Rapid method for evaluating reversed-phase high-performance liquid chromatography column stability

A procedure is presented for the rapid evaluation of HPLC stationary phase stability at pH 8.4 or 10.1 using a temperature of 60 degrees C. Mobile phase (MeOH-0.1 mol l(-1) aqueous NaHCO3, 50:50, v/v) is continuously passed through the column with periodic injections of a test solution until the several chromatographic parameters of the resulting chromatograms are degraded. The tests were applied to several commercial and laboratory-made stationary phases. After degradation two of these phases, one commercial and one laboratory-made, were examined by elemental analysis and scanning electron microscopy to elucidate the degradation process.     

Influence of injection parameters on column performance in nanoscale high-performance liquid chromatography

Summary The influence of the injection volume and the sample solvent on column efficiency has been evaluated in packed nano liquid chromatography using columns 150μ i.d. Evaluation of column performance was by means of reduced plate height (h) versus reduced velocity (v) for four polyaromatic hydrocarbon test compounds (PAHs). When compounds are dissolved in a weak solvent (such as MeCN: H₂O, 30∶70), and whatever the injection volume −60 or 200 nL-a gain in efficiency can be observed due to the well-known on-column focusing phenomenon, but keeping constant solute retention factors. Under optimized conditions (flow rate: 150 nLmin⁻¹, solvent sample MeCN: H₂O, 30∶70, injection volume 200 nL), a reduced plate height of 1.83 has been obtained for a 15 μm C₁₈ packing corresponding to 36000 plates m⁻¹, which illustrates the absence of any extracolumn band broadening under nano LC conditions.     

Peak symmetry as a measure of column performance in high-performance liquid chromatography

Summary During a column-packing method development study, 114 reverse phase and 40 silica gel columns were packed. A relationship between peak symmetry and column efficiency was observed which was found to be useful when evaluating column performance.     

Chemically-bonded chiral column packings for high-performance liquid chromatography

Summary N-Protected aminoacids have been chemically bonded to aminopropylated silica gel, and the resulting chiral phases have been applied to the LG resolution of racemic mixtures of polar compounds. Best results were obtained with surfacebound N-formyl-isoleucine and N-formylvaline, with which baseline resolutions of a range of aminoacid and aminoalcohol derivatives were achieved.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Atypical silica-based column packings for high-performance liquid chromatography

Column packings widely used for high-performance liquid chromatography (HPLC) mostly are based on porous silica microspheres with certain pore sizes and pore size distributions. Such materials have the most desirable compromise of properties that provide for effective and reproducible separations over a wide range of operating conditions. To provide desired separation characteristics, several manufacturers specially synthesize the silica particles for these packings. While such column packing materials have general utility for a wide range of needs, special silica-based particles have been synthesized with different physical conformations for special separation goals. This presentation describes some atypical types of silica-based particles with unique separation properties that enlarge the capabilities of HPLC methods.     

Flow-through pore characteristics of monolithic silicas and their impact on column performance in high-performance liquid chromatography

In order to elucidate the role of the flow-through characteristics with regard to the column performance in high-performance liquid chromatography (HPLC) native and n-octadecyl bonded monolithic silica rods and columns, respectively of 100 mm length and 4.6 mm ID with mesopores in the range between 10 and 25 nm and macropores in the range between 0.7 and 6.0 μm were examined by mercury intrusion/extrusion, scanning electron microscopy, image analysis and permeability. The obtained data of the flow-through pore sizes and porosity values as well as surface-to-volume ratio of the stationary phase skeleton enabled to predict their influence to the chromatographic separation efficiency. Our data demonstrate that mercury porosimetry is a reliable technique to obtain all the characteristic parameters of the flow-through pores of silica monoliths. An important result of our examination was that the surface-to-volume ratio of monolithic silica skeletons had more significant impact to the separation process, rather than the average flow-through pore sizes. We could also show the essential differences between the particulate and monolithic stationary phases based on theoretical computation. The results, obtained from other characterization methods also indicated the structural complexity of monolithic silica samples. Permeability of columns is a generally applicable parameter to characterize all chromatographic phases no matter the chemistry or format. The correlation coefficient obtained for mercury intrusion and permeability of water was 0.998, though our investigation revealed that the surface modification is more likely influencing the obtained results. Further, the assumption of the cylindrical morphology of flow-through pores is not relevant to the investigated monolithic silica columns. These results on the morphology of the flow-through pores and of the skeletons were confirmed by the image analysis as well. Our main finding is that the flow-through pore sizes are not relevant for the estimation of the chromatographic separation efficiency of monolithic silica columns.     

Chemical stability of reversed phase high performance liquid chromatography silica under sodium hydroxide regeneration conditions

Chromatographic sorbents used within the purification of peptide or protein based active pharmaceutical ingredients (APIs) are commonly subjected to caustic regeneration procedures, so-called CIP treatments. While polymeric materials remain unaffected by this treatment, silica-based sorbents are at an intrinsic risk of dissolution under high pH conditions, such as, e.g. 0.1M NaOH. It is common misconception that silica-based materials simply cannot be subjected to alkaline conditions above pH 9. Moreover, most studies covering the chemical stability of HPLC sorbents above pH 9 have been limited to the chromatographic conditions used for the separations themselves. Such studies have used buffered mobile phases up to pH 11 or 12. Very little focus has been put on the stability of the stationary phases when subjected to shorter but harsher pH conditions required for regeneration purposes, such as 0.1M NaOH (pH 13). Knowledge about the amount of so-called leachables, degradation products originating from the stationary phase, is of growing importance for the registration of pharmaceuticals for human use and is addressed in this work. This study compares the chemical stability of different commercially available reversed phase silica materials (C18) that are used in industrial scale preparative HPLC. The silica materials were subjected to NaOH regeneration conditions and it is shown that some materials are able to withstand 0.1M NaOH conditions without significant harm. It is demonstrated that contaminants present in the effluent in the range of 10-50 microg/mL can lead to significant contamination of API product fraction.     

Mesoporous polybutadiene-modified zirconia for high-temperature packed capillary liquid chromatography: column preparation and temperature programming stability

In the present study, three different methods for packing of 3 microm PBD-ZrO2 particles in 0.5 mm i.d. glass-lined stainless steel columns have been examined. The two first methods were based on a traditional downstream high-pressure technique using tetrachloromethane (Method I) or aqueous Triton X-100 (Method II) as slurry solvents, while Method III was an upstream high-pressure flocculating method with stirring, using isopropanol both as the slurry and packing solvent. Method I was found to be superior in terms of efficiency, producing 0.5 mm i.d. x 10 cm columns with almost 90,000 plates m(-1) for toluene (R.S.D. = 8.7%, n = 3), using a slurry concentration of 600 mg ml(-1), ACN-water (50:50 (v/v)) as the packing solvent and a packing pressure of 650 bars. For Method I, the slurry concentration, column i.d., column length and initial packing pressure were found to have a significant effect on column efficiency. Finally, the long-term temperature stability of the prepared columns was investigated. In isothermal mode, using ACN-20 mM phosphate buffer, pH 7 (50:50 (v/v)) as the mobile phase, the columns were found to be stable for at least 3,000 void volumes at 100 degrees C. At this temperature, the solute efficiencies changed about 5-18% and the retention factors changed about 6-8%. In temperature programming mode (not exceeding 100 degrees C), on the other hand, a rapid decrease in both column efficiency and retention factors was observed. However, when the columns were packed as initially described, ramped up and down from 50 to 100 degrees C for 48 h and refilled, fairly stable columns with acceptable efficiencies were obtained. Although not fully regaining their initial efficiency after refilling, the solute efficiencies changed about 19-28% (32-37%) and the retention factors changed about 4-5% (13-17%) after running 3,000 (25,000) void volumes or 500 (3,900) temperature programs.     

Variability of column selectivity for reversed-phase high-performance liquid chromatography compensation by adjustment of separation conditions

Reversed-phase columns are widely used in assays based on high-performance liquid chromatography (HPLC). When such assays are repeated over time, it is often necessary to replace the column. In such cases, the selectivity of columns from different production batches may prove sufficiently variable to result in a failed separation. It is possible to compensate for differences in column selectivity by making small changes (adjustments) in separation conditions. The present paper describes an efficient procedure for choosing adjusted conditions and discusses its general applicability.     

Simultaneous assay of olanzapine and fluoxetine in tablets by column high-performance liquid chromatography and high-performance thin-layer chromatography

This paper describes validated high-performance liquid chromatographic (LC) and high-performance thin-layer chromatographic (TLC) methods for the simultaneous estimation of olanzapine and fluoxetine in pure powder and tablet formulations. The LC separation was achieved on a Lichrospher 100 RP-180, C18 column (250 mm, 4.0 mm id, 5 microm) using 0.05 M potassium dihydrogen phosphate buffer (pH 5.6 adjusted with o-phosphoric acid)-acetonitrile (50 + 50, v/v) as the mobile phase at a flow rate of 1 mL/min and ambient temperature. The TLC separation was achieved on aluminum sheets coated with silica gel 60F254 using methanol-toluene (40 + 20, v/v) as the mobile phase. Quantitation was achieved by measuring ultraviolet absorption at 233 nm over the concentration range of 10-70 and 40-280 microg/mL with mean recovery of 99.54 +/- 0.89 and 99.73 +/- 0.58% for olanzapine and fluoxetine, respectively, by the LC method. Quantitation was achieved by measuring ultraviolet absorption at 233 nm over the concentration range of 100-800 and 400-3200 ng/spot with mean recovery of 101.53 +/- 0.06 and 101.45 +/- 0.35% for olanzapine and fluoxetine, respectively, by the TLC method with densitometry. These methods are simple, precise, and sensitive, and they are applicable for simultaneous determination of olanzapine and fluoxetine in tablet formulations.     

Characterization of a novel diol column for high-performance liquid chromatography

For the investigation of a diol phase (Inertsil Diol column) in hydrophilic interaction chromatography, urea, sucrose and glycine were used as test compounds. The chromatographic conditions were investigated for optimal column efficiency. The column temperature used in common reversed-phase liquid chromatography could also be used for the separation and the flow-rate should be adjusted to 0.3-0.5 ml/min to optimize column efficiency. It is suggested that the velocity of the hydrophilic interaction is slower than the hydrophobic interaction in RPLC. The addition of trifluoroacetic acid is effective for the retention of glycine, but ineffective for urea and sucrose. The diol phase exhibited sufficient chemical stability even if exposed to water in high percentage, and could be applied with isocratic elution for the separation/analysis of amino acids and glucose.     

线性非理想条件下液相色谱柱末端峰形规律

 色谱流出曲线的二阶中心矩μ２和三阶中心矩μ３以及描述峰形非对称程度的偏态系数∑ｋ＝μ３／μ１．５２是反映色谱峰形的重要参数。从液相色谱过程动力学方程出发，运用电子计算机证明了在线性非理想条件下高效液相色谱体系中不同保留值组分在柱末端峰形的分布基本一致的结论。     

Comprehensive two-dimensional gas chromatography using a high-temperature phosphonium ionic liquid column

A high-temperature ionic liquid, trihexyl(tetradecyl)phosphonium bis(trifluoromethane)sulfonamide, was used as the primary column stationary phase for comprehensive two-dimensional gas chromatography (GC × GC). The ionic liquid (IL) column was coupled to a 5% diphenyl/95% dimethyl polysiloxane (HP-5) secondary column. The retention characteristics of the IL column were compared to polyethylene glycol (DB-Wax) and 50% phenyl/50% methyl polysiloxane (HP-50+). A series of homologous compounds that included hydrocarbons, oxygenated organics, and halogenated alkanes were analyzed with each column combination. This comparison showed that the ionic liquid is less polar than DB-Wax but more polar than HP-50+. The most unique feature of the IL × HP-5 column combination is that alkanes, cyclic alkanes, and alkenes eluted in a narrow band in the GC × GC chromatogram; whereas, these compounds occupied a much larger portion of the DB-Wax × HP-5 and the HP-50+ × HP-5 chromatograms. Each column combination was used to analyze diesel fuel. The IL × HP-5 chromatogram displayed narrow bands for three major compound classes in diesel fuel: saturates, monoaromatics, and diaromatics. The IL column was used at temperatures as high as 290 °C for several months without any noticeable changes in column performance.     

Comparison of reversed-phase column materials for high-performance liquid chromatography of proteins

Nine reversed-phase materials with various bonded phases from different suppliers were studied for the separation of hydrophilic proteins with two solvent systems. Protein retention, resolution and recovery were not correlated with the nature of the hydrocarbonaceous ligand. Peak volumes increased with molecular weight, which led to broad, irregular peaks for the larger proteins on some columns. Four columns that performed equally well were selected for the purification of hydrophobic Sendai virus membrane proteins. In this case, more distinct differences were found between columns. Recovery of the membrane proteins strongly depended on the combination of column and solvent systems.     

Evaluation of high-performance liquid chromatography column retentivity using macromolecular probes I

The application of macromolecular probes is proposed for evaluation of HPLC column retentivity. The idea is tested with a set of different commercial silica C₁₈ reversed-phases. For comparison, porous glass C₁₈ and polystyrene/divinylbenzene column packings are also included. Polar, mainly silanophilic interactions are evaluated. The retention volumes of a series of narrow molar mass distribution polystyrenes (PS) and poly(methyl methacrylate)s (PMMA) in toluene eluent are compared. Toluene is a weak mobile phase concerning silica gel surface and it promotes adsorption of PMMA on silanols, while PS is not adsorbed from toluene. Simultaneously, toluene is a thermodynamically good solvent for both polymers so that extensive partition in favour of stationary phase is not probable. Differences in retention behaviour of PS and PMMA indicate presence of abundant free silanols on the surface of some reversed-phases. These silanols are accessible even for large macromolecules of PMMA. Pore diameter and pore volume of the column packing can be semiquantitatively evaluated from the elution data of PS in toluene in the course of retentivity tests.     

Effect of temperature and flow-rate on analysis of basic compounds in high-performance liquid chromatography using a reversed-phase column

The peak shape and retention of some basic probes together with a neutral reference compound were investigated as a function of temperature and flow-rate using a reversed-phase HPLC column at both pH 3.0 and pH 7.0. The retention of bases often showed an anomalous increase with temperature; retention mechanisms are complex as shown by studies of the effect of buffer cation concentration on retention. Considerable improvements in column efficiency for bases may result from operation at elevated temperature. Improvements did not seem attributable either to incidental changes in the retention factor, or (in this particular study where low sample masses were utilised) to the influence of sample load. The optimum flow-rate for highest efficiency is generally lower for basic compounds than neutrals, and due to the steepness of the Van Deemter curves obtained, high flow-rates appear to be particularly detrimental in the chromatography of basic compounds.     

Effect of column loading on the accuracy of analyzing pentalgin N tablets by gradient high-performance liquid chromatography

Two approaches for the high-performance liquid chromatographic analysis of multicomponent medical preparations were experimentally studied by an example of Pentalgin N tablets. One of them was the simultaneous determination of all components of the preparation; another was the independent analysis of concentrated and diluted solutions for determining the presentpresent in small and large amounts, respectively. The performance characteristics of the studied procedures were determined in the analysis of model solutions containing all active and adjuvant substances of the tablets. In the simultaneous determination of all components, the relative error was smaller because of the more sustained performance of the chromatographic column in the absence of its overload with the sample observed in the independent analysis.