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.     

Appropriate column configurations for the rapid analysis and semipreparative purification of the radiolabeled drug flutamide by high-performance liquid chromatography

Flutamide, marketed as Eulexin, is used for treatment of metastic prostatic carcinoma. Purity of a radiolabeled batch for metabolism studies was first determined by reversed-phase HPLC on a 5 microm, 150x4.6 mm analytical column. The separation was then scaled up to give a semipreparative column (5 microm, 250x10 mm) purification procedure. Fraction analysis was done on a short rapid analysis (5 microm, 50x3.0 mm) column. Analysis of the final product was performed on the analytical column. All columns were YMC-Pack ODS-AQ. The analytical work involved large mass injections in order to have the required amounts of radioactivity needed for accurate impurity profile determinations, and the preparative work involved masses much larger than the calculated scale-up values. Ultraviolet and radiochromatograms of the drug on the various column configurations are compared. A 95.7% recovery of product was obtained, with radiochemical purity increased from 95.0 to 99.8%.     

High-speed analyses using rapid resolution liquid chromatography on 1.8-microm porous particles

The potential of high-speed analyses by rapid resolution liquid chromatography (RRLC) and RRLC/MS on 1.8-microm porous particles packed into short columns operated at high flow-rate was investigated and compared with the performance of 5-microm porous particles packed into conventional columns. Using similar chemistries, the ease of conversion from conventional HPLC to an RRLC method was demonstrated. In order to display the practicality of RRLC separations, the analysis of pesticides in crops and catechins in Japanese green tea was selected. Using the Japanese Food Hygiene Law method, which employs a conventional 5-microm RP column (250 mm x 4.6 mm) for quantification of pesticides in crops, the analysis time was 25 min under isocratic conditions. Using the RRLC method on the short (50 mm x 4.6 mm) column packed with 1.8-microm porous particles, the same separation could be performed in 0.8 min with the RRLC/MS method without a loss in resolution. At the highest flow rate, compared to the conventional method, the time could be reduced by a factor of 31. In gradient elution, the fastest separation of catechins in Japanese green tea was achieved by RRLC on 50-mm x 4.6-mm id or 50-mm x 2.1-mm id RRLC columns packed with 1.8-microm particles. The analysis time at 5 mL/min was less than 1 min. Compared to the conventional HPLC method on a 150-mm column packed with 5-microm particles, time was reduced by a factor of 15. The effect of other experimental parameters such as the column temperature, acquisition rate of the detector and the influence of cell volume on chromatographic performance was also investigated. After the optimization, the analysis precision under the fastest RRLC conditions was examined. RSDs of retention time and peak area were 0.2% and 0.47%, respectively.     

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.     

High-performance liquid chromatography of seized drugs at elevated pressure with 1.7 microm hybrid C18 stationary phase columns

High-performance liquid chromatography (HPLC) separation of drugs at elevated pressure with 1.7 microm hybrid C18 stationary phase columns was investigated. This technique, which uses instrumentation engineered to handle the narrow peaks and high back pressures generated by 1.7 microm particle columns, provided significantly better resolution and/or faster analysis than conventional HPLC and capillary electrophoresis (CE). The use of 2mm internal diameter (i.d.) columns of 3-10 cm length has been evaluated for the separation of basic and neutral drugs, drug profiling, and general screening (including acidic drugs). For these applications, compared to conventional HPLC and CE, it provided up to 12x and 3x faster analyses, respectively. Precision was excellent for both isocratic and gradient analyses. For retention time and peak area, RSDs of < or =0.1% were obtainable. Fifteen anabolic steroids and esters were well separated in a 2.5 min gradient. For drug profiling, compared to HPLC and CE, approximately twice as many peaks were resolved. HPLC at elevated pressure is also well suited as a general screening technique. Twenty-four solutes of varying drug classes including narcotic analgesics, stimulants, depressants, hallucinogens, and anabolic steroids were fully separated in a 13.5 min gradient.     

High performance liquid chromatographic determination of copper(II) and nickel(II) by using solvent extraction and bis(acetylpivalylmethane)ethylenediimine as complexing reagent

The reagent bis(acetylpivalylmethane)ethylenediimine has been examined for the HPLC separation of copper(II), nickel(II), palladium(II) and oxovanadium(IV) chelates on reversed phase HPLC columns (250 x 4 mm) packed with Hypersil ODS, 5 mu and (150 x 3.9 mm) Nova Pak C(18) with guard column. The complexes are eluted with a binary mixture of methanol and water or methanol, acetonitrile and water. Detection is achieved with a UV detector. The solvent extraction procedure is used for the determination of copper and nickel simultaneously at microgram levels, corresponding to ng levels and pg levels respectively, per injection. The method has been applied to the determination of copper and nickel in a coin, nickel-aluminum alloy and water samples.     

Chromatographic and column stability at pH 7 of a C18 dimethylurea polar stationary phase

Chromatographic evaluations of a C18 dimethylurea phase in 150 mm x 3.9 mm HPLC columns were performed using the Tanaka and Engelhardt test mixtures. The applicability of the new C18 dimethylurea phase was also evaluated with a mixture of some herbicides and their metabolites. An artificial aging procedure was also performed by passing a potassium phosphate mobile phase buffered at pH 7.0 through C18 50 mm x 3.9 mm dimethylurea columns. The column stability was evaluated by means of the chromatographic parameters obtained for the separation of some compounds from the Neue test mixture, using apolar, polar and highly basic analytes.     

Ultrahigh-pressure dual online solid phase extraction/capillary reverse-phase liquid chromatography/tandem mass spectrometry (DO-SPE/cRPLC/MS/MS): a versatile separation platform for high-throughput and highly sensitive proteomic analyses

Capillary RPLC/ESI-MS (cRPLC/ESI-MS) is one of the most powerful analytical tools for current proteomic research. The development of cRPLC techniques coupled online to a mass spectrometer has focused on increasing the separation efficiency, detection sensitivity, and throughput. Recently, the use of high-pressure (over 10,000 psi) LC systems that utilize long, small inner diameter capillary columns has gained much attention for proteomic analyses. In this study, we developed an ultrahigh-pressure dual online SPE/capillary RPLC (DO-SPE/cRPLC) system. This LC system employs two online SPE columns and two capillary columns (75 microm inner diameter x 1 m length) in a single separation system, and has a maximum operating pressure of 10,000 psi. This DO-SPE/cRPLC system is capable of providing high-resolution separation in addition to several other advantageous features, such as high reproducibility in terms of the LC retention time, rapid sample injection, online desalting, online sample enrichment of dilute samples, and increased throughput as a result of essentially removing the column equilibration time between successive experiments. We coupled the DO-SPE/cRPLC system online to a tandem mass spectrometer to allow high-throughput proteomic analyses. In this paper, we demonstrate the efficiency of this DO-SPE/cRPLC/MS/MS system by its use in the analyses of proteomic samples exhibiting different levels of complexity.     

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.     

Comparison between monolithic conventional size, microbore and capillary poly(p-methylstyrene-co-1,2-bis(p-vinylphenyl)ethane) high-performance liquid chromatography columns Synthesis, application, long-term stability and reproducibility

Novel monolithic supports (MS/BVPE) were prepared by thermally initiated free radical copolymerisation of p-methylstyrene (MS) and 1,2-bis(p-vinylphenyl)ethane (BVPE). The polymer was synthesised in fused silica capillaries (80 mm x 0.2 mm and 80 mm x 0.53 mm) and in borosilicate glass columns (90 mm x 1.0 mm and 90 mm x 3.0 mm) to yield different HPLC column designs. A comparison of those column dimensions regarding morphology as well as separation efficiency and applicability in bioanalysis is presented. The efficiency towards proteins as well as oligonucleotides was found to be considerably improved with decreasing column I.D. While a 5-protein mixture was baseline separated on all investigated column designs, the separation of small biomolecules like oligonucleotides or peptides on microbore and conventional size glass columns was strongly restricted in terms of resolution due to extensive peak broadening or the occurrence of peak asymmetry. Monolithic MS/BVPE capillary columns up to 0.53 mm I.D., however, proved to be applicable to the fractionation of the whole spectrum of biopolymers, including proteins, peptides, oligonucleotides as well as double-stranded DNA fragments. Due to the fact that reliable chromatography makes great demand on the robustness of the stationary phase, monolithic MS/BVPE capillaries were subjected to a comprehensive reproducibility study including run-to-run as well as batch-to-batch reproducibility.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Separation of natural product using columns packed with Fused‐Core particles

Three HPLC columns packed with 3 μm, sub‐2 μm, and 2.7 μm Fused‐Core (superficially porous) particles were compared in separation performance using two natural product mixtures containing 15 structurally related components. The Ascentis Express^TM C18 column packed with Fused‐Core particles showed an 18% increase in column efficiency (theoretical plates), a 76% increase in plate number per meter, a 65% enhancement in separation speed and a 19% increase in back pressure compared to the Atlantis T3^TM C18 column packed with 3 μm particles. Column lot‐to‐lot variability for critical pairs in the natural product mixture was observed with both columns, with the Atlantis T3 column exhibiting a higher degree of variability. The Ascentis Express column was also compared with the Acquity^TM BEH column packed with sub‐2 μm particles. Although the peak efficiencies obtained by the Ascentis Express column were only about 74% of those obtained by the Acquity BEH column, the 50% lower back pressure and comparable separation speed allowed high‐efficiency and high‐speed separation to be performed using conventional HPLC instrumentation.     

Performance of new prototype packed columns for very high pressure liquid chromatography

The reduced heights equivalent to a theoretical plate (HETP) of naphtho[2,3-a]pyrene were measured at room temperature on two sets of new prototype columns designed to be used in very high pressure liquid chromatography (VHPLC). The mobile phase used was pure acetonitrile. The columns are 50, 100, and 150 mm long. Those of the first set are 2.1 mm I.D., those of the second set, 3.0 mm I.D. The performance of these new columns were compared to those of the first generation of VHPLC columns, commercially available in 2.1 mm I.D. The prototype and commercial columns behave similarly at low reduced linear velocities (ν<5$\nu <5$), when the heat effects are negligible. At high flow rates, the shorter prototype columns have a twice better efficiency and less steep C-branches than the commercial columns. In contrast, the C-branch of the 150 mm long prototype columns are slightly steeper than those of the commercial columns. The important contribution to the reduced HETP that is due to the heat effects at high flow rates can in part be accounted for by a band broadening model governed by a flow mechanism with the shortest prototype columns. The sole heat effects cannot, however, explain the mediocre reduced HETPs of the 2.1 and 3.0 I.D. 150 mm long prototype columns. It seems that radial heterogeneity of the flow rate of the long prototype columns is significantly larger than that of the short columns. The contribution of the packing heterogeneity adds up to that of the heat effects to yield a poor column efficiency when sub-2μm$\text{sub-}2\mu \text{m}$ are packed into thin, long column tubes.     

Evaluation of monolithic and sub 2 microm particle packed columns for the rapid screening for illicit drugs--application to the determination of drug contamination on Irish euro banknotes

A study comparing recently available 100 x 3 mm id, 200 x 3 mm id monolithic reversed-phase columns with a 50 x 2.1 mm id, 1.8 microm particle packed reversed-phase columns was carried out to determine the most efficient approach (using traditional van Deemter analysis and a modern kinetic plot approach) for the rapid screening of samples for 16 illicit drugs and associated metabolites. A plot of column backpressure versus plate number (N) showed a significant advantage of using the monolithic phases, with the 20 cm monolithic column exhibiting a maximum 15,000 plates at a column backpressure of approximately 70 bar, compared to approximately 7000 plates at 150 bar for the 5 cm 1.8 microm particle packed column. Optimum linear velocities were found to be 0.40 mm s(-1), 0.52 mm s(-1) and 0.98 mm s(-1) for the three above columns, respectively. The 20 cm monolithic column was subsequently applied to the separation and determination of illicit drug contamination on Irish euro banknotes, using methanol extraction followed by LC-MS/MS. Method performance data showed that the new LC-MS/MS method was significantly more sensitive than previous GC-MS/MS based methods for this application, with detection limits in the pg note(-1) region, based upon a 20 microL standard injection. All of the notes examined tested positive for trace quantities of cocaine, with benzoylecgonine detected on 12 of the 45 notes sampled. Traces of heroin were also detected on three of the 45 notes.     

Application of Convective Interaction Media (CIM) disk monolithic columns for fast separation and monitoring of organic acids

The separation of organic acids on the anion-exchange monolithic support, commercially available as Convective Interaction Media (CIM), is presented in this study. It is demonstrated that citric, isocitric, pyruvic, fumaric, malic, and alpha-ketoglutaric acid can be successfully separated using a CIM monolithic column of suitable user-adjustable length. The effect of the mobile phase composition on the separation is investigated. CIM monolithic columns of adjustable length from 3 to 18 mm are compared regarding the resolution and the back pressure. It is shown that the CIM monolithic column of 12 mm in length enables a good separation of all six organic acids within 3 min and exhibits a linear dependence of back pressure versus flow rate. The resolution and the dynamic binding capacity are found to be flow-unaffected. A filtrated sample of bioprocess supernatant is analyzed without previous pretreatment, which indicates the possibility of online monitoring of small molecules during the bioprocess using CIM monolithic columns.     

Monolithic silica columns with various skeleton sizes and through-pore sizes for capillary liquid chromatography

Reduction of through-pore size and skeleton size of a monolithic silica column was attempted to provide high separation efficiency in a short time. Monolithic silica columns were prepared to have various sizes of skeletons (approximately 1-2 microm) and through-pores (approximately 2-8 microm) in a fused-silica capillary (50-200 microm I.D.). The columns were evaluated in HPLC after derivatization to C18 phase. It was possible to prepare monolithic silica structures in capillaries of up to 200 microm I.D. from a mixture of tetramethoxysilane and methyltrimethoxysilane. As expected, a monolithic silica column with smaller domain size showed higher column efficiency and higher pressure drop. High external porosity (> 80%) and large through-pores resulted in high permeability (K = 8 x 10(-14) -1.3 x 10(-12) m2) that was 2-30 times higher than that of a column packed with 5-mirom silica particles. The monolithic silica columns prepared in capillaries produced a plate height of about 8-12 microm with an 80% aqueous acetonitrile mobile phase at a linear velocity of 1 mm/s. Separation impedance, E, was found to be as low as 100 under optimum conditions, a value about an order of magnitude lower than reported for conventional columns packed with 5-microm particles. Although a column with smaller domain size generally resulted in higher separation impedance and the lower total performance, the monolithic silica columns showed performance beyond the limit of conventional particle-packed columns under pressure-driven conditions.     

An improved method for tracking and reducing the void volume in nano HPLC–MS with micro trapping columns

Tandem mass spectrometry coupled to HPLC is the state of the art technique in proteomic research. Here we describe a highly sensitive nano liquid chromatography system (nano HPLC) for analysis of protein digests. Using preconcentration in a column-switching set-up, we were able to inject large sample volumes (250 µL) without significant loss of sensitivity. The major problem with this type of preconcentration is usually the occurrence of void volumes. In order to diagnose void volumes a simple and easy test was developed by which the UV trace and the pressure profile in the separation column were monitored. Part by part replacement of connection tubing restored a void volume-free system. A major pre-requisite for handling samples in the femtomol range was found to be the use of protein/peptide-saturated columns tryptic digests of cytochrome C were injected directly onto the reversed-phase nano separation column (75 µm inner diameter) and the separation results were compared with chromatograms obtained from separations using column switching. By using column switching we were able to inject large sample volumes in a short time period without losing resolution.     

Fast high performance liquid chromatography analysis in lipidomics: Separation of radiolabelled fatty acids and phosphatidylcholine molecular species using a monolithic C18 silica column

HPLC procedures using conventional C₁₈ columns are usually used to separate simple and complex lipid mixtures but these methods of separation remain often laborious and very slow. Here, monolithic columns were successfully applied to separate lipids - radiolabelled fatty acid mixtures and individual phosphatidylcholine (PC) molecular species. For that, isocratic elution was performed using two Chromolith™ Performance RP-18e columns connected in series. Detection was achieved by online measurement of radioactivity for radiolabelled fatty acids and by UV absorbance at 205 nm for PC molecular species. The performances of such silica rods were compared to conventional reverse-phase silica columns. Monolithic stationary phase separated radiolabelled fatty acids and PC molecular species two times and four times faster, respectively. In each analysis, monolithic columns allowed better separation efficiency per unit of time, with lower inlet pressure. The main advantages of this method for lipid separation are that, under isocratic conditions, it is simpler and much faster, while remaining accurate and selective when compared to conventional methods. Therefore, monolithic columns may represent a powerful tool for the near future in the field of lipidomics.     

通过柱切换技术与高效液相色谱联用的限进性柱对盐酸贝那普利的在线富集性能

以自制的限进性填料柱为预处理富集柱,Luna C₁₈柱为分析柱,通过柱切换技术将限进性填料柱与高效液相色谱联用(RAM-HPLC),研究了盐酸贝那普利的在线富集效果。考察了进样体积与峰面积、系统总压力的关系,以及常规进样与大体积进样的差别。当进样体积在100 μL以内时,峰面积随进样体积的增加而增加;当进样体积大于80 μL时,系统总压力变化明显。考虑对整个系统的保护,选择80 μL作为最大进样体积。同一浓度的样品进样20 μL与进样80 μL所得峰面积之间的线性关系良好。RAM柱对盐酸贝那普利具有良好的富集作用,能够有效提高HPLC的灵敏度,而且具有简单、经济的特点。     

Capillary ion chromatography of inorganic anions on octadecyl silica monolith modified with an amphoteric surfactant

A reversed-phase monolithic silica based capillary column (Onyx C(18), 150 mm x 0.1 mm) was modified with the amphoteric surfactant, N-dodecyl-N,N-(dimethylammonio)undecanoate (DDMAU) and evaluated for the separation and determination of inorganic anions using on-column capacitively coupled contactless conductivity detection (C(4)D). The chromatographic performance of the column was evaluated and under optimal conditions separation efficiencies of 56,200 plates per meter or 7025 plates per column (at detection point) were observed (for iodide). Direct plumbing of the capillary column to the micro-injector and on-column detection eliminated extra-column band broadening, thus allowing accurate analysis of van Deemter curves obtained for the monolithic capillary column. The calculated value for the C-term in the obtained van Deemter curve was between 3 and 4 ms for inorganic anions, allowing for the utilisation of relatively high flow rates without significant losses in efficiency. The performance of the C(4)D detector was investigated and compared for detection on an open tubular capillary column and on the modified monolithic silica capillary column. The on-column detection approach did not result in any significant decrease in peak sensitivity for the monolith compared to responses recorded for open tubular capillary columns, and in addition meant the system could be applied to rapid separations by simple variation in apparent column length. The proposed chromatographic system allowed for detection of common anions at sub-ppm level with a 10 nL injection volume. Additionally, on-column detection allowed visualisation of the development of the separation at any point in time and evaluation of the longitudinal uniformity of the ion-exchange coating.