Estimation of the band broadening contribution of HPLC equipment to column elution profiles

Summary Methods to determine the contribution of the chromatographic equipment to the total band broadening which involve replacing the column by a union or a capillary tube are not suitable as they involve a fundamental change in the chromatographic system. The linear extrapolation method, based on the estimation of the relative influence of the instrument variance on solutes with different capacity factors, is a more attractive alternative method since the column remains in the chromatographic system. This method is only valid when a number of conditions are satisfied. By meeting these conditions the error in the instrument variance by using the linear extrapolation method was determined. At the same time, ways to minimise these errors were studied. Use of the linear extrapolation method in combination with conventional columns of 4.6 mm i.d. appears to yield inaccurate results. In combination with microbore columns the method can be used, provided the columns have a maximum length of 5cm and contain a packing material with a particle size of 2 or 3μm. The error in the determined instrument variance is then of the order of 2μl².     

Microbore liquid chromatographic determination of cadralazine and cephalexin in plasma with large-volume injection

The application of microbore systems (15 cm X 1 mm I.D. columns filled with Nucleosil C18, 5 microns particle size) to the determination of cephalexin and cadralazine in plasma was investigated. Factors such as mobile phase flow-rate, detector flow-cell volume and injection volume were examined with regard to the needs of routine drug analysis. Mobile phase flow-rates of 50-60 microliters/min were used. A flow cell with an optical path length of 6 mm and an intermediary volume (2.4 microliters) was selected for UV detection in order to obtain sufficient sensitivity. Large volumes of non-eluting solvent containing the drug were injected on the column. The addition of an ion-pairing reagent to samples containing cephalexin and cefroxadin prior to the injection was found to improve the chromatographic performance. The blood sample size required for analysis with microbore columns was smaller than that with conventional columns. The analysis time was similar and the limit of quantitation was also similar, provided that large sample volumes were injected on the microbore column.     

Semi-micro size-exclusion chromatography: Molecular-weight measurement of poly (ethylene terephthalate) and separation of oligomers and prepolymers

Summary Several polystyrene gels of different pore sizes were packed into a 500 mm×2.1 mm I.D. column. Semi-micro size-exclusion chromatography (SEC) using these columns was carried out with a system consisting of a triple piston pump, a micro loop injector and a flow cell with 1.0-μl cell volume constructed for semi-micro HPLC, because the dead volume of the injector and the cell volume of flow cell for conventional HPLC caused a significant loss in column efficiency. The effects of sample amount, injection volume and mobile phase flow rate on column efficiency and retention volume were examined and the optimized operational variables of the sample amount (below 500 μg), the injection volume (less than 15 μl) and the flow rate range (30–70 μl/min) determiend for semi-micro SEC. Oligostyrene, epoxy resin, phenol-formaldehyde resin and phthalates were analyzed by the optimized semi-micro SEC system under the given conditions. In addition, molecular weight distribution of four different poly(ethylene terephthalate) films was successfully measured by using a mixture of chloroform and hexafluoroisopropanol as the eluent.     

Microbore columns in quantitative column liquid chromatography

Summary Some quantitative implications of the use of microbore columns in column liquid chromatography are investigated. Although for several pumps the flow rate stability at 50 l min^–1 is slightly worse than that of pumps operating at 1 ml min^–1, the quantitative performance with respect to repeatability and reproducibility of response factors equals that of conventional liquid chromatography. Thermostatting is strongly recommended for microbore column operation.     

Applications of Fast High Performance Liquid Chromatography in Pharmaceutical Analysis

Abstract

Fast HPLC offers a definite time advantage for analyses such as content uniformity which requires, in general, a routine analysis of as many as 30 samples and for the analysis of dosage forms of developmental drugs. The columns for Fast HPLC can be used with a minimal amount of modification to conventional HPLC hardware. The resolution obtainable with a Fast HPLC column is poorer than that of a conventional column. (All references to conventional columns in the article imply, unless otherwise indicated, those columns 15-30 cm in length and 3.9-4.6 mm i.d.) Attempts to modify a mobile phase to improve the resolution of a closely eluting peak resulted in retention times not too different from typical conventional columns. Although microbore columns offered improved resolution and a savings in both solvent and sample consumption, the utility of these columns is limited by the need for specialized hardware. Since the sample volume is not a limiting factor in a typical pharmaceutical analysis and overall cost savings from lesser solvent consumption are not significant in a majority of cases as solvent can be recycled, it is concluded that the Fast HPLC columns can play a more important role than the currently available microbore columns in laboratories engaged in pharmaceutical analysis, particularly at the product development stage.     

Determination of myristicin in rat serum samples using microbore high performance liquid chromatography with column-switching

Summary A microbore high-performance liquid chromatographic method with column-switching was developed for the analysis of myristicin from rat serum without prepurification. Deproteinization, fractionation, concentration and separation of analyte were carried out by appropriate switching of columns and using solvent mixtures. The method showed excellent precision, accuracy and speed with a detection limit of 10 ng mL⁻¹ from 25 μL of serum. The total analysis time per sample was 25 min and the coefficients of variation for intra- and inter-assay were less than 1.8%.     

Reproducibility of retention indices measurement of alkylbenzenes on crosslinked methyl silicone phase fused silica capillaries

Summary Reproducibility of the Kovats retention indices of alkylbenzenes was studied on conventional “PONA” fused-silica capillary columns coated with crosslinked dimethyl silicone phase, with a film thickness of 0.5 μm. The data were compared with those obtained on conventional OV-101 columns. The tested PONA columns showed reproducible chromatographic properties, capacity ratios and high efficiencies. Having a standard deviation of s=0.03 i.u. of the retention indices on a single column, the column-to-columns differences were found to be up to 0.3 i.u. Higher indices were determined for crosslinked columns, the difference being in most cases about one i.u.     

Microbore HPLC column performance and temperature programming capabilities

Microbore columns (1 mm i.d.) are compared to conventional 4.6 mm i.d. columns with respect to speed, efficiency and sensitivity. If column lengths, particule diameter, packing efficiency, mobile phase, and linear velocity are the same, most chromatographic properties such as speed (analysis time), efficiency, pressure drop, and sensitivity are the same. The major differences are the column volumes, the void volumes, and the volumetric flow rates for equal linear flow velocities. The low flow rates of microbore columns (30 to 200 μl/min), conserve solvent and make for easier interfacing to other instruments such as MS, NMR, or FTIR. Temperature programming of microbore HPLC columns produces faster analyses for large k′ values, increased sensitivity (due primarily to sharper peak shape), and increases the range of compounds which can be handled in an isocratic mode.     

A comparative study of microbore reverse-phase columns

A procedure for packing reverse-phase microbore columns using moderate pressure is described. Columns packed from different manufacturer's tubing are compared with commercially available columns. A method for coupling the column to the injection valve is suggested. The effect of detector cell volume on overall efficiency of the system is also examined.     

High-sensitivity phenylthiohydantoin amino acid analysis using conventional and microbore chromatography

Reversed-phase microbore high-performance liquid chromatography was investigated for high-sensitivity analysis of phenylthiohydantoin (PTH) amino acids. A mixed nitrile alkylsilane bonded phase was developed and ternary gradient elution conditions were devised for resolution of the common PTH amino acids. Elution conditions were developed with a conventional 150 X 4.6 mm I.D. column and transferred to a 150 X 1 mm I.D. microbore column. The performance of these columns was evaluated in terms of PTH amino acid resolution, enhanced sample detectability, and retention time precision. For this work a general purpose high-performance liquid chromatograph was modified to reduce extra column band broadening and a preformed gradient elution technique was developed to achieve rapid analysis times at microbore flow-rates. The microbore high-performance liquid chromatographic system is useful for high-sensitivity analysis of PTH amino acids in micro-sequencing applications.     

Fast LC separation of a myoglobin digest: a case study using monolithic and particulate RP 18 silica capillary columns

A method was developed for the fast separation of a myoglobin digest using a monolithic RP 18 silica capillary column of 100 μm I.D. The results were compared with those obtained with a particulate RP 18 silica capillary column of 100 μm I.D. at a flow-rate between 0.6 and 1.2 μl/min. The digest was analyzed at the monolithic column at a flow-rate up to 2.8 μl/min. This high flow-rate could not be applied to the particulate column due to the high back-pressure. When the starting composition of the gradient was changed from 0 to 20% and a gradient steepness of 16%/min was used, the analysis time was less than 4 min. A positive Mascot identification score of 115 was achieved for the MS–MS data. When a lower gradient steepness was employed, the chromatographic resolution and the peak capacity did not increase for most compounds. The intraday repeatability for the retention time of the monolithic column was better than 1.5% at 2.8 μl/min and even less than 0.5% using a flow-rate of 0.6 or 1.0 μl/min. For the particulate column, it was between 0.5 and 1.4% for a flow-rate of 0.6 μl/min, probably due to the high column back-pressure. The interday reproducibility for the retention time of the monolithic column was less than 0.9% using a flow-rate of 1.0 μl/min.     

2D LC Separation and Determination of Bradykinin in Rat Muscle Tissue Dialysate with On-Line SPE-HILIC-SPE-RP-MS

A 2D liquid chromatography (LC) system using hydrophilic interaction chromatography (HILIC) and reversed phase columns has been employed for comprehensive (LC × LC) separation of rat muscle tissue micro-dialysate. Incorporation of an on-line reverse-phase solid phase extraction (SPE) enrichment column in front of the first dimension enabled aqueous samples with high salt concentrations to be injected directly without compromising the chromatographic performance of the HILIC column. Since the SPE enrichment column allowed injection of large sample volumes (e.g. 450 μL), a capillary HILIC column (inner diameter 0.3 mm) could be employed instead of a larger column which is often used in the first dimension to load sufficient amounts of sample. The two chromatographic dimensions were connected using a column selector system with 18, 1.0 mm I.D. C₁₈ “transition” SPE columns. A PLRP C₁₈ column was used in the second dimension. The 2D LC system’s performance was evaluated with a tryptic digest mixture of three model proteins. Good trapping accuracy (HILIC→transition SPE→RP recovery >95%) and repeatability (within-and between day retention time RSDs of first and second dimension chromatography >1%) was achieved. A dialysis sample of rat muscle tissue was separated with the 2D system, revealing complexity and large differences in concentrations of the various compounds present, factors which could potentially interfere with the quantification and monitoring of two target analytes, arg-bradykinin and bradykinin. Subsequently, “Heart-cut” 2D LC-electrospray–mass spectrometry (ESI–MS) with post-column on-line standard injection was employed to monitor arg-bradykinin and bradykinin levels as a function of various muscle conditions. The method’s quantification precision was RSD = 3.4% for bradykinin.     

Electrochromatography and micro high-performance liquid chromatography with 320 μm I.D. packed columns

Electrochromatography is a chromatographic method in which the mobile phase (liquid or supercritical fluid) is “pumped” through a stationary phase in a microbore or capillary column by electroosmosis using an electric field. The technique permits separation of charged and uncharged compounds with higher resolution and superior efficiency when compared with micro-HPLC with an identical column. It is desirable to work with packed capillary columns with wide diameter in electrochromatography in order to improve detectability and column loadability. This study shows that we have moved a step forward towards this goal in spite of problems and difficulties, due to Joule heating, frit making and column packing in using wide-diameter columns. The paper demonstrates that the pressure pump of micro-HPLC with a commercially available 320 μm I.D. column can be replaced by the electroosmotic “pump” of capillary zone electrophoresis. Experiments were carried out in a chromatographic system under both electroosmosis and pressure-driven flow with 320 and 50 μm I.D. columns packed with 3- and 5-μm ODS. The advantage of electrochromatography over conventional micro-HPLC is shown.     

The new generation of L. C. columns; A critical evaluation of their systems compatibility and performance

Summary The demand for faster, more economic and convenient high-efficiency LC analyses has led to the development of microbore and fast columns. However, to utilize their benefits, the low column dispersions produced by these new technologies necessitate low external variances in the total system.This paper describes the determination of the external variance of microbore, fast and conventional LC systems by a column method. Compatibility with the relevant column technologies is evaluated by a method involving the comparison of the external and column dispersions.Direct comparison of chromatograms of an 8-component mixture, on a set of columns of various diameters and lengths, shows the relative speed, economy and sensitivity obtainable in practice.The problem of reduced sample loadability is considered and the practical solution of peak compression illustrated.Part of this work was presented during a poster session at the 15th International Symposium on Chromatography in Nürnberg, October 1984.     

Comparison of different types of stationary phases for the analysis of soy isoflavones by HPLC

Nowadays, there are new technologies in high-performance liquid chromatography columns available enabling faster and more efficient separations. In this work, we compared three different types of columns for the analysis of main soy isoflavones. The evaluated columns were a conventional reverse phase particle column, a fused-core particle column, and a monolithic column. The comparison was in terms of chromatographic parameters such as resolution, asymmetry, number of theoretical plates, variability of retention time, and peak width. The lower column pressure was provided by the monolithic column, although lower chromatographic performance was achieved. Conventional and fused-core particle columns presented similar pressure. Results also indicate that direct transfer between particle and monolithic columns is not possible requiring adjustment of conditions and a different method optimization strategy. The best chromatographic performance and separation speed were observed for the fused-core particle column. Also, the effect of sample solvent on the separation and peak shape was evaluated and indicated that monolithic column is the most affected especially when using higher concentrations of acetonitrile or ethanol. Sample solvent that showed the lowest effect on the chromatographic performance of the columns was methanol. Overall evaluation of methanol and acetonitrile as mobile phase for the separation of isoflavones indicated higher chromatographic performance of acetonitrile, although methanol may be an attractive alternative. Using acetonitrile as mobile phase resulted in faster, higher resolution, narrower, and more symmetric peaks than methanol with all columns. It also generated the lower column pressure and flatter pressure profile due to mobile phase changes, and therefore, it presents a higher potential to be explored for the development of faster separation methods.     

Adsorption in a stratified column bed packed with porous particles having partially fractal structures and a distribution of particle diameters

Stratified column bed systems whose sections are formed by packing adsorbent particles with a partially fractal structure are proposed and studied. The simulation results clearly show that the breakthrough times and the shape of the breakthrough curves obtained from stratified column beds are significantly larger and sharper than those obtained from conventional columns. The stratified column beds provide, to the designer and user of chromatographic column systems, more degrees of freedom with respect to the number of parameters and variables that could be controlled in the design, construction, and operation of efficient chromatographic adsorption systems. Furthermore, the results suggest that the stratified column beds could provide a higher dynamic adsorptive capacity than conventional columns when it is required to increase the column throughput.     

Comprehensive two-dimensional liquid chromatography in the analysis of antioxidant phenolic compounds in wines and juices

A novel comprehensive two-dimensional liquid chromatographic (LC×LC) system was developed for the quantification of antioxidant phenolic compounds in wine and juice. The system allows parts of the sample that are well separated in the first column to pass directly to the detector after the first column, while the rest of the sample proceeds to the second column. The optimised LC×LC system employed a combination of two C18 columns, the latter column with an ion-pair reagent (tetrapentylammonium bromide). The relative standard deviations (RSD) for the retention times were better than 0.01% in the first dimension and on average 6.3% in the second. The RSD values of the peak volumes varied from 3% (protocatechuic acid) to 13% (caffeic acid, n = 3, 10 μg/ml).     

Polybutadiene-coated zirconia packing materials in solvating gas chromatography using carbon dioxide as mobile phase

Summary In this paper, practical considerations of column efficiency, separation speed, thermal stability, and column polarity of capillary columns packed with polybutadiene-coated zirconia were investigated under solvating gas chromatography (SGC) conditions using carbon dioxide as mobile phase. When compared with results obtained from conventional porous octadecyl obtained from conventional porous octadecyl bonded silica (ODS) particles, PBD-zirconia particles produced greater change in mobile phase linear velocity with pressure than conventional ODS particles under the same conditions. The maximum plate number per second (N_t) obtained with a 30 cm PBD-zirconia column was approximately 1.5 times higher than that obtained with an ODS column at 100 °C. Therefore, the PBD-zirconia phase is more suitable for fast separations than conventional ODS particles in SGC. Maximum plate numbers per meter of 76,900 and 63,300 were obtained using a 57 cm×250 μm i.d. fused silica capillary column packed with 3 μm PBD-zirconia at 50 °C and 100 °C, respectively. The PBD-zirconia phase was stable at temperatures up to 320 °C under SGC conditions using carbon dioxide as mobile phase. Polarizable aromatic compounds and low molecular weight ketones and aldehydes were eluted with symmetrical peaks from a 10 cm column packed with 3 μm PBD-zirconia. Zirconia phases with greater inertness are required for the analysis of more polar compounds by SGC.     

Direct analysis of clomipramine in human plasma by microbore high performance liquid chromatography with column-switching

Summary An automated microbore, liquid chromatographic method with column-switching was developed for the determination of clomipramine from human plasma samples. After direct injection of samples (60 μL), plasma proteins and clomipramine were separated in size-exclusion mode using 20% acetonitrile in 20 mM phosphate buffer (pH 7.0) on Capcell Pak MF Ph-1 precolumn (10×4 mm I.D.). By valve switching, a fraction containing clomipramine was directed to an intermediate column for subsequent main separation on a microbore C₁₈ column (250×1.5 mm I.D.) using 50% acetonitrile in 20 mM phosphate buffer (pH 2.5) at 0.1 mL min⁻¹. The method was advantageous for rapidity (total analysis time: 15 min), reproducibility (C.V.<4.8%), and increased sensitivity (1 ng mL⁻¹). The linearity of response was good (r ²≥0.999) over the concentration range 1–250 ng mL⁻¹.     

纳升液相色谱仪器的研究进展

小型化是液相色谱分离技术发展的重要趋势之一,包括仪器外形尺寸的小型化、分离材料粒径的小型化以及色谱柱内径的小型化。色谱柱内径的减小能够降低样品和流动相的消耗,具有更高的质量灵敏度,特别适合用于复杂样品体系的分离分析。纳升液相色谱一般是指使用内径小于100 μm的毛细管色谱柱,流速范围在每分钟几十至几百纳升的色谱技术。由于流速很低,色谱柱体积很小,柱外效应显著,因此对色谱仪器系统各个模块的性能以及系统柱外效应的优化提出了较高的要求。纳升液相色谱的输液装置需要能够准确稳定地输送纳升级流速,具有梯度输液模式,且拥有一定的耐压能力,以适应不同规格的色谱柱类型;进样装置需要能够进行准确重复的进样过程,进样体积及进样方式适合毛细管色谱柱,同时不产生明显的柱外效应;检测装置需要具有较高的灵敏度,且具有较小的柱外扩散;管路与连接系统需要稳定、可靠、易操作,并能够最大限度地减小柱外体积,适配纳升级流速。鉴于目前大多数纳升液相色谱系统与质谱检测器联用,因而本文主要从输液装置、进样装置、管路与连接3个方面对相关技术领域的研究论文、技术专利以及仪器厂商的宣传文件等进行了检索与归纳,综述了这些模块的技术路线与研究进展,同时简要介绍光学吸收型检测装置的优化思路与研究进展,并对部分商品化的纳升液相色谱系统进行了对比。