Monolithic HPLC Columns Research Articles

Repeatability of monolithic HPLC columns while using a flow program

Fast HPLC methods are becoming more and more important. Using monolithic HPLC columns for fast separations, a flow program can be applied for further decrease in the total run time. An interesting issue was whether the flow program affects repeatability. The investigated method was a generic assay for the oral antidiabetic drugs glibenclamide and glimepiride in the presence of two of their degradation products. A flow program ranging from 5.0 to 9.9 mL/min had been set up to decrease the run time to approximately 1.7 min. Within-day RSD% (n = 40) for both retention times and peak areas were less than 1%. At flow rates higher than 7 mL/min, repeatability was impaired to some extent. It became mainly noticeable through the day-to-day precision (n = 60) which showed RSD% up to 2%. However, further investigations indicated that this was rather related to pump inefficiency at high flow rates than to the flow program as such. Presuming the use of appropriate equipment, qualified for high flow rates, the application of a flow program for shortening the run time is absolutely reasonable and does not affect repeatability.

Automated on-line column-switching HPLC–MS/MS method for measuring environmental phenols and parabens in serum

We developed a method using on-line solid phase extraction (SPE) coupled to high performance liquid chromatography–isotope dilution tandem mass spectrometry (HPLC–MS/MS) to measure the serum concentrations of seven environmental phenols and five parabens: bisphenol A; ortho-phenylphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; benzophenone-3; triclosan; and methyl-, ethyl-, propyl-, butyl-, and benzyl-parabens. The phenols and parabens present in serum were retained and concentrated on a C18 reversed-phase size-exclusion SPE column, back-eluted from the SPE column while the eluate was diluted through a mixing Tee (analyte peak focusing), separated using a pair of monolithic HPLC columns, and detected by isotope dilution-MS/MS. Sample preparation did not require protein precipitation, only dilution of the serum with 0.1 M formic acid. This method, which combines an on-line SPE with analyte peak focusing feature and the selective atmospheric pressure photoionization MS detection, resulted in limits of detection ranging from 0.1 to 0.5 ng/mL for most of the analytes. The high throughput and adequate sensitivity with yet a relative low serum volume used (100 μL) confirm that analytically it is possible to measure simultaneously these phenols and parabens with the precision and accuracy at sub-parts-per-billion levels required for biomonitoring. However, important additional factors, including validated sample collecting, handling, and storing protocols, as well as toxicokinetic data, are required if these measures are used for exposure assessment.

Evaluation of monolithic HPLC columns for various pharmaceutical separations: Method transfer from conventional phases and batch to batch repeatability

Methods developed on conventional particle-packed C18 columns for pilocarpine, propranolol, glibenclamide, glimepiride, insulin and their respective degradation products or related compounds were transferred from the conventional Superspher 100 RP-18e column to Chromolith Performance RP-18e columns. All transfers were successful applying the same chromatographic conditions, except for insulin where the acetonitrile content of the mobile phase was reduced by 0.5%. The intraday and interday precisions for both retention time and peak area were evaluated over a wide concentration range. Results were found to be equal, or slightly better on Chromolith Performance with RSD% < 1.1% in all cases. Monolithic batch to batch repeatability of both retention time and peak area, compared for monolithic columns from different batches gave an RSD% of less than 1.3%. The separation of each drug and its related products was investigated on monolithic columns at flow rates from 1 to 9 ml/min, and superior resolution was always obtained using monolithic over conventional columns at the same flow rate. A total of seven monolithic columns from four different batches were used in this study.

Poly(cyclooctene)-based monolithic columns for capillary high performance liquid chromatography prepared via ring-opening metathesis polymerization

Monolithic columns for capillary HPLC were prepared via ring-opening metathesis polymerization (ROMP) from cis-cyclooctene (COE), tris(cyclooct-4-enyl-1-oxy)methylsilane (CL) as monomers, 2-propanol and toluene as porogens and RuCl 2(Py) 2(IMesH 2)(CHC 6H 5) (Py = pyridine, IMesH 2 = 1,3-dimesityl-4,5-dihydroimidazolin-2-ylidene) as initiator within the confines of 200 μm i.d. fused silica columns. For evaluation of the novel monolithic capillary HPLC columns, a protein standard consisting of six proteins in the molecular weight range of 5800–66000 g/mol, i.e. ribonuclease A, insulin, albumin, lysozyme, myoglobin and β-lactoglobulin, was used. Reproducibility of synthesis was checked by determining the relative standard deviation (RSD) in retention times ( t R), which was found to be in the range of 2.9–3.9% for all analytes. Variations in polymer kinetics were realized by adding different amounts of free pyridine and had a significant influence on the monolith's morphology, the backpressure and retention times. On the contrary, variations in monomer content and COE to CL ratio showed only minor changes on these parameters. Long-term stability of 1000 runs at 50 °C showed excellent stability of the columns and no significant alteration in separation performance was observed in combination with slightly decreased retention times (approx. 1.6–7.2% for all analytes).

Enantiomer separation on monolithic silica HPLC columns using chemically bonded methylated and methylated/acetylated 6-O-tert-butyldimethyl- silylated β-cyclodextrin

A new 2,3-methylated 3*-monoacetylated 6-O-tert-butyldimethylsilylated beta-CD derivative was synthesized and chemically bonded onto aminopropyl derivatized monolithic silica HPLC columns. In this CD derivative, only one of seven methyl groups in 3-position was substituted by an acetyl group. Its applicability as a chiral stationary phase for HPLC was tested and compared with exclusively 2,3-methylated 6-O-tert-butyldimethylsilylated beta-CD immobilized onto aminopropyl-modified monoliths. Thirty-two chiral compounds from different chemical classes and different functionalities were tested under RP conditions. Fourteen compounds were resolved into their enantiomers by methylated 6-O-tert-butyldimethylsilylated beta-CD. By use of methylated/acetylated 6-O-tert-butyldimethylsilylated beta-CD as the chiral stationary phase 7 analytes were successfully stereodifferentiated.

Evaluation of ring-opening metathesis polymerization (ROMP)-derived monolithic capillary high performance liquid chromatography columns

Novel monolithic capillary HPLC columns were prepared via ring opening metathesis polymerization (ROMP) within the confines of fused silica columns with 200 μm i.d. using norborn-2-ene (NBE), 1,4,4a,5,8,8a-hexahydro-1,4,5,8, exo, endo-dimethanonaphthalene (DMN-H6) as monomers, 2-propanol and toluene as porogens and RuCl 2(PCy 3) 2(CHPh) as initiator. Using the monolithic capillary HPLC columns, different sets of analytes (i.e. standard systems) were used for the evaluation of the monolithic columns: (i) a protein standard consisting of six proteins in the range of 5000–66 000 g/mol, (ii) an insulin–albumin standard, and (iii) a peptide standard obtained from a tryptic digest of cytochrome C. With these three different standard systems the reproducibility of synthesis in terms of separation performance proved to be 1–2% relative standard deviation in t R. Variation of polymerization parameters had a significant influence on the monolithic morphology and therefore separation efficiency and back pressure. The maximum analytical loading capacity of ROMP-derived monolithic capillary columns for albumin was found to be 30–125 ng, depending on the monomer content. Long-term stability studies showed no alteration in separation performance.

Automated On-Line Column-Switching HPLC-MS/MS Method with Peak Focusing for the Determination of Nine Environmental Phenols in Urine

We developed a method using isotope dilution on-line solid-phase extraction (SPE) coupled to high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for the determination in urine of nine environmental phenolic compounds: Bisphenol A; 4-tert-octylphenol; o-phenylphenol; 2,4-dichlorophenol; 2,5-dichlorophenol; 2,4,5-trichlorophenol; 2,4,6-trichlorophenol; benzophenone-3 (2-hydroxy-4-metoxybenzophenone); and triclosan (2,4,4'-trichloro-2'-hydroxyphenyl ether). A unique fully automated column-switching system, constructed using 1 autosampler, 2 HPLC pumps, and a 10-port switching valve, was designed to allow for concurrent SPE-HPLC operation with peak focusing. The phenols present in 100 microL of urine were retained and concentrated on a C18 reversed-phase size-exclusion SPE column. Then, the phenols were "back-eluted" from the SPE column and diluted through a mixing Tee before being separated from other urine matrix components using a pair of monolithic HPLC columns. The phenols were detected by negative ion-atmospheric pressure chemical ionization-MS/MS. The efficient preconcentration of the phenols by the SPE column, analyte peak focusing by the dilution, and minimal ion suppression in the LC/MS interface by the buffer-free mobile phases resulted in limits of detection as low as 0.1-0.4 ng/mL for most analytes. The method was validated on spiked pooled urine samples and on urine samples from 30 adults with no known occupational exposure to environmental phenols. The method can be used for quick and accurate analysis of large numbers of samples in epidemiologic studies for assessing the prevalence of human exposure to environmental phenols.

Comparison of performance of C18 monolithic rod columns and conventional C18 particle-packed columns in liquid chromatographic determination of Estrogel and Ketoprofen gel

The performance of monolithic HPLC columns Chromolith™ (made by Merck, Germany) and conventional C18 columns Discovery (Supelco, Sigma–Aldrich, Prague, Czech Republic) was tested and the comparison for two topical preparations Ketoprofen gel and Estrogel gel was made. The composition of mobile phases – for Ketoprofen analysis a mixture of acetonitrile, water and phosphate buffer adjusted to pH 3.5 (40:58:2) and for Estrogel analysis a mixture of acetonitrile, methanol, water (23:24:53) – was usually not optimal for analyses at all types of columns. Thus an adjustment of components ratio was necessary for sufficient resolution of the compounds analysed. Various flow rates (1.0–5.0 ml/min) and mobile phases (usually increasing ratio of water content) were applied. Determination of active substances, preservatives and impurities and comparison of retention times and system suitability test parameters was accomplished. For Estrogel gel, following chromatographic conditions were found: using Chromolith Flash RP-18e monolith column, mobile phase was acetonitrile, methanol, water (13:24:63, v/v/v) and flow-rate 3.0 ml/min. Using monolith column ChromolithSpeedROD RP-18e, the mobile phase was acetonitrile, methanol, water (18:24:58, v/v/v) and flow-rate 4.0 ml/min. For the monolith column Chromolith Performance RP-18e, the mobile phase was acetonitrile, methanol, water (23:24:53, v/v/v), flow-rate 3.0 ml/min. Analysis of Ketoprofen gel gave the best results using following analytical conditions: for monolith column Chromolith Flash RP-18e, mobile phase as a mixture of acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) was used, at flow-rate 2.0 ml/min. For ChromolithSpeedROD RP-18e monolith column, acetonitrile, water, phosphate buffer pH 3.5 (35:63:2, v/v/v) was used as a mobile phase at flow-rate 3.0 ml/min. Chromolith Performance RP-18e gave the best results using mobile phase acetonitrile, water, phosphate buffer pH 3.5 (30:68:2, v/v/v) at the flow-rate 5.0 ml/min. It was proved that monolith columns, due to their porosity and low back-pressure, can save analysis time by about a factor of three with sufficient separation efficiency. Thus, for example 11 min long analysis can be performed in 4 min with comparable results.

Stereoselective determination of allethrin by two-dimensional achiral/chiral liquid chromatography with ultraviolet/circular dichroism detection

A two-dimensional achiral/chiral HPLC method with circular dichroism (CD) detection was optimized for the stereochemical resolution and determination of the elution order of the eight stereoisomers of synthetic allethrin. A monolithic silica HPLC column (Chromolith, Merck, 100 mm × 4.6 mm i.d.) was put orthogonally to an enantioselective OJ Daicel column (250 mm × 4.6 mm i.d.) by means of a switching valve. The resolution of cis and trans diastereoisomers on the silica column was obtained by using a mobile phase consisting of n-hexane:tert-butyl methyl ether (96:4) (v/v) at a flow rate of 1 ml min−1. The cis and trans peaks were then switched to the enantioselective OJ column separately in two subsequent injections. The resolution of the four trans stereoisomers was accomplished by using n-hexane:tert-butyl methyl ether (90:10) (v/v), while the mobile phase composition for the four cis stereoisomers consisted of n-hexane:isopropanol (99.3:0.7) (v/v). The CD based detection system allowed the determination of the elution order on the basis of the CD signals of the single stereoisomers, together with the injection of pure stereoisomers. Under the final conditions, the validated method was applied to the determination of stereoisomeric composition and absolute configuration of the prevailing stereoisomers of real samples, i.e. commercial batches of different sources of d-allethrin.

Stereoselective determination of allethrin by two-dimensional achiral/chiral liquid chromatography with ultraviolet/circular dichroism detection

A two-dimensional achiral/chiral HPLC method with circular dichroism (CD) detection was optimized for the stereochemical resolution and determination of the elution order of the eight stereoisomers of synthetic allethrin. A monolithic silica HPLC column (Chromolith, Merck, 100 mm × 4.6 mm i.d.) was put orthogonally to an enantioselective OJ Daicel column (250 mm × 4.6 mm i.d.) by means of a switching valve. The resolution of cis and trans diastereoisomers on the silica column was obtained by using a mobile phase consisting of n-hexane: tert-butyl methyl ether (96:4) (v/v) at a flow rate of 1 ml min −1. The cis and trans peaks were then switched to the enantioselective OJ column separately in two subsequent injections. The resolution of the four trans stereoisomers was accomplished by using n-hexane: tert-butyl methyl ether (90:10) (v/v), while the mobile phase composition for the four cis stereoisomers consisted of n-hexane:isopropanol (99.3:0.7) (v/v). The CD based detection system allowed the determination of the elution order on the basis of the CD signals of the single stereoisomers, together with the injection of pure stereoisomers. Under the final conditions, the validated method was applied to the determination of stereoisomeric composition and absolute configuration of the prevailing stereoisomers of real samples, i.e. commercial batches of different sources of d-allethrin.

Applications of silica‐based monolithic HPLC columns

The recent invention and successive commercial introduction of monolithic silica columns has motivated many scientists from both academia and industry to study their use in HPLC. The first paper on monolithic silica columns appeared in 1996. Currently about 200 papers have been published relating to applications and characterization of monolithic silica columns, including monolithic capillaries. This review attempts to give an overview covering various aspects of this new column type in the field of high throughput analysis of drugs and metabolites, chiral separations, analysis of pollutants and food-relevant compounds, as well as in bioanalytical separations such as in proteomics. Some of the applications are described in greater detail. The numerous publications dealing with the physicochemical and chromatographic characterization of monolithic silica columns are briefly summarized.

HPLC Determination of Tetracyclines in Lamb Muscle Using an RP-C18 Monolithic Type Column

Four antibiotics, oxytetracycline (OTC), tetracycline (TCC), chlortetracycline (CTC) and doxycycline (DXC), were separated and quantified in lamb muscle extracts using a commercial RP-C18 monolithic HPLC column and a DAD detector. Operating an isocratic flow of 2 mL min−1 the HPLC separation of the four compounds was obtained within 6 min with a good peak symmetry and an acceptable separation factor (1.27) for the critical peak pair OTC-TCC. Values of the normalized numbers of theoretical plates (N cm−1) were comprised between 338–780 while the limits of quantitation (S/N=10) were within 12–65 μg kg−1.

Computational fluid dynamics simulations yielding guidelines for the ideal internal structure of monolithic liquid chromatography columns

A theoretical calculation of the separation performance of a (hypothetical) micro-structured monolithic LC column is presented, confirming that the polydispersity effect in parallel bundle columns can theoretically be eliminated to a very large extent by radially redistributing the mobile phase fluid at regular intervals. It is demonstrated that the flow can be redistributed in such a way that the advantage coming from the suppression of the polydispersity effect largely exceeds the losses caused by the additional pressure-drop and band broadening. The presently considered micro-structured column would allow to perform N>100 000 plate separations in a few hundred of seconds, i.e., about an order of magnitude faster than the best possible packed bed and monolithic HPLC columns, while offering the same mass loadability. This clearly demonstrates that the currently available LC columns are still far away from the absolute resolution limit of the ideal, fully optimised LC column.

High-throughput selected reaction monitoring liquid chromatography–mass spectrometry determination of methylphenidate and its major metabolite, ritalinic acid, in rat plasma employing monolithic columns

This work presents a high-throughput selected reaction monitoring (SRM) LC–MS method for the determination of methylphenidate (MPH), a central nervous stimulant, and its de-esterified metabolite, ritalinic acid (RA) in rat plasma samples. A separation of these two compounds was achieved in 15 s by employing a 3.5-ml/min flow-rate, a porous monolithic column and a TurboIonSpray source compatible with relatively high flow-rates. In addition, a relatively fast autosampler and a new data acquisition system resulted in a time lag of less than 17 s between consecutive injections. Overall, 768 protein-precipitated rat plasma samples (eight 96-well plates) containing both MPH and RA were analyzed within 3 h and 45 min. The partial method validation described in this report included an assessment of linearity, intra and inter-assay precision and accuracy, and method robustness. Deuterated internal standards for the target compounds, d 3-MPH and d 5-RA, were employed. The calibration curves ranged from 0.1 to 50 ng/ml for MPH and from 0.5 to 50 ng/ml for RA. The limit of quantification (LOQ) for MPH and RA was 0.1 and 0.5 ng/ml, respectively. For both analytes, the intra- and inter-assay precision (relative standard deviation, % C.V.) and accuracy (relative error) did not exceed 15% for the quality control samples (QCs) QC1, QC2 or QC3 (0.3, 1.5 and 40 ng/ml for MPH and 0.15, 15 and 40 ng/ml for RA) for either analyte and did not exceed 20% at the lower limit of quantitation (LOQ) level. No carry-over from the autosampler was detected. The retention times remained constant throughout the experiment. Baseline resolution of MPH and RA was consistently observed throughout the plates analyzed. The described method demonstrates the feasibility for employing monolithic HPLC columns to effect rapid bioanalytical SRM LC–MS analysis of representative biological samples.

Monolithic silica liquid chromatography columns for the determination of cyclooxygenase II inhibitors in human plasma

Methods employing monolithic HPLC columns for the determination of the cyclooxygenase II inhibitors rofecoxib ( I) and 3-isopropoxy-4-(4-methanesulfonylphenyl)-5,5′-dimethyl-5H-furan-2-one (DFP, III) in human plasma are described. Each analyte, together with an internal standard was extracted from the plasma matrix using solid-phase extraction in the 96-well format. The analytes were chromatographed on a Chromolith Speed Rod monolithic HPLC column (4.6×50 mm). Analyte detection for rofecoxib was via fluorescence following post-column photochemical derivatization. Detection for III was based on the native fluorescence of the compound. The precision, accuracy, and linearity of the methods were found to be comparable to those obtained using methods employing conventional packed HPLC columns. Use of the monolithic column permitted mobile phase flow-rates of up to 6.5 ml/min to be employed in the assays. The use of elevated flow-rates enabled the per sample analysis time to be reduced by up to a factor of 5 compared with assays based on packed HPLC columns. The results of experiments aimed at evaluating the ruggedness and reproducibility of monolithic columns employed in bioanalytical methods are presented.

Comparative study on the column performance of microparticulate 5-μm C 18-bonded and monolithic C 18-bonded reversed-phase columns in high-performance liquid chromatography

In this paper we report on the results of a comparative study on the performance of Purospher RP 18e, 5 μm, columns and prototypes of monolithic columns named SilicaROD from Merck, Darmstadt, Germany. The studies were performed on HPLC equipment with minimum extra column contribution. The plate height linear velocity dependency of the Purospher RP 18e column showed a minimum of H of about 10–15 μm at a linear velocity of 1 mm/s. The H versus u curves of the monolithic columns followed the same course. Yet, the curves remained flat up to a linear velocity of about 7 mm/s, where the Purospher RP 18e column could not be operated anymore due to the extremely high back-pressure. In conclusion the monolithic HPLC columns showed a column performance equivalent to a column with about 5 μm porous particles and were operated at flow-rates up to 9 ml/min on a conventional HPLC equipment without notable loss in column efficiency. The column back-pressure drop at the monolithic columns was three- to five-times lower than that on the 5-μm particulate column.