Analytical Currents Synopses of significant analytical articles from other publications
Characterizing two different methods to add GOx to the
opioid peptides Opioid peptides such as enkephalin and endorphin bind to opioid re ceptors in the brain and are known to be important in control ling pain and emotional states. As part of a research program to develop opioid pep tides to minimize obstetrical complications, Dominic M. Desiderio and colleagues at the University of Tennessee have used tan dem MS to characterize synthetic enkephalin-related peptides that were de signed to have specific opioid receptorbinding properties. The researchers used low-energy colli sion-induced dissociation tandem MS to study five opioid peptides in ovine plasma in an effort to determine the placental transfer of the peptides to the fetus. Liq uid secondary ion MS was used for sam ple desorption-ionization, and a hybrid (EjBEgqQ) tandem mass spectrometer was used to collect the product ion spec tra. A protonated molecular ion, [M + H ] \ was observed for each of the three linear peptides as well as for the two disulfide bond-containing peptides. (Rapid Com mun. Mass Spectrom. 1995, 9, 264-75)
Luminol-based glucose sensor Most glucose sensors based on the glu cose oxidase (GOx)-horseradish peroxi dase (HRP) enzyme couple are electro chemical sensors, but peroxidase can also catalyze the reactions of a variety of indi cators for light detection. Continuing pre liminary work in which they demon strated a fiber-optic biosensor for hydro gen peroxide based on HRP-catalyzed chemiluminescence of luminol, Mark A. Arnold and Xiangji Zhou of the University of Iowa developed a fiber-optic chemiluminescence-based glucose biosensor with a PMT detector. The peroxide biosensors consisted of fiber-optic cables placed inside a tube con taining luminol and HRP; the tube was covered with a Teflon membrane at one end to allow peroxide to permeate from the sample solution. The researchers used
outside of the membrane: chemical immo bilization on the Teflon membrane or in corporation of a commercial membrane with embedded GOx. Under optimal con ditions, the steady-state signal varied lin early with the square of the peroxide con centration. The sensors had a linear dy namic range of 2-18 mmol/L glucose with response times of less than 60 s and were demonstrated as viable for glucose determination in whole blood. (Anal. Chim. Acta 1995,304,147-56)
Enzymatic assay of β-galactoside by CE-LIF New approaches to enzymatic assays involve reducing reaction volumes and im proving detection sensitivity. Because CE can separate compounds of very simi lar structure and laser-induced fluores cence (LIF) detection can improve sensi tivity, developing an enzymatic assay that incorporates these techniques should re sult in a highly sensitive assay that can monitor the formation of products and loss of substrate simultaneously, handle small volumes easily, require short analysis times, and allow easy quantitation. Nor man J. Dovichi and co-workers at the Uni versity of Alberta (Canada) have demon strated the advantages and sensitivity of enzymatic assay by CE-LIF using β-gal actoside. The researchers used the fluorogenic substrate fluorescein-di-P-D-galactopyranoside and monitored the generation of the fluorescent products fluoresceinmono-p-D-galactopyranose and fluores cein. After incubation of the enzymatic re action mixture was complete, a picoliter volume containing the fluorescent products was electrokinetically injected into the CE capillary and then detected by postcolumn LIF. By analyzing as little as 40 pL of the en zymatic mixture, they obtained a detection limit of 6.5 χ ΙΟ-14 Μ β-galactoside or 1.6 molecules based on the detection of fluores cein- di-p-D-galactopyranoside. (Anal. Biocheni. 1995,226,147-53)
Purifying DNA Preparation of the template is often a bot tleneck in large DNA sequencing projects, and although PCR-based DNA isolation
offers the possibility of greatly increasing throughput in template preparation, meth ods currently used to purify the PCRamplified DNA fragments are expensive and cumbersome. Leroy Hood and col leagues at the University of Washington have developed a reliable and inexpensive high-throughput method based on gel fil tration. Their method uses a mini spin-column made from a 96-well microtiter plate with a membrane bottom. Sephacryl-500HR fil tration media is added to each well, the plate is centrifuged, and then the process is repeated until the final column height is about three-fourths of the depth of the well. PCR mixture is then applied to the center of each column, the plate is centri fuged, and the flow-through and eluate combined. The final product is examined by gel electrophoresis to determine purity and yield. On average, the researchers were able to recover 50% of the PCR prod ucts, which can be used in fluorescent or radioactive sequencing reactions, and they routinely obtained 400 bp of reliable "reads" from either fluorescent dye primer or dye terminator sequencing reactions from DNA purified by this method. The purified PCR products can also be used in other enzymatic manipulations, such as generating DNA probes for hybridization protocols. (Anal. Biochem. 1995,226,8590)
Multipurpose DEMS Work involving dif ferential electro chemical MS (DEMS) has proved useful for elucidating the re action mechanisms of many compounds at different electrode systems. Despite nu merous advantages of the technique, how ever, its widespread use has been ham pered because the mass spectrometer has been dedicated to the DEMS technique; it could not be used for other applications. R. F. Savinell and colleagues at Case Western Reserve University have de signed a system in which the mass spec trometer can be switched easily between different applications. Analytical Chemistry, June 1, 1995 3 4 5 A
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The experimental setup incorporates a the tubular cells designed for work in peared to act synergistically. At low values dual inlet system. One inlet is connected high-resistance solvents can achieve excel for both, the observed species were prima to a vacuum recipient at which the electro lent quality electrochemical responses, rily mono- and divalent cationic analyte ionchemical cell is attached when the appa they tend to be mechanically unstable and solvent clusters, but as the parameter val ratus operates in the DEMS mode; the sec cannot be easily used under inert atmo ues increased, the species were declusond consists of a capillary inlet that can tered and double charges were reduced to spheres. In addition, because the elec be used for other MS applications. Modes trodes must be manually (and therefore ir- single charges. At high values, the bare can be switched without shutting down singly charged metal ion dominated. (Appl. reproducibly) adjusted, it is difficult to the vacuum system by opening and clos obtain a minimum iR drop. Alan M. Bond Spectrosc. 1995,49,324-34) ing two valves. The researchers con and colleagues at La Trobe University nected a thermogravimetry analyzer to (Australia) have designed a SEESR cell Reactive ion bombardment the capillary inlet and determined weight with a special Ag/AgCl reference elec for chemical modification of loss as well as thermal decomposition trode fixed in the center of a Pt working surfaces products as a function of temperature. electrode coil. The collision of atoms, molecules, and ions (J. Electrochem. Soc. 1995,142,1183-89) The 0.2-mL cell allowed simultaneous with surfaces is of particular interest to rapid-scan voltammetric ESR experiments researchers involved in investigating con trolled surface chemical modification. Piezoelectric pesticide to be performed directly in the cavity of a sensor spectrometer over a wide temperature By selecting an appropriate atomic, molec ular, or ionic reagent at the optimum col Enzyme-linked immunosorbent assays range in low or high dielectric solvents lision energy, it should be possible to and enzyme immunoassays, originally de and facilitated the study of electrochemi signed for biomedical applications, have cally generated radicals that are highly achieve selective chemical modification of the outermost atomic monolayers of a been developed in recent years as rapid sensitive to oxygen or moisture. The screening tests for the determination of proximity of the reference electrode to the surface. To study this possibility, R. G. Cooks and colleagues at Purdue Univer pesticides and other contaminants in envi working electrode, resulting in small cell ronmental samples. Piezoelectric for time constants, allowed the ESR signal to sity and 3M Corporate Research Laborato mats for the immunoassays provide signal be measured at fast scan rates. The re ries have used reactive collisions of low energy (< 100 eV) mass-selected ions to transduction by registering changes in searchers also demonstrated that lower mass on the quartz crystal resonator sur ing the temperature to 130 Κ does not de chemically modify fluorinated self-assem bled monolayer surfaces composed of alface when analytes bind to the antibodies stroy the cell; thus, frozen-solution spec immobilized on it. Kenji Yokoyama and co tra of electrochemically generated radicals kanethiolate chains (CF3[CF2]n[CH2]2S) workers at the University of Tokyo and can also be obtained. (J. Electrochem. Soc.bound to gold. Japan Women's University have fabricated 1995,142,862-67) Typical experiments were performed quartz crystal immunosensors for the si with 1-nA/cm2 beams and submonolayer multaneous determination of triazine her doses of reactant ions. The modified sur Elements of bicides in water. face was characterized by in situ chemi electrospray Because atrazine and other triazine cal sputtering with 60-eV Xe+ and by inde ESI sources are herbicides have a low molecular weight, widely used for MS pendent high mass resolution TOFdirect herbicide measurement with piezo SIMS (15-25 keV, Ga+) experiments. of large biomoleelectric sensors would be difficult be cules and other del Treatment with SPCÇ produced a surcause they would not produce a very large face from which Xe+' sputtering liberated icate molecules frequency shift when the herbicides CF|5C1+ ions, suggesting chlorine for fluobecause they can bound to the antibodies for them. The re produce intact molecular ions by relatively rine halogen exchange at the surface. searchers performed a competitive immu gentle means. ESIMS can also be used The researchers concluded that selective noassay using protein-labeled atrazine as chemical modification of monolayer surfor elemental analysis of solutions to pro the competing ligand for the antibody and vide valence and speciation details on the faces can be achieved by using reactive mixed the labeled atrazine solution with ion complexes, but the results are depen ions beams, which lead to new covalent spiked water samples. The protein label bonds at the surface and in the scattered dent on the source and interface operat ing made the mass changes more appar ing parameters. Gary Horlick and George ions. (J. Am. Soc. Mass Spectrom. 1995, 6, ent and allowed a detection limit of 187-94) R. Agnes of the University of Alberta 0.001 ng/mL and a linear dynamic range (Canada) have investigated the effects of of 0.01-1 ng/mL. Several other triazines varying ESI source and interface variables Improving the sensitivity of cross-reacted with the antibody and on the composition of ion clusters for al LC/C-IRMS were also detectable when spiked into the kali metals, alkaline earth metals, and co High-precision isotope ratios are widely samples. {Anal. Chim. Acta 1995, 304, balt in methanolic solutions. used in biomedical science for tracer appli139-45) cations. LC coupled with combustion isoCurtain gas flow rate for the ionization tope ratio MS can achieve precisions of source and voltage biases for the electroA fixed SEESR cell spray capillary tip, the front plate, the sam ± 0.5% in the flow injection mode and Research aimed at extending the capabili pling plate, the skimmer, and the barrel ion ± 1% in the LC mode but suffers from seties of the simultaneous electrochemi extractor lens were studied. The research verely limited sensitivity. J. Thomas cal-electron spin resonance (SEESR) ers found that, of these, curtain gas flow rate Brenna and Richard J. Caimi of Cornell method has met with difficulties because University have improved the sensitivity of and sampling plate voltage bias affected of cell design and performance. Although ion cluster composition the most and ap this technique by more than 2 orders of 346 A
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magnitude by modifying the instrumenta tion to optimize flows. The researchers added separate flow controllers to allow independent adjust ment of critical helium carrier flow, con structed a pneumatic nebulizer to facilitate aerosol spray deposition as an alternative to conventional dip coating, and pretreated the transport material to enhance analyte combustion. The resulting increase in sen sitivity allowed them to determine a se ries of underivatized vitamins, including tocopherols, retinyl acetate, and ergocalciferol, using both normal and reversedphase LC. The researchers emphasize that the amount of material actually trans ferred to the IRMS system remains low because of the absolute coating and sam pling splits. (J. Mass Spectrom. 1995,30, 466-72)
Behavior of TBP on basalt and quartz Tri-M-butyl phosphate (TBP) is often used to extract uranium and plutonium in the processing of nuclear fuels. Because of poor disposal practices, TBP and radio nuclides have been buried together in uncontained landfills, making TBP a signif icant environmental contaminant at some Department of Energy sites. Two of these contaminated sites are underlain with ba salt, an inhomogenous mineral with a highly variable surface morphology, which makes it difficult to assess contami nants adhering to the surface, a process that is important to remediation strate gies. Gary S. Groenewold and colleagues at the Idaho National Engineering Labora tory have used static SIMS with a molec ular primary ion gun to evaluate how TBP adsorbed on basalt and quartz is af fected by the composition of the mineral surface. They found that when TBP is adsorbed on Fe(II) surfaces, it undergoes concomi tant H~ abstraction and reduction as well as elimination of two C4Hg molecules to form an ion at ml ζ 137+; on Fe(III) sur faces, TBP undergoes ΡΓ abstraction and elimination of two C4H8 molecules to form an ion at mlζ 153+; on quartz or other nonreducing surfaces, it undergoes protonation and elimination of three C4H8 molecules to form H4PO4. These conclusions are sup ported by model studies that used FeO, Fe 2 0 3 , TBP, and tributyl phosphite, and in dicate that static SIMS of TBP has the po tential to characterize the chemical nature of mineral surfaces as well as the mode of contaminant-surface interaction. (J. Am. Soc. Mass Spectrom. 1995,6,165-74)
Measuring arsenic speciation Because toxic ar senic compounds are often used for industrial and agri cultural purposes, such as insecticides and wood preserva tives, it is important to quantitate the vari ous arsenic species that eventually find their way into the environment. Although capillary zone electrophoresis (CZE) of fers rapid and efficient separation of ar senic species, it lacks a sensitive univer sal detector for these compounds. Joseph Caruso and colleagues at the University of Cincinnati have coupled indirect UV de tection with CZE to measure arsenate, arsenite, dimethylarsinate (DMA), and monomethylarsonate (MMA) extracted from fly ash. Using sodium chromate as the electro lyte solution, they were able to obtain ab solute detection limits of 5.2 pg arsenate, 3.5 pg arsenite, and 15.6 pg for both DMA and MMA. The researchers note that because the electroosmotic flow var ies with the pH of the electrolyte solution, choosing suitable pH conditions for CZE separations with indirect UV detection is very important. Future work will focus on coupling CZE with ICPMS to provide high sensitivity and low-level elementspecific detection. (/. Chromatogr. Set. 1995,33,177-80)
Dendrimers as pseudo-stationary phases for EKC Electrokinetic chromatography is a sepa ration technique based on differences in the distribution equilibria of sample com ponents between an aqueous mobile phase and a pseudo-stationary phase. Although charged micelles are the most widely used pseudo-stationary phases, interest has fo cused recently on the use of dendrimers for this application. These three-dimen sional, highly structured macromolecules are stable under a wide variety of experi mental conditions, and their size and struc ture can be controlled during synthesis. Pirn G.H.M. Muijselaar and co-workers at Eindhoven University of Technology (The Netherlands) and DSM Research have in vestigated the electrophoretic properties of diaminobutane-based carboxylic acidterminated dendrimers (OAB-dendr(COOH)J and their potential as pseudostationary phases in electrokinetic chroma tography.
They observed different selectivities for aromatic sample compounds with SDS mi celles and OAB-dendr-(COOK)64 as the pseudo-stationary phase and attribute the differences to the characteristic chemical structures of the surface and the interior of the micelles and dendrimers. On the basis of these results, the researchers suggest that electrokinetic chromatography may be an attractive technique for studying the in teraction between dendrimers and small or ganic molecules. (J. High Résolut. Chromatogr. 1995,18,121-23)
Determining VOCs in breath The presence of volatile organic hydrocarbons, such as ethanol or tetrachloroethylene, in exhaled breath can be used as an indicator of exposure to these compounds. Standard methods for determination of VOCs in ambient air cannot be used for analyzing human breath because of differences in water and C0 2 content and because the periodic nature of breathing may allow ambient air to be pulled into the sampling system. In addition, excess exogenous compounds such as acetone and dimethyl sulfide can be present as a result of disease. Andrew Β. Lindstrom and Joachim D. Pleil of the U.S. Environmen tal Protection Agency have developed a set of three complementary GC/MS meth ods for determining VOCs in exhaled breath collected in evacuated stainless steel canisters. The first method is used to screen for C0 2 ; this value is then used to determine the dilution with nonalveolar air through comparison with known levels of C0 2 in breath. The sample is then reanalyzed for volatile endogenous compounds such as acetone, isoprene, ethanol, and dimethylsulfide. Finally, the sample is analyzed for trace-level VOCs at the sub-part-per-billion by volume level. The three methods can all be performed on the same sample, and to gether they allow complete constituent characterization. (J. Chromatogr. B, 1995, 665,271-79)
Separating PCBs on a single column Poly chlorinated biphenyls (PCBs) are generally lipophilic and relatively stable, but not all congeners bioaccumulate at the same rate or are equally toxic. Complete separation of PCB congeners has been achieved by multidimensional GC, but the single-column media usually used in routine quantitative work, 5% phenyl methylpolysiloxane, fails to separate some congeners, such as PCB 138 from PCB Analytical Chemistry, June 1, 1995 347 A
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163. Barbara R. Hillery and colleagues at ization, platform atomization, and probe didn't eliminate the convection compoThe American University and the National nent for cadmium and lead. Using an end- atomization techniques for the direct deInstitute of Standards and Technology termination of these toxic elements in polcapped ICC increased sensitivity by an evaluated the use of a novel />,£-cyanobi- average factor of 3.2 but didn't reduce the luted seawater. phenyl stationary phase for GC separation convection component. Plugging the inThey used pyrolytic graphite-coated of these congeners. jection port of an end-capped ICC gave (PGC) cuvettes for wall atomization, toThe researchers used />-cyanobiphenyl- sensitivity increases proportional to the tally pyrolytic graphite L'vov platforms in^-allyloxy methylpolysiloxane coated on a pressure increase, suggesting that convec- serted in PGC cuvettes for platform atfused-silica capillary column to separate tion had been eliminated. (J. Anal. At. omization, and totally pyrolytic graphite the hexachlorobiphenyl congeners PCB Spectrom. 1995,10,187-95) cuvettes with graphite probes for probe 138,163, and 164 and other pairs of conatomization. The researchers reported geners that coelute on conventional colthat the best results for all elements were Using IMS to characterize umns. MS detection was used to quantiobtained using totally PGC tubes, peak outgassing from tate the PCBs in four standard reference height signal processing, and a chemical semiconductors materials (threefishoils and whale blubmodifier (10 pg Pd as nitrate). Arsenic The increasing use of polymers in semiber). The stationary phase is reportedly conductor technology has resulted in add- measurements were made on seawater more polar than the 5% phenyl methylthat was directly injected onto the plating volatile organic compounds to the list polysiloxane, and PCB retention on it is re- of common semiconductor contaminants, form, whereas dilution with ultrapure walated to the planarity and polarity of the ter was preferable for lead measurements which has traditionally included only parcongener. The researchers concluded that ticles and metal ions. These organic mole- and dilution with 25% ammonium nitrate the/>,/>-cyanobiphenyl column could be was required for cadmium measurements. cules can cause severe problems in epiused as a complement to the conventional taxial growth, oxidation kinetics, and wet Detection limits obtained were 1.7 pg/L separation media and note that the abilfor arsenic, 1.0 pg/L for cadmium, and 1.3 chemical treatment of the wafer. In order ity to separate PCB 138,163,164, and 158 to select appropriate polymeric materials pg/L for lead; recoveries were 100% ±5% provides an important advantage befor all elements. (J. Anal. At. Spectrom. for semiconductor equipment, K. L. cause these congeners are subject to bio1995,10,149-54) Budde and colleagues at Siemens AG accumulation. (J. High Résolut. Chro- (Germany) have used ion mobility specmatogr. 1995,18,89-96) trometry to investigate the outgassing Using IR to probe methanol characteristics of polypropylene (PP), surface chemistry polycarbonate (PC), perfluoroalkoxy The catalytic oxidation of methanol at noAtomization polymer (PFA), polyvinylidenefluoride ble metal electrodes is a technologically at elevated (PVDF), acrylonitrile butadiene styrene important process because complete oxipressure in copolymer (ABS), and polytetrafluoroeth- dation to carbon dioxide and water yields GFAAS ylene (PTFE). six electrons per molecule of methanol, Since the graphite which makes it attractive for use in fuel Each polymer was heated from 60 °C furnace was first cells. This process is complicated, howto 20 °C below its softening temperature developed, rewith continuous monitoring of the amount ever, by the formation of adsorbed intersearchers have inmediates such as carbon monoxide, which of outgassing; the outgassing compovestigated atomization at increased presblock catalytic surface sites and inhibit nents were also identified separately. sures. In principle, increasing the presPTFE and PFA showed the lowest amount energy-producing pathways. Carol Korzesure for a diffusion-limited system should niewski and Jungwon Shin of the Univerof outgassing over the entire temperature proportionally increase the time-integrated absorbance, assuming that the analyte's range, although PVDF and PC are almost sity of Michigan have used IR spectrosas good over a limited temperature range. copy to probe site-dependent aspects of chemical interactions are unchanged and methanol surface chemistry at a highly The researchers are investigating the that convection is not a significant loss correlation between this elevated tempera- corrugated platinum electrode. mechanism. Because research has yielded conflicting results, James M. Harnly and ture outgassing behavior and that at IR spectral features of the adsorbed room temperature. (J. Electrochem. Soc. carbon monoxide reaction intermediate Clare M. M. Smith of USDA used an in1995,142,888-97) strument comprising a continuum source, were measured in parallel experments an échelle spectrometer, and a linear photowith Pt(335) and Pt(lll) electrodes and diode array to investigate the effect of eleconfirm that methanolic CO formation is ETAAS and toxic metals in vated atomization pressures. inhibited at Pt(lll) at potentials in the hypolluted seawater drogen adsorption region. The researchThey measured absorbances for alumi- Electrothermal atomic absorption specers also found that methanol dissociative num, cadmium, chromium, manganese, trometry (ETAAS) is frequently used to and lead at pressures as high as 6 atm in a determine the toxic elements arsenic, cad- chemisorption proceeds to a limited extent in this potential region at the Pt(335) standard integrated contact cuvette mium, and lead in seawater because the (ICC) (tube ends open), in an end-capped technique is sensitive, analyses can be au- surface, for which independent vibrational spectral features for carbon monoxide at ICC (tube ends restricted), and with the tomated, and sample preparation is relastep and terrace sites were detected. This injection port open and plugged. In a stantively simple. Direct analyses can be diffidard ICC, the maximum signal increase cult, however, because of the presence of information should be useful in studying site-dependent aspects of methanol surwas limited to a factor of 3 at 6 atm behigh salt-matrix interferences. Alfredo face electrochemistry. (J. Phys. Chem. cause of a strong convection component. Sanz-Medel and colleagues at the UniverPlugging the injection port reduced but sity of Oviedo (Spain) compared wall atom- 1995,99, 3419-22) 348 A
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