A practical approach to transferring linear gradient elution methods

Attempts to theoretically address the problems involved in transferring linear gradient elution methods have been somewhat ad hoc due to the simplifying assumptions usually made in conventional gradient elution theory. Until now, all equations based on the k* parameter of linear gradient elution theory used as the basis for predicting the separation selectivity have not explicitly included the effect of the dwell volume (VD). Using an exact equation for predicting k*, that is, one which fully accounts in an a priori fashion for VD, we find a set of simple yet exact equations which unequivocally must be satisfied to transfer an optimized linear gradient elution method from one system (column or instrument or both) to another. These relationships absolutely mandate that a change in the instrument dwell volume requires a proportional change in the column volume; in turn, a change in the column volume requires a proportional change in the flow rate and/or gradient time to maintain a constant gradient steepness. Although we are not the first to suggest these guidelines, this work provides a complete theoretical foundation for these exact guidelines for the maintenance of gradient selectivity for the case of transferring a linear gradient elution method between different columns packed with the same particles and/or between different instruments.     

A parallel pore and surface diffusion model for predicting the adsorption and elution profiles of lispro insulin and two impurities in gradient-elution reversed phase chromatography

Lispro insulin (LPI), a widely used insulin analog, is produced on tons per year scale. Linear gradient reversed phase chromatography (RPC) is used in the production to separate LPI from two impurities, which differ from LPI by a single amino acid residue. A chromatography model for the ternary separation in this RPC process is unavailable from the literature. In this study, a parallel pore and surface diffusion model is developed and verified for LPI and the two impurities. The LPI can be recovered with high yield (≥95%) and high purity (>99.5%). A new method, which requires a small amount of materials and an order of magnitude fewer experiments, has been developed to estimate the solvent-modulated isotherm parameters. A modified reversed phase modulator model is developed to correlate the adsorption isotherms of LPI and impurities. A strategy has been developed for estimating the intrinsic pore diffusivity and surface diffusivity. Since the adsorption affinities decrease by more than three orders of magnitude as organic fraction (φ) increases from 0.19 to 0.40, the apparent diffusivities based on a pore diffusion model or a surface diffusion model can also vary by several orders of magnitude. For this reason, a pore diffusion model or a surface diffusion model with a constant apparent diffusivity cannot predict closely the chromatograms over the same range of organic fractions, concentrations, and loadings. The parallel pore and surface diffusion model with constant diffusivities can predict closely the frontal and elution profiles over a wide range of organic fractions (0.19-0.40), LPI concentrations (0.05-18 g/L), linear velocities (<10 cm/min), and loading volume (0.0004-13 CV). For large loading stepwise and linear gradient elution, the peaks of LPI and the impurities are strongly focused by self-sharpening and gradient focusing effects as a result of the steep decrease of adsorption affinity from the loading φ (0.19) to elution φ (≥0.27). When the ratio of diffusion rate to convection rate is greater than 10, spreading due to diffusion is largely compensated by the focusing effects. As a result, a pore diffusion model with a constant pore diffusivity can predict closely the elution profiles in stepwise and linear gradient elution. The experimental yield values (≥95%) can be predicted to within ±1% by the model.     

Unusual signal enhancement in reversed-phase chromatography of polystyrene monitored by UV detector

Summary Gradient elution of polystyrene standards on reversed phase C18 columns by methanol/tetrahydrofuran or methanol/dichloromethane mixtures yielded a strange effect of the molecular weight of the sample on the specific peak are (mAUs per g sample injected). The effect did not occur when pure dichloromethane was used as an eluent. Further to this, dependence of specific peak area on flow rate was observed in gradient elution with methanol/tetrahydrofuran mixtures. It was found that these effects were due to polymer elution at the verge of precipitation. Depending on the dwell time of the sample in the column, opalescence occurred that added to the UV signal used for monitoring the elution.     

Improvement of separation in zonal preparative thin-layer chromatography by gradient elution

Summary Complex extracts of the plants Azulan and Hemorigen were separated by zonal micropreparative thin-layer chromatography in sandwich chambers of the ES and DS type which permitted zonal application of large volumes of sample, without auxiliary equipment. Application from the edge of the layer, in the frontal chromatography mode, markedly improved the separation efficiency and capacity owing to displacement effects which narrow the initially broad zones. Further improvement of separation efficiency and purity of fractions, revealed by densitometry, was observed using stepwise gradient elution. This was confirmed by extraction of some of the separated fractions from the layer and rechromatography; the composition of these fractions were generally simpler than for the corresponding isocratic chromatograms.     

Optimisation technique for stepwise gradient elution in reversed-phase liquid chromatography

An optimisation technique of reversed-phase liquid chromatographic separations based on gradient elution with a stepwise variation pattern of the volume fraction phi of the organic modifier in the water-organic mobile phase is presented. It uses a non-linear least-squares programme with a Monte-Carlo search for initial estimates in order to determine the best variation pattern that leads to the optimum separation of a mixture of solutes. The validity of the above methodology was tested by separating eight catechol-related solutes with mobile phases modified by methanol or acetonitrile and variation patterns of two, three or four steps in the psi values. It was found in all cases a very satisfactory accuracy of the predicted gradient elution times, which is of the same order with the accuracy of the retention times predicted under isocratic or linear gradient conditions. In addition, it was shown that the proposed optimisation technique is both effective and flexible but well-shaped chromatograms are obtained under electrochemical detection only if steps with increasing psi are used and the change in psi is programmed to occur at the intermediate of the predicted peaks.     

A gradient anion exchange chromatographic method for the speciation of arsenic in lobster tissue extracts

A gradient anion exchange chromatographic technique was developed for the separation of arsenobetaine (AsB), arsenocholine (AsC), arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMAA) and dimethylarsinic acid (DMAA) in one chromatographic run. This technique used low residue ammonium carbonate buffer and the inductively coupled plasma-mass spectrometry (ICP-MS) chromatograms showed little baseline drift. Gradient elution improved peak shape and peak separation. The separation was completed in approximately 27 min with low detection limits (0.017-0.029 mug As kg(-1)). Baseline resolution of all the arsenic species evaluated was achieved when the concentration of AsC was less than approximately 12.5 mug As kg(-1). This technique was successfully applied to different extracts of a standard reference material, TORT-2, and lobster tissue. AsB was found to be the major arsenic species present. AsC, DMAA, MMAA and As(V) were also found, although MMAA was not detected in all of the TORT-2 extracts. Two unknown peaks found may be due to the presence of arsenosugars or other arsenic species. Discrepancy between extraction recoveries previously determined using flow injection-ICP-MS and the high-performance liquid chromatography-ICP-MS was observed in some cases. The differences may be due to the extraction technique and/or conditions at which the extractions were performed.     

Characterization of unstable ion-exchange chromatographic separation of proteins.

Very fine separation of proteins by stepwise elution ion-exchange chromatography is very often a unstable process. To characterize the unstability of such processes the elution volume variations were examined by the model equation which contained the ion-exchange capacity and the number of adsorption sites. The data needed for the model calculation were obtained from gradient elution experiments. As a model separation system stepwise elution of a model protein (beta-lactoglobulin) near the isoelectric point on a weak cation-exchange chromatography column was chosen. The elution volume varied significantly with a small change in the ion-exchange capacity. It was found that the ionic strength of the elution buffer must be adjusted in order to compensate a change in the elution volume due to the ion-exchange capacity variations. The ionic strength and the pH of the elution buffer were also found to be important variables affecting the elution volume. In this model separation system, it was indicated that the pH should be within +/-0.1 unit and the ionic strength within +/-0.002 mol/l in order to meet the criteria (+/-5% elution volume variation). It is recommended that gradient elution data be obtained for predicting elution volume variations in stepwise elution. By using the gradient elution data the process diagnosis can be performed, and the important information on the process stability can be obtained.     

The gradient volume principle for fast evaluation of optimum conditions for isocratic analysis

Summary The paper describes by simple experiments in a pragmatical way by easy rules of thumbs gradient optimization. Besides selection of the stationary phase and initial and final conditions the two other important variables are program time and eluent flow rate. It is demonstrated, that when the product of both, the gradient volume, is kept constant, the solutes are always eluted with the same eluent composition at column outlet. At constant gradient volume, peak broadening depends on flow rate and on the eluent properties (viscosity) at which the solutes elute, and on the time the solutes spend in the column. Because peak broadening increases with increasing gradient volume, the peak capacity in gradient elution shows an optimum at gradient volumes around 15 empty column volumes (program times 45 to 60 min at flow rates of 1 ml/min with standard columns).Gradient elution can also be used for fast evaluation of optimum eluent composition for isocratic analysis. This procedure requires a calibration of the equipment for determination of eluent composition at column outlet. The sample is chromatographed in a standard gradient run of 10 to 15 empty column volumes. The eluent composition at which the solute of interest elutes during the gradient is used for isocratic analysis, where the k' value of this solute will then be around 2.Part of Ph. D. Thesis H. Elgass, Saarbrücken, 1978, present address Hewlett-Packard, Waldbronn, FRG. In part presented at Eastern Analytical Symposium, New York, 1982.     

High-field asymmetric waveform ion mobility spectrometry coupled with liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-FAIMS-MS/MS) multi-component bioanalytical method development, performance evaluation and demonstration of the constancy of the compensation voltage with change of mobile phase composition or flow rate

The feasibility of developing a multi-component bioanalytical method using high-field asymmetric waveform ion mobility spectrometry coupled with liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-FAIMS-MS/MS) is demonstrated using nefazodone and its two metabolites as model compounds. The performance of the bioanalytical method for the three analytes, with three different compensation voltage (CV) values, is assessed using standard curves and quality control samples, which exhibited good accuracy, precision and ruggedness. The number of analytes with different CV values that can be quantitated simultaneously depends on the acquisition cycle time, which is a function of the FAIMS residence time (fixed), chromatographic peak width and selected reaction monitoring (SRM) dwell time. It is established that CV, the FAIMS selectivity parameter, is reproducible for at least 16 h, thus ensuring the constancy of the CV during a large-batch sample analysis. It is also established that change in mobile phase composition or of flow rate does not cause a shift in CV. Thus, CV values determined from a CV scan via infusion of a sample can be used for an LC/ESI-FAIMS-M/MS method based on isocratic or gradient elution.     

Peak spreading in linear gradient elution chromatography with a thin monolithic disk

The peak spreading of DNAs of various sizes [12-mer, 20-mer, 50-mer and 95-mer poly(T)] in linear gradient elution (LGE) chromatography with a thin monolithic disk was investigated by using our method developed for determining HETP in LGE. Electrostatic interaction-based chromatography mode (ion-exchange chromatography, IEC) was used. Polymer-based monolithic disks of two different sizes (12 mm diameter, 3mm thickness and 0.34 mL; 5.2 mm diameter, 4.95 mm thickness and 0.105 mL) having anion-exchange groups were employed. For comparison, a 15-μm porous bead IEC column (Resource Q, 6.4mm diameter, 30 mm height and 0.97 mL) was also used. The peak width did not change with the flow velocity for the monolithic disks where as it became wider with increasing velocity. For the monolithic disks the peak width normalized with the column bed volume was well-correlated with the distribution coefficient at the peak position K(R). HETP values were constant (ca. 0.003-0.005 cm) when K(R)>5. Much higher HETP values which are flow-rate dependent were obtained for the porous bead chromatography. It is possible to obtain 50-100 plates for the 3mm monolithic disk. This results in very sharp elution peaks (standard deviation/bed volume=0.15) even for stepwise elution chromatography, where the peak width is similar to that for LGE of a very steep gradient slope.     

Prediction of Retention Time of Solutes under Linear Gradient Elution Conditions in RP-HPLC

A model for prediding retention time of solutes undeir linear gradient elulioa conditions has been established. In this model,the theorelkal expressions under different eluflon modes were derived and tested with the retention behaviors of p-hydroxy-benzaldehyde, vanilUn, biphenyl, phenanthrene in gradient elution. With halting into account the dwell time of the instrumeatal system, the theoretically predicted retention times agreewell with those experimentally determined.     

High performance liquid chromatographic determination of theophylline metabolites in human liver microsomes

A high performance liquid chromatographic (HPLC) technique for the determination of three metabolites of theophylline, 3-methylxanthine (3-MX), 1-methylxanthine (1-MX) and 1,3-dimethyluric acid (1,3-DMU) in human liver microsomes is described. The analytes were extracted from human liver microsomes with methylene chloride/isopropanol and stepwise gradient elution was employed for the resolution of peaks. The limits of quantitation were 15 ng/mL for 3-MX, 20 ng/mL for 1-MX and 20 ng/mL for 1,3-DMU. The calibration range was linear for the three metabolites and the calibration ranges were 15-250 ng/mL for 3-MX, 20-250 ng/mL for 1-MX and 250-4000 ng/mL for 1,3-DMU. The absolute recovery ranged from 63-84% for 3-MX, 65-79% for 1-MX and 77-89% for 1,3-DMU over the calibration curve range. Accuracy for all three metabolites was within +/- 10% and adequate selectivity was demonstrated by the lack of interfering peaks in blank chromatograms. The within-run and interday precision were within 10% RSD for all three metabolites tested at two concentrations. The advantage of this method over previous methods is that the use of quaternary ammonium ion pair reagents in the mobile phase has been obviated. Also, unlike a previous radiometric HPLC method, the need for radiolabelled theophylline has also been eliminated. The method was used to characterize theophylline metabolism in human liver microsomes for immunoinhibition studies and to investigate the interaction of theophylline with selected quinolone antibiotics.     

Fast-GC-conventional quadrupole mass spectrometry in essential oil analysis

This study reports on the compatibility of a conventional quadrupole MS (qMS) as detector for Fast-GC in terms of separation, identification, and quantitation when applied to the analysis of peppermint essential oil (e.o.), adopted as representative of this field. The influence of Fast-GC carried out on a 10 mx0.1 mm id narrow bore column with temperature programmes from 20 to 60 degrees C/min on the results of a qMS in total ion chromatograms (TIC) at different speeds (from 999 and 11 111 amu/s) and SIM modes was evaluated on ten differently abundant components characterizing peppermint e.o. Separation measure (S), peak capacity (n), and half height peak width were taken as separation parameters; match quality, number of scans per peak (NP), spectral skewing, and TIC area repeatability were used for identification. Quantitation was in SIM mode and NP, dwell time, SIM area repeatability and calibration curves, LOD, and LOQ of the selected components were measured. The results show that the peppermint e.o. markers can successfully be analysed qualitatively and quantitatively by F-GC-qMS up to temperature programmes of 60 degrees /min provided that a suitable scan speed is applied. Fast-GC-qMS reduces analysis time by a factor greater than ten and gives results that are qualitatively reliable and quantitatively comparable to those obtained by conventional GC-qMS.     

制备型液相色谱法分离纯化3-甲基吡啶光氯化产物中的2-氯-5-三氯甲基吡啶

利用制备型高效液相色谱从3-甲基吡啶光氯化产物中分离纯化得到2-氯-5-三氯甲基吡啶,对制备色谱的洗脱方式、洗脱剂组成及浓度、进样量等参数进行了优化。使用的制备柱为Zorbax-C18柱,以乙腈-水为流动相,采用速度梯度洗脱方式进行洗脱,用二极管阵列检测器在240 nm波长下检测,进样体积为100 μL。该方法的制备回收率为82.7%,相对标准偏差为4.0%（n=5）,产品纯度为99.01%。     

不同产地龙葵药材的高效液相色谱-蒸发光散射检测指纹图谱

采用高效液相色谱-蒸发光散射检测器(HPLC-ELSD)来研究不同产地龙葵药材品质的差异,为龙葵药材质量标准的制定提供参考依据。使用Phenomenex C₁₈色谱柱,流动相为乙腈-20%(v/v)甲醇水溶液(含0.03%(v/v)三乙胺),梯度洗脱,蒸发管温度为40 ℃。利用"中药色谱指纹图谱相似度评价系统"对所采集的色谱图进行相似度比对,选择相似度在0.9~1.0之间的色谱图归一化拟合成龙葵的标准指纹图谱,并利用对照品进行色谱峰指认。研究结果表明,不同产地来源的龙葵药材之间具有显著的差异,依据HPLC-ELSD采集到的非挥发类成分可对中药材进行区分。该方法为龙葵药材的真实性、优良性和稳定性评价提供了一种新的、具有广阔应用前景的技术手段。     

Universal response model for a corona charged aerosol detector

The universality of the response of the Corona Charged Aerosol Detector (CoronaCAD) has been investigated under flow-injection and gradient HPLC elution conditions. A three-dimensional model was developed which relates the CoronaCAD response to analyte concentration and the mobile phase composition used. The model was developed using the response of four probe analytes which displayed non-volatile behavior in the CoronaCAD and were soluble over a broad range of mobile phase compositions. The analyte concentrations ranged from 1μg/mL to 1mg/mL, and injection volumes corresponded to on-column amounts of 25ng to 25μg. Mobile phases used in the model were composed of 0-80% acetonitrile, mixed with complementary proportions of aqueous formic acid (0.1%, pH 2.6). An analyte set of 23 compounds possessing a wide range of physicochemical properties was selected for the purpose of evaluating the model. The predicted response was compared to the actual analyte response displayed by the detector and the efficacy of the model under flow-injection and gradient HPLC elution conditions was determined. The average error of the four analytes used to develop the model was 9.2% (n=176), while the errors under flow-injection and gradient HPLC elution conditions for the evaluation set of analytes were found to be 12.5% and 12.8%, respectively. Some analytes were excluded from the evaluation set due to considerations of volatility (boiling point <400°C), charge and excessive retention on the column leading to elution outside the eluent range covered by the model. The two-part response model can be used to describe the relationship between response and analyte concentration and also to offer a correction for the non-linear detector response obtained with gradient HPLC for analytes which conform to the model, to provide insight into the factors affecting the CoronaCAD response for different analytes, and also as a means for accurately determining the concentration of unknown compounds when individual standards are not available for calibration.     

Stepwise gradient of buffer concentration for capillary electrochromatography of peptides on sulfonated naphthalimido-modified silyl silica gel

The advantage of using a stepwise gradient of buffer concentration in CEC was demonstrated with the mixed-mode stationary phase, 3-(4-sulfo-1,8-naphthalimido)propyl-modified silyl silica gel (SNAIP). Before the application of a stepwise gradient, the effect of buffer concentration on the separations of six peptides and tryptic digests was investigated. Bubble formation caused by Joule heating at currents up to 95 microA was successfully suppressed by using SNAIP column even without pressurization, which contributed to a stepwise gradient of buffer concentration. Utilizing the stepwise gradient improved and shortened the separation of six peptides as compared to the separation under an isocratic elution.     

Gradient thin-layer chromatography of plant extracts

Summary A modified Niederwieser chamber for stepwise gradient elution, consisting of a PTFE capillary to store the series of eluents and a horizontal glass sandwich chamber with a glass distributor was used for the chromatographic separation of complex plant extracts (Seboren, Hemorigen and Pectosol) used in therapy. Densitograms demonstrate markedly improved separations of the extracts in comparison to isocratic elution.Produced by Polish Reagents, Melgiewska 18, 20-234 Lublin, Poland     

离子色谱线性梯度淋洗分离阴离子的计算机模拟

建立了使用氢氧化钠淋洗液时用计算机模拟离子色谱梯度淋洗分离阴离子的新方法,研究了在线性梯度淋洗过程中不同时间阴离子在色谱柱中的位置及所在位置的淋洗液浓度,得出阴离子在不同时间的容量因子、迁移速度,通过积分得到保留时间。再根据离子色谱峰峰形变化的规律,得到色谱峰峰形的参数。模拟色谱图保留值与实验值的相对误差小于5%,模拟色谱图的色谱峰峰形与实验所得到的色谱图的色谱峰峰形也非常接近。     

Predicting the behaviour of polydisperse polymers in liquid chromatography under isocratic and gradient conditions

In this paper we describe how the existing theories to describe retention and peak width in isocratic and gradient-elution liquid chromatography can be expanded to describe the retention behaviour of natural and synthetic repetitive polymers, which feature distributions of molecules with different masses (and often different structures) rather than unambiguous molecular formulas. For polydisperse samples, it is vital that the model accommodates (isocratic) elution of sample components before the onset of a gradient, elution during the gradient, and elution after the completion of the gradient. The expanded models can readily be implemented in standard spreadsheet software, such as Excel. We have created such spreadsheets based on the conventional model for retention in reversed-phase liquid chromatography (RPLC) and on two different models for retention in normal-phase liquid chromatography. The implementation allows an easy visualization of the theoretical concept. Up to three different polymeric series can be entered, with a total of up to 100 peaks being computed and displayed in isocratic or gradient-elution chromatograms. Also visualized are "retention models" (diagrams of isocratic retention vs. composition) and "calibration curves" (retention or elution composition vs. molecular mass or degree of polymerization). The coefficients in the isocratic retention model may be correlated, as has often been observed in RPLC. It is shown that under certain conditions such a correlation corresponds to the existence of so-called critical (isocratic) conditions, at which all the members of a given polymeric series (same composition and end groups, different number of repeat units) show co-elution.