Analytical Currents Synopses of significant analytical articles from other publications
CEing amplified DNA The polymerase chain reaction (PCR) uses DNAmanipulating en zymes to amplify a desired DNA sequence and is especially helpful for analyses involving samples with a limited amount of DNA. Because the amplification is enzymatic, the effi ciency of each PCR assay can vary. Bruce R. McCord and colleagues at the Univer sity of Virginia and the FBI Academy eval uated the use of CE with laser fluores cence detection to quantitate the PCR am plification products and compared their method with hybridization blots and spectrophotometric determinations. Using a coated open-tubular capillary with a non-gel sieving buffer and a fluores cent intercalating dye, they obtained sep arations within 20 min. Unincorporated PCR primer oligonucleotides and PCR byproducts were separated from the de sired PCR-amplified DNA, and peak migra tion time for an internal standard had an RSD < 0.1%. The researchers concluded that CE with fluorescence detection can be used to monitor the quality and quan tity of the PCR product. (/. Chromatogr. Β 1994, 658, 271-80) Comparing g e l electrophoresis techniques for protein s e p a r a t i o n Traditional planar sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) requires time-consuming gel preparation and visualization techniques but allows a large number of samples to be analyzed at the same time. Capillary SDS gel electrophoresis, a one-dimensional ver sion of SDS PAGE, allows direct quantita tion by on-column UV detection, fast sin gle sample analysis, and the use of nanoliter sample volumes. Andras Guttman and Judith Nolan of Hafslund Nycomed Pharma (Austria) and Beckman Instruments compared the re-
sults of planar SDS PAGE and capillary SDS gel electrophoresis for 65 different proteins with respect to performance, pre cision, and efficiency. They found that the advantage of capillary SDS gel electro phoresis is that the sieving material (noncross-linked gel) in the capillary column can be replaced after each analysis, mak ing the technique less labor intensive than planar SDS PAGE. They also found that the polyethylene oxide-based sieving ma trix used in the experiments gives separa tion performance and estimated molecu lar weights that are slightly different from those obtained by planar SDS PAGE. (Anal Biochem. 1994, 221, 285-89)
Detecting metals in pore water In aquatic environ ments where there is a good supply of organic material, such as lakes, riv ers, estuaries, and coastal waters, the sedi ment-water interface is characterized by marked chemical changes. Nutrients and metal ions can be released from the rap idly decaying organic materials, and the reduction of organic components can re sult in a sharp redox boundary. To under stand the relationships between the vari ous chemical changes, it is essential that the components be spatially defined. Wil liam Davison and colleagues at Lancaster University (U.K.) and the University of Ox ford (U.K.) have used polyacrylamide gel probes to obtain depth-resolved iron and magnesium profiles in pore waters. Both Fe(II) and Mn(II) diffuse freely within the gel matrix and, with the use of a 0.4-mm-thick gel, complete equilibration is achieved in 6 min. The gels are treated with NaOH or KOH to immobilize the Fe and Mn as their insoluble oxyhydroxides, and the concentration of Fe and Mn in the gel is measured using atomic ab sorption or proton-induced X-ray emis sion spectrometry. (Environ. Set. Technol. 1994.2S, 1623-32)
Quantifying water in biomolecules by FT-IR Because of their potential use as drugs, the pharmaceutical specification of pep tides, enzymes, and other proteins are of interest. Determining water content, a minimum requirement for pharmaceuti cal preparations, may be difficult because these biomolecules are hygroscopic and difficult to dry completely. Noting that the OH stretching bands of water in the IR region are extremely in tense and can be used to measure the wa ter absorbed in organic solvents, Teiki Iwaoka, Fumiko Tabata, and Shinya Tsutsumi of Sankyo Co., Ltd. Oapan) devel oped a method for determining water in biomolecules that involves extraction into an organic solvent under conditions that do not induce aggregation, followed by FT-IR measurement of the intensity of the OH stretching band at 3600 cm"1. The extraction into acetonitrile was per formed in a glove box that was purged with nitrogen to avoid errors caused by water in ambient air. The results for the analysis of peptides and enzymes were compared with those obtained by Karl Fisher titration, and measurements for loss of weight on drying in vacuo were also investigated. (Appl. Spectrosc. 1994, 48, 818-21)
CE and NMR coiled together NMR spectroscopy can provide detailed information about molecular structure and has been successfully used as an on-line detector for LC. Coupling NMR spectros copy to CE, however, has not been report ed—primarily because of the inherently low sensitivity of NMR spectroscopy and because the NMR cell volumes typically used in LC (25-200 pL) are orders of magnitude too large to be used for CE. Jonathan V. Sweedler and colleagues at the University of Illinois have designed an rf microcoil wrapped directly around a fused-silica capillary that provides a detec tion cell of ~ 5 nL and detection limits in the nanogram range for short (< 1 min) NMR acquisitions.
Analytical Chemistry, Vol. 66, No. 20, October 15, 1994 1 0 0 3 A
Analytical
Currents
The rf microcoil is formed with copper wire and typically has 17 turns for a total detection length of 1.1 mm. The coil and capillary are mounted so that they are perpendicular to the static magnetic field over the detection region. This arrange ment provides a good filling factor for de tection in the cell but causes significant susceptibility-induced line broadening. For a thin-walled capillary the linewidth is > 200 Hz; for a thick-walled capillary the linewidth is narrowed to 11 Hz. Uniform wrapping of the coil and positioning of the capillary/coil far from nonunity sus ceptibility materials reduces linewidth. (/. Am. Chem. Soc. 1994,116, 7929-30)
Negative CI in the quadrupole ion trap The quadrupole ion trap mass spec trometer is increas ingly being used as a sensitive, small, and low-cost detector for GC. Most work with the quadrupole ion trap has involved the use of positive ions, although limited studies of negative ions have been reported. D. W. Berberich and R. A. Yost of the University of Flor ida have characterized the experimental parameters necessary for successful nega tive chemical ionization (CI) with re agent ion formation within the ion trap. They investigated the effects of ap plied voltage and reaction times on the for mation of negative ions via both electron capture and negative ion-molecule reac tions and discovered that mass-selected ejection of undesired reagent ions and se lective mass storage of only negative ions are required for practical negative CI. This can be achieved by the application of rf and dc voltages to the ring electrode to control the m/z of stored ions, as well as by the application of a supplemental rf volt age applied across the endcap electrodes to selectively eject ions of a particular m/z. (J. Am. Soc. Mass Spectrom. 1994, 5, 757-64)
Tracing the oil source A ground survey completed 22 years after the tanker Arrow sank off Nova Scotia and released ~ 2.5 million gallons of oil showed that natural weathering processes have removed most of the oil along the contaminated shoreline. Scientists are in terested in analyzing samples of any re maining oil to verify that it came from the Arrow spill and to obtain information about 1004 A
the oil's residual composition and changes in its characteristics as a result of expo sure. Concentrations of key components or compound classes used to evaluate the behavior, fate, and toxicity of the spilled oil remaining in the environment could also be determined. Because most saturated hydrocarbons and PAHs have been lost from the weath ered oil, Zhendi Wang, Merv Fingas, and Gary Sergy of Environment Canada fo cused on characterizing biomarker com pounds by GC/MS. They found that the relative abundance ratios of selected pairs of triterpanes and steranes, espe cially the ratio of C 29 /C 30 hopane that are displayed as major peaks in the m/z 191 ion chromatograms, are apparently inde pendent of weathering effects and can be used to identify the source of the Arrow oil samples. (Environ. Sci. Technol. 1994, 28,1733-46)
One parameter is not enough The solvent strength of the mo bile phase in RPLC is often measured by using ET(30), 2,6,-diphenyl-(2,4,6-triphenyWV-pyridino) phenolate, as a solvent retention indicator. However, while investigating ET(33), a dichloro analogue with more stable pH dye interaction characteristics, Jung Hag Park, Andrew Dallas, Phoebe Chau, and Peter Carr at the University of Minne sota discovered that plots of the transition energy ET for ET(33) versus ΕΎ for ET(30) were not linear as the mobile phase composition varied, even though both dyes remained completely unprotonated under all phase compositions in the study. The organic solvents used in the aque ous mobile phases were methanol, acetonitrile, isopropanol, and tetrahydrofuran, and each was mixed with water over the full range of composition. Nonlinearities in the plots of In k' vs. ET for ET(33) and In k' vs. ET for ET(30) strengthened the group ' s conclusion that a single-parame ter solvent strength scale is insufficiently general for RPLC. (/. Chromatogr. A 1994, 677,1-9)
Quantitating PCBs in marine benthic copepods Because of their high hydrophobicity, PCBs in aqueous solutions strongly associ ate with organic carbon, clays, and silts
Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
and eventually deposit in estuarine sedi ments. The effect of these PCBs on benthic meiofauna, invertebrates that range in size from 0.063 to 1.00 mm and comprise an obligate food source for many fishes and shrimp, has not been well characterized, primarily because of a lack of techniques for accurate and precise quantification of the contaminant burdens in organisms this small. Edward F. Wirth and colleagues at the University of South Carolina have developed a microextrac tion method that uses 100 pL of total ex traction volume and as few as 20 copepods ( ~ 25 pg of dry tissue mass) to quantify PCB body burdens in two species of meiobenthic copepods. The copepods were homogenized and extracted in a 100-pL closed vial with alka line media and isooctane spiked with an internal standard (hexachlorobenzene) to create a colloidal suspension. After centrifugation, Aroclor 1254 was directly de termined from the organic solvent phase by GC-ECD. Spiked samples of Aroclor 1254 yielded recoveries of 95% ± 12 % (n = 11). Body burdens of sedimentexposed copepods were measured over a 12-day period, and predicted body bur dens with 95% confidence intervals were calculated. (Environ. Sci. Technol. 1994, 28,1609-14)
Balancing the synthesis of conducting polymers Conducting poly mers show poten tial as lightweight rechargeable batteries or in applications such as electrochemical gas detection. However, the composition of the or ganic medium used for synthesizing them affects their electroactive and physical characteristics. Patrice Topart and Mira Josowicz of the Universifât der Bundeswehr (Germany) used an electrochemical quartz crystal microbalance to determine the effects of changing the water content of the background electrolyte during synthesis of poly(/V-vinylcarbazole) films. The frequency shifts showed that the polymerization efficiency decreased with increased water content in the background medium and became constant when water content was 10% or more by volume. The degree of cross-linking, the doping level, and the morphology of the films varied simultaneously with water content. (Talanta 1994,41, 909-16)
