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.     

Synthesis of spherical porous silicas in the micron and submicron size range: challenges and opportunities for miniaturized high-resolution chromatographic and electrokinetic separations

Classical silica technology has reached its limit with respect to an ultimate minimum particle size of about 2 microm in diameter. Here, a novel process is presented which allows one to synthesize porous silica beads and control their particle diameter in situ, within the range of 0.2-2.0 microm. As a result, no sizing is required and losses of silica are avoided. Furthermore, the process enables one to control in situ the pore structural parameters and the surface chemistry of the silica beads. Even though surface funtionalized silicas made according to this process can principally be applied in fast HPLC the column pressure drop will be high even for short columns. In addition, the column efficiency, expressed in terms of the theoretical plate height is about H-2d(p) in the best case and limited by the A and C term of the Van Deemter equation. In other words the gain in total plate number when using 1-2 microm silica beads in short columns is minimal as compared to longer columns packed with 5 microm particles. Capillary electrochromatography (CEC) as a hybrid method enables the application of micron size as well as submicron size particles. This consequently enhances column efficiency by a factor of 5-10 when compared to HPLC. The use of short CEC columns packed with submicron size silicas provides the basis for fast and efficient miniaturized systems. The most significant feature of CEC as compared to HPLC is that the former allows one to resolve polar and ionic analytes in a single run. An alternative method for miniaturization is capillary electrophoresis (CE) which generates extremely high efficiencies combined with fast analysis. Its application, however, is limited to ionic substances.     

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.     

Packed-column hydrodynamic chromatography using 1-μm non-porous silica particles

150×3 mm I.D. columns, packed with 1-μm non-porous spherical silica particles, were used to separate soluble synthetic polymers by hydrodynamic chromatography. The columns exhibited a plate height of about 1.4 μm allowing very fast and efficient separations of polymers in the molecular mass range 10³−2·10⁶ g/mol. The migration behaviour of polymers could be well described by a simple theoretical model. The applicability of packed bed HDC for the fast separation of polymers was illustrated with separations of polystyrene and poly(methyl methacrylate) mixtures.     

Pressure drop effects on selectivity and resolution in packed column supercritical fluid chromatography

The influence of pressure drop on retention, selectivity, plate height and resolution was investigated systematically in packed supercritical fluid chromatography (SFC) using pure carbon dioxide as the mobile phase. Numerical methods developed previously which enabled the prediction of pressure gradients, diffusivities, capacity factors, plate heights and resolutions along the length of the column were used for the model calculations. The effects of inlet pressure and supercritical fluid flow rate on selectivity and resolution are studied. In packed column SFC with pure carbon dioxide as the mobile phase, the pressure drop can have a significant effect on resolution. The flow rate is shown to have a larger effect than generally realized. The calculated data are shown to be in good agreement with the experimental results. Finally, the variation of the chromatographic parameters along a 5.5 meter long model SFC column is illustrated. The possibilities and limitations of using long packed columns in SFC are discussed. It is demonstrated that long columns with large plate numbers do not necessarily yield better separations.     

Packed-column hydrodynamic chromatography using 1-μm non-porous silica particles

150×3 mm I.D. columns, packed with 1-μm non-porous spherical silica particles, were used to separate soluble synthetic polymers by hydrodynamic chromatography. The columns exhibited a plate height of about 1.4 μm allowing very fast and efficient separations of polymers in the molecular mass range 10³−2·10⁶ g/mol. The migration behaviour of polymers could be well described by a simple theoretical model. The applicability of packed bed HDC for the fast separation of polymers was illustrated with separations of polystyrene and poly(methyl methacrylate) mixtures.     

Monolithic silica rod columns for high-efficiency reversed-phase liquid chromatography

Chromatographic properties of a new type of monolithic silica rod columns were examined. Silica rod columns employed for the study were prepared from tetramethoxysilane, modified with octadecylsilyl moieties, and encased in a stainless-steel protective column with two polymer layers between the silica and the stainless-steel tubing. A 25 cm column provided up to 45,000 theoretical plates for aromatic hydrocarbons, or a minimum plate height of about 5.5 μm, at optimum linear velocity of ca. 2.3 mm/s and back pressure of 7.5 MPa in an acetonitrile-water (80/20, v/v) mobile phase at 40°C. The permeability of the column was similar to that of a column packed with 5 μm particles, with K(F) about 2.4×10(-14) m(2) (based on the superficial linear velocity of the mobile phase), while the plate height value equivalent to that of a column packed with 2.5 μm particles. Generation of 80,000-120,000 theoretical plates was feasible with back pressure below 30 MPa by employing two or three 25 cm columns connected in series. The use of the long columns enabled facile generation of large numbers of theoretical plates in comparison with conventional monolithic silica columns or particulate columns. Kinetic plot analysis indicates that the monolithic columns operated at 30 MPa can provide faster separations than a column packed with totally porous 3-μm particles operated at 40 MPa in a range where the number of theoretical plates (N) is greater than 50,000.     

Designing Fixed Bed Column and Batch Studies of Chitosan Nanoparticles for Defluoridation of Drinking Water

The aim of present investigation was to prepare nanoparticles of chitosan and perform batch and column studies with them to study their defluoridation capacity. The nanoparticles of chitosan were characterized by techniques like FTIR, SEM, TEM, etc. Effect of initial fluoride concentration, adsorbent dose, pH and temperature were studied in the batch studies. Effect of bed height, flow rate, and inlet concentration on the column performance were studied. Performance of packed columns were described through the concept of breakthrough curves and column parameters were predicted as a function of bed heights. The breakthrough curves were defined by the Adams-Bohart and Wolborska models.     

Study of the influence of the aspect ratio on efficiency, flow resistance and retention factors of packed capillary columns in pressure- and electrically-driven liquid chromatography

The influence of the aspect ratio, rho (rho = column diameter/particle diameter), on column parameters such as efficiency, retention factors and flow resistance was studied in both high-performance liquid chromatography and capillary electrochromatography with packed capillary columns. In order to compare the true efficiencies of different columns, a procedure to account for external band broadening was applied. High efficiencies (reduced plate height h approximately 2) were obtained with capillary columns with internal diameters of 150-, 100-, and 75-microm, packed with 10-microm particles. In contrast to previous reports in the literature, no significant improvements in efficiency or flow resistance were observed when the aspect ratio of such columns was decreased. Our observations suggest that the wall effect in these types of columns is not significant. When the aspect ratio was decreased by increasing the particle size, a decrease in reduced plate height was observed. However, the results of flow resistance measurements showed that the latter effect should be attributed to differences in packing and particle batch quality rather than to differences in the aspect ratio.     

New dynamic axial compression columns with means for monitoring floating adapter position. Column design and utilisation in low pressure LC on soft packings

Summary The design of new dynamic, axial-compression columns with a system for continous packed bed adjustment and monitoring of the floating adapter position is described. The columns are meant for liquid chromatography at low pressures (up to 8 bar) in aqueous and organic media with stationary phases of all types. The columns have adapter position pickups for continuous automatic monitoring of the bed height (original “swellographic” monitoring). The column described with gas pressurisation was tested with soft Sephadex G-10 and G-25. In spite of the reduction in external porosity there was no dramatic increase in back-pressure. The column proved to provide long-term stability of the packed bed and improvement in resolution. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

Compressibility effects in packed and open tubular gas and supercritical fluid chromatography

The influence of the pressure drop on the efficiency and speed of analysis in packed and open tubular supercritical fluid chromatography (SFC) is described: methods previously developed to describe the effects of mobile phase compressibility on the performance of open tubular columns in SFC have been extended to packed columns. The Horvath and Lin equation has been used to elucidate the influence of variations in velocity, diffusivity, and capacity factor along the column on the overall efficiency of packed column SFC. In packed columns, in contrast with the situation in open tubular columns, because the increase in velocity is no longer compensated by an increase in diffusion coefficients, the increase in both linear velocity and capacity factor which result from a significant pressure drop cause the plate height to increase along the column. The effect of fluid decompression along the length of the column on the speed of analysis in SFC has been studied and numerical expressions derived which enable calculation of compressibility correction factors for the plate height. Both the f₁ and f₂ correction factors remain very close to unity for acceptable pressure drops, which means that the pressure drop has virtually no effect on the number of plates generated per unit time for an unretained component. For retained species, the decompression of the mobile phase across the column causes the capacity factor to increase and hence leads to increased analysis times.     

Silica particles encapsulated poly(styrene-divinylbenzene) monolithic stationary phases for micro-high performance liquid chromatography

In the paper we demonstrate a new approach for the preparation and application of continuous silica bed columns that involve encapsulation (entrapment) of functionalized silica microparticles, which can be used as packing material in micro high performance liquid chromatography (micro-HPLC) and capillary electrochromatography (CEC). Like traditional packed columns, these capillaries possess characterized silica particles that offer high phase ratio and narrow pore size distribution leading to high retention and separation efficiency, respectively. More importantly, immobilization of the microparticles stabilizes the separation bed and eliminates the need for retaining frits. The developed capillary columns were fabricated in exactly the same way as a packed capillary column (slurry packing) but with an additional entrapment step. This immobilization of the packed bed was achieved by in situ polymerization of styrene and divinylbenzene in presence of decanol as a porogen and azobisisobutyronitrile as thermal initiator. Silica particles with different particle sizes and pore sizes ranging from 60 to 4000 A were studied. In addition different modified silica was used, including C-18 reversed phase, anion exchange and chiral stationary phases. Efficient separation of polyphenolic compounds, peptides, proteins and even DNA mutation were achieved using the developed technique depending on the properties of the silica particles used (particles pore size). For example, using 3 microm ProntoSIL C-18 particles with 300 A pore size, separation efficiencies in the range of 120,000-200,000 plates/m were obtained for protein separation, in a 6 cm x 200 microm i.d. capillary column. Using encapsulated silica C-18 with 1000 A pore size, separation of DNA homo and hetero duplexes were achieved under denaturing HPLC conditions for mutation detection. In addition, nucleotides were separated using anion exchange material encapsulated with poly(styrene-divinylbenzene) (PS/DVB), which indicated that the chromatographic properties of the silica packing material were still active after polymerization. The prepared capillary columns were found to be stable and could easily be operated continuously up to a pressure of 350 bar without column damage and capillary can be cut to any desired length.     

Are axial and radial flow chromatography different?

Radial flow chromatography can be a solution for scaling up a packed bed chromatographic process to larger processing volumes. In this study we compared axial and radial flow affinity chromatography both experimentally and theoretically. We used an axial flow column and a miniaturized radial flow column with a ratio of 1.8 between outer and inner surface area, both with a bed height of 5 cm. The columns were packed with affinity resin to adsorb BSA. The average velocity in the columns was set equal. No difference in performance between the two columns could be observed. To gain more insight into the design of a radial flow column, the velocity profile and resin distribution in the radial flow column were calculated. Using mathematical models we found that the breakthrough performance of radial flow chromatography is very similar to axial flow when the ratio between outer and inner radius of the radial flow column is around 2. When this ratio is increased, differences become more apparent, but remain small. However, the ratio does have a significant influence on the velocity profile inside the resin bed, which directly influences the pressure drop and potentially resin compression, especially at higher values for this ratio. The choice between axial and radial flow will be based on cost price, footprint and packing characteristics. For small-scale processes, axial flow chromatography is probably the best choice, for resin volumes of at least several tens of litres, radial flow chromatography may be preferable.     

Monolithic Silica Columns for HPLC,Micro‐HPLC,and CEC

Two types of monolithic silica columns derivatized to form an ODS phase, one prepared in a fused silica capillary (SR‐FS) and the other prepared in a mold and clad with an engineering plastic (poly‐ether‐ether‐ketone) (SR‐PEEK), were evaluated. The column efficiency and pressure drop were compared with those of a column packed with 5‐μm ODS‐silica particles and of an ODS‐silica monolith prepared in a mold and wrapped with PTFE tubing (SR‐PTFE). SR‐FS gave a lower pressure drop than a column packed with 5‐μm particles by a factor of 20, and a plate height of 20 μm at a linear velocity below 1 mm/s. SR‐PEEK showed higher flow‐resistance than the other monolithic silica columns, but they still showed a minimum plate height of 8–10 μm and a lower pressure drop than popular commercial columns packed with 5‐μm particles. The evaluation of SR‐FS columns in a CEC mode showed much higher efficiency than in a pressure‐driven mode.     

Towards the column bed stabilization of columns in capillary electroendosmotic chromatography. Immobilization of microparticulate silica columns to a continuous bed

This article discusses a novel method generating a continuous bed inside the CEC column. The column bed composed of microparticulate reversed-phase silica is completely immobilized by a hydrothermal treatment using water for the immobilization process. This process eliminates the manufacture of frits of both ends of the column and all problems associated with their preparation. Fundamental studies on operational parameters will be presented such as the dependence of the immobilization on the column temperature, the type of stationary phase and the column back pressure. The immobilized CEC columns show the same high column efficiency as packed columns with frits.     

Chromatographic evaluation of perfusion on charcoal in uraemia

Chromatography on Bio-Gel P-2 and high-performance liquid chromatography on RP-18 columns monitored with UV, fluorescence and electrochemical detectors have been used to evaluate the efficiency of granular, uncoated, active charcoal to remove from the ultrafiltrates of uraemic patients those organic substances accumulated in the blood that are not easily removed by dialysis. Chromatography on Bio-Gel P-2 and high-performance liquid chromatography on RP-18 columns carried out isocratically and monitored with an electrochemical detector seem very useful for clinical investigation as they increase the information obtained from routine haematochemical analyses such as blood urea nitrogen, creatinine, uric acid and electrolytes (calcium, phosphorus, sodium and potassium).     

Evaluation of the secondary consolidation of columns for liquid chromatography by ultrasonic irradiation

The consolidation of packed analytical chromatography columns was carried out under ultrasonic irradiation. Columns were first packed using a conventional high pressure downward slurry method. Then, they were subjected to further bed consolidation in the presence of ultrasonic vibration. This process of further bed consolidation is referred to as secondary consolidation. Secondary consolidation was observed to occur more readily in solvents of low viscosity and at low flow-rates (low pressures). Column efficiency was not observed to be a factor affecting the process of secondary consolidation of the packed bed.     

Lab on a chip packing of submicron particles for high performance EOF pumping

The packing of submicrometer sized silica beads inside a microchannel was enabled by a novel method which avoids the complication and limitations of generating a frit using conventional approaches and the restriction of flow using a submicrometer sized weir. A micrometer sized weir and two short columns of 5 μm and 800 nm silica beads packed in succession behind the weir together functioned as a high pressure frit to allow the construction of a primary packed bed of 390 nm silica beads. This packed bed microchannel was tested as an EOF pump, wherein it exhibited superior performance with regards to pressure tolerance, i.e., sustaining good flow rate under extremely high back pressure, and maximal pressure generation. Under a modest applied electric field strength of 150 V/cm, the flow rate against a back pressure of 1200 psi (～8.3 MPa) was 40 nL/min, and the maximal pressure reached 1470 psi (～10 MPa). This work has demonstrated that it is possible to create a high performance packed bed microchannel EOF pump using nanometer sized silica beads, as long as proper care is taken during the packing process to minimize the undesirable mixing of two different sized particles at the boundaries between particle segments and to maximize the packing density throughout the entire packed bed.     

Addition of modifier in supercritical fluid chromatography using a microbore reciprocating pump

Summary Solubilities of polar analytes in supercritical CO₂ can be enhanced by the addition of polar modifiers. The addition of modifier into an SFC system using a low cost reciprocating pump has been studied. Two different mixing chambers were evaluated for mixing the supercritical CO₂ with modifier. It appeared that a mixing chamber with a packed bed was enough to reduce baseline noise from the modifier pump. Results from the effect of pressure and temperature with various modifier flow rates were obtained. High percentages of modifier (>15%) at a low CO₂ pressure (2000 psi) caused baseline instability. In addition, different I.D. columns were tested with the system and the effect of modifier compressiblity on detector noise was also studied. Several pharmaceutical compounds were separated to demonstrate system performance.     

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.