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.     

Evaluation of packed capillary liquid chromatography columns and comparison with conventional-size columns

Apart from extracolumn effects peak dispersion in liquid chromatographic columns is caused by the column inlet, the packed bed, and the column outlet. A strategy applicable for independent evaluation of the individual sources of column band broadening was developed on the basis of the linear extrapolation method (LEM). This method was applied to compare the performance of packed capillary LC columns from various commercial suppliers with conventional-size columns. The columns differed widely in their performance with respect to peak shapes and widths for standard substances. The capillary columns were found well packed, but in some cases overall performance would benefit from improving the design of the area between the packed bed and the connecting capillaries, containing frits as well as dead volumes.     

Packing capilllary electrochromatography columns using vacuum--a preliminary study

This paper introducesa novel method for packing Capillary Electrochromatography Columns (CEC). Using vacuum packing methodology, silica particles as small as 1 microm were successfully packed into the capillary columns with 75 microm inner diameter. The columns are verystable and show no noticeable loss in efficiency after 200 sample injections. The performance of these vacuum packed capillary columns was evaluated with a mixture of aromatic and non-aromatic compounds. A 24 cm long capillary column can produce peak efficiencies of around 45,000 plates for benzene.     

Life-time studies with capillary electrochromatography columns operated under different conditions

A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k(cec), peak symmetry coefficients, lambda(sym), and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 microm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 microm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 - 8.0, similar to those employed to study the retention behaviour of peptides that we have previously reported. The relative standard deviation (RSD) of the retention factors (k(cec) values) of these n-alkylbenzenes, acquired with an eluent of (25 mM Tris-HCl, pH 8.0,)-acetonitrile (1:4, v/v), when the CEC capillary columns were used for the first time (virgin values), were 4% (based on data acquired with 4 CEC capillary columns) for the n-octyl bonded silica capillary columns, and 6% (based on 8 columns) for n-octadecyl bonded silica capillary columns. The RSD values of the k(cec) values of the n-alkylbenzenes for one set of replicates (n=6) with one CEC capillary column was < 0.5%. The theoretical plate numbers, N, for the virgin CEC capillary columns were ca. 60,000, whilst the observed N values for all new CEC capillary columns were > or = 40,000 for n-octyl bonded silica capillary columns and > or = 50,000 for n-octadecyl bonded silica capillary columns. The peak symmetry coefficients, lambda(sym), of the n-alkylbenzenes for virgin CEC capillary columns and for CEC capillary columns used for more than 1,000 injections were always in the range 0.95-1.05. The experimental results clearly document that the life-time performance of the CEC capillary columns depends on the eluent composition, as well as the nature of the analytes to which the CEC capillary columns are exposed.     

Comparison of packed and capillary columns for quantitative gas chromatography of triglycerides in milk fat

Summary Quantitative gas chromatography of triglycerides in conjunction with established triglyceride formulae can be used to determine various milk fat parameters. Since the evaluation of, for example, iodine number or content of non-milk fats (foreign fats) in milk fat requires only the separation of triglycerides by carbon number and since repeatabilities, especially of the highboiling triglycerides, have been less acceptable with capillary columns in the past, packed columns have been used exclusively. There is, however, an increasing demand for the use of capillary instead of packed columns and to that end the present investigation has been carried out. To achieve a suitable resolution, with this particularly exacting high-temperature application, a short 5 m capillary column of extreme temperature stability has been used. As well as modification of various analytical conditions different injection techniques have been investigated. On-column, PTV and split injection were compared with regard to repeatability. The cold-on-column injection technique was found to produce the best results, being comparable to the excellent precision of a packed column. Thus, a method is now available by means of which the determination of milk fat parameters by the triglyceride formulae, such as the amount of foreign fats in milk fat, can now be carried out with a standardized capillary column.     

Effects of column length, particle size, gradient length and flow rate on peak capacity of nano-scale liquid chromatography for peptide separations

The effects of the column length, the particle size, the gradient length and the flow rate of a nanoLC system on peptide peak capacity were investigated and compared. Columns packed with 1.7 microm and 3 microm C(18) materials into pieces of 75 microm capillary tubing of various lengths were tested with different gradient lengths and flow rates. While increasing the length of a column packed with the 1.7 microm material helped improve peptide peak capacity at the whole range of the tested gradient lengths (24-432 min), little improvement in peak capacity was observed with the increase of the length of a column packed with the 3 microm material unless a gradient longer than 50 min was carried out. Up to 30% of peak capacity increase was observed when a column's length is doubled, with little reduction in the throughput. In most cases, more than 50% of the increase in peak capacity was obtained with the reduction in the particle size from 3 microm to 1.7 microm. With the same backpressure generated, a shorter 1.7-microm-particle column outperformed a longer column packed with the 3 microm material. In a flow rate range of 100-700 nl/min, increasing the flow rate improved peak capacity for columns packed with 1.7 microm and 3 microm materials.     

Capillary electrochromatography using continuous-bed columns of sol-gel bonded silica particles with mixed-mode octadecyl and propylsulfonic acid functional groups

Continuous-bed columns containing sol-gel bonded 3 microm silica particles with mixed-mode octadecyl and propylsulfonic acid functional groups (ODS/SCX) were prepared by first packing the ODS/SCX particles into a fused-silica capillary, then filling the packed capillary with a siliceous sol-gel, curing the sol-gel, and finally drying the column with supercritical carbon dioxide. The performance of the sol-gel bonded ODS/SCX columns was evaluated for capillary electrochromatography using acetonitrile aqueous mobile phase containing phosphate buffer. The columns were mechanically strong and permeable. Both EOF velocity and current increased linearly with elevation of the applied electric field strength. The EOF velocity was high at low pH and nearly constant over a range of pH 2-9. Higher buffer concentration resulted in higher current and lower EOF velocity. The acetonitrile content had no significant effect on the EOF. Without thermosetting the column, no bubble formation was noticed with currents up to 2.5 microA. The minimum plate height of a 25/34 cm x 75 microm I.D. sol-gel bonded 3 microm ODS/SCX column was 5.7 microm (1.75 x 10(5) plates per meter) at an optimum EOF velocity of 0.92 mm s(-1). Mixtures of test aromatic compounds and aromatic hydrocarbon homologues gave symmetrical peaks when using a low pH mobile phase. The retention and elution order of aromatic compounds represented a typical reversed-phase separation mechanism similar to conventional ODS columns. The run-to-run and column-to-column retention factor reproducibility was better than 2.5% and 8.0% RSD, respectively.     

Comparison of different packing methods for capillary electrochromatography columns

A study was carried out in which 50 microm I.D. fused-silica capillaries were packed with 3 microm octadecylsilane bonded silica, from the same batch, by four methods; liquid slurry and carbon dioxide supercritical carrier, each with and without the use of an ultrasonic probe. A neutral test mixture was analysed by capillary column in reversed-phase mode, and the reproducibility of the electroosmotic flow and of migration time, column efficiency and retention factors, was determined. Initially results suggested that there was no significant difference between properties of columns packed by different methods, and a more thorough statistical evaluation confirmed this; differences observed in the column performance were attributed to random variations between replicate columns, and not between packing methods. However, the variation was least when applying the ultrasonication during liquid slurry.     

Evaluation of frontal chromatograms

A novel pressure-balanced injection valve was evaluated for use with ultrahigh pressure liquid chromatography (UHPLC) at pressures up to 120 MPa (1,200 bar). Fused-silica capillaries (30-33 cm x 100 microm I.D.) packed with nonporous 1.5 microm isohexylsilane-modified (C6) silica particles were employed to study maximum pressure, injection reproducibility, injection time, and sample amount consumed for an injection. The new valve was more reproducible, convenient, and required much less sample than previously used injection systems. The effect of column diameter on efficiency and sensitivity was studied. The 100 microm I.D. columns demonstrated approximately 40% lower efficiency but 10-fold higher sensitivity than the 29 microm I.D. columns. Columns packed with nonporous C6 particles produced higher efficiencies than columns packed with a 1.5 microm porous octadecylsilane-modified (C18) material.     

Capillary electrochromatography using fibers as stationary phases.

Fiber-packed capillary columns have been evaluated in chromatographic performance in capillary electrochromatography (CEC). The change of electroosmotic flow (EOF) velocity and selectivity using different kinds of fiber materials was examined. Although the EOF velocity among the different fiber packed columns was almost the same, retention of parabens was larger on the Kevlar-packed column than on the Zylon-packed one, and was larger on the as-span-type fiber-packed column than on the high-modulus-type packed one. Using 200 microm ID x 5 cm Kevlar packed column combined with a 100 microm ID x 20 cm precolumn capillary and a 530 microm ID x 45 cm postcolumn capillary, the separation of three parabens within 30 s was achieved. Other compounds were also separated in a few minutes by the fiber-packed CEC method.     

How to utilize the true performance of monolithic silica columns

Ways of utilizing the true separation efficiency of monolithic silica (MS) columns were studied. The true performance of MS columns, both regular-sized (rod-type clad with PEEK resin, 4.6 mm ID, 10 cm) and capillary sized (in 100 or 200 microm ID fused silica capillary, 25-140 cm) was evaluated by calculating the contribution of extra-column effects. HETP values of 7-9 microm were observed for solutes having retention factors (kvalues) of up to 4 for rod columns and up to 15 for a capillary column. The high permeability of MS columns allowed the use of long columns, with several connected together in the case of rod columns. Narrow-bore connectors gave good results. Peak variance caused by a column connector ranges from 50 to 70% of that caused by one rod-type column for up to three connectors or four columns in 80% methanol, but the addition of a 4th or 5th connector to add a 5th and 6th column, respectively, caused a much greater increase in peak variance, especially for long-retained solutes, which is greater than the variance caused by one rod column. Rod columns seem to show slightly lower efficiency at a pressure higher than 10 MPa or so. The use of acetonitrile-water as a mobile phase better preserved the ability of individual rod columns to generate up to 100,000 theoretical plates with 14 columns connected. Methods for eliminating extra-column effects in micro-HPLC were also studied. Split injection and on-column detection resulted in optimum performance. A long MS capillary measuring 140 cm produced 160,000 theoretical plates. The column efficiency of a capillary column was not affected by the pressure, showing advantages over the rod columns that exhibited peak broadening caused by connectors and pressure.     

Tryptic digest mapping by gradient capillary electrochromatography

A high performance liquid chromatography (HPLC) system complemented with T-split, capillary detection cell and a high voltage power supply was used for peptide mapping by gradient electrochromatography and nanoliquid chromatography (nano-LC). With capillary columns of 100 microm ID, 6 cm packed with octadecylated 1.5 microm silica particles, the typical analysis time was approximately 10-15 min. The resolution of a tryptic digest of cytochrome c obtained by electrochromatography at 100 kV/m was superior compared to the analysis by nano-LC. Bubble formation caused by Joule heating at currents up to 100 microA was successfully suppressed by using a resistor capillary of 25 microm ID connected to the outlet of the packed column.     

Comparison of the efficiency of microparticulate and monolithic capillary columns

A comparison is made between the efficiency of microparticulate capillary columns and silica and polymer-based monolithic capillary columns in the pressure-driven (high-performance liquid chromatography) and electro-driven (capillary electrochromatography) modes. With packed capillary columns similar plate heights are possible as with conventional packed columns. However, a large variation is observed in the plate heights for individual columns. This can only be explained by differences in the quality of the packed bed. The minimum plate height obtained with silica monolithic capillary columns in the HPLC mode is approximately 10 microm, which is comparable to that of columns packed with 5-microm particles. The permeability of wide-pore silica monoliths was found to be much higher than that of comparable microparticulate columns, which leads to much lower pressure drops for the same eluent at the same linear mobile phase velocity. For polymer-based monolithic columns (acrylamide, styrene/divinyl benzene, methacrylate, acrylate) high efficiencies have been found in the CEC mode with minimum plate heights between 2 and 10 microm. However, in the HPLC mode minimum plate heights in the range of 10 to 25 microm have been reported.     

Influence of temperature on the behaviour of small linear peptides in capillary electrochromatography

The influence of temperature, T, on the retention times, peak widths, peak symmetry coefficients and theoretical plate numbers of two small linear peptides, [Met5]enkephalin and [Leu5]enkephalin, has been studied with capillary electrochromatography (CEC) capillary columns of 100 microm I.D. and 250 mm packed length with a total length of 335 mm, containing 3 microm Hypersil n-octadecyl bonded silica. With increasing column temperature from 15 to 60 degrees C, the electroosmotic flow (EOF) and the column efficiencies increased, whereas the retention coefficients (Kcec) of both peptides decreased. A linear relationship was found between the EOF value and the square root of the temperature over this temperature range, with a linear regression correlation of 0.998. Non linear Van 't Hoff plots (In Kcec versus 1/T) were observed for these peptides between 15 and 60 degrees C, suggesting that a phase-transition occurred with the n-octadecyl chains bonded on the silica surface, affecting the CEC retention behaviour of these peptides. In CEC systems, the Kcec values of peptides incorporate contributions from both electrophoretic migration and chromatographic retention. Positive and negative Kcec values can, in principle, thus arise with these charged analytes. However, the Kcec values of the enkephalin peptides under all temperature conditions studied were positive with an eluent composed of water-50 mM NH4OAc/AcOH, pH 5.2-acetonitrile (5:2:3, v/v) and therefore the chromatographic component dominates the retention process with these small peptides under these conditions.     

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.     

Ultrahigh-pressure liquid chromatography using a 1-mm id column packed with 1.5-microm porous particles

The evolution of chromatography has led to the reduction in the size of the packing materials used to fabricate HPLC columns. The increase in the backpressure required has led to this technique being referred to as ultrahigh-pressure liquid chromatography (UHPLC) when the column backpressure exceeds 10000 psi (approximately 700 bar). Until recently, columns packed with sub-2-microm materials have generally fitted into two classes; either short (less than 5 cm) columns designed for use on traditional HPLC systems at pressures less than 5000 psi (350 bar), or capillary columns (inner diameters less than 100 microm). By using packing materials with diameters <2 microm to fabricate UHPLC columns, there is an increase in efficiency and a decrease in the analysis time that are directly proportional to the size of the packing material. In order to realize and exploit the increase in efficiency, however, the columns must maintain lengths typically associated with analytical columns (15-25 cm). We have packed 1 mm diameter, 150 mm in length columns with 1.5 microm packing material, and evaluated their performance in UHPLC. The pressure required to achieve optimum linear velocities in plots of plate height versus linear velocity was in the vicinity of 1104 bar (16000 psi). The 1.5 microm particle-packed column was compared with the more traditional 150 mm long analytical columns packed with 3 microm materials. This column showed an efficiency that was approximately twice that observed with the 3 microm packed column and a concomitant reduction in the analysis time, theoretically predicted.     

Application of nanoscale packed capillary liquid chromatography (75 μm id) and capillary zone electrophoresis/electrospray ionization mass spectrometry to the analysis of macrolide antibiotics

Nanoscale separation techniques, nanoscale packed capillary columns (75 μm id), and capillary zone electrophoresis (CZE), on-line with electrospray mass spectrometry (ESI/MS), were applied to the separation of a series of ten macrolide antibiotics. Both techniques use sub-microliter-per-minute flow rates through the analytical column and therefore require an electrospray probe that incorporates coaxial sheath flow. Positive ion electrospray mass spectra of these compounds yielded mainly protonated molecules. Fragmentation to yield structurally significant fragment ions was achieved by collision-induced dissociation (CID) at increased skimmer voltages. Separations were achieved using both techniques, with CZE/ESI/MS showing improved peak shapes and detection limits combined with faster analysis times. Nanoscale packed capillary columns provided better chromatographic resolution and was less susceptible to peak broadening caused by overloading of the analytes.     

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography

The impact of column inner diameter on chromatographic performance in temperature gradient liquid chromatography has been investigated in the present study. Columns with inner diameters of 0.32, 0.53, 3.2 and 4.6 mm were compared with respect to retention and efficiency characteristics using temperature gradients from 30 to 90 degrees C with temperature ramps of 1, 5, 10 and 20 degrees C min(-1). The columns were all of 15 cm length and were packed with 3 microm Hypersil ODS particles. Alkylbenzenes served as model compounds, and the mobile phase consisted of acetonitrile-water (50:50, v/v). The study revealed that the column ID is not a critical limiting factor when performing temperature programming in LC, at least for columns narrower than 4.6 mm inner diameter in the temperature interval 30-90 degrees C. The retention times for all components on all columns were highly comparable, with similar peak profiles without any signs of peak splitting. The use of mobile phase pre-heating when using the larger bore columns was avoided by starting the temperature gradients close to ambient. However, the relative apparent efficiency was inversely proportional to column inner diameter, making the capillary columns generally more functional towards temperature gradients than the larger bore columns with respect to chromatographic efficiency. In addition, the capillary columns possessed higher robustness towards temperature programming than the conventional columns.     

一种多通道匀浆装填毛细管色谱柱的新装置及应用

针对目前毛细管色谱柱装柱效率低、不同批次装填的毛细管色谱柱之间性能差异大的问题,我们发展了一种多通道匀浆装填毛细管色谱柱的新装置。该装置以液相色谱泵提供压力、采用磁力搅拌保持匀浆液均匀分散,一次可装填多达6根毛细管色谱柱。以牛血清白蛋白(BSA)的胰蛋白酶酶切肽段混合物为样本,选择峰容量、蛋白覆盖率、3个特定离子的保留时间以及毛细管色谱柱柱压为指标,在毛细管液相色谱-质谱联用系统上对装填的反相毛细管色谱柱的性能进行了评价。分别考察了一次装填的6根毛细管色谱柱、两次装填的12根毛细管色谱柱以及一次装填1根与一次装填6根毛细管色谱柱的性能及稳定性。实验结果表明:同一批次装填的6根毛细管色谱柱的性能相近;不同批次装填的12根毛细管色谱柱的峰容量和覆盖率没有明显的区别,但保留时间和毛细管色谱柱柱压的稳定性较差;一次装填1根和一次装填6根毛细管色谱柱柱性能的稳定性与两次分别装填6根毛细管色谱柱的稳定性相近,即采用本装置可显著提高毛细管色谱柱的装填效率且每次装填毛细管色谱柱的数量不会对柱性能产生影响。     

宽口径填充毛细管液相色谱柱柱尾结构的改进

 设计并评价了尾锥形填充毛细管液相色谱柱,与粘结型柱尾比较,前者柱效高且van Deemter曲线高流速部分的流速-柱效关系明显改善。在折合流速为10～15时,前者的柱效比后者高出30%～50%。谱带的抛物线状的流形在尾锥形柱内得到改善,使色谱峰的对称性有明显提高。比较了两种结构的色谱柱在芳烃快速分析中的分离效果,结果是尾锥形柱的分离性能优于粘结型柱。