Fast HPLC with a silica-based monolithic ODS column

Fast high-performance liquid chromatography is becoming routine in laboratories that require high throughput or for combinatorial libraries. Reduced analysis time is commonly achieved by using shorter columns and higher flow rates. Shorter columns require smaller particles in order to maintain efficiency. However, smaller particles increase backpressure, which limits both column length and higher flow rates for typical LC pumps. This disadvantage has been addressed by the emergence of monolithic liquid chromatographic columns (1). Unlike particle-base columns, monolithic columns consist of a continuous rod-shaped porous network with a bimodal pore distribution. In this study, a commercially available 50- x 4.6-mm silica-based octadecyl silane monolithic column (Chromolith SpeedROD RP18e, EM Science, Gibbstown, NJ) was used to separate a seven-component test mixture with a wide range of polarity. The primary goals of this investigation were: (a) to study the chemistry (selectivity) of the new silica-based monolithic columns and (b) to study their run-to-run and column-to-column performance (retention times and peak areas). The selectivity (alpha factor) is a ratio of partition coefficients and, if comparable for a variety of solutes, would mean that methods could be readily transferred between particulate and monolithic columns.     

Retention behaviour of beta-blockers in HPLC using a monolithic column

The chromatographic behaviour of a new HPLC-stationary phase (Chromolith RP-18e) is described for separation of beta-blockers. The effects of the different chromatographic conditions (buffer system, pH value, content of organic modifier, injection volume and flow rate) on the separation behaviour were studied. At higher flow rates the peaks seemed to be more symmetrical than at lower flow rates. The use of buffer or salt in the mobile phase for this separation is found to be very essential and the counter-anion type of this buffer or salt significantly affected the retention behaviour of beta-blockers while the cation type did not play the same important role.     

Fast chiral separation by ligand-exchange HPLC using a dynamically coated monolithic column

The preparation and application of dynamically coated ligand-exchange chromatography phases for enantioseparation is described. The phases were prepared by pumping a solution of N-decyl-L-4-hydroxyproline, N-hexadecyl-L-4-hydroxyproline, or N-2-hydroxydodecyl-L-4-hydroxyproline through a commercially available monolithic RP-18 column. These coatings are stable against desorption for months at ambient temperature when aqueous mobile phases are used. The columns were applied to the chiral separation of amino acids, glycyl dipeptides and diastereomeric dipeptides, and tripeptides. The chiral selector can be removed or changed easily by washing the column with ACN or methanol. Ultrafast separations in the range of seconds were achieved using high flow rates.     

A hybrid FIA/HPLC system incorporating monolithic column chromatography

We have combined the generation of solvent gradients using milliGAT pumps, chromatographic separations with monolithic columns and chemiluminescence detection in an instrument manifold that approaches the automation and separation efficiency of HPLC, whilst maintaining the positive attributes of flow injection analysis (FIA), such as manifold versatility, speed of analysis and portability. As preliminary demonstrations of this hybrid FIA/HPLC system, we have determined six opiate alkaloids (morphine, pseudomorphine, codeine, oripavine, ethylmorphine and thebaine) and four biogenic amines (vanilmandelic acid, serotonin, 5-hydroxyindole-3-acetic acid and homovanillic acid) in human urine, using tris(2,2′-bipyridyl)ruthenium(III) and acidic potassium permanganate chemiluminescence detection.     

Parabens determination with a hybrid FIA/HPLC system with ultra-short monolithic column

The combination of an ultra-short C18 monolithic column (5 mm long) with a flow injection analysis (FIA) scheme results in a versatile and efficient system that has been used for the chromatographic determination of four preservatives — methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP). The separation is carried out by using two carriers, A and B, consisting of a mixture of ACN: water in different proportions. The described procedure is able to separate the analytes in only 150 s. The applicable concentration range, detection limit and the relative standard deviation were the following: for MP from 1.6 × 10^-5 to 1.1 × 10^-3 M; 4.8 × 10^-4 M; 0.65%; for EP between 3.7 × 10^-5 and 2.0 × 10^-3 M; 1.2 × 10^-5 M; 1.2%; for PP between 3.9 × 10^-5 and 2.0 × 10^-3 M; 1.2 × 10^-5 M; 1.2%; and for BT between 6.0 × 10^-5 and 2.0 × 10^-3 M; 1.8 × 10^-5 M; 1.8%. The method was applied and validated satisfactorily for the determination of these parabens in commercial cosmetics samples, comparing the results with those obtained by HPLC reference method.     

A novel monolithic column for capillary electrochromatographic separation of oligopeptides

A monolithic column was prepared using l-phenylalanine as template and a covalent approach through the formation of Schiff base with o-phthalaldehyde (OPA). OPA, allylmercaptan, l-phenylalanine, and triethylamine were stirred at first, then methacrylic acid, 2-vinylpyridine, ethyleneglycol dimethacrylate, α,α-azobisisobutyronitrile, and 1-propanol were added to the reaction mixture. The resulting material was introduced into a capillary column. Following thermal polymerization, the template was then extracted with a mixture of HCl and methanol. The column was employed for the capillary electrochromatographic separation of oligopeptides. A capillary column of 75 (50) cm × 75 μm ID with a mobile phase of phosphate buffer (pH 7.0, 40 mM)/methanol (5%, v/v), an applied voltage of +15 kV, and detection at 214 nm, could baseline separate angiotensin I, angiotensin II, [Sar¹, Thr⁸] angiotensin, oxytocin, vasopressin, tocinoic acid, β-casomorphin bovine, β-casomorphin human, and FMRF amide within 20 min. The separation behavior of the templated polymer was also compared with that of the non-templated polymer. As a result, it can be concluded that the electrochromatographic separation of this set of peptides was mediated by a combination of electrophoretic migration and chromatographic retention involving hydrophobic, hydrogen bonding, electrostatic as well as the Schiff base formation with OPA in the cavity of the templated polymer.     

Determination of tadalafil in pharmaceutical preparation by HPLC using monolithic silica column

The simple, reliable and reproducible HPLC and extraction methods were developed for the analysis of tadalafil in pharmaceutical preparation. The column used was monolithic silica column, Chromolith Performance RP-18e (100 mm × 4.6 mm, i.d.). The mobile phase used was phosphate buffer (100 mM, pH 3.0)-acetonitrile (80:20, v/v) at the flow rate of 5 mL min⁻¹ with UV detection at 230 nm at ambient temperature. Extraction of tadalafil from tablet was carried out using methanol. Linearity was observed in the concentration range from 100 to 5000 ng mL⁻¹ for tadalafil with a correlation coefficient (R²) 0.9999 and 100 ng mL⁻¹ as the limit of detection. The values of linearity range, correlation coefficient (R²) and limit of detection were 50-5000 ng mL⁻¹, 0.9999-50 ng mL⁻¹, respectively for sildenafil. Parameters of validation prove the precision of the method and its applicability for the determination of tadalafil in pharmaceutical tablet formulation. The method is suitable for high throughput analysis of the drug.     

Chromatographic characterisation of monolithic capillary columns for liquid chromatography based on methyltrimethoxysilane as sole precursor

A set of monolithic capillary columns for liquid chromatography, synthesized according to a recently developed protocol which uses methyltrimethoxysilane (MTMS) as a sole precursor, is characterised by various chromatographic tests to assess its physico-chemical properties. The new stationary phase material shows a hydrophobicity (assessed on the basis of methylene selectivity) comparable to commercial C(8) columns. The MTMS-based columns exhibit a reduced affinity towards planar molecules such as PAHs, compared to C(18) modified columns, which can be explained by a retention mechanism that is more governed by adsorption rather than partitioning. In comparison to commercial products an only moderate silanol activity was observed, even without any endcapping procedure applied. Selectivity between hydrophobic test compounds showed to be uniform between the columns investigated in this study, whereas retention factors differed up to 20% (batch-to-batch reproducibility) between columns produced under the same conditions. For most of the materials investigated in this study, size exclusion towards even only slightly larger molecules such as triphenylene was observed. It was demonstrated that inclusion of a micelle-forming detergent such as Brij in the synthetic protocol could partially overcome this problem.     

Enantioseparation of dansyl amino acids by HPLC on a monolithic column dynamically coated with a vancomycin derivative

In this work a chiral stationary phase was prepared by dynamically coating a monolithic reversed-phase HPLC column with a vancomycin-derivative as chiral selector. A hydrophobic alkyl-chain was attached to the vancomycin molecule, providing the immobilization of the chiral selector on the reversed-phase material. Dansyl amino acids were chosen as model analytes for testing the separation power of the dynamically coated phase. All investigated compounds were separated into their enantiomers. Compared with a conventionally packed vancomycin-CSP, a reversal of the enantiomer elution order was obtained.     

Determination of zonisamide by a coated monolithic column

In this paper, a monolithic ODS-silica gel column dynamically coated with cetylpyridinium chloride (CPC) was used to demonstrate the high-speed and efficient separation of zonisamide (1,2-benzisoxazole-3-methanesulfonamide, ZNS), its raw material (1,2-benzisoxazole-3-methanecarbonic acid) and intermediate (sodium 1,2-benzisoxazole-3-methanesulfonate) in drugs. Using a 40 mmol/L sodium perchlorate solution (pH 7.0) containing 10% acetonitrile as eluent, the analytes were eluted with a sharp and symmetrical peak within 3.0 min, detected with UV detection at 210 nm. The column demonstrates excellent stability over time, and exhibits unusual selectivity for pharmaceutical analysis. Thus, by this developed method, zonisamide in drug samples can be determined rapidly with high recoveries and good selectivity.     

Analysis of ecstasy with a monolithic reversed-phase column

Summary A new, rapid, and precise liquid chromatographic method has been developed for simultaneous identification and quantification of amphetamine,N-methyl-3,4-methylenedioxymetamphetamine,N-ethyl-3,4-methylenedioxymetamphetamine, andN-methyl-1-(3,4-methyl-enedioxyphenyl)-2-butanamine in the presence of other constituents. The compounds were separated on a monolithic column with a gradient prepared from acetonitrile and 20mm monobasic potassium buffer at a flow rate of 1.5 mL min⁻¹. Quantitation was performed with metoclopramide as internal standard. Use of different flow rates was investigated and enabled reduction of the separation time from 11 to 3.5 min for seven substances. The method was then applied to ten seized tablets to identify and quantify their active ingredients.     

A rapid HPLC method for the determination of carboxylic acids in human urine using a monolithic column

A rapid HPLC method for the determination of carboxylic acids in urine samples using a Chromolith Performance RP/18e 100/4.6 with Chromolith Guard Cartridge RP/18e 10/4.6 (Merck KgaA, Darmstadt, Germany) was developed. The method facilitates the simultaneous determination of aromatic hydrocarbon metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA) from styrene and ethylbenzene, hippuric acid (HA) from toluene and 2-, 3-, 4-methylhippuric acids (MHA) from xylene. 3-Hydroxybenzoic acid (3-HBA) was used as internal standard. A chromatographic run is completed within less than 5 min for styrene, ethylbenzene and toluene metabolites, and within 10 min for xylene metabolites. The detection limits are 9 mg L^–1 urine for MA, 1.25 mg L^–1 urine for PGA, 4.9 mg L^–1 urine for HA, 22 mg L^–1 urine for 2-MHA, and 18.5 mg L^–1 urine for 3-MHA.No significant differences of the MA, PGA and HA concentrations in human urine samples obtained by HPLC chromatography on LiChrosorb RP 18 and on Chromolith RP/18e columns were found. The results were evaluated by using ANOVA.Abbreviations MA mandelic acid - PGA phenylglyoxylic acid - HA hippuric acid - MHA methylhippuric acid - 3-HBA 3-hydroxybenzoic acid - ANOVA analyses of variance - GC gas chromatography - HPLC high-performance liquid chromatography     

Fast HPLC for quality control of Harpagophytum procumbens by using a monolithic silica column: method transfer from conventional particle-based silica column

The applicability of a monolithic C18-bonded silica column for the rapid HPLC separation of ingredients in medicinal plants and their phytopharmaceutical preparations has been evaluated in the author's laboratory. In this presentation, an existing method for the determination of the iridoid glycoside harpagoside in Harpagophytum procumbens (Devil's Claw) was successfully transferred from a conventional particle-based C18 silica column to a monolithic silica column. The very high porosity of the stationary phase allows chromatography with a much lower backpressure than on conventional columns. Therefore, the flow rate could be easily increased from 0.8 mL/min (particle-based column) to 5 mL/min (monolithic column) and the run-time reduced from 30 to 5 min (that is a reduction about 85% !), without losing any chromatographic resolution of the compound of interest. The amount of harpagoside was measured with the original method on a conventional particle-based silica column and on the adapted method on a monolithic silica column. The statistical mean t-test showed no significant differences of the variances and the means indicating that the fast HPLC method is an acceptable alternative. The shorter analysis time makes the method very valuable for commercial quality control of Harpagophytum extracts and its pharmaceutical preparations.     

Structural radial heterogeneity of a silica-based wide-bore monolithic column

The radial distribution of the main characteristics (elution time and standard deviation) of the elution profiles of a flat injected band recorded at the exit of a monolithic column were determined. These distributions provide the radial distributions of the average mobile phase velocity, the elution time and the maximum height of the peak of an analyte, the column efficiency and the analyte concentration. The band profiles were measured at the exit of a 10-mm i.d., 100-mm long silica-based monolithic column. An on-column local electrochemical amperometric detector allowed the recording of the elution profiles at different spatial positions throughout the column cross-section. The local spatial distribution of the mobile phase velocity does not follow a piston-flow behavior but exhibits radial heterogeneity. The local efficiency near the wall is lower than that near the column center. The radial distribution of the maximum concentration of the peaks varies throughout the column exit section, partially due to the radial variations of the column efficiency. These results might explain the rather large value of the A term of the Van Deemter or the Knox equations reported previously for monolithic columns.     

Faster axial band dispersion in a monolithic silica column than in a particle-packed column

The contribution of molecular diffusion to peak broadening was studied in a reversed-phase HPLC system, consisting of a monolithic silica C18 column and methanol-water mobile phase. Study on the band broadening effect of holding a solute in a column or elution at very low linear velocity of mobile phase allowed facile determination of the contribution of the molecular diffusion term. Less obstruction against molecular diffusion, or the faster axial band dispersion in a monolithic silica column than in a particle-packed column, was found both in mobile phase and in stationary phase.     

整体柱的制备及应用进展

对近期不同类型整体柱制备方面的进展情况进行了综述,概括了整体柱应用于不同色谱模式的情况,展望了整体柱的发展趋势。     

A novel ionic liquid monolithic column and its separation properties in capillary electrochromatography

A novel ionic liquid (IL) monolithic capillary column was successfully prepared by thermal free radical copolymerization of IL (1-vinyl-3-octylimidazolium chloride, ViOcIm⁺Cl⁻) together with lauryl methacrylate (LMA) as the binary functional monomers and ethylene dimethacrylate (EDMA) as the cross-linker in binary porogen. The proportion of monomers, porogens and cross-linker in the polymerization mixture was optimized in detail. The resulting IL-monolithic column could not only generate a stable reversed electroosmotic flow (EOF) in a wide pH range (2.0–12.0), but also effectively eliminate the wall adsorption of the basic analytes. The obtained IL-monolithic columns were examined by scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR). These results indicated that the IL-monolithic capillary column possessed good pore properties, mechanical stability and permeability. The column performance was also evaluated by separating different kinds of compounds, such as alkylbenzenes, thiourea and its analogues, and amino acids. The lowest plate height of ∼6.8 μm was obtained, which corresponded to column efficiency (theoretical plates, N) of ∼147,000 plates m⁻¹ for thiourea. ILs, as a new type of functional monomer, present a promising option in the fabrication of the organic polymer-based monolithic columns in CEC.     

基于金属介导和离子液体的新型绿原酸印迹整体柱的制备及评价

选用1-乙烯基-3-乙基咪唑四氟硼酸盐([VElm]BF4,一种离子液体)作为功能单体,以Co2+为介导离子,结合1-丁基-3-甲基咪唑四氟硼酸盐([BMIM]BF4)/二甲基亚砜(DMSO)二元致孔体系制备了绿原酸印迹整体柱。经过对制备参数的考察,确定最佳比例为绿原酸:Co2+:[VElm]BF4:EDMA(乙二醇二甲基丙烯酸酯)(摩尔比)=1:1:5:20,[BMIM]BF4:DMSO=3:1(V/V),最大印迹因子达2.10。通过优化色谱条件,最终在乙腈:20 mmol/L乙酸钠缓冲液(pH 4.2)=70:30(V/V)时实现了绿原酸及其类似物的完全分离。由此可见,以离子液体为功能单体及致孔剂,在金属介导策略下制备的分子印迹聚合物可实现绿原酸的特异性识别及分离。     

Preparation and characterisation of ribonuclease monolithic bioreactor

In gene therapy and DNA vaccination, RNA removal from DNA preparations is vital and is typically achieved by the addition of ribonuclease into the sample. Removal of ribonuclease from DNA samples requires an additional purification step. An alternative is the implementation of immobilized ribonuclease. In our work, ribonuclease was covalently coupled onto the surface of methacrylate monoliths via epoxy or imidazole carbamate groups. Various immobilization conditions were tested by changing immobilization pH. Ribonuclease immobilized on the monolith via imidazole carbamate groups at pH 9 was found to be six times more active than the ribonuclease immobilized on the monolith via epoxy groups. Under optimal immobilization conditions the Michaelis-Menten constant, Km, for cytidine-2,3-cyclic monophosphate, and turnover number, k3 were 0.52 mM and 4.6s(-1), respectively, and mirrored properties of free enzyme. Enzyme reactor was found to efficiently eliminate RNA contaminants from DNA samples. It was active for several weeks of operation and processed 300 column volumes of sample. Required residence time to eliminate RNA was estimated to be around 0.5 min enabling flow rates above 1 column volume per min.     

Study of the mass transfer kinetics in a monolithic column

The purpose of this work is to investigate the mass transfer kinetics of butylbenzoate on a monolithic RPLC column, with methanol-water (65:35, v/v) as the mobile phase. We used the perturbation method, measuring the height equivalent to a theoretical plate (HETP) of the peaks obtained as the response to small pulses of solute injected on a concentration plateau. The equilibrium isotherm of butylbenzoate was previously determined by frontal analysis. It is well accounted for by a liquid-solid extended multilayer BET isotherm model. The equilibrium data derived from the pulse method are in excellent agreement with those of frontal analysis in the accessible concentration range of 0 to 8 g/dm3. Plots of the HETP of small pulses. injected on eight different plateau concentrations, were acquired in a wide range of mobile phase flow velocities. The axial dispersion and the mass transfer kinetic coefficients were derived from these data. The validity of these measurements is discussed. The mass kinetics of butylbenzoate depends strongly on the plateau concentration. Processes involving adsorptive interactions between the solute and the stationary phase, e.g. surface diffusion and adsorption-desorption kinetics, combine in series to the external mass transfer kinetics and to effective pore diffusivity.