Development of a New Capillary Electrophoresis‐based Fibre Optic SensorStokes, David L.; Sepaniak, Michael J.; Vo‐Dinh, Tuan
doi: 10.1002/(SICI)1099-0801(199707)11:4<187::AID-BMC671>3.0.CO;2-0pmid: 9256994
A new fluorescence‐based fibre optic sensor is described which combines the sensitivity offered by laser‐induced fluorescence with the selectivity offered by capillary electrophoresis (CE). A single optical fibre directly probes the terminus of a 5–8 cm separation capillary. The linear geometry associated with this sensor necessitates a ‘single reservoir’ design, thus presenting major challenges to overcome in comparison to the conventional two‐reservoir configuration common to a typical laboratory setup. Some of the challenges confronted by the design features presented in this work include the reduction of gravity‐driven hydrostatic flow, the ejection of electrolytic gases evolved at the detection‐side electrode and the establishment a suitable compromise between detectability and separation performance. The success of such design features demonstrates the feasibility of a CE‐based sensor which offers several amenities particularly useful for in situ sensing. Such attributes include selectivity, diminutive size, flexibility, reusability, high sensitivity, speed, and remote control. Detailed descriptions of sensor fabrication are included, including two variations on a general design concept. In addition, the single‐fibre optical detection system is described. Separation characteristics of the new CE‐based sensor are presented, highlighted by an observed separation efficiency of up to 8000 theoretical plates (for a 5 cm capillary). The separation of a three‐component mixture of the laser dyes, Rhodamine 6G, fluorescein isothyocyanate and sodium fluorescein, is demonstrated. © 1997 John Wiley & Sons, Ltd.
Chiral Separations of Enantiomeric Pharmaceuticals by Capillary Electrophoresis Using Sulphobutyl Ether β‐Cyclodextrin as Isomer SelectorXie, Guang‐hua; Skanchy, David J.; Stobaugh, John F.
doi: 10.1002/(SICI)1099-0801(199707)11:4<193::AID-BMC672>3.0.CO;2-8pmid: 9256995
Capillary electrophoresis has developed into an extremely useful technique for the separation of optical isomers. High efficiencies and the availability of many types of isomer selectors allowing rapid and inexpensive methods development make capillary electrophoresis (CE) an attractive alternative to gas chromatography (GC) and high‐pressure liquid chromatography (HPLC) for the determination of chiral purity. In this research the separation of the enantiomers of some chiral pharmaceuticals was investigated using anionic sulphobutyl ether‐β‐cyclodextrins as isomer selectors. These chiral selectors have a large countercurrent mobility, making them inherently advantageous as selectors as compared to neutral cyclodextrins. The effects of pH, buffer composition and selector concentration on the chiral separation of these compounds was investigated. All of the compounds studied were successfully resolved by the suphobutyl ether β‐cyclodextrins (SBE‐β‐CDs) typically with run times of less than 20 min using low concentrations of the SBE selector. © 1997 John Wiley & Sons, Ltd.
Measurement of Total and Bioactive Interleukin‐2 in Tissue Samples by Immunoaffinity–Receptor Affinity ChromatographyPhillips, Terry M.
doi: 10.1002/(SICI)1099-0801(199707)11:4<200::AID-BMC674>3.0.CO;2-Hpmid: 9256996
The detection and measurement of cytokines is an important issue in the clinico‐pathological diagnosis of several clinical entities, including organ transplant rejection. Existing techniques, although sensitive, measure only total cytokine concentrations and cannot measure bioactivity. A chromatographic system combining immunoaffinity chromatography with an immobilized receptor detection cartridge has been developed for measuring total and bioactive interleukin (IL)‐2 concentrations in tissue extracts prepared from biopsy materials taken from renal transplant recipients during both rejection and drug‐induced nephrotoxic episodes. The technique employs a short high‐pressure chromatography column packed with antibody‐coated glass beads for the initial analyte separation and concentration, followed by detection of bioactive molecules through their interactions with specific, immobilized receptors. This system compares favourably with both conventional bioassays and immunoassays for measuring IL‐2 in tissue samples. © 1997 John Wiley & Sons, Ltd.
Size‐selective Derivatizations with Polymer Immobilized ReagentsSzulc, Michael; Swett, Peter; Krull, Ira S.
doi: 10.1002/(SICI)1099-0801(199707)11:4<207::AID-BMC676>3.0.CO;2-Rpmid: 9256998
A reagent immobilized on a macroporous polymer support was prepared for size selective derivatizations. These derivatization reagents showed two distinct reaction zones. Sterically bulky analytes were denied access to some of the surface of the support, so that when reagent on the outer surface was exhausted from the support, only analytes that had full access to all regions of the support could react. These reagents were applied to the derivatization of different amines in the presence of a high concentration of a bulky analyte, adamantanamine. Pore size measurements and determination of molecular dimensions also support a size‐selective derivatization mechanism. The reagents were also applied to the derivatization of proteins. Proteins with several sites available for tagging shows a reduced number of products with size selective reagents, reflecting reactions only at sites accessible to the reagent. © 1997 John Wiley & Sons, Ltd.
Analysis of Bile Acids and Bile Alcohols in Urine by Capillary Column Liquid Chromatography–Mass Spectrometry using Fast Atom Bombardment or Electrospray Ionization and Collision‐induced DissociationYang, Y.; Griffiths, W. J.; Nazer, H.; Sjövall, J.
doi: 10.1002/(SICI)1099-0801(199707)11:4<240::AID-BMC686>3.0.CO;2-6pmid: 9257002
Solid‐phase extraction and group separation by anion exchange chromatography were combined with capillary column liquid chromatography–mass spectrometry (LC/MS) to permit a thorough characterization of bile acids and intact conjugates of bile alcohols in human urine. Groups of compounds were separated according to acid strength and were analysed on a capillary column, 0.25×500 mm, packed with 5 μm particles of Chromasil C18, and connected via a fused silica capillary to the continuous‐flow fast atom bombardment (CF‐FAB) or electrospray (ES) sources of an AutoSpec‐TOFFPD hybrid mass spectrometer. Acetonitrile:water mixtures containing 30 mm ammonium acetate pH 7.2 were used as mobile phases, with 5% glycerol added for FAB ionisation. Bile acids were analysed directly or after derivatization of carboxyl groups with 4‐aminobenzenesulphonic acid. Negative‐ion spectra (m/z 1000 or 800 to 300 or 100) were recorded using the point detector or, in the case of ES ionization, the focal plane array detector (FPD). Deprotonated molecules of bile acids containing a sulphonic acid group were detected with a spectral signal to noise ratio of 5:1 when about 90 fmol were injected onto the column of the LC/CF‐FAB system. The corresponding peak in the reconstructed ion chromatogram gave a signal‐to‐noise ratio of about 25:1. The sensitivity could be increased 20–50 times by using ES ionization and the FPD. Bile acids without a sulphonic acid group gave about 70% of the signal of sulphonic acids using ES ionization. The capillary column LC/MS systems were evaluated by analyses of urine from an infant with cholestatic liver disease. More than 150 different bile acids and bile alcohol conjugates were detected, some of which were partially characterized using collision induced dissociation (CID) of the deprotonated molecules and B/E linked scans. A number of compounds were detected for the first time, e.g. di‐, tri‐, and tetrahydroxycholestanoic acids conjugated with N‐acetylhexosamine and cholestenediol, cholestenetriol and cholestanetriol doubly conjugated with sulphuric acid and glucuronic acid. The relative merits of ES and FAB ionization are discussed. © 1997 John Wiley & Sons, Ltd.