off-line spectrophotometry) used in China. The similar contents of formaldehyde were obtained for most samples by the two methods, but little higher for some samples obtained by the proposed method. The average relative standard deviation (RSD) obtained by the on-line method was 3.2% which is lower than 29.5% obtained by the standard method.

A unified classification of stationary phases for packed column supercritical fluid chromatography

The use of supercritical fluids as chromatographic mobile phases allows to obtain rapid separations with high efficiency on packed columns, which could favour the replacement of numerous HPLC methods by supercritical fluid chromatography (SFC) ones. Moreover, despite some unexpected chromatographic behaviours, general retention rules are now well understood, and mainly depend on the nature of the stationary phase. The use of polar stationary phases improves the retention of polar compounds, when C18-bonded silica favours the retention of hydrocarbonaceous compounds. In this sense, reversed-phase and normal-phase chromatography can be achieved in SFC, as in HPLC. However, these two domains are clearly separated in HPLC due to the opposite polarity of the mobile phases used for each method. In SFC, the same mobile phase can be used with both polar and non-polar stationary phases. Consequently, the need for a novel classification of stationary phases in SFC appears, allowing a unification of the classical reversed- and normal-phase domains. In this objective, the paper presents the development of a five-dimensional classification based on retention data for 94–111 solutes, using 28 commercially available columns representative of three major types of stationary phases. This classification diagram is based on a linear solvation energy relationship, on the use of solvation vectors and the calculation of similarity factors between the different chromatographic systems. This classification will be of great help in the choice of the well-suited stationary phase, either in regards of a particular separation or to improve the coupling of columns with complementary properties.

Determination of tetracyclines residues in honey by on-line solid-phase extraction high-performance liquid chromatography

An automated system using on-line solid-phase extraction (SPE) high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection was developed for the determination of tetracyclines (TCs), such as tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC), metacycline (MC), and doxycycline (DC) in honey. One milliliter diluted honey sample was injected into a conditioned C18 SPE column and the matrix was washed out with water for 3 min. By rotation of the switching valve, TCs were eluted and transferred to the analytical column by the chromatographic mobile phase. Chromatographic conditions were optimized. TCs were separated in less than 8 min with a gradient elution using a mixture of 0.8% formic acid and acetonitrile. The UV detection was performed at 365 nm. The conditions for on-line SPE, including solvent and total time for loading sample and washing matrix were also optimized. Time for extraction and separation decreased greatly. For the five kinds of TCs, the limits of detection (LODs) at a signal-to-noise of 3 ranged from 5 to 12 ng g −1. The relative standard deviations (R.S.D.) for the determination of TCs ranged from 3.4 to 7.1% within a day and ranged from 3.2 to 8.9% in 3 days, respectively.

Analysis of a G protein-coupled receptor for neurotensin by liquid chromatography–electrospray ionization–mass spectrometry

The type 1 neurotensin receptor (NTS1) belongs to the G protein-coupled receptor (GPCR) family. GPCRs are involved in important physiological processes, but for many GPCRs ligand binding sites and other structural features have yet to be elucidated. Comprehensive analyses by mass spectrometry (MS) could address such issues, but they are complicated by the hydrophobic nature of the receptors. Recombinant NTS1 must be purified in the presence of detergents to maintain solubility and functionality of the receptor, to allow testing of ligand, or to allow G protein interaction. However, detergents are detrimental to MS analyses. Hence, steps need to be taken to substitute the detergents with MS-compatible polar/organic solvents. Here we report the characterization of NTS1 by electrospray ionization (ESI)–MS with emphasis on methods to transfer intact NTS1 or its proteolytic peptides into compatible solvents by protein precipitation and liquid chromatography (LC) prior to ESI–MS analyses. Molecular mass measurement of intact recombinant NTS1 was performed using a mixture of chloroform/methanol/aqueous trifluoroacetic acid as the mobile phase for size exclusion chromatography–ESI–MS analysis. In a separate experiment, NTS1 was digested with a combination of cyanogen bromide and trypsin and/or chymotrypsin. Subsequent reversed phase LC–ESI–tandem MS analysis resulted in greater than 80% sequence coverage of the NTS1 protein, including all seven transmembrane domains. This work represents the first comprehensive analysis of recombinant NTS1 using MS.

Analysis of tricyclic antidepressant drugs in plasma by means of solid‐phase microextraction‐liquid chromatography‐mass spectrometry

Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 microm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 degrees C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 degrees C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96-98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm x 2.1 mm, 5 microm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plasma.

Simultaneous determination of aflatoxins, ochratoxin A andFusariumtoxins in maize by liquid chromatography/tandem mass spectrometry after multitoxin immunoaffinity cleanup

A liquid chromatography/tandem mass spectrometry method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, fumonisins (B(1), B(2)), deoxynivalenol, zearalenone, T-2 and HT-2 toxins in maize. A double extraction approach, using a phosphate-buffered solution followed by methanol, was applied to achieve effective co-extraction of the 11 mycotoxins under investigation having quite different polarities and chemical structures. A new multitoxin immunoaffinity column containing antibodies for all these mycotoxins was used to clean up the extract. Detection and quantification of the 11 mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS) using, as chromatographic mobile phase, a linear gradient of methanol/water containing 0.5% acetic acid and 1 mM ammonium acetate. Method performances were quite satisfactory for all tested mycotoxins at contamination levels close to or below the relevant EU maximum permitted or recommended levels. Limits of detection in maize ranged from 0.3 to 4.2 microg/kg. Recoveries higher than 79% were obtained for all tested mycotoxins with relative standard deviations less than 13%.

Voltammetric Behavior ofp-Nitrophenol and Its Trace Determination in Human Urine by Liquid Chromatography with a Dual Reductive Mode Electrochemical Detection System

The determination of trace p-nitrophenol (PNP) concentrations in human urine has been successfully achieved by high performance liquid chromatography dual electrode detection (LC-DED) in the reduction-reduction mode. Initial cyclic voltammetric studies were undertaken to investigate the electrochemical behavior of PNP at a glassy carbon electrode over a wide pH range; the redox processes giving rise to the signals have been deduced. Further, deductions regarding the behavior in the flow cells were made from hydrodynamic voltammetric data. PNP eluting from the analytical LC column is first reduced to p-hydroxylaminophenol, at the first generator electrochemical cell. This species then undergoes chemical oxidation to give a quinoneimine species which is then detected at the downstream detector electrode using an applied potential of −0.1 V. The optimum chromatographic mobile phase consisted of 40% acetonitrile, 60% water, containing 25 mM o-phosphoric acid, at a flow rate of 0.5 mL/min; this was used in conjunction with a Hypersil C18 column. Hydrodynamic voltammetric studies were undertaken to investigate the dual electrode behavior of PNP, and an applied potential of −2.0 V at the generator cell and −0.10 V at the detector cell were found to be optimum. The response was found to be linear over the range 7.0 ng to 500 ng on column, with an associated R2 value of 0.9981; the limit of detection was found to be 1.0 ng PNP on column. No interferences were seen for a number of common drugs or for the principal electrochemically active components of human urine or serum. The developed assay was successfully applied to the determination of trace concentrations of PNP in human urine samples, exhibiting coefficients of variation of 7.1% (n=7), with a mean recovery of 94.7% for urine fortified at 522 ng mL−1.

Determination of andrographolide and dehydroandrographolide in rabbit plasma by on-line solid phase extraction of high-performance liquid chromatography

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining andrographolide and dehydroandrographolide in rabbit plasma. Plasma samples (100 μL) were injected directly into a C18 SPE column and the biological matrix was washed out for 6 min using 15% aqueous methanol. By rotation of the switching valve, andrographolide and dehydroandrographolide were eluted in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase consisted of methanol:acetonitrile (ACN):water (50:10:40; v/v). The UV detection was performed at 225 nm. The calibration curves showed excellent linear relationship ( R ≥ 0.9993) over the concentration range of 0.05–5.0 μg mL −1. The within- and between-day precisions (R.S.D.) of two analytes were in the range of 1.2–6.5% and the accuracies were between 92.0% and 102.1%. Their recoveries were all greater than 94%. The limits of detection were 0.019 μg mL −1 for andrographolide and 0.022 μg mL −1 for dehydroandrographolide. This method was successfully applied to the plasma concentration–time curve study after oral administration of Andrographis paniculata Nees extract in rabbit.

Purification method development for chiral separation in supercritical fluid chromatography with the solubilities in supercritical fluid chromatographic mobile phases

A comprehensive approach was applied to develop a chiral purification method for an analyte that was found to be unusually difficult to scale-up in supercritical fluid chromatography (SFC). This was performed by studying major factors such as the solubility of an analyte in SFC mobile phases, impurity profiles, and cycle time. For this case study, the solubility in SFC mobile phase was measured by a packed column technique, coupled with a novel trapping mechanism to enhance measurement precision in SFC conditions. The solubility studies in SFC mobile phases suggested a couple of possible SFC mobile phases, in which the analyte would potentially be most soluble. The SFC methods were developed to purify a sample containing 15% of an impurity, after considering impurity profiles and cycle times of several potential methods in addition to SFC mobile phase solubility. An equal volume mixture of acetonitrile and ethanol was chosen for the final purification method, since this mixture demonstrated the relatively high SFC solubility among all solvent combinations with enhanced resolution between the analyte and the impurity as well as the shortest run time. The solubility of the compound was also determined in various organic solvents using a high throughput solubility screening system to better understand relative change of solubility from neat solution to SFC mobile phases.

Mixture–mixture design for the fingerprint optimization of chromatographic mobile phases and extraction solutions for Camellia sinensis

A composite simplex centroid–simplex centroid mixture design is proposed for simultaneously optimizing two mixture systems. The complementary model is formed by multiplying special cubic models for the two systems. The design was applied to the simultaneous optimization of both mobile phase chromatographic mixtures and extraction mixtures for the Camellia sinensis Chinese tea plant. The extraction mixtures investigated contained varying proportions of ethyl acetate, ethanol and dichloromethane while the mobile phase was made up of varying proportions of methanol, acetonitrile and a methanol–acetonitrile–water (MAW) 15%:15%:70% mixture. The experiments were block randomized corresponding to a split–plot error structure to minimize laboratory work and reduce environmental impact. Coefficients of an initial saturated model were obtained using Scheffe-type equations. A cumulative probability graph was used to determine an approximate reduced model. The split–plot error structure was then introduced into the reduced model by applying generalized least square equations with variance components calculated using the restricted maximum likelihood approach. A model was developed to calculate the number of peaks observed with the chromatographic detector at 210 nm. A 20-term model contained essentially all the statistical information of the initial model and had a root mean square calibration error of 1.38. The model was used to predict the number of peaks eluted in chromatograms obtained from extraction solutions that correspond to axial points of the simplex centroid design. The significant model coefficients are interpreted in terms of interacting linear, quadratic and cubic effects of the mobile phase and extraction solution components.

Comparison of LC/MS and SFC/MS for Screening of a Large and Diverse Library of Pharmaceutically Relevant Compounds

The search for greater speed of analysis has fueled many innovations in high-performance liquid chromatography (HPLC), such as the use of higher pressures and smaller stationary-phase particles, and the development of monolithic columns. Alternatively, one might alter the chromatographic mobile phase. The low viscosity and high diffusivity of the mobile phase in supercritical fluid chromatography (SFC) allows higher flow rates and lower pressure drops than is possible in traditional HPLC. In addition, SFC requires less organic, or aqueous-organic, solvent than LC (important in preparative-scale chromatography) and provides an alternative, normal-phase retention mechanism. But fluids that are commonly used as the main mobile-phase component in SFC, such as CO2, are relatively nonpolar. As a result, SFC is commonly believed to only be applicable to nonpolar and relatively low-polarity compounds. Here we build upon recent work with SFC of polar and ionic compounds and peptides, and we compare the LC/MS and SFC/MS of a diverse library of druglike compounds. A total of 75.0% of the library compounds were eluted and detected by SFC/MS, while 79.4% were eluted and detected by LC/MS. Some samples provided strong peaks that appeared to be related to the purported compound contained in the sample. When these were added to the "hits", the numbers rose to 86.7 and 89.9%, respectively. A total of 3.7% of the samples were observed by SFC/MS, but not by LC/MS, and 8.1% of the samples were observed by LC/MS, but not by SFC/MS. The only compound class that appeared to be consistently detected in LC/MS, but not in SFC/MS under our conditions, consisted of compounds containing a phosphate, a phosphonate, or a bisphosphonate. The SFC/MS method was at least as durable, reliable, and user-friendly as the LC/MS method. The APCI source required less cleaning during the SFC/MS separations than it did during LC/MS.

Viscosity measurements of methanol–water and acetonitrile–water mixtures at pressures up to 3500 bar using a novel capillary time-of-flight viscometer

A new type of viscometer based on the Poiseuille flow principle has been developed that is capable of measuring solution viscosities at ultrahigh pressures. The capillary time-of-flight (CTOF) viscometer has been used to measure the viscosity of methanol–water and acetonitrile–water mixtures in decade volume% increments from atmospheric pressure to 3500 bar (50,000 psi), at 25 °C. This instrument works by utilizing a relatively small pressure drop (∼200 bar) across a capillary which has both inlet and outlet pressurized so that the average column pressure can be significantly elevated (up to 3500 bar). Measurements from the CTOF viscometer match high-pressure viscosity data collected previously using falling-body viscometers of the Bridgman design. This manuscript serves to bring viscosity data at ultrahigh pressures for the two most common liquid chromatographic mobile phases into the chromatographic literature.

Separations of Long-chain Hydrocarbons and Mink Oils Using Pressure Programming Supercritical Fluid Chromatography

The advantages of supercritical fluid chromatography (SFC) have recently been realized in particular its rapidity, flexibility, and ability to allow the analysis of substances which cannot be analysed by gas chromatography (GC). The separating power of open-tubular column GC is unparalleled, but its applicability is restricted by the limited volatility and thermal stability of many organic compounds; less volatile compounds can be analysed by high performance liquid chromatography (HPLC), but long analysis times and very small column diameters are required for efficient separations because of the limitations of solute diffusion in the mobile phase. SFC overcomes these difficulties and permits high resolution at low temperatures with short analysis times. Above its critical point a substance such as carbon dioxide has properties which make its use as a chromatographic mobile phase very favourable. The ability of supercritical fluids to dissolve substances was first recorded in 1879, when Hannay and Hogarth studied the solubility of cobalt and iron chlorides in supercritical ethanol. Numerous applications of extraction with supercritical solvents have been described, and the underlying theory fully understood. Following a suggestion by Lovelock in 1958 that a supercritical fluid might be used as a mobile phase in chromatography, Klesper et al. first deminstrated SFC by the separation of nickel porphyrins using supercritical chlorofluoromethanes as mobile phases. Sie and Rijnders and Giddings developed the technique further, both practically and theoretically, and many applications have been reported. Supercritical fluids may be defined as fluids that are at temperatures and pressures above their critical temperatures and critical pressures. Supercritical fluids are now widely used in extraction, fraction, and chromatography. Supercritical fluid separations of the triglycerides in mink oils have not been reported yet, although other triglycerides in fish oil, rapeseed oil, soybean oil, and butterfat were separated and those of butterfat were identified using massspectrometry. In addition to their critical temperatures and pressures, the chromatographically important properties of supercritical fluid are the density, viscosity, and the diffusion coefficients of solutes. Above its critical point, a substance has density and solvating power approaching that of a liquid, but viscosity similar to that of a gas, and diffusivity intermediate between those of a gas and liquid. Hence, the supercritical fluids have properties which make their use as chromatographic phase very favourable. As long as intermolecular interactions are sufficiently strong, supercritical fluids are able to dissolve a variety of solutes, even those with high molecular mass and low volatility. The density of supercritical fluids, and hence the solubility and chromatographic retention of solutes, can easily be changed by changing the applied pressure. This paper deals with the supercritical fluid separations of long-chain hydrocarbons and mink oils that are very difficult to be separated by either GC or HPLC, using pressure programming technique.

DETERMINATION OF NINE COMPONENTS IN BAYER LIQUORS BY HIGH PERFORMANCE ION CHROMATOGRAPHY WITH CONDUCTIVITY DETECTOR

DETERMINATION OF NINE COMPONENTS IN BAYER LIQUORS BY HIGH PERFORMANCE ION CHROMATOGRAPHY WITH CONDUCTIVITY DETECTOR

Presence of imidazole in loading buffer prevents formation of free radical in immobilized metal affinity chromatography and dramatically improves the recovery of herpes simplex virus type 1 gene therapy vectors

We have recently shown that immobilized metal affinity chromatography (IMAC) is an effective technique for purification of herpes simplex virus type 1 (HSV-1) gene vector engineered to display cobalt affinity tag on the envelope. However, the tagged HSV-1 viruses were severely inactivated by oxidative hydroxyl free radicals when crude HSV-1 supernatant was applied on an immobilized cobalt column and eluted by a low pH buffer. Furthermore, we have reported that virus inactivation could be prevented by inclusion of high concentration of ascorbate in chromatographic mobile phase. In this paper we report that when elution of bound virus was attempted by inclusion of imidazole in elution buffer, rather than lowering the pH of elution buffer, similar inactivation was also observed. The results also demonstrated that virus inactivation was dramatically reduced by inclusion of 20 mM imidazole in the loading buffer. Electron spin resonance (ESR) experiments suggest that imidazole prevents hydroxyl free radical generation from the cobalt complexes. This is the first report describing the role of imidazole in preventing free radical formation in an IMAC column. From a practical stand point, our results imply that inclusion of appropriate amount of imidazole in the loading buffer is an effective strategy for improving the recovery yield of active products and for enhancing product quality during IMAC purification.

Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: Oxidative damage by hydroxyl free radicals and its prevention

Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it.

Evaluation of Methanol-Water and Acetonitrile-Water Binary Mixtures as Eluents for Temperature-dependent Inclusion Chromatography

In the present study the solubility of beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin at sub-zero and elevated temperatures (-10 and +30 degrees C) for a given composition of methanol/water and acetonitrile/water binary mixtures (Xs = 0.16) was studied. Moreover, the freezing temperature profiles of acetonitrile-based chromatographic mobile phases were measured, and the obtained results were compared with data available in the literature. Furthermore, the effect of the macrocycles concentration on the liquid-phase freezing points was determined. The low solubility of native beta-cyclodextrin in a methanol/water mixture at sub-zero temperature as well as the non-linear behavior of acetonitrile/water mixtures that were observed concerning the freezing point profile are discussed from a practical point of view.