Visualising the onset of viscous fingering in chromatography columns

Viscous fingering is an important fluid transport phenomenon that manifests itself when two fluids having different viscosities move in the same direction. Their interface is unstable and a complex fingering pattern may arise. This phenomenon is important in chromatography because it may lead to a decrease or even a failure in separations. The onset of viscous fingering was visually observed by packing a glass column with particles having the same refractive index as the mobile phase and injecting plugs of dye solutions having viscosities different from that of the mobile phase. Severe fingering effects are observed if the viscosity difference exceeds 0.17 cP. However, for smaller viscosity differences, band distortions are observed that may affect retention data, band efficiency, and band resolution. Careful attention should be paid to matching the mobile phase viscosity and that of the injection plug when accurate chromatographic information is required.     

Unusually high efficiency of the separation of polymers by hydrodynamic chromatography on hollow capillary columns

A series of polystyrene standards was separated by means of hydrodynamic chromatography on a open capillary with a length of 5 m (active zone is 4.5 m) and an inner diameter of 2 μm. Unusually high efficiency in the separation of polymers with masses of up to 500 kDa was noticed: 1.5 × 10⁶ theoretical plates at a retention time of ～70 min for a sample of polystyrene with a mass of 390 kDa. It was established that the column efficiency decreases rapidly for polymers with a mass above 500 kDa. A calibration curve for polystyrene standards was constructed. It was shown that, in contrast to the models described in the literature, it is described by a simple two-term expression without quadratic terms. It is concluded that hydrodynamic chromatography is a technique complementary to exclusion chromatography, especially in the separation of high-molecular polymers.     

Temperature gradients in HPLC columns due to viscous heat dissipation

Summary Temperature effects in HPLC columns due to viscous heat dissipation are examined. For the case when the thermostatted column wall and mobile phase at the column inlet are at the same temperature an explicit solution of the heat transport equation is given. The predicted temperature profile is parabolic at large distances from the column entrance; the magnitude of the effect is proportional to the square of the mobile phase velocity, and is of the order of a few degrees centigrade. At the upper end of the column a relaxation occurs over a length of a few centimers. Experimental results confirm the validity of the predictions made and indicate that the various assumptions and approximations are justified. Plate height curves obtained with two mobile phases with differing viscosities show a much smaller efficiency for the less viscous mobile phase. The curves show an upward curvature at high reduced velocities. Both phenomena can be related to thermal effects. It is concluded that viscous heat dissipation constitutes an obstacle to obtaining higher speed and efficiency in HPLC by the use of smaller particles. Possible remedies, such as the use of smaller bore columns or special thermostatting devices, look troublesome from the experimental point of view.     

Visualising viscous fingering in chromatography columns: high viscosity solute plug

The interface between two fluids that have different viscosities and are percolating through a porous bed is unstable. Sooner or later, a flow instability termed viscous fingering (VF) develops. This phenomenon is important in chromatography because the solute plug does not have the same viscosity as the mobile phase. Because the sample is often much more viscous than the mobile phase, it is the interface at the rear of the sample band that is usually unstable. This situation is frequent in many modes of chromatography, e.g., in preparative and in multidimensional chromatography, in size exclusion chromatography, in frontal analysis, and in other physicochemical measurements (e.g., determination of adsorption isotherms and of mass transfer parameters). When the solute plug is more viscous than the mobile phase, we observed that the solute band compressed. When the viscosity contrast increased up to 0.30 cP, fingers appeared to trail behind the solute plug. The development of fingers then became more substantial as the viscosity contrast increased. To avoid effects associated with VF, the mobile phase and the solute plug should have nearly the same viscosity.     

A simple terminator for high efficiency liquid chromatography columns

A comparison is made between a flat bottom column terminator and a cone terminator on. 1.27 cm O.D. and 2.54 cm O.D. columns. The cone terminator results in a doubling of the column efficiency, is simple and inexpensive, and is universally adaptable to any diameter column.     

Theoretical calculation of the retention enthalpy effect on the viscous heat dissipation band broadening in high performance liquid chromatography columns with a fixed wall temperature

The problem of the additional band broadening arising from the trans-column temperature profile under conditions of significant viscous heat dissipation has been extended by including the retention enthalpy effect. This effect can significantly aggravate the trans-column migration velocity profile of retained species as compared to the already known zero-retention enthalpy case. An approximate analytical solution for the accompanying additional band broadening could be established. The solution is valid in nearly all practically relevant cases and shows how the additional band broadening varies as a function of the generated heat, the retention coefficient and the retention enthalpy. Surveying all possible HPLC operating conditions, it was found that the inclusion of the retention enthalpy effect leads to significantly larger viscous heating plate height contributions than those predicted by the zero-retention enthalpy solution (up to a factor of 10 or more).     

The influence of pressure on the separating properties of columns in gas chromatography

The Giddings model taking into account the dependence of the coefficients of the van Deemter equation on pressure was used to study changes in the efficiency of a hollow capillary column as the inlet and outlet carrier gas pressures changed. The observed dependence of height equivalent to a theoretical plate (HETP) in the coordinates of inlet and outlet pressures can be approximated by a surface having the shape of a folded sheet of paper, when minimum HETP values are situated along the bend line. Any surface section is actually a van Deemter curve in the corresponding coordinates. The dependence of the minimum HETP on inlet and outlet pressures, which determines the optimum parameters of column service, is of the greatest interest. It was shown that, over the range of pressures studied, the minimum HETP should monotonically decrease as the pressure increases. Experimental model verification showed close correspondence between the inlet and outlet pressures and the values predicted by the model. At the same time, the experimentally found improvement of the efficiency of the column was smaller than that predicted theoretically. Possible reasons for the discrepancy between theory and experiment are considered.     

Characterization of the efficiency of microbore liquid chromatography columns by van Deemter and kinetic plot analysis

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro‐liquid chromatography system. Fully porous, core–shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub‐2 μm fully porous as well as the 2.7 μm core–shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56–2.69) before correction for extra‐column contribution compared to normal‐bore columns. Moreover, the influence of extra‐column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub‐2 μm fully porous particle packed column for ultra‐fast liquid chromatography.     

Analysis of the cinchona alkaloids by liquid chromatography. Reversed-phase chromatography on octadecylsilyl columns

Summary The complete separation of the major cinchona alkaloids and their dihydro derivatives is achieved in less than 30 minutes, using a Hypersil 5m octadecylsilyl column in the reversed-phase mode. Excellent peak symmetry is obtained by incorporation of a long chain amine additive in the acidic mobile phase.     

A study of the efficiency of monolithic silica gel capillary columns for gas chromatography

The efficiency and dynamic characteristics of seven silica-gel-based monolithic capillary columns were analyzed by separating on them a mixture of five light hydrocarbons. For helium carrier gas flowing at an optimum velocity, the height equivalent to a theoretical plate was found to be 0.15–0.20 mm, values comparable to those typical of packed capillary columns. An analysis of the Van Deemter curves for the columns under study demonstrated that the main contribution to the smearing of the chromatographic zone comes from the diffusional processes in the mobile phase while the mass transfer between the mobile and stationary phases plays only a minor role. At the same time, the parameter A in the Van Deemter equation, which characterizes the degree of column packing uniformity, was found to be negative. This result contradicts the classical theory of chromatography and calls for further studies of monolithic capillary columns.     

Chromatographic efficiency of polar capillary columns applied for the analysis of fatty acid methyl esters by gas chromatography

The chromatographic efficiency that could be achieved in temperature‐programmed gas chromatography was compared for four capillary columns that are typically applied for analysis of fatty acid methyl esters (FAME). Three different carrier gases, hydrogen, helium and nitrogen, were applied. For each experiment, the carrier gas velocities and the temperature rates were varied with a full 9 × 3 design, with nine levels on the carrier gas velocity and temperature rates of 1, 2 or 3°C/min. Response surface methodology was used to create models of chromatographic efficiency as a function of temperature rate and carrier gas velocity. The chromatographic efficiency was defined as the inverse of peak widths measured in retention index units. The final results were standardized so that the efficiencies that could be achieved within a certain time frame, defined by the retention time of the last compound in the chromatogram, could be compared. The results show that there were clear differences in the efficiencies that could be achieved with the different columns and that the efficiency decreased with increasing polarity of the stationary phase. The differences can be explained by higher resistance to mass transfer in the stationary phase in the most polar columns.     

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.     

Prediction of retention times and efficiency in linear gradient programmed pressure analysis on capillary columns

A method for the prediction of the retention time and the resolution of chromatographic peaks in different experimental conditions by starting from few experimental data measured in isothermal and isobaric analyses was published previously. In this paper, the same mathematical model was implemented for calculating the retention times and the column efficiency in programmed pressure runs. Some models originated from the Golay equation and reported in the literature are compared, and a new modified equation for the calculation of the peak width at half height is proposed. The procedure for the prediction of the retention time and the peak width at half height at programmed pressure of the carrier gas and different column temperature and linear gradient by using retention data of different compounds obtained in few isobaric runs is described. The prediction of the retention time and the separation efficiency of compounds with different polarity gave good results for the programmed pressure runs with linear gradient. The effect of the variation of the initial parameters of the experimental analyses and of the mathematical model on the accuracy of the prediction has been evaluated.     

Open tubular columns: The influence of changing the liquid phase film thickness on column efficiency

Summary The factors characterizing the influence of the variation of the liquid phase film thickness on the mass transfer terms in the Golay equations describing the performance of open tubular columns are investigated and numerical values are given.Paper dedicated to Professor S. R. Lipsky on his sixtieth birthday.     

The influence of carrier gas pressure on the retention of sorbates on monolithic capillary columns in gas chromatography

The influence of carrier gas pressure on the retention factor k′ of light hydrocarbons C₁–C₄ in a monolithic capillary column based on divinylbenzene was studied. It was shown that, for monolithic columns and nonideal carrier gases, the pressure dependence of lnk′ was nonlinear over a wide pressure range and could be described by the classic Everett equation. It was concluded that the competitive adsorption model failed to describe the experimental data correctly, especially for strongly retained sorbates and/or heavy carrier gases. Original Russian Text ? A.A. Korolev, V.E. Shiryaeva, T.P. Popova, A.V. Kozin, A.A. Kurganov, 2009, published in Zhurnal Fizicheskoi Khimii, 2009, Vol. 83, No. 4, pp. 776–783.     

Enzyme activity determination by radio gas chromatography on capillary columns

Androgen 5α-reductase from the foreskin of 48 boys in prepubertal age was analyzed by evaluation of the apparent K_m-and V_max- values after tissue incubation. Reaction rates were calculated after determination of specific radioactivity of distinct metabolites, by comparison to the radioactivity of the precursor. Reaction products were separated by radio gas chromatography on capillary columns. The chromatography system was based on multicolumn equipment with column switching facilities and a variable splitter at the outlet of the separation column. A radioactivity monitor (gas proportional counter) was used for measurement of radioactivity in the column effluents. An electron capture detector was used for mass detection of steroid acyl derivatives.     

Separations of aldehydes by means of ion-exchange chromatography on bisulfite columns

Mixtures of carbonyl compounds were chromatographed on Dowex-I columns in the bisulfite form. Sodium bisulfite solutions of increasing concentrations were used as eluants. The studied compounds, including acetaldehyde, formaldehyde, 5-hydroxymethylfurfural, furfural and vanillin, were separated completely from each other. The eluate was also analysed quantitatively.