The use of Bond-Elut for the estimation of serum bile pigments bonded covalently to albumin

A simple and precise method has been devised for the quantitation of biliprotein (delta-bilirubin or albumin bound bilirubin) in serum. In the presence of caffeine/benzoate, Bond-Elut (C8, 200 mg) extracts unconjugated and conjugated bilirubin but not pigments that are covalently bonded to albumin which pass through the column and can be quantitated by a standard diazo method. Following elution from the Bond-Elut column with methanol-acetonitrile (50:50, v/v) unconjugated and conjugated bilirubin can be quantitated either as total pigments or individually by HPLC.     

The Effects of an Ionic Mobile Phase Modifier and Thermal History on a Reversed-Phase Liquid Chromatographic System

Abstract

The addition of millimolar quantities of potassium chloride to a totally aqueous mobile phase increased retention and reduced band broadening with an RP-8 column and polar solute as compared to pure water. This effect was minimized after cycling the solvent-conditioned column through a temperature gradient. These effects are explained by a dynamic surface model in which entrapped conditioning solvent as well as imbibed ionic species all play a part in structuring the interfacial region where retention occurs.     

Control of adsorption and solubility in gradient high performance liquid chromatography 1. Principles of sudden transition gradients and elution characteristics of copolymers from styrene and methacrylates

Summary Proper retention of polymers in high performance liquid chromatography often requires injection into a starting eluent which is not a solvent for the sample under investigation. In this case, the polymer is precipitated at the top of the column. Subsequent gradient elution has to be performed by addition of an eluent with sufficient chromatographic strength and solvent power. In normal phase chromatography, it must be a solvent of high polarity. With the gradient elutions reported so far, polarity and dissolution power were simultaneously increased.The present paper reports the separate control of solvent strength and chromatographic power by applying gradient programs which include sudden addition of a moderately polar solvent. The amount of the latter does not suffice for elution, which is performed by subsequent, controlled addition of a highly polar nonsolvent. Sudden transition gradients of this kind work with, e.g.,iso-octane as a nonpolar starting eluent, tetrahydrofuran as a solvent of intermediate polarity, and methanol as a strongly polar nonsolvent. They have been applied to copolymers from styrene and ethyl methacrylate, methyl methacrylate, or methoxyethyl methacrylate.     

The Separation of Conjugated and Unconjugated Bilirubin in Bile by High-Performance Liquid Chromatography

Abstract

A fast and simple method was developed for the separation of unconjugated bilirubin and its mono- and di-glucuronide conjugates from bile by high-performance liquid chromatography (HPLC). Unconjugated bilirubin was separated on a reversed-phase column using acetonitrile-water (70:30 v/v) as the mobile phase, while the conjugates were separated on a μ-Bondapak-carbohydrate column employing acetonitrile-water (90:10 v/v) as the eluent. The application of this method was demonstrated by the analysis of the bile pigments in rat bile.     

Quantitative fractionation of serum bilirubin species by reversed-phase high-performance liquid chromatography

A rapid high-performance liquid chromatographic method is described for separating and quantifying four bilirubin species present in serum: bilirubin, bilirubin monoglucuronide, bilirubin diglucuronide, and bilialbumin. Sample preparation consists of dilution with ascorbic acid and dimethylsulfoxide and filtration to remove solid materials. The diluted serum was injected directly onto a wide-pore (300 A) reversed-phase column and the bilirubin species eluted with a water-isopropanol gradient. Excellent agreement was found between the total bilirubin concentration measured by the high-performance liquid chromatographic method and a diazotized sulfanilic acid procedure.     

Chromatographic behaviour of low molar-mass polyesters in normal-phase high-performance liquid chromatography

Summary The normal-phase chromatographic retention behaviour of polyesters on bare silica and on a polymer-based polyamine (PA) column has been studied with a variely of binary mobile phases under isocratic conditions. The dependence of experimental retention data on the degree of polymerization (p) and on mobile phase composition (φ) was characterized by to an approach developed by Jandera et al. The bulky repeating unit and the relatively highly polar end groups of the polyesters both had a large influence on retention behaviour. The two effects in combination explain the molar-mass-independent retention observed experimentally at a particular mobile phase composition for all the mobile phase—stationary phase combinations investigated. These conditions were found to be independent of the type of end group. End group separation on a silica column improves when the polarity of the less polar solvent is increased. End group separation is better on the PA column because of a greater difference between the adsorption energy of the alcohol and acid end groups. Better prediction of retention data on the PA column was achieved by use of an approach which assumes two different types of adsorption site. Results enabled further understanding of retention behaviour in normalphase gradient polymer-elution chromatography (NPGPEC) and explained both the dependence of the order of elution onp and differences between the end-group selectivity of different systems.     

Development and evaluation of a spiral tube column for counter-current chromatography

An improved type-J counter-current chromatography (CCC) planet centrifuge with two spiral tube columns (volume 2×15 mL, β value 0.3-0.7, tubing 0.8 mm id) was developed and evaluated for its retention ability of four typical different solvent systems including heptane-methanol (1:1, v/v) (A), hexane-ethyl acetate-methanol-water (1:1:1:1, v/v) (B), n-butanol-acetic acid-water (4:1:5, v/v) (C), PEG1000-K(2)HPO(4)-water (12.5:12.5:75, w/w) (D) under eight different operation modes. The results indicated that the spiral tube column could significantly increase the retention of four typical solvent systems compared with a traditional multilayer coil column with similar parameters (volume 35 mL, β value 0.3-0.7, tubing 0.8 mm id). The retention of stationary phase (S(f)) for the less polar system (A) and moderately polar solvent system (B) can be increased by about 10%, and for the polar system (C) and aqueous two-phase system (ATPS) (D) by 30-40%. The preliminary applications of this spiral tube column to the separation of small molecular compounds such as moderately polar theaflavins, polar anthocyanins and dipeptides were successful. Acceptable resolution can be obtained between cytochrome c and myoglobin, lysozyme and myoglobin when it was applied on protein separation; however, it still needs to be improved with regard to its column efficiency.     

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.     

Characterization and prediction of retention in gradient-elution normal phase HPLC with ternary gradients

Summary Chromatographic behaviour of phenylurea herbicides and phenols on a silica gel column with ternary gradient elution using mobile phases containing dry 2-propanol, n-heptane and dioxane was investigated. With dried solvents and the temperature controlled to ±0.1 °C, the data from repeated experiments measured on the same column after ten months of use were reproducible. Algorithms were suggested for characterization and prediction of retention in ternary gradients at a constant ratio of the two polar solvents (elution strength gradients) and at a constant sum of their concentrations (selectivity gradients). These were based on two- and three-parameter equations describing the dependence of the sample retention factors (isocratic) on the concentration of the polar solvent in binary mobile phases. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Analysis of hydrophilic metabolites by high-performance liquid chromatography-mass spectrometry using a silica hydride-based stationary phase

A novel silica hydride-based stationary phase was used to evaluate the retention behavior in the aqueous normal-phase (ANP) mode of standards representing three classes of metabolites. The effects on retention behavior of amino acids, carbohydrates and small organic acids were examined by altering the column temperature, and by adding different additives to both the mobile phase and sample solvent. Gradient mode results revealed the repeatability of retention times to be very stable for these compound classes. At both 15 and 30 degrees C, excellent RSD values were obtained with less than 1% variation for over 50 injections of an amino acid mixture. The ability to separate the 19 nonderivatized amino acid standards, organic acids and carbohydrates was demonstrated as well as the potential for this material to separate polar metabolites in complex fluids such as urine.     

Quantitative aspects of gradient HPLC of copolymers from styrene and ethyl methacrylate

Summary Prerequisite of quantitative evaluation in chromatography is equivalence of sample composition and detector signal. This includes complete retention and proper elution of all sample constituents. In polymer HPLC, complete retention requires a poor starting eluent, a sufficiently active column, and a low ratio of injection volume to column volume. On small pore columns, insufficient retention caused the polymer to elute either in the interstitial volume (sample exclusion), together with the sample solvent, or immediately after the solvent plug.Stat-copoly(styrene/ethyl methacrylate) samples are more difficultly retained thanstat-copoly(styrene/acrylonitrile) specimes. With the former copolymer it could be shown that incomplete retention did not cause sample demixing. In order to gain complete retention, non-exclusion HPLC of polymers should be performed with columns whose solvent volume is at least 50 times as large as the injection volume. This consequence is of practical importance in chromatographic cross-fractionation where rather large volumes of SEC eluate are injected into the apparatus for gradient HPLC.     

Gradient elution in normal-phase high-performance liquid chromatographic systems

Gradient elution is widely used for separation of complex samples in reversed-phase HPLC systems, but is less frequently applied in normal-phase HPLC, where it has a notoriously bad reputation for poor reproducibility and unpredictable retention. This behaviour is caused by preferential adsorption of polar solvents used in mixed mobile phases, which may cause significant deviations of the actual gradient profile from the pre-set program. Another important source of irreproducible retention behaviour is gradual deactivation of the adsorbent by adsorption of even traces of water during normal-phase gradient elution. To avoid this phenomenon, carefully dried solvents should be used. Finally, column temperature should be carefully controlled during normal-phase gradient elution if reproducible results are to be obtained. Working with dry solvents at a controlled constant temperature and using a sophisticated gradient-elution chromatograph, reproducibility of the retention data in normal-phase gradient elution better than 2% may be achieved even over several months of column use. The retention data in gradient elution can be calculated accurately if appropriate corrections are adopted for the gradient dwell volume and for the preferential adsorption of the polar solvents using experimental adsorption isotherms. The average error of prediction for the corrected calculated gradient retention data was lower than 2% for a silica gel column and lower than 3% for a bonded nitrile column, which may be suitable for the optimization of separation. Further, a simple approach is suggested for rapid estimation of changes in the retention induced by a change in the gradient profile in normal-phase HPLC. For such a rough estimation, it is not necessary to know the parameters of the dependence of the solute retention factors on the composition of the mobile phase.     

Separation of cationic aracyl derivatives of betaines and related compounds

Cationic aracyl esters of betaines can be formed by alkylation with aracyl halides or trifluoromethanesulfonates. HPLC on a non-endcapped strong cation exchange (SCX) column gave high retention of these derivatives. Cation exchange HPLC may be carried out on a normal-phase (silica or alumina) column using a polar organic solvent (acetonitrile, propan-2-ol) containing an aqueous buffer with an organic cation and a hydrophilic anion. Selectivity is affected by the choice of organic solvent and buffer, e.g. alcohols decrease the retention times of hydroxybetaines such as carnitine. Retention is reduced by increasing the water content and the buffer concentration. Capillary electrophoresis migration times are affected by the choice of buffer anion, with low pH citrate buffers favoured.     

Gas chromatographic analysis of organic solvent mixtures on capillary columns of different polarity

Summary A gas chromatographic method for the analysis of organic solvents in chemical products is described. The analysis is performed by the use of a polar column, Supelcowax 10, and a non-polar column CP-Sil-5CB. Samples containing a non-volatile matrix or water were analysed by headspace analysis. The identification of the solvents in a sample, based on GC retention times on one column, is confirmed by GC of the sample on the second column. The method has been found to be suitable for the routine analysis of solvent mixtures.     

多羟基化合物键合亲水色谱固定相的制备及其分离性能

用硅胶与氨丙基三甲氧基硅烷反应,再与δ-葡萄糖酸内酯反应,制备了一种多羟基化合物键合的新型亲水色谱固定相。以水-有机溶剂(乙醇、乙腈、四氢呋喃)为流动相,通过改变流动相中有机溶剂的种类及浓度、缓冲盐浓度和柱温,考察了该固定相对6种强极性中药组分的保留行为和保留机理。当水的比例在0～40%(v/v)范围时,溶质的保留随着流动相中水的比例的增大而减小,属于典型的亲水色谱分离模式;而当流动相中水的比例在0～100%(v/v)范围内变化时,溶质的保留随着水的比例变化呈“U”形曲线,属于亲水色谱和反相色谱的混合保留机理。缓冲盐的浓度和pH效应说明,所选用的中药组分与所制备的固定相间还存在弱的静电作用。该固定相对6种中药组分以及丹参注射液具有良好的分离性能,表明其在强极性中药有效成分的分离及其他强极性物质的分离分析中具有一定的应用前景。     

The Use of Perfluorinated Carboxylic Acids in the Reversed-Phase HPLC of Peptides

Abstract

The relative effectiveness of trifluoroacetic acid (TFA), pentafluoropropanoic acid (PFPA), heptafluorobutyric acid (HFBA) and undecafluorocaproic acid (UFCA) as hydrophobic counter-ions in the reversedphase high performance liquid chromatography (RP-HPLC) of peptides was assessed. Four solvent systems were compared each containing 0.01M of a perfluorocarboxylic acid throughout. Twelve standard peptides and proteins were loaded onto the RP-HPLC column which was eluted with a linear gradient of 20-58.4% aqueous acetonitrile over 90 minutes. The retention times of the peptide standards were different in each solvent system. In progressing from TFA to PFPA, HFBA and UFCA all the peptides showed greater retention times. However, the effect was most marked with peptides having the greatest number of basic groups. By exploiting this behaviour a different type of chromatography can be introduced into the RP-HPLC purification of peptides. For instance, column fractions obtained from the use of the TFA solvent system can be re-chromatographed in a solvent system containing HFBA. It is possible by this procedure to purify naturally occurring peptides on the basis of their overall positive charges. At 0.01M each acid solution is sufficiently U.V. transparent to permit monitoring of column effluents at 210 nm. TFA, PFPA, HFBA and UFCA solvent systems are also completely volatile and this property facilitates the bioassay, radioimmunoassay and amino acid analysis of column fractions.     

Characterization of Amino-, Cyano-alkyl Bonded Silica Columns in Normal-Phase Liquid Chromatography by Using Steroids

Abstract

In order to characterize the chemically bonded phases in HPLC analysis, the retention behavior of fifteen steroids including estrogen, androgen, progestogen and corticoid were systematically examined using dioxan as the stronger component in an n-hexane-binary system. A linear relationship between the logarithm of the capacity ratio and logarithm of the molar concentration of the binary solvent was confirmed for amino- and cyano-type bonded as well as non-bonded silica gel columns. Based on the retention indices of these phases, the retentivity of the packing materials was determined as follows: the amino-type is similar to and the cyano-type is weaker (0.7 times) than bare silica gel when using dioxan as the stronger component. The specific retentivity of an amino column for polar steroids containing phenolic and alcoholic hydroxyl groups suggests a molecular interaction associated with hydrogen bonding between the polar packing surface and solute compounds. The selectivity of amino packing was found to be larger than cyano packing whose retention selectivity is similar to a bare silica gel.     

Analysis of linear and cyclic oligomers in polyamide-6 without sample preparation by liquid chromatography using the sandwich injection method. III. Separation mechanism and gradient optimization

The first six linear and cyclic oligomers of polyamide-6 can be quantitatively determined in the polymer using HPLC with the sandwich injection method and an aqueous acetonitrile gradient. In this final part of the triptych concerning the determination of the oligomers in polyamide-6, the irregular elution behavior of the cyclic monomer compared to the cyclic oligomers was investigated. We also optimized the separation of the involved polyamide oligomers, with respect to gradient steepness, stationary phase, column temperature and mobile phase pH. The irregular elution behavior of the cyclic monomer could be caused by its relatively large exposed/accessible hydrophobic surface, which permits relatively easy penetration into the hydrophobic stationary phase giving extra retention. The dipole moment of the different oligomers was used as a measure for this exposed/accessible hydrophobic area to correlate the retention factors using quantitative structure-retention relationships. We also studied the retention behavior of the polyamide, which is injected each run directly onto the column and modifies the stationary phase. Using a 250-microl post gradient injection zone of formic acid on a 250x3 mm Zorbax SB-C18 column, the polyamide could be effectively removed from the stationary phase after each separation. The linear solvent strength (LSS) model was used to optimize the separation of the first six linear and cyclic oligomers. As the LSS model assumes a linear correlation between the modifier concentration and the logarithm of the retention factor and the cyclic monomer and dimer show extreme curvation of this relation in the eluting region, we investigated different models to predict gradient elution from isocratic data. A direct translation of the isocratic data to gradient retention times did not yield adequate retention times using the LSS model. It was found that the LSS model worked acceptably if gradient retention times were used as input data. Even for fast non-linearly eluting components, an average error of 0.4 resolution units of 4sigma was obtained. Using the LSS model in combination with different column temperatures and mobile phase pH values, a separation of the first six linear and cyclic oligomers was accomplished.     

Solid-phase extraction and determination of dansyl derivatives of unconjugated and acetylated polyamines by reversed-phase liquid chromatography: improved separation systems for polyamines in cerebrospinal fluid, urine and tissue

A sensitive and simple liquid chromatographic assay with fluorometric detection for unconjugated and acetylated polyamines in biological fluids is described. After precolumn derivatization with dansyl chloride, unconjugated polyamines and acetylated polyamines were extracted by elution from a Bond-Elut C18 column and then separated on a reversed-phase column with gradient elution. The complete analysis of unconjugated putrescine, spermidine, and spermine in either hydrolyzed urine, cerebrospinal fluid or tissue could be accomplished within 20-26 min, while the simultaneous analysis of unconjugated polyamines and monoacetylpolyamines could be completed within 40 min. Unhydrolyzed urine and cerebrospinal fluid required a Bond-Elut cation-exchange clean-up before dansylation. Standard curves for the assay were linear up to 20 nmol/ml, and the within-day and day-to-day coefficients of variation were between 1.1 and 4.6% and between 1.6 and 11.8%, respectively. Results obtained with the method were compared with results obtained with a well established modified amino acid analyzer method for urine, tissue and cerebrospinal fluid samples. The correlation coefficients between these two methods were in the range 0.933-0.996. Detection limits between 50 and 150 fmol were achieved for unconjugated and acetylated polyamines. Of more than twenty drugs and amines tested for possible interference with the assay, only normetanephrine was found to have the same retention time as the internal standard 1,6-diaminohexane.