A comparative study of large volume injection techniques for microbore columns in HPLC

Summary This paper focuses attention on the potentially larger signal-to-noise ratios produced by microbore columns in comparison with conventional columns. The increased chromatographic signals by the application of microbore columns are due to the lower chromatographic dilution of elution profiles which are proportional to the square of the column inner radius. Generally less than 1μl sample should be injected into microbore systems to obtain the full benefit of the column performance. However, since more sample can be loaded on conventional columns compared to microbore columns the advantage of improved signal-to-noise ratio can only be realised in situations where very little sample is available. To inject more than 1μl sample, at the same time avoiding extra band-broadening effects, suitable injection techniques must be available. In this study three injection methods for microbore systems that meet this condition, are studied and compared.     

Practical aspects of fast HPLC separations for pharmaceutical process development using monolithic columns

A large number of samples can be generated during pharmaceutical process development. Fast separation for these samples is usually challenging due to the complexity of sample matrix, which requires high efficiency as well as high speed. Monolithic columns (E. Merck, Germany) were investigated as a possible tool for reducing separation time in reversed-phase HPLC without significantly sacrificing efficiency or resolution. Both van Deemter plots and separations of alkyl benzenes and in-process samples showed that monolithic columns were suitable for fast separations without significantly compromising resolution. Practical parameters including the pressure drop, retention factor, selectivity, and tailing factor of monolithic columns (Chromolith type) were compared to those of conventional YMC 150 mm × 4.6 mm (3-μm particles) and 250 mm × 4.6 mm (5-μm particles) packed columns. The batch-to-batch reproducibility of the 100 mm × 4.6 mm Chromolith columns from five randomly ordered batches was also compared to the 250 mm × 4.6 mm YMC particle-packed columns. Fast and efficient separations of complicated process samples including crude drug substances, reaction mixtures, and crystallized mother liquors were demonstrated for both monolithic columns and conventional packed columns. The analysis times were decreased by three to seven times on the coupled monolithic columns, while maintaining the comparable resolution to typical 5-μm particle-packed 250 mm × 4.6 mm columns.     

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.     

Liquid Chromatographic Analysis of Antiepileptic Drugs: An Overview

Abstract

Extraction of antiepileptic drugs (AEDs) can be carried out by different procedures which include: (i) protein precipitation, (ii) liquid-liquid and (iii) liquid-solid extraction. The latter yields a cleaner sample and shows efficiency comparable to the other procedures; recovery ranges from 90 to 105% for most AEDs.

Types of column include: (i) conventional, (ii) microbore and (iii) high-speed. Compared with conventional and high-speed columns, microbore columns, which have a diameter of 1–2 mm, allow to achieve the highest sensibility (up to double values) and to reduce solvent consume (up to 70 %). High-speed columns, characterized by a length of 3–10 cm and by a reduced packing particle size (? 3μm), make the analysis time more rapid by at least 50%.

Reversed phase chromatography is the most versatile technique as compared to the normal phase and to the precision and accuracy. These parameters, in fact, resulted close to those of the fluorescence polarization immunoassay technique (FPIA), which was the most precise and accurate among the methods compared (79).     

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.     

Optimization for Minimum Analysis Time of the Separation of Benzene and Deuterated Benzene by Reverse Phase Microbore and Recycle HPLC,and the Utility of D2O

Abstract

Optimization for minimum analysis time is demonstrated for HPLC separations of different degrees of difficulty. The separation of benzene and perdeuterobenzene can be performed la less than ten minutes, separation of benzene and monodeuterobenzene takes several hours.

The use of deuterium oxide and long microbore columns is not recommended since two-column recycle chroiaatography offers shorter analysis times than a long single-pass column.     

Microbore reversed-phase chromatography of proteins with conventional gradient equipment for high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography with microbore columns (50 X 1.0 mm) was used effectively for the separation and analysis of proteins down to 1 ng at flow-rates of 0.1-0.2 ml/min. With the use of standard low-pressure gradient HPLC equipment, the peak volumes were five times smaller when compared with a conventional column at equal chromatographic efficiencies and analysis time. The sensitivity of detection was further increased by a reduction in solvent peaks, resulting in a 20-fold overall increase.     

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.     

Demonstration of the Feasibility of Radially Compressed Microbore HPLC Columns

Abstract

This work involved the development of radially compressed, microbore high performance liquid chromatography (HPLC) columns. The design of the overall system and the column are described, and the problems associated with the design features are reported. Variables examined during the course of this work included the column material, column length, packing method, flow rate, radial compression pressure, and internal column pressure. Efficiencies (expressed as plates/meter) are shown for various combinations of those variables and are compared to those obtained using a commercial, steel microbore column.     

Potential of microbore HPLC within a multiresidue method for the trace analysis of plant-protective substances in water

The performance of microbore HPLC as a "measurement channel" within a true multiclass/multiresidue method for monitoring plant protectants in raw and potable water is demonstrated. The method has a modular design and consists of a non-selective sampling and preparation line generating 250 microL of an "extract" from a 100-mL water sample; this extract can be introduced to up to four measurement channels, as required by the analytical task. The microbore HPLC channel can be used to quantify 34 plant protectants in the 0.1 microg L(-1) concentration range by use of diode-array detection at seven different wavelengths. A solvent change is necessary to link sample preparation to microbore HPLC; this uses 50 microL of the "extract" and is accomplished directly in an autosampler vial. Performance characteristics were evaluated for tap water spiked at 0.2 microg L(-1). Average recoveries were between 65 and 100% and method detection limits were 0.07 microg L(-1) or better. The ability to provide comparable and accurate results was proven by participation in an interlaboratory comparison trial. The procedure for preparing microbore columns from 750 microm i.d. PEEK tubing is described in detail to enable the reader to prepare his own columns. The reproducibility of this preparation procedure was proven by an analysis-of-variance test.     

Potential of microbore HPLC within a multiresidue method for the trace analysis of plant-protective substances in water

The performance of microbore HPLC as a “measurement channel” within a true multiclass/multiresidue method for monitoring plant protectants in raw and potable water is demonstrated. The method has a modular design and consists of a non-selective sampling and preparation line generating 250 μL of an “extract” from a 100-mL water sample; this extract can be introduced to up to four measurement channels, as required by the analytical task. The microbore HPLC channel can be used to quantify 34 plant protectants in the 0.1 μg L^–1 concentration range by use of diode-array detection at seven different wavelengths. A solvent change is necessary to link sample preparation to microbore HPLC; this uses 50 μL of the “extract” and is accomplished directly in an autosampler vial. Performance characteristics were evaluated for tap water spiked at 0.2 μg L^–1. Average recoveries were between 65 and 100% and method detection limits were 0.07 μg L^–1 or better. The ability to provide comparable and accurate results was proven by participation in an interlaboratory comparison trial. The procedure for preparing microbore columns from 750 μm i.d. PEEK tubing is described in detail to enable the reader to prepare his own columns. The reproducibility of this preparation procedure was proven by an analysis-of-variance test.     

Fast and universal HPLC method for determination of permethrin in formulations using 1.8-μm particle-packed column and performance comparison with other column types

An HPLC method has been developed for the fast separation and quantification of permethrin using C18 column packed with 1.8 μm particles. The method is specific with good resolution to degradation products and to other present components. It has acceptable validation results. The run time is 4.5 min (or may be within 1.6 min is rapid resolution mode) with an organic solvent consumption of 3.6 mL per run. The method has been applied to samples of formulations for various uses: mattress cleaner, shampoo, and veterinary powder. The performance of the applied column is compared with other common column types. The relationships between linear velocity of the mobile phase (u) and resolution factor (Rs), back-pressure (ΔP), and efficiency (H) are presented. The experimental data shows the advantages of 1.8-μm particle columns to be a significant reduction in solvent consumption (by factor of 4.4 and 1.5) and a reduction in run-time (by factor 4.7 and 1.5), and the weaknesses are a high back-pressure and lower efficiency. Finally, it has been shown that use of 1.8-μm particle packed columns with conventional HPLC systems is possible, but with limitations in mobile phase flow-rate.     

毛细管电色谱和加压毛细管电色谱的进展与应用

毛细管电色谱(CEC)以内含色谱固定相的毛细管为分离柱,以电渗流为驱动力,既可以分离带电物质也可以分离中性物质。它结合了毛细管电泳和高效液相色谱两者的优点,兼具高柱效、高分辨率、高选择性和高峰容量的特点,同时具有色谱和电泳的双重分离机理。然而,“纯粹”的电色谱在实际应用中有着天然的弱点,即: 在电流通过毛细管柱中的流动相时容易产生气泡(焦耳热作用),从而使电流中断和电渗流停止,毛细管柱必须被重新用流动相润湿后方能再次使用。加压毛细管电色谱(pCEC)将液相色谱中的压力流引入CEC系统中,不仅解决了气泡、干柱等问题,而且实现了定量阀进样和二元梯度洗脱。CEC和pCEC作为微分离领域的两种前沿技术,满足了当前复杂样品分析和分析仪器微型化的需求,近年来获得了广泛的关注。本文综述了这两种技术近来的发展,包括仪器、色谱固定相的发展,总结了其在生命科学、药物分析、食品安全以及环保样品分析等方面的应用进展,评述了各方法的特点,并展望了CEC和pCEC今后的发展和应用前景。     

Use of partially porous column as second dimension in comprehensive two-dimensional system for analysis of polyphenolic antioxidants

In the present work, a comprehensive LC system using a microbore HPLC column in the first dimension and a partially porous column in the second dimension was developed and applied to the separation of polyphenolic components in a red wine sample. The performance of the partially porous short column (3.0 cm) was compared to that of a monolithic column, of comparable dimensions. The results obtained demonstrated the possibility to use partially porous columns to obtain fast analyses, using high flow rates, under repetitive gradient conditions and with very brief reconditioning times. A conventional HPLC system was used since the backpressure generated by the shell-packed column, even at very high flow rates, was well within the operational limits. The use of an increased column temperature (60 degrees C) allowed a further pressure-drop decrease, with no stationary phase degradation, or loss in column performance.     

A Simple Solvent Programmer for Liquid Chromatography. I

Abstract

Equations have been derived from which the dimensions of a solvent gradient generator, coupled to open tubular, micro-packed, semi-micro packed or conventional HPLC columns, may be calculated for a desired gradient volume. Packed and open-tubular generators have been considered. Calculations, using the derived equations, predict that a generator of particular dimensions is needed for each column type. These dimensions are practically feasible for all column types except the conventional column.     

Sampling considerations in supercritical fluid chromatography

Summary Packed column supercritical fluid chromatography, like HPLC, utilizes a sample loop to introduce materials onto the column for analysis. Unlike HPLC the mobile phase in SFC cannot be used to dissolve the sample. In practice, this causes a solvent peak, which can create a problem in the chromatographic interpretation. This paper describes one approach to solving this problem. A valving scheme is used to extract materials with the supercritical CO₂ mobile phase and introduce them onto the column with no external handling. The viability of this method is demonstrated and separations of the CO₂ extracts for several materials are shown on various columns. Comparisons are made for coal and coffee extracts using this on-line method and conventional off-line CH₂Cl₂ extracts. Advantages of the on-line procedure as they apply to chromatography and high information detectors are also discussed.     

The role of UHPLC in pharmaceutical development

Pharmaceutical separations can be divided into three categories: high throughput, high productivity, and high resolution. These categories contain specific pharmaceutical applications, each of which has distinct separation goals. Traditionally, these goals have been achieved utilizing conventional HPLC with typical column dimensions and particle sizes. The recent introduction of ultra-HPLC (UHPLC) has provided a new potential for method development and analysis. Pharmaceutical chemists must determine the impact of this emerging technology. UHPLC is achieved by using sub-2 microm particle size column packing at increased linear velocities. In order to utilize this technology, mobile phase viscosity must be minimized or the chromatography system must be redesigned to withstand an increased backpressure. Today, there are many commercially available UHPLC systems capable of exceeding conventional pressure limits of 400 bar. The advantage of UHPLC over conventional HPLC is the capability to increase the speed without sacrificing efficiency. In comparison to traditional HPLC, our research showed that UHPLC can decrease run times up to 7 x. In addition, for high resolution applications, UHPLC achieved significant efficiency advantages over traditional HPLC. This paper will evaluate the potential roles for utilizing UHPLC in the pharmaceutical industry.     

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.     

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.     

Separtion of Menthol Isomers by Normal Phase High Performance Liquid Chromatography (1)

Abstract

An HPLC method has been developed for the separtion of the four isomers of methol using isocaratic and normal phase ethyl acetate/isooctane systems. This method has used to detect and measure these isomers in peppermint techniques. It is more rapid than GC which in addition requires unstable columns for similar analysis. Beacause solvent and column are normal phase and isocratic, the method and sample preparation are very simple.