- Email: [email protected]
Miniaturisation in LC versus CE
trends in analytical chemistry,
~01.14, nod,
1995
Golay award 19% During the 17th International Symposium on Capillary Chromatography and Electrophoresis in Wintergreen, VA, USA, the Golay Medal was presented to Professor Pat Sandra by Dr. J. Hinshaw, representing the sponsoring Perkin Elmer organization. Sandra was elected by the Golay Award Committee for his accomplishments in micro separations. More than 200 of his publications have had a large impact with subjects
Miniaturisation
such as immobilized stationary phases for GC, high temperature GC, micro LC, SFC/SFE, and coupled techniques. More recently, he has focused on methods for automated sample preparation/introduction and electrophoretic techniques. Less well known within analytical circles is his work in organic synthesis and the important areas of environmental analysis, natural product research and biomedical applications. Altogether he has published over 100 very well cited papers in a large number of prominent journals covering these fields. In this way he has pro-
moted the importance of micro-separations far beyond the field of analytical chemistry.
New VWVVVsite AOAC INTERNATIONAL has announced its World Wide Web HomePage which can be accessed at http:llwww.aoac.orgi Currently, the site provides information about the organization, including: a calendar of upcoming activities: membership information with an online membership application; descriptions of and ordering information for AOAC publications; details and registration information for the upcoming Annual Meeting and Exposition; and AOAC Education Programs and Sections. Soon to be added is information on AOAC’s methods validation programs. Also included are links to other sites of interest to analytical scientists.
in LC versus CE
A report on the Conference on Miniaturisation in Liquid Chromatography versus Capillary Electrophoresis held in Ghent, Belgium, 23 May 1995. This symposium was organised following the first meeting on miniaturised systems as an initiative of Dr. Smits (BASF) in 1993. Despite the fact that the lectures were mainly in Dutch a large audience proved that there is growing awareness of the importance and advantages of scalingdown existing analytical techniques. Fifteen universities and colleges from Belgium and the Netherlands and more than 30 companies in the pharmaceutical, biochemical, and clinical research areas were represented by over 140 participants. Many
posters covered the potential of capillary electrophoretic techniques in pharmaceutical, environmental and clinical research and demonstrated the profits of scaling down in LC. Extended abstracts of the contributions will be published in a special issue of Biomedical Chromatography. A technical exhibition and forum illustrated industrial investigations into optimisation of narrowand microboreHPLC columns and their applications using adapted instrumental systems. The advantages and limitations of miniaturising chromatoexisting graphic applications were clearly represented by H. Claessens (Technical University, Eindhoven). He stressed that when the internal diameter is reduced, especially below 2.0 mm, the demands put on all the chromatographic components become greater.
The packing techniques, injection systems, detector, and all connections need careful optimisation to minimise extra-column variance. He examined the influence of the contributors to extra-column variance on resolution and peak shape, and evaluated their connection with the capacity factors of the eluting analytes. P. Sandra (University of Ghent) emphasised that the future lies with the electrodriven systems of micropacked LC, to combine the efficiency and speed of cGC (capillary GC) with the inertness of LC packings. The feasibilities of micropacked LC were illustrated with separations of cis- and trans-isomers of polyunsaturated fatty acids which give better results than cGC. Only small amounts of expensive polymeric chiral stationary phases (e.g., cellulose derivatives) are needed to pack columns with very high plate numbers applied in the SFC (supercritical fluid
VI
chromatography) mode, resulting in higher resolutions in analysis times which are very short compared to LC. The gain in efficiency with electrodriven systems with open tubular columns of 50 to 100 m I.D. was proved with a mixture of amino acids in a MEKC (microemulsion electrokinetic chromatography) mode using dodecylcarbonylvaline (DCA). Very promising results were presented from the area of phospholipid discrimination. The prospects of NACE (non-aqueous capillary electrophoresis) were assessed: it gives uncommonly high selectivities (e.g., using vancomycine). E Van Damme (Dow Deutschland, Rheinmtinster) highlighted the potential of micro-LC of macromolecules in the critical mode. By careful tuning of the eluent composition and temperature, the retention of a polymer was determined only by the number and polarity of its endgroups. This permitted separation and characterisation of macromolecules, as illustrated for mixtures of oligo(caprolactone)-diols and -trials and polybutadiene diols. Further identification of eluent fractions was achieved by direct hyphenation of the microcolumn outlet to a GPC (gel permeation chromatography) system. Miniaturisation, with the reduction of solvent consumption, may involve health benefits for researchers, as exemplified by L. de Thibault de Boesinghe (University of Ghent) who stressed the precautions and safety requirements that should be taken in handling commonly used chemicals. Y. Michotte (Free University of Brussels) made clear that miniaturisation in clinical research is also related to down scaling of the sampling procedures. The use of a microdialysis probe allows simultaneous sampling of free fractions of endogenous compounds, drugs and metabolites in viva in various tissues. Very sensitive detection techniques (e.g., electrochemical or capillary UV detection) are needed to monitor the low concentrations of dialysates following direct analysis by micro-LC or CZE (capillary zone electrophoresis). For studies of the biometaholism of, e.g., anti-epileptic drugs in rats, a microbore column of 1 mm I.D. was preferred, and gave accurate results with good mass sensitivity. Calibration of the dialysis yield was
trends in analytical chemistry, vol. 14, no. 8, 1995
improved by monitoring the loss of an internal standard through the dialysis membrane. M. De Smet (Janssen Research Foundation, Beerse) commented on the use of micro-LC and CZE as routinely applied in the different stages of the industrial development Down-scaled of drugs. chromatography is used in the backup validation of production lines, in quality control of raw materials, in the synthesis of new drugs, and monitoring of decomposition products. This lecturer sought for the maximum enhancement of the efficiency of chromatographic methods for discriminating between isomeric analogues in synthesis pathways and for quantifying impurities up to a limit of 0.05%. The group of C. Gooijer (Free University, Amsterdam) exploited the inherent advantages (photostability, cheapness and durability) of a diode laser emitting at 670 nm. Tailor-made labelling agents such as dicarbocyanine squaraine and moieties with appropriate functional groups were used to prepare various groups of chemicals for the absorption of the red laser light. Laser induced fluorescence (LIF) allows analytes containing thiol, aldehyde, acid or amine groups derivatised with a suitable label, to be quantified down to the pmol range after micro-LC or CZE separation. The derivatisation procedures must be performed in at least the nmol concentration range because of side reactions. A mixture of 18 amino acids, derivatised with a
Affinity chromatography recognition
A report on the 11th International
ognition
held at San Antonio, TX,
Continuing a biannual tradition over 20 years old, more than 200 people from around the globe attended the 11th International Symposium on Affinity Chromatography and Biomolecular Recognition at Trinity University in sunny San Antonio, Texas, 25-31 May. The International
dicarbocyanine label, could be separated by MEKC. An alternative detection technique was presented by L. Nagels (University of Antwerp) who investigated the possibilities of a miniaturised potentiometer in CZE. A wall-jet type detector was constructed, with electrodes that were xly,z positioned at the capillary outlet using a microscope. Macro-electrodes were coated with a PVC membrane containing specific carrier molecules for ion exchange with the eluting anionic analytes. A macrocyclic pentamine was preferred to a quaternary ammonium carrier molecule for the quantification of a homologous series of aliphatic acids (ClClO). Detection limits down to 200 fmol were reached. In his concluding remarks, J. Chervet (LC Packings, Amsterdam) pointed out that, despite the great efforts made by supplying industries, there is still a need for adequate hardware. The symposium deserves a sequel to promote new achievements and technologies in the miniaturisation field to be spread into the domains of education and research. A. VAN OVERBEKE and W.R.G. BAEYENS A. Van Overbeke and WRG. Baeyensure af the Laboratory of Drug Analysis, Dept. of PharmaceuficaiAnalyis, University of Ghenf, Harelbekestraat 72, B-9000 Ghent, Belgium.
and biomolecular
Society for Molecular Recognition, 2 academic institutions, 20 industrial participants and 9 industrial exhibitors helped sponsor this symposium highlighting new discoveries in applications and fundamental properties of biological recognition phenomena. The program was organized along traditional oral and poster sessions with special sessions dedicated to young investigators (35 years old and younger). From its outset the meeting was dedicated to the memory and work of Professor Chris Anfinsen (Johns Hopkins University), the
~01.14, nod,
1995
Golay award 19% During the 17th International Symposium on Capillary Chromatography and Electrophoresis in Wintergreen, VA, USA, the Golay Medal was presented to Professor Pat Sandra by Dr. J. Hinshaw, representing the sponsoring Perkin Elmer organization. Sandra was elected by the Golay Award Committee for his accomplishments in micro separations. More than 200 of his publications have had a large impact with subjects
Miniaturisation
such as immobilized stationary phases for GC, high temperature GC, micro LC, SFC/SFE, and coupled techniques. More recently, he has focused on methods for automated sample preparation/introduction and electrophoretic techniques. Less well known within analytical circles is his work in organic synthesis and the important areas of environmental analysis, natural product research and biomedical applications. Altogether he has published over 100 very well cited papers in a large number of prominent journals covering these fields. In this way he has pro-
moted the importance of micro-separations far beyond the field of analytical chemistry.
New VWVVVsite AOAC INTERNATIONAL has announced its World Wide Web HomePage which can be accessed at http:llwww.aoac.orgi Currently, the site provides information about the organization, including: a calendar of upcoming activities: membership information with an online membership application; descriptions of and ordering information for AOAC publications; details and registration information for the upcoming Annual Meeting and Exposition; and AOAC Education Programs and Sections. Soon to be added is information on AOAC’s methods validation programs. Also included are links to other sites of interest to analytical scientists.
in LC versus CE
A report on the Conference on Miniaturisation in Liquid Chromatography versus Capillary Electrophoresis held in Ghent, Belgium, 23 May 1995. This symposium was organised following the first meeting on miniaturised systems as an initiative of Dr. Smits (BASF) in 1993. Despite the fact that the lectures were mainly in Dutch a large audience proved that there is growing awareness of the importance and advantages of scalingdown existing analytical techniques. Fifteen universities and colleges from Belgium and the Netherlands and more than 30 companies in the pharmaceutical, biochemical, and clinical research areas were represented by over 140 participants. Many
posters covered the potential of capillary electrophoretic techniques in pharmaceutical, environmental and clinical research and demonstrated the profits of scaling down in LC. Extended abstracts of the contributions will be published in a special issue of Biomedical Chromatography. A technical exhibition and forum illustrated industrial investigations into optimisation of narrowand microboreHPLC columns and their applications using adapted instrumental systems. The advantages and limitations of miniaturising chromatoexisting graphic applications were clearly represented by H. Claessens (Technical University, Eindhoven). He stressed that when the internal diameter is reduced, especially below 2.0 mm, the demands put on all the chromatographic components become greater.
The packing techniques, injection systems, detector, and all connections need careful optimisation to minimise extra-column variance. He examined the influence of the contributors to extra-column variance on resolution and peak shape, and evaluated their connection with the capacity factors of the eluting analytes. P. Sandra (University of Ghent) emphasised that the future lies with the electrodriven systems of micropacked LC, to combine the efficiency and speed of cGC (capillary GC) with the inertness of LC packings. The feasibilities of micropacked LC were illustrated with separations of cis- and trans-isomers of polyunsaturated fatty acids which give better results than cGC. Only small amounts of expensive polymeric chiral stationary phases (e.g., cellulose derivatives) are needed to pack columns with very high plate numbers applied in the SFC (supercritical fluid
VI
chromatography) mode, resulting in higher resolutions in analysis times which are very short compared to LC. The gain in efficiency with electrodriven systems with open tubular columns of 50 to 100 m I.D. was proved with a mixture of amino acids in a MEKC (microemulsion electrokinetic chromatography) mode using dodecylcarbonylvaline (DCA). Very promising results were presented from the area of phospholipid discrimination. The prospects of NACE (non-aqueous capillary electrophoresis) were assessed: it gives uncommonly high selectivities (e.g., using vancomycine). E Van Damme (Dow Deutschland, Rheinmtinster) highlighted the potential of micro-LC of macromolecules in the critical mode. By careful tuning of the eluent composition and temperature, the retention of a polymer was determined only by the number and polarity of its endgroups. This permitted separation and characterisation of macromolecules, as illustrated for mixtures of oligo(caprolactone)-diols and -trials and polybutadiene diols. Further identification of eluent fractions was achieved by direct hyphenation of the microcolumn outlet to a GPC (gel permeation chromatography) system. Miniaturisation, with the reduction of solvent consumption, may involve health benefits for researchers, as exemplified by L. de Thibault de Boesinghe (University of Ghent) who stressed the precautions and safety requirements that should be taken in handling commonly used chemicals. Y. Michotte (Free University of Brussels) made clear that miniaturisation in clinical research is also related to down scaling of the sampling procedures. The use of a microdialysis probe allows simultaneous sampling of free fractions of endogenous compounds, drugs and metabolites in viva in various tissues. Very sensitive detection techniques (e.g., electrochemical or capillary UV detection) are needed to monitor the low concentrations of dialysates following direct analysis by micro-LC or CZE (capillary zone electrophoresis). For studies of the biometaholism of, e.g., anti-epileptic drugs in rats, a microbore column of 1 mm I.D. was preferred, and gave accurate results with good mass sensitivity. Calibration of the dialysis yield was
trends in analytical chemistry, vol. 14, no. 8, 1995
improved by monitoring the loss of an internal standard through the dialysis membrane. M. De Smet (Janssen Research Foundation, Beerse) commented on the use of micro-LC and CZE as routinely applied in the different stages of the industrial development Down-scaled of drugs. chromatography is used in the backup validation of production lines, in quality control of raw materials, in the synthesis of new drugs, and monitoring of decomposition products. This lecturer sought for the maximum enhancement of the efficiency of chromatographic methods for discriminating between isomeric analogues in synthesis pathways and for quantifying impurities up to a limit of 0.05%. The group of C. Gooijer (Free University, Amsterdam) exploited the inherent advantages (photostability, cheapness and durability) of a diode laser emitting at 670 nm. Tailor-made labelling agents such as dicarbocyanine squaraine and moieties with appropriate functional groups were used to prepare various groups of chemicals for the absorption of the red laser light. Laser induced fluorescence (LIF) allows analytes containing thiol, aldehyde, acid or amine groups derivatised with a suitable label, to be quantified down to the pmol range after micro-LC or CZE separation. The derivatisation procedures must be performed in at least the nmol concentration range because of side reactions. A mixture of 18 amino acids, derivatised with a
Affinity chromatography recognition
A report on the 11th International
ognition
held at San Antonio, TX,
Continuing a biannual tradition over 20 years old, more than 200 people from around the globe attended the 11th International Symposium on Affinity Chromatography and Biomolecular Recognition at Trinity University in sunny San Antonio, Texas, 25-31 May. The International
dicarbocyanine label, could be separated by MEKC. An alternative detection technique was presented by L. Nagels (University of Antwerp) who investigated the possibilities of a miniaturised potentiometer in CZE. A wall-jet type detector was constructed, with electrodes that were xly,z positioned at the capillary outlet using a microscope. Macro-electrodes were coated with a PVC membrane containing specific carrier molecules for ion exchange with the eluting anionic analytes. A macrocyclic pentamine was preferred to a quaternary ammonium carrier molecule for the quantification of a homologous series of aliphatic acids (ClClO). Detection limits down to 200 fmol were reached. In his concluding remarks, J. Chervet (LC Packings, Amsterdam) pointed out that, despite the great efforts made by supplying industries, there is still a need for adequate hardware. The symposium deserves a sequel to promote new achievements and technologies in the miniaturisation field to be spread into the domains of education and research. A. VAN OVERBEKE and W.R.G. BAEYENS A. Van Overbeke and WRG. Baeyensure af the Laboratory of Drug Analysis, Dept. of PharmaceuficaiAnalyis, University of Ghenf, Harelbekestraat 72, B-9000 Ghent, Belgium.
and biomolecular
Society for Molecular Recognition, 2 academic institutions, 20 industrial participants and 9 industrial exhibitors helped sponsor this symposium highlighting new discoveries in applications and fundamental properties of biological recognition phenomena. The program was organized along traditional oral and poster sessions with special sessions dedicated to young investigators (35 years old and younger). From its outset the meeting was dedicated to the memory and work of Professor Chris Anfinsen (Johns Hopkins University), the