BODIPY dyes used for DNA sequencing High-throughput, genome-scale DNA detection assays rely on families of fluorescent dyes that demonstrate physical stability, minimal emission spectral overlap, high-fluorescence intensity, and uniform electrophoretic mobilities. Despite being physically stable, the fluorescein and rhodamine dyes currently available do not fulfill the remaining criteria. Michael L Metzker and colleagues at Baylor College of Medicine have characterized single- and double-dye primers using four spectrally resolvable dipyrrometheneboron difluoride (BODIPY) dyes that propriate for DNA sequencing For two of the dyes, a three-base separation between the donor and acceptor dyes optimized the signal enhancement, whereas the other two dyes required a sixbase separation. Unlike the conventional universal dye primers, the BODIPY universal dye primers provided raw data
from which accurate visual interpretations could be made prior to computer analysis of the data. In addition, the BODIPY dye set was sensitive enough that a 16-fold dilution of DNA template was correctly analyzed, which indicates that the direct loading of sequencing reactions without a concentration step will be possible. (Science 1996,271,1420-22)
Chemical structures of four BODIPY dyes for automated DNA sequencing. (Adapted with permission of the American Association for the Advancement of Science.)
Predicting universal retention indices for reversed-phase LC The problem with trying to construct a retention index system for reversed-phase LC is the mobile phase: Not only every stationary phase, but also every mobile phase, requires a new index base. One solution may be to eliminate the model entirely and rely on the relationships defined by the data. Because differences in retention behavior of different compounds in different mobile phases are determined by different combinations of molecular forces and interactions, it may be possible to find retention characteristics for compounds and mobile phases in one data set and relate them to other data sets. Charles E. Reese and colleagues at Duke University applied principal component analysis (which can reveal the rank of a data set and produce abstract factors intrinsic to that data set) and target transfor-
The new technique is based on sparkgap AES, in which atomic emission is produced by creating a high-voltage discharge between two electrodes (one of which is often the sample). For spark-gap AE microscopy, the sample is used as one electrode, and the probe tip serves as the counter electrode. A high-amplitude bias pulse is used to induce a visible spark, and Although scanning tunneling microscopy (STM) is commonly used by material scien- collection of the emitted light with a CCD array allows spectral analysis and identifitists to image metals and semiconductors, cation of the elements in the material the resulting images lack compositional or chemical information. The desire to simulta- directly below the tunneling tip. The use of tungsten eliminate serineously image topography and obtain elemental composition prompted M. L. Myrick ous problems resulting from tip erosion and colleagues at the University of South and continued imaging is possible even afCarolina to modify a scanning tunneling mi- ter sparking croscope to allow in situ elemental analyThe researchers found that because of sis of the surface being imaged. the extreme proximity of tip and sample, relatively low voltages can be used for spark formation, which allows sampling of very small (a few um2) regions. Further improvements in spatial resolution are necessary they say to make the new technique competitive with other surface Retention prediction for compound 1 on analysis techniques. column 1 (A), column 2 (B), and column 3 (J. Phys. Chetn. 1996(C). (Adapted with permission of Preston 100 3646-51) Optics for spectral analysis of the high-voltage spark. Publications.)
Spark-gap AE microscopy combines imaging and elemental analysis
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mation factor analysis (which can relate one data space to another by vector rotation) to retention data from three different reversed-phase columns. They found that the data from the three columns shared a common factor space and that three factors are sufficient to describe the retention data. The eigenvector matrix associated with analytes from singular value decomposition was character-
istic of the retention behavior of the compounds and independent of the mobile phase and column, and the same is true for the mobile-phase eigenvector. On the basis of these observations, they developed a reference retention index system for chromatographic solutes and solvents across different columns with a mean retention error of prediction of 4%. (J. Chromatogr. .ci. 1196,34,101-10)
Diagram of the interface: 1, ion source block; 2, ceramic block; 3, heating wires; 4, thermocouple; 5, Vespel ferrule; 6, SFC/MS probe; 7, capillary column; 8, end of transfer line. (Adapted with permission of Huthig GmbH.)
tance between the restrictor and ion source was > 10 mm were higher interface temperatures required for efficient transfer of the more polar compounds. A distance of —11 mm from the beginning of Most coupled SFC/MS systems are based the heated zone was found to result in maximum sensitivity. When noise caused on quadrupole mass analyzers, which are relatively simple and inexpensive but have by C0 2 was removed from the total ion current chromatogram, S/N was further limited resolving power and mass range. improved by a factor of 4. Under optiMagnetic sector instruments can provide higher resolving power, greatly expanded mized conditions, the estimated detection limit for M-pentadecane is —30 ppm, mass range, and better sensitivity. However, these instruments are expensive, and and spectra obtained in electron impact ionization mode were very similar to liinterfacing them with a chromatographic inlet is complicated. Manuel A. A. Mertens brary spectra. and colleagues at Eindhoven University They also used the system to separate of Technology (The Netherlands) and and identify a set of low molecular mass, Koninklijke/Shell-Laboratorium (The thermally unstable peroxides that can be Netherlands) have described an interfound in many large-scale industrial proface for coupling a magnetic sector mass cesses; they were able to distinguish more spectrometer to a supercritical fluid chro- than 50 different compounds. However, bematograph bcised on direct introduction cause of the restricted scan range, strucof the CO; mobile phase into the ion ture elucidation was sometimes troublesome. With additional information from GC/MS, the majority of peaks could be They optimized the interface using a identified. The SFC/MS system also demtest mixture of low-to-medium mass compounds dissolved in hexane. For relatively onstrated good performance for high molecular mass compounds such as polyacrylow-boiling compounds in the mixture, the restrictor temperature had virtually no lates and polymer additives. (J. High Resolut. Chromatogr. 1996,19,17-22) influence on S/N. Only when the dis-
Interfacing a magnetic sector MS to SFC
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Enantiomeric separations using CEC In capillary electrochromatography, a relatively new technique, an electricfieldis applied between both ends of a 5- to 500-um i.d. capillary column with a stationary phase that is either bonded to the capillary walls or packed as small particles. The mobile phase is driven through the capillary by electroosmosis, and solutes are separated according to how they partition between the phases and the ratio of charge to friction coefficient (electrophoretic mobility) Richard N Zare and colleagues at Stanford University and Ecole Nationale Surjerieure de Chimie de Parrs (France) studied some fundamental aspects of CEC bv separating neutral compounds in caoillaries Dacked with ootadecylsilica particles They found no loss of efficiency up to a linear electroosmotic velocity of 1.3 mm/s for retained analytes with capacity factors of 0.72.5. Neutral enantiomers of the drug chlorthalidone were successfully separated by adding the chiral agent hydroxypropyl-P-cyclodextrin (HP3CD) to the acetonitrile/ phosphate buffer mobile phase and using an ODS column and by using a chiral HPBCD stationary phase with an archiral acetonitrile/ phosphate buffer mobile phase. The separation using the HPBCD column achieved higher selectivity and resolution in less time (/ Chromatogr A 1996 727 145-56)
Influence of % acetonitrile on separation of chlorthalidone on HPfiCD-bonded silica. (Adapted with permission of Elsevier Science.)
