Thomas Welsch

Coupling of a microbore column with a column packed with non-porous particles for fast comprehensive two-dimensional high-performance liquid chromatography

To reduce the analysis time in comprehensive two-dimensional HPLC columns packed with 1.5 μm non-porous particles were applied in the second dimension. Preservation of the efficiency under coupling conditions was realized by adapting the peak volumes in the first dimension. To achieve this an instrumental set-up was designed and used which combines a micro-column in the first dimension and a column packed with non-porous particles in the second dimension. A theoretical approach to estimate the optimum column diameter ratio was verified experimentally. As an example a mixture of phenols was separated using a tetrachlorophthalimidopropyl silica in the first dimension and a 1.5 μm RP-18 packing in the second dimension. The total time for the two-dimensional analysis of this mixture with an analysis time of 16 min in the first dimension was 70 min analysing 27 fractions on the second dimension column.

Two-dimensional high performance liquid chromatography for the separation of complex mixtures of explosives and their by-products

SummaryThe insufficient ability of one dimensional HPLC to separate complex mixtures such as environmental samples can be overcome by using two dimensional systems combining columns with alternative/orthogonal selectivities. Such a system for the separation of complex mixtures of explosives, their by-products and degradation products from environmental samples was developed and evaluated. It makes use of the different retention characteristics of an alkyl modified silica and a safrol modified silica in the reversed phase mode. The high peak capacity of two dimensional systems predicted by theory was realized employing a flexible switching technique and utilizing differences in the elution strength of the mobile phases. Thus, peak compression on the head of the second column was exploited. The efficiency of the two dimensional system was demonstrated for the separation of a complex mixture of nitroaromatic reference compounds. Furthermore, the system was applied to separate and identify nitro and nitroamino organic compounds in a groundwater sample from a former ammunition plant.

Improvement of the long-term stability of polyimide-coated fused-silica capillaries used in capillary electrophoresis and capillary electrochromatography.

Swelling of the polyimide coating of fused-silica capillaries in acetonitrile-containing buffers was found to be the reason for several problems in capillary electrophoresis (CE) and capillary electrochromatography (CEC). Scanning electron microscopy photographs of the ends of raw fused-silica tubing showed that the coating becomes soft and increases its volume after longer contact with such buffers. As a consequence, separation efficiency can deteriorate, the capillary ends can clog or break off. To prevent swelling of the polyimide coating, fused-silica capillaries used in CE or CEC were heated at 300 degrees C for a longer period of time which improved their long-term stability in comparison to raw fused-silica tubing.

Preparation of On-Column Frits in Packed Fused Silica Capillaries by Sol-Gel Technology

Existing methods for preparing frits in packed fused silica capillaries as used for electrochromatography and micro HPLC are not applicable to all silica based packing materials and involve a high thermal stress for both the stationary phase and the fused silica tubing including the polyimide coating. A new procedure for the production of such on-column frits under mild conditions by a sol-gel type reaction of polydimethoxysiloxane (PDMOS) is described in this paper. Reaction conditions were established for optimum mechanical stability and high permeability of the frits. Frits produced in this manner showed no noticeable effect on the overall efficiency.

Silanol effects in reversed-phase liquid chromatography

Abstract A large number of butyl-, hexyl-, octyl- and octadecylsilica stationary phases were synthesized by the usual low-temperature and also novel high-temperature silylation methods. The phases were characterized by measuring their silanol concentrations, organic functional group concentrations and wettabilities and, additionally, by reversed-phase liquid chromatographic retention measurements. Unusual retention data, not expected on the basis of the solvophobic retention theory, were observed and are explained. Separation selectivity and peak asymmetry are interpreted by taking into account the surface concentrations of both the silanol groups and the organic functional groups. The peak shapr is better, but the separation selectivity is lower, when access to all silanol groups of a stationary phase is equally easy or equally difficult.

Temperature of the eluent: a neglected tool in high-performance liquid chromatography?

The influence of eluent temperature on efficiency in high-performance liquid chromatography with special reference to the role of viscous heat dissipation is investigated for water-jacket and air-bath thermostatted columns. When separations are performed on well-thermostatted large-diameter columns packed with small particles or on analytical columns operated at high flow-rates, the additional thermostatting of the eluent to a specific temperature below the column temperature turns out to have some advantages. The precooled eluent causes the development of a temperature profile in a radial direction which is opposed to and compensates the radial temperature profile in the column caused by viscous heat dissipation. For columns that are thermostatted in an air bath, a similar effect is observed, but the strong dependence of the column temperature on the eluent temperature results in a poor reproducibility if the eluent is not thermostatted itself.

Development of a dual capillary column GC method for the trace determination of C2-C9 hydrocarbons in ambient air

An analytical method based on a dual capillary gas chromatographic technique combining the advantages of GasPro PLOT and a non polar narrow bore WCOT column was developed for the analysis of air samples containing C2–C9 NMHCs. A refocusing step was not required due to the fast heating rate of the sample preconcentration trap and the resolving power of the PLOT column for C2 and C3 NMHCs. Water had to be removed from the air samples to avoid plugging of the columns if the initial GC oven temperature was below ambient temperature. To dry air samples, a scrubber and a cryogenic technique were employed. The interferences caused by carbon dioxide were reduced by purging the loaded sample preconcentration trap with helium. The dual column system was compared to a method employing a refocusing device and a single narrow bore WCOT column. Both systems provided a high degree of precision. However, the dual column approach was superior to the single column system due to better resolution of low molecular weight components.

Influence of viscous heat dissipation on efficiency in high-speed high-performance liquid chromatography

The effect of viscous heat dissipation on efficiency in high-speed high-performance liquid chromatography was investigated for columns packed with non-porous 1.5 μm particles. When these columns are operated at high flow-rates and therefore high-pressure drops, viscous heat is generated leading to axial and longitudinal temperature gradients within the column which are translated into secondary gradients of the mobile phase viscosity, the flow velocity, the diffusion rate, and the retention factor. This resulted in additional band broadening which can destroy the separation quality as demonstrated by chromatograms and plate height curves. Thermostatting of the eluent to a specific temperature below the column wall temperature compensated for this effect. The necessary degree of precooling decreased with increasing column temperature.

(Micellar) electrokinetic chromatography: an interesting solution for the liquid phase separation dilemma

High-performance liquid chromatography (HPLC) is a well-established method in modern analysis. The method is simple, very robust and is applicable to the majority of components to be analyzed in contrast to gas chromatography. Low efficiency and small peak capacity are sore points of HPLC when complex mixtures have to be separated. The reason for this dilemma is the small diffusion coefficient of the analytes in the liquid mobile phase compared to a gaseous phase. This review, complemented by exemplary calculated data and some latest results of our own research, illustrates the dilemma of liquid phase chromatography to achieve high efficiencies under reasonable conditions. It is shown that (micellar) electrokinetic chromatography, offering fast and efficient separations, is a very promising solution for this dilemma. Additional features of this method are possibilities of on-line analyte concentration, coupling to mass spectrometry and the easy change of selectivities by applying various separation additives. The pros and cons of electrokinetic chromatography are pointed out and some application examples are given.

Moderation of the electroosmotic flow in capillary electrophoresis by chemical modification of the capillary surface with tentacle-like oligourethanes.

The surface chemistry of the inner wall of fused-silica capillaries is one important means to control the magnitude as well as the direction of the electroosmotic flow and the adsorption activity. A method was developed to change the surface characteristics of fused-silica capillaries by binding tentacle-like oligourethane groups onto the inner surface. The electroosmotic flow at a buffer pH of 6-9 was reduced by 15 to 40% compared to that in a bare fused-silica tubing, dependent on the type of coating. Sample adsorption is diminished at the same time resulting in a separation of proteins with higher resolution and good migration time precision. At a pH below 4.5 the electroosmotic flow is reversed into the anodic direction, which offers further possibilities for the separation of positively charged analytes as demonstrated for the separation of aromatic and biogenic amines.