Advantages of ultra performance liquid chromatography over high-performance liquid chromatography: comparison of different analytical approaches during analysis of diclofenac gel

Miniaturization embracing instrumentation, column particle size, and column dimensions is one of the major current trends in separation techniques. This leads to shortening of analysis time and great savings in solvent consumption. Ultra performance liquid chromatography (UPLC) is one of the new developments in liquid chromatography. An ultra-high pressure system allows using of small particle-packed columns with small diameter, which has a positive effect on both system efficiency and analysis time. An analytical method for determination of the active substance diclofenac, the degradation product 1-(2,6-dichlorphenyl)-2-indolinone, and the preservatives methylparaben and propylparaben was used for testing and comparing LC systems. Various octadecylsilica-based analytical columns were examined. Acquity UPLC BEH C18 (2.1 x 50 mm, 1.7 microm) and (2.1 x 100 mm, 1.7 microm) were tested for UPLC. The following analytical columns were used in a test for HPLC: Purospher RP 18e (125 x 4.0 mm, 5 microm), Zorbax Eclipse XDB C18 (75 x 4.6 mm, 3.5 microm), Zorbax Eclipse SB C18 (50 x 4.6 mm, 1.8 microm), as was a monolithic column (Chromolith Performance RP-18e (100 x 4.6 mm). Results of a System Suitability Test (SST) were calculated and compared for each chromatographic peak. System efficiency and analysis duration were compared with regard to solvent consumption and system maintenance     

Simultaneous determination of econazole nitrate, main impurities and preservatives in cream formulation by high performance liquid chromatography

A reversed-phase high performance liquid chromatographic (RP-HPLC) method for determination of econazole nitrate, preservatives (methylparaben and propylparaben) and its main impurities (4-chlorobenzyl alcohol and alpha-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol) in cream formulations, has been developed and validated. Separation was achieved on a column Bondclone^® C18 (300 mm × 3.9 mm i.d., 10 μm) using a gradient method with mobile phase composed of methanol and water. The flow rate was 1.4 mL min⁻¹, temperature of the column was 25 °C and the detection was made at 220 nm. Miconazole nitrate was used as an internal standard. The total run time was less than 15 min. The analytical curves presented coefficient of correlation upper to 0.99 and detection and quantitation limits were calculated for all molecules. Excellent accuracy and precision were obtained for econazole nitrate. Recoveries varied from 97.9 to 102.3% and intra- and inter-day precisions, calculated as relative standard deviation (R.S.D.), were lower than 2.2%. Specificity, robustness and assay for econazole nitrate were also determined. The method allowed the quantitative determination of econazole nitrate, its impurities and preservatives and could be applied as a stability-indicating method for econazole nitrate in cream formulations.     

Characterization of new types of stationary phases for fast and ultra-fast liquid chromatography by signal processing based on AutoCovariance Function: A case study of application to Passiflora incarnata L. extract separations

In this study, a comparative investigation was performed of HPLC Ascentis^® (2.7 μm particles) columns based on fused-core particle technology and Acquity^® (1.7 μm particles) columns requiring UPLC instruments, in comparison with Chromolith™ RP-18e columns. The study was carried out on mother and vegetal tinctures of Passiflora incarnata L. on one single or two coupled columns. The fundamental attributions of the chromatographic profiles are evaluated using a chemometric procedure, based on the AutoCovariance Function (ACVF). Different chromatographic systems are compared in terms of their separation parameters, i.e., number of total chemical components (m_tot), separation efficiency (σ), peak capacity (n_c), overlap degree of peaks and peak purity. The obtained results show the improvements achieved by HPLC columns with narrow size particles in terms of total analysis time and chromatographic efficiency: comparable performance are achieved by Ascentis^® (2.7 μm particle) column and Acquity^® (1.7 μm particle) column requiring UPLC instruments. The ACVF plot is proposed as a simplified tool describing the chromatographic fingerprint to be used for evaluating and comparing chemical composition of plant extracts by using the parameters D% – relative abundance of the deterministic component – and c_EACF – similarity index computed on ACVF.     

Enhanced capabilities of separation in Sequential Injection Chromatography--fused-core particle column and its comparison with narrow-bore monolithic column

In the Sequential Injection Chromatography (SIC) only monolithic columns for chromatographic separations have been used so far. This article presents the first use of fused-core particle packed column in an attempt to extend of the chromatographic capabilities of the SIC system. A new fused-core particle column (2.7 μm) Ascentis^® Express C18 (Supelco™ Analytical) 30 mm × 4.6 mm brings high separation efficiency within flow rates and pressures comparable to monolithic column Chromolith^® Performance RP-18e 100-3 (Merck^®) 100 mm × 3 mm. Both columns matches the conditions of the commercially produced SIC system - SIChrom™ (8-port high-pressure selection valve and medium-pressure Sapphire™ syringe pump with 4 mL reservoir - maximal work pressure 1000 PSI) (FIAlab^®, USA). The system was tested by the separation of four estrogens with similar structure and an internal standard - ethylparaben. The mobile phase composed of acetonitrile/water (40/60 (v/v)) was pumped isocratic at flow rate 0.48 mL min⁻¹. Spectrophotometric detection was performed at wavelength of 225 nm and injected volume of sample solutions was 10 μL. The chromatographic characteristics of both columns were compared. Obtained results and conclusions have shown that both fused-core particle column and longer narrow shaped monolithic column bring benefits into the SIC method.     

Comparative study of new shell-type,sub-2 μm fully porous and monolith stationary phases,focusing on mass-transfer resistance

Today sub-2 μm packed columns are very popular to conduct fast chromatographic separations. The mass-transfer resistance depends on the particle size but some practical limits exist not to reach the theoretically expected plate height and mass-transfer resistance. Another approach applies particles with shortened diffusion path to enhance the efficiency of separations. In this study a systematical evaluation of the possibilities of the separations obtained with 5 cm long narrow bore columns packed with new 2.6 μm shell particles (1.9 μm nonporous core surrounded by a 0.35 μm porous shell, Kinetex™, Core-Shell), packed with other shell-type particles (Ascentis Express™, Fused-Core), totally porous sub-2 μm particles and a 5 cm long narrow bore monolith column is presented. The different commercially available columns were compared by using van Deemter, Knox and kinetic plots. Theoretical Poppe plots were constructed for each column to compare their kinetic performance. Data are presented on polar neutral real-life analytes. Comparison of a low molecular weight compounds (MW = 270–430) and a high molecular weight one (MW ∼ 900) was conducted. This study proves that the Kinetex column packed with 2.6 μm shell particles is worthy of rivaling to sub-2 μm columns and other commercially available shell-type packings (Ascentis Express or Halo), both for small and large molecule separation. The Kinetex column offers a very flat C term. Utilizing this feature, high flow rates can be applied to accomplish very fast separations without significant loss in efficiency.     

Comparison of UPLC and HPLC for Analysis of 12 Phthalates

Recent technological advances have resulted in the availability of reversed-phase chromatographic media of particle size 1.7 μm and a liquid-handling system that can be used to operate columns packed with these materials at much higher pressures. This technology, UPLC, has significant theoretical advantages in speed, resolution, and sensitivity of analysis, especially time saving and solvent consumption. The work discussed in this paper with new analytical method used for separation of 12 phthalates and the results were compared with those obtained by use of HPLC. Differences between the techniques, system suitability test data, and advantages and disadvantages of UPLC are discussed.     

Determination of methylparaben,propylparaben, clotrimazole and its degradation products in topical cream by RP-HPLC

Summary Reversed-phase, high-performance liquid chromatographic (RP-HPLC) methods with UV detection were developed and validated for determination of compounds in a topical cream. The first method describes determination of the active component clotrimazole and two preservatives present in the cream; methylparaben and propylparaben. The second method describes determination of two degradation products of clotrimazole, imidazole and (2-chlorophenyl) diphenylmethanol, in a topical cream after long-term stability tests. Chromatographic separation was on a Purospher RP-18e column; the mobile phase in Method1 for separation of clotrimazole, methylparaben and propylparaben comprises acetonitrile and water (70:30 v/v). For determination of degradations products-imidazole and (2-chlorophenyl) diphenylmethanol—the optimum composition of mobile phase in Method2 was acetonitrile and water (75:25 v/v) apparent pH^* 2.7. Analysis time was <10 min for both methods. The methods were found to be applicable for routine analysis of the active compound clotrimazole, preservatives and degradation products in the pharmaceutical product: topical cream 1% Clotrimazol Cream. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Rapid high performance liquid chromatography method development with high prediction accuracy,using 5 cm long narrow bore columns packed with sub-2 μm particles and Design Space computer modeling

Many different strategies of reversed phase high performance liquid chromatographic (RP-HPLC) method development are used today. This paper describes a strategy for the systematic development of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC) methods using 5 cm × 2.1 mm columns packed with sub-2 μm particles and computer simulation (DryLab^® package). Data for the accuracy of computer modeling in the Design Space under ultrahigh-pressure conditions are reported. An acceptable accuracy for these predictions of the computer models is presented. This work illustrates a method development strategy, focusing on time reduction up to a factor 3–5, compared to the conventional HPLC method development and exhibits parts of the Design Space elaboration as requested by the FDA and ICH Q8R1. Furthermore this paper demonstrates the accuracy of retention time prediction at elevated pressure (enhanced flow-rate) and shows that the computer-assisted simulation can be applied with sufficient precision for UHPLC applications (p > 400 bar). Examples of fast and effective method development in pharmaceutical analysis, both for gradient and isocratic separations are presented.     

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.     

Practical assessment of frictional heating effects and thermostat design on the performance of conventional (3 μm and 5 μm) columns in reversed-phase high-performance liquid chromatography

A practical investigation of frictional heating effects in conventional C18 columns was undertaken, to investigate whether problems found for sub-2 μm columns were also present for those of particle size 3 μm and 5 μm and different internal diameter. The influence of a water bath, a still air heater, and a forced air heater on performance was investigated. Heating effects were substantial, with a decrease in k of almost 15% for toluene over the flow rate range ∼0.4–2.3 mL/min with a 15 cm × 0.46 cm ID column packed with 3 μm particles. Heating effects on retention increased with increasing solute k, with increase in the column ID, with decrease in the column particle size, and with decrease in the set column oven temperature. While the water bath minimised axial temperature gradients and thus its effect on k, radial temperature gradients were potentially serious with this system, especially at high mobile phase velocity, even with columns containing 5 μm particles. In contrast to the effects of axial temperature gradients in 4.6 mm columns, very little difference in Van Deemter plots was noted between the three different thermostats with 2 mm ID columns, even when 3 μm particles were used. However, the efficiency of 2 mm columns for peaks of low or moderate k (k < 4) can be compromised by the extra dead volume introduced by the heating systems, even with conventional HPLC systems with otherwise minimised extra column volume.     

Development and validation of a simple stability-indicating high performance liquid chromatographic method for the determination of miconazole nitrate in bulk and cream formulations

A simple and stability-indicating high performance liquid chromatographic method was developed and validated for the determination of miconazole nitrate in bulk and cream preparations. The extraction step for cream samples consisted in a warming, cooling and centrifugation procedure that assures the elimination of the lipophilic matrix component, in order to avoid further precipitation in the chromatographic system. Separation was achieved on a ZORBAX Eclipse XDB - C18 (4.6 mm × 150 mm, 5 μm particle size) column, using a mobile phase consisting of water, methanol and acetonitrile, in a flow and solvent gradient elution for 15 min. The column was maintained at 25 °C and 10 μL of solutions were injected. UV detection was performed at 232 nm, although employment of a diode array detector allowed selectivity confirmation by peak purity evaluation. The method was validated reaching satisfactory results for selectivity, precision and accuracy. Degradation products in naturally aged samples could be simultaneously evaluated, without interferences in the quantitative analysis.     

Radial heterogeneity of some analytical columns used in high-performance liquid chromatography

An on-column electrochemical microdetector was used to determine accurately the radial distribution of the mobile phase velocity and of the column efficiency at the exit of three common analytical columns, namely a 100 mm × 4.6 mm C18 bonded silica-based monolithic column, a 150 mm × 4.6 mm column packed with 2.7 μm porous shell particles of C18 bonded silica (HALO), and a 150 mm × 4.6 mm column packed with 3 μm fully porous C18 bonded silica particles (LUNA). The results obtained demonstrate that all three columns are not radially homogeneous. In all three cases, the efficiency was found to be lower in the wall region of the column than in its core region (the central core with a radius of 1/3 the column inner radius). The decrease in local efficiency from the core to the wall regions was lower in the case of the monolith (ca. 25%) than in that of the two particle-packed columns (ca. 35–50%). The mobile phase velocity was found to be ca. 1.5% higher in the wall than in the core region of the monolithic column while, in contrast, it was ca. 2.5–4.0% lower in the wall region for the two particle-packed columns.     

Hydrodynamic chromatography for the size classification of micron and sub-micron sized packing materials

Hydrodynamic chromatography (HDC) was used as a size classification and purification method for porous bridged ethyl hybrid (BEH) packing materials (particles) in the micron to sub-micron range. Using packed column HDC, a batch of particles with size 0.76 ± 0.26 μm was fractionated to yield classified material of 1.05 ± 0.16 μm, reducing the relative standard deviation from 33% to 15%. Subsequent chromatographic evaluation of this packing material showed significant improvement in column performance and decrease in flow resistance over the unclassified material. Comparing a column packed with the classified versus non-classified material, the effective flow resistance of the two columns was decreased by 58% and the minimum HETP for the packing material was improved from 4 to 2.5 μm.     

Effects of extra-column band spreading,liquid chromatography system operating pressure,and column temperature on the performance of sub-2-μm porous particles

The effects of extra-column band spreading, LC system operating pressure, and separation temperature were investigated for sub-2-μm particle columns using both a conventional HPLC system as well as a UPLC^® system. The contributions of both volume- and time-based extra-column effects were analyzed in detail. In addition, the performance difference between columns containing 2.5 and 1.7-μm particles (same stationary phase) was studied. The performance of these columns was compared using a conventional HPLC system and a low dead volume UPLC system capable of routine operation up to 1000 bar. The system contribution to band spreading and the pressure limitations of the conventional HPLC system were found to be the main difficulties that prevented acceptable performance of the sub-2-μm particle columns. Finally, an increase in operating temperature needs to be accompanied by an increase in flow rate to prevent a loss of separation performance. Thus, at a fixed column length, an increase in temperature is not a substitute for the need for very high operating pressures.     

Rapid optimization of dual-mode gradient high performance liquid chromatographic separation of Radix et Rhizoma Salviae Miltiorrhizae by response surface methodology

An approach for rapid optimization of dual-mode gradient high performance liquid chromatography (HPLC) by response surface methodology (RSM) was developed for fast simultaneous separation of hydrophilic and hydrophobic components in Radix et Rhizoma Salviae Miltiorrhizae (Danshen) and its preparations. The aim of this study was to achieve a high throughput RSM optimization using a short ultra-high performance liquid chromatographic (UHPLC) column to simultaneously optimize flow rate and solvent gradient, and then transfer the optimized method to conventional HPLC for routine analytical purposes. The optimization was designed with Box Behnken design (BBD) and the global Derringer's desirability was used for describing the multicriteria response variables. Sixty-two designed experiments were performed by UHPLC with a short sub-2 μm column (2.1 mm × 50 mm, 1.7 μm) and a total running time of only 5 h. The predicted gradient profile was further transferred to a long UHPLC column (2.1 mm × 100 mm, 1.7 μm) and a conventional HPLC columns (2.1 mm × 100 mm, 3.5 μm and 4 mm × 100 mm, 5 μm, respectively). Compared to the published methods, the newly developed dual-mode gradient is faster and more efficient at simultaneously separating hydrophilic and hydrophobic components in Danshen and its preparations.     

Fast gradient screening of pharmaceuticals with 5 cm long, narrow bore reversed-phase columns packed with sub-3 μm core-shell and sub-2 μm totally porous particles

The performance of 5 cm long narrow-bore columns packed with 2.6-2.7 μm core-shell particles and a column packed with 1.7 μm totally porous particles was compared in very fast gradient separations of polar neutral active pharmaceutical compounds. Peak capacities as a function of flow-rate and gradient time were measured. Peak capacities around 160-170 could be achieved within 25 min with these 5 cm long columns. The highest peak capacity was obtained with the Kinetex column however it was found that as the flow-rate increases, the peak capacity of the new Poroshell-120 column is getting closer to that obtained with the Kinetex column. Considering the column permeability, peak capacity per unit time and per unit pressure was also calculated. In this comparison the advantage of sub-3 μm core-shell particles is more significant compared to sub-2 μm totally porous particles. Moreover it was found that the very similar sized (d_p = 2.7 μm) and structured (ρ = 0.63) new Poroshell-120 and the earlier introduced Ascentis Express particles showed different efficiency. Results obtained showed that the 5 cm long narrow bore columns packed with sub-3 μm core-shell particles offer the chance of very fast and efficient gradient separations, thus these columns can be applied for fast screening measurements of routine pharmaceutical analysis such as cleaning validation.     

Determination of methylparaben,propylparaben, triamcinolone acetonide and its degradation product in a topical cream by RP-HPLC

A novel reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the determination of active component triamcinolone acetonide, its degradation product triamcinolone (occurring in formulation after long-term stability tests) and two preservatives presented in the cream-methylparaben and propylparaben, using hydrocortisone as an internal standard.The chromatographic separation was performed on a Supelco Discovery C18 column; the mobile phase for separation of all compounds consists of a mixture of acetonitrile and water (40:60 v/v). The analysis time was less than 9 min, at a flow rate of 0.6 mL min(-1) and detection at 240 nm. The method was found to be applicable for routine analysis (stability tests, homogeneity) in the pharmaceutical product topical cream Triamcinolon cream 0.1%.     

Simple capillary flow porometer for characterization of capillary columns containing packed and monolithic beds

A simple capillary flow porometer (CFP) was assembled for through-pore structure characterization of monolithic capillary liquid chromatography columns in their original chromatographic forms. Determination of differential pressures and flow rates through dry and wet short capillary segments provided necessary information to determine the mean diameters and size distributions of the through-pores. The mean through-pore diameters of three capillary columns packed with 3, 5, and 7 μm spherical silica particles were determined to be 0.5, 1.0 and 1.4 μm, with distributions ranging from 0.1 to 0.7, 0.3 to 1.1 and 0.4 to 2.6 μm, respectively. Similarly, the mean through-pore diameters and size distributions of silica monoliths fabricated via phase separation by polymerization of tetramethoxysilane (TMOS) in the presence of poly(ethylene glycol) (PEG) verified that a greater number of through-pores with small diameters were prepared in columns with higher PEG content in the prepolymer mixture. The CFP system was also used to study the effects of column inner diameter and length on through-pore properties of polymeric monolithic columns. Typical monoliths based on butyl methacrylate (BMA) and poly(ethylene glycol) diacrylate (PEGDA) in capillary columns with different inner diameters (i.e., 50–250 μm) and lengths (i.e., 1.5–3.0 cm) were characterized. The results indicate that varying the inner diameter and/or the length of the column had little effect on the through-pore properties. Therefore, the through-pores are highly interconnected and their determination by CFP is independent of capillary length.     

Fast simultaneous spectrophotometric determination of naphazoline nitrate and methylparaben by sequential injection chromatography

Fast simultaneous determination of naphazoline nitrate and methylparaben in pharmaceuticals using separation method based on a novel reversed-phase sequential injection chromatography (SIC) is described in this contribution as an alternative to classical HPLC. A Chromolith™ Flash RP-18e (25 mm × 4.6 mm) column (Merck^®, Germany) and a FIAlab^® 3000 system (USA) with a six-port selection valve and 5.0 ml syringe pump were used for sequential injection chromatographic separations in our study. The mobile phase used was methanol/water (40:65, v/v), pH 5.2 adjusted with triethylamine 0.8 μl ml⁻¹ and acetic acid, at flow rate 0.9 ml min⁻¹. UV detection provided by DAD detector and two wavelengths were simultaneously monitored for increasing sensitivity of determination. Detector was set up at 220 nm for naphazoline nitrate and 256 nm for methylparaben and ethylparaben (IS). There is no necessity to use pre-adjustment of sample of nasal drops (only dilution with mobile phase) so the time of the whole analysis is very short. The validation parameters have shown good results: linearity of determination for both components (naphazoline nitrate and methylparaben), correlation coefficient >0.999; repeatability of determination (R.S.D.) in the range 0.5-1.6% at three different concentration levels, detection limits 0.02 μg ml⁻¹ (naphazoline nitrate) and 0.20 μg ml⁻¹ (methylparaben and ethylparaben), and recovery from the pharmaceutical preparations in the range 100.06-102.55%. The chromatographic resolution between peaks of compounds was more than 4.0 and analysis time was less than 4 min under the optimal conditions. The advantages and drawbacks of SIC against classical HPLC are discussed showing that SIC can be an advantageous alternative in many cases.     

Hydrophilic interaction liquid chromatography – charged aerosol detection as a straightforward solution for simultaneous analysis of ascorbic acid and dehydroascorbic acid

Ascorbic acid (AA) and dehydroascorbic acid (DHA) are small polar molecules difficult to be retained in conventional chromatographic RP systems. Hydrophilic interaction liquid chromatography (HILIC) using Obelisk R (100 × 3.2 mm, 5 μm, Sielc) analytical column and isocratic elution by ammonium acetate buffer pH 4.2 was found to be successful at this task, while other tested HILIC columns – Obelisk N (100 × 3.2 mm, 5 μm, Sielc) and Luna HILIC (100 × 3.0 mm, 3 μm, Phenomenex) were unsuccessful for the purposes of analysis. Charged aerosol detection (CAD) has recently become a new alternative universal detection system in HPLC, and was extremely convenient for the simultaneous analysis of AA and DHA without the need of subtraction approach and oxidation/reduction step. CAD response was found linear in defined range in spite of the fact that CAD is designated as non-linear detection method. A simple and fast HILIC-CAD method was applied for the analysis of pharmaceutical preparations containing AA. Method validation was performed including parameters of precision, accuracy, linearity, limit of detection and limit of quantitation (LOQ). The method was fast, accurate and precise for both detectors with LOQ_AA 5 μg/ml for UV detection and 10 μg/ml for CAD, respectively. DHA was detected only by CAD within tested concentration range with LOQ_DHA 1 μg/ml.