Showing papers in "Analytical Sciences in 1993"

High-Precision Lead Isotope Ratio Measurement by Inductively Coupled Plasma Multiple Collector Mass Spectrometry

Abstract: An inductively coupled plasma (ICP) ion source coupled to a magnetic sector mass analyser equipped with seven Faraday detectors has been used to measure the lead isotope ratios in solutions of Sanshiro Pond sediment collected at the University of Tokyo, airborne particulates collected at Shinjuku in Tokyo and Merck multielement standard product number 97279494. A thallium correction technique was utilised to allow a simultaneous correction for mass bias. This work followed an earlier interlaboratory comparison study of the above-mentioned solutions using ICP quadrupole mass spectrometry, and has demonstrated a considerable improvement in analytical precision. The following isotope ratio measurements were recorded. Pond sediment solution containing 82ng ml-1 lead: 206Pb/204Pb=17.762±0.014; 206Pb/ 207Pb=1.1424±0.0009; 208Pb/204Pb=37.678±0.034. Airborne particulate solution containing 45 ng ml-1 lead: 206Pb/ 204Pb=17.969±0.006; 206Pb/207Pb=1.1528±0.0003; 208Pb/204Pb=37.915±0.021. Merck multielement standard solution containing 100ng ml-1 lead: 206Pb/204Pb=19.255±0.015; 206Pb/207Pb=1.2238±0.0004; 208Pb/204Pb=38.476±0.021 (All errors are given as ±2 standard deviations).

Determination of Femto-gram Amounts of Zinc and Lead in Individual Airborne Particles by Inductively Coupled Plasma Mass Spectrometry with Direct Air-Sample Introduction

Abstract: Heavy metals in airborne particles are important subjects to investigate in environmental science and public health as well as clean-environment control technology. Chemical composition of airborne particles has conventionally been analyzed by time-consuming processes in which the particles collected on a filter in an air sampler are digested with acids before their averaged composition is determined by such sensitive analytical methods as atomic absorption spectrometry (AAS) and inductively coupled plasma atomic emission spectrometry (ICP-AES). Previously, we have developed a particle analyzer by ICP-AES, in which air samples are directly introduced into the ICP and the elemental content in individual particles can be continuously determined by measuring the pulseheight distribution of flush signals emitted from the ICP.s'2 Furthermore, the particle analyzer has been combined with laser-light scattering measurements in order to simultaneously measure the content of a given element and the size of individual aerosol particles.3 Though the system was able to analyze the aerosol particles in the micrometer to sub-micrometer range, depending on the element and its content, its sensitivity was not sufficient to measure actual aerosol samples. Inductively coupled plasma mass spectrometry (ICP-MS) has recently been demonstrated to be a more sensitive analytical method for heavy metals than ICP-AES. A channel electron multiplier is currently being used in a high-gain pulse-counting mode for a sensitive detection of ions in ICP-MS.4 For particle measurements we need a signal consisting of a single pulse for each particle with a height corresponding to the content of an analyte element in the particle. This means that the detector must be used in an analog mode. Although Hutton et al, have described a dual-mode (pulse-counting and analog) detection system for ICP-MSS, their analog mode was used for the lower sensitivity by decreasing the gain of the multiplier in order to expand the determination range. In the present work, we obtained a single pulse for each particle by replacing a high-speed pre-amplifier in a commercial ICP-MS with a laboratory-made pre-amplifier

Prompt γ-Ray analysis of elements using cold and thermal reactor guided neutron beams

Abstract: Basic examination of elemental analysis by a reactor-neutron-induced prompt γ-ray analysis (PGA) using a system installed at the JRR-3M (upgraded Japan Research Reactor No. 3) cold and thermal neutron beam guides is reported. Analytical sensitivities and detection limits for 71 elements were measured. Boron, Gd, Sm, and Cd are the most sensitive elements with detection limits down to 1 to 7ng. The decay γ-rays of some elements such as F, Al, V, Eu, and Hf have a higher analytical sensitivity compared to their respective prompt γ-rays. In addition, the analytical sensitivity of several heavy elements through detection of characteristic X-rays was higher than through the prompt γ-ray detection. The present system is useful for the determination of, for example, ppb level of B in graphite and Be, ppb level of Gd, Sm, Eu, and Dy in high purity cerium(IV) oxide, and ppm to % level of H and Hf in Zr and zircaloy. This system is also applicable to trace determination of Cd and Hg in environmental samples and also to major and minor elementsdetermination in various kinds of samples that cannot be destroyed or dissolved.

High-resolution proton nuclear magnetic resonance studies on chondroitin sulfates

Abstract: The proton nuclear magnetic resonance (1H-NMR) spectra of chondroitin sulfates (chondroitin, chondroitin 4-sulfate, chondroitin 6-sulfate and dermatan sulfate) have been obtained at 500MHz and 333K in deuterium oxide. The J connectivities of sugar ring protons of chondroitin sulfates are revealed by the uses of homonuclear two-dimensional (2D) multiple-relayed-chemical-shift-correlated spectroscopy (multiple-relayed COSY), homonuclear Hartman-Hahn spectroscopy (2D HOHAHA), and multiple-quantum-filtered COSY. Formation of sulfate ester on N-acetylgalactosamine hydroxy group causes predictable 0.4-0.6ppm deshielding of the proton directly attached to the sulfation site. Furthermore, the partial structure of low-sulfated chondroitin sulfate chain isolated from human urinary trypsin inhibitor was determined from the obtained 1H-NMR data.

Electrochemiluminescence Detection of Primary Amines Using Tris(bipyridine)ruthenium(III) after Derivatization with Divinylsulfone

Abstract: Aliphatic primary amines are industrial chemicals with a wide variety of applications. For the determination of these substances, HPLC has recently been used; it has several advantages over other analytical methods, including high selectivity and sensitivity. On the other hand, an electrochemiluminescence (ECL) method has been developed as a highly sensitive method for HPLC detection.)-3 However., there is still no established methodology for the determination of aliphatic primary amines. The Tris(bipyridine)ruthenium(II) (Ru(bpy)32+) ECL system is useful for a selective, sensitive determination of oxalates4'5, indoles1'6, and trialkylamines2'' in biological and pharmaceutical samples. We previously reported that by using this system alicyclic tertiary amines8 were detectable at the pmol level, althouth the CL intensity of aliphatic primary amines was very low compared with that of the tertiary amines; and they could only be detected at a level of about 1000-times lower than that of tertiary amines.' In this paper we discuss the CL detection of some primary amines using a cycloaddition reaction of the amines with divinyl sulfone (DVS) caused by electrogenerated Ru(bpy)33t Furthermore, precolumn derivatization HPLC of the amines using LC postcolumn reaction detection with this CL system is described.

Triiodide Ion-Selective Electrode Based on Manganese(III)-Tetraphenylporphine

Abstract: A new sensitive and selective electrode for triiodide ion has been developed. The electrode was constructed by incorporating Mn(III)-tetraphenylporphine into a plasticized poly(vinyl chloride) membrane. The electrode shows super-Nernstian response over the range 10-5 to 10-3M triiodide ion in the pH range of 2 to 9, with an anionic slope of 87 mV/ concentration decade. The electrode exhibits high specificity for triiodide ion over other anions (salicylate, benzoate, propionate, perchlorate, iodide, bromide, chloride, fluoride, nitrate and bicarbonate). The electrode has been applied to measure triiodide ion generated from iodine. The results have indicated that iodine can be determined by simple potentiometric measurements. A similar method may be applied to determine oxidizing agents, bromine and hydrogen peroxide, which oxidize iodide ion to iodine.

Electrode modification by long-chain, dialkyl disulfide reagent having terminal dinitrophenyl group and its application to impedimetric immunosensors

Abstract: A long-chain, dialkyl disulfide having a terminal dinitrophenyl (DNP) group was newly synthesized for modification of a surface of gold with chemisorbed, self-assembled organic monolayers. The resulting monolayer was characterized by infrared spectroscopy of reflection absorption spectroscopy mode, by cyclic voltammetric measurement and by electrical impedance measurement. Coupled with impedance measurements, a possible application of the modified electrode system to impedimetric immunosensors was demonstrated. The sensor gave electrical response to anti-DNP antibody at 10-103ng cm-3 concentration.

A Comparative Study of Gas Chromatography with Atomic Absorption and Atomic Emission Detection for the Speciation Analysis of Organotin

Abstract: Two techniques, megabore column gas chromatography quartz furnace atomic absorption spetrometry (MCGC-QFAAS) and MCGC graphite furnace AAS (MCGC-GFAAS), were developed for the determination of pentylated organotin species and compared with the existing techniques, packed column (PC) GC AAS and capillary column (CC) GC-atomic emission spectrometry (AES). Particular attention was given to the design of the interface between a GC column and the detector cell (GF or QF) as well as optimization of the operational variables of the interface and detector. The accuracy of the developed techniques is discussed on the basis of an intermethod comparison analysis of 15 water samples.

Preparation of Poly(thionine)-Modified Electrode and Its Application to an Electrochemical Detector for the Flow-Injection Analysis of NADH.

Abstract: Poly(thionine)-modified electrodes have been prepared using basal-plane pyrolytic graphite, glassy carbon and In-Sn oxide conducting glass as electrode substrates by an electrooxidative polymerization of thionine in both aqueous and acetonitrile media. It was found that the prepared films, the surface concentration of which can be controlled over the range of ca. 10-10-10-8mol cm-2 by appropriately choosing the electropolymerization conditions (e.g., solvent, concentration of thionine and electrolysis time), are electroactive (the formal potential is ca. 0.0V vs. Ag/AgCl at pH 7.0) and chemically stable. The modified electrodes exhibited excellent electrocatalysis for NADH oxidation in neutral aqueous solutions, with an activation overpotential which is ca. 400mV lower than that of a bare electrode. Further, the modified glassy carbon electrode was found to be promising as an amperometric detector for the flow-injection analysis of NADH, typically with a dynamic range of 5μM-1mM.

Application of Supercritical Fluid Extraction and Chromatography to Assay Fat-Soluble Vitamins in Hydrophobic Ointment

Abstract: The determination of retinol palmitate and tocopherol acetate in a hydrophobic ointment by a coupled supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) system is described. The ointment, triturated with diatomaceous earth powder, was subjected to SFE with carbon dioxide, and the extract containing fat-soluble vitamins was trapped on a trimethylsilyl silica-gel column by reducing the fluid pressure. The trapped components were analyzed on an octadecylsilyl silica-gel column by SFC using carbon dioxide containing ethanol as a mobile phase and UV absorption monitoring at 284 nm. In this method, retinol palmitate and tocopherol acetate in preparations could be determined within ten minutes without any manual products after placing the sample into the system

Lithium ion-selective electrodes based on 1-10-phenanthroline derivatives

Abstract: The preparation of 1, 10-phenanthroline derivatives and 4, 7-diphenyl-1, 10-phenanthroline derivatives as neutral carriers for ion-selective electrodes and the properties of the title electrodes are described in detail. A log KPotLi, Na value of -3.1 was obtained for a Li+-selective PVC membrane electrode based on 2, 9-dibutyl-1, 10-phenanthroline. This value is superior to those reported so far. The electrodes also showed excellent selectivity coefficients for Li+ relative to K+, Mg2+, and Ca2+ The effects of substituents at the 2- and 9- positions of the carriers on the selectivity are discussed.

Determination of a Biguanide Metformin, by High-Performance Liquid Chromatography with Precolumn Fluorescence Derivatization

Abstract: A sensitive method is described for determining a biguanide drug, metformin, by high-performance liquid chromatography with precolumn fluorescence derivatization. Metformin and phenylguanidine, as an internal standard, react with a novel fluorogenic reagent, desyl chloride, or benzoin to afford the corresponding fluorescent derivatives, which are separated by reversed-phase chromatography on a TSKgel ODS-120T column with linear-gradient elution. The applicability of the method to the determination of metformin in human serum is also demonstrated. The calibration curve of metformin was linear over the range of 0-100nmol ml-1 in human serum. The detection limit (S/N=3) was 1.3pmol per 20-μl injection volume.

Micro High-Performance Frontal Analysis for Simultaneous Determination of Unbound and Total Carbamazepine Concentrations in Protein Binding Equilibrium

Abstract: Micro high-performance frontal analysis (MHPFA) method has been developed for the simultaneous determination of unbound and total carbamazepine (CBZ) concentrations in protein binding equilibrium. When a 300-μl portion of 4-12μg/ml CBZ and 593μM human serum albumin (HSA) mixed solution was directly injected into a small bore ISRP column (10cm×2.0mm i.d.), CBZ gave a trapezoidal peak with a plateau separated from HSA. The CBZ concentration calculated from the plateau height agreed well with the unbound concentration determined by the conventional ultrafiltration-HPLC method, while the CBZ concentration calculated from the peak area coincided with the total (bound plus unbound) concentration. The within-run and day-to-day reproducibilities were satisfactory (relative standard deviation, <1.27%, n=5). Compared with the injection volume (1.4ml) required for the HPFA using a conventional size (15cm×4.6mm i.d.) ISRP column, MHPFA could reduce the sample size to about one-fifth (300μl).

Simultaneous Determination of Sub .MU.g g-1 Levels of Impurities in High Purity Iron by Inductively Coupled Plasma-Atomic Emission Spectrometry after Circulation of Eluant through Anion Exchange Resin Mini-Column.

Abstract: Boron, Mo, Nb, Sn, Ta, W and Zr were separated from an iron matrix using a horizontal mini-column of an anion exchange resin. The flow of solutions through the mini-column was controlled by a peristaltic pump. The elements adsorbed on the resin were simultaneously eluted by the circulation of an eluant and determined by inductively coupled plasma-atomic emission spectrometry. Quantitative recoveries were obtained for all 7 elements in the acid-soluble fraction and for 5 elements (not B or Sn) in the acid-insoluble fraction. A part of B in the acid-insoluble fraction was lost during the ashing and the fusion but the loss was compensated by using the calibration solutions prepared with the same procedure as that for the samples

Semiconductor Laser Spectrometry.

Abstract: A semiconductor laser is more compact and less expensive than a conventional laser. Therefore, scientists prefer to use this light source in analytical spectroscopy. However, the emitting wavelength of the semiconductor laser is located in the near-infrared or deep-red region. Only a limited number of compounds are fluorescent in these spectral regions; we have already developed many analytical reagents and procedures for application. Inorganic species such as potassium ions and oxygen gas, organic molecules such as dyes and surfactants, and biological molecules such as metabolites, enzymes, proteins, and deoxyribonucleic acid have been determined by this method. For example, amino acids are determined at zeptomole levels after labeling them with a dye fluorescent in the deep-red region and separation by capillary electrophoresis. Thus semiconductor laser spectrometry is advantageous for practical trace analysis.

Determination of Lithium and Niobium in Lithium Niobate by Inductively Coupled Plasma Atomic Emission Spectrometry after Fusion with Ammonium Hydrogensulfate

Abstract: The photorefractive and piezoelectric properties of lithium niobate (LiNbO3) crystals have been widely investigated. The Li/ Nb ratios of crystals usually shifted from stoichiometry during the process of crystallization. It has therefore been necessary to develop a procedure for a high-precision determination of lithium and niobium in order to evaluate the constitution of LiNbO3 crystals. Inductively coupled plasma atomic emission spectrometry (ICP-AES) is especially well suited for the determination of lithium and niobium, due to its high sensitivity and wide dynamic range. Ceramic samples were decomposed by acid under pressure using a PTFE pressure vessel1'2, or fusion with alkali metal salts.3'4 In order to develop a procedure for a simple, high-precision determination of lithium and niobium in LiNbO3, a fusion method using ammonium hydrogensulfate (NH4HSO4)5 was investigated and employed. Because this method is expected to yield a short sample-preparation, the effects of the flux were scarcely found based on the analytical values by ICP-AES.

Palladium as a Matrix Modifier in Graphite-Furnace Atomic Absorption Spectrometry of Group IIIB - VIB Elements.

Abstract: The performances of palladium as a matrix modifier for group IIIB-VIB elements in graphite-furnace atomic absorption spectrometry were compared to those of other modifiers hitherto reported. The application of palladium and mixed modifiers containing palladium to biological, mineral sediment or natural water samples is summarized. The addition of palladium generally makes a higher thermal pretreatment temperature possible without any sensitivity loss, thus reducing the interferences of coexisting materials. Mass spectrometry connected to graphite-furnace atomic absorption spectrometry enabled real-time observations of the vaporized species in the furnace and revealed the vaporization and atomization process of arsenic and selenium in the presence of palladium. Both elements form compounds PdnMmOl (M=As or Se), which are then decomposed to PdM and finally to atomic vapor of M.The performances of palladium as a matrix modifier for group IIIB-VIB elements in graphite-furnace atomic absorption spectrometry were compared to those of other modifiers hitherto reported. The application of palladium and mixed modifiers containing palladium to biological, mineral sediment or natural water samples is summarized. The addition of palladium generally makes a higher thermal pretreatment temperature possible without any sensitivity loss, thus reducing the interferences of coexisting materials. Mass spectrometry connected to graphite-furnace atomic absorption spectrometry enabled real-time observations of the vaporized species in the furnace and revealed the vaporization and atomization process of arsenic and selenium in the presence of palladium. Both elements form compounds PdnMmOl (M=As or Se), which are then decomposed to PdM and finally to atomic vapor of M.

Comparison of avidin and ovomucoid as chiral selectors for the resolution of drug enantiomers by high-performance liquid chromatography

Abstract: The effects of the pH, organic modifiers and buffer salt of the mobile phase upon the retention and separation characteristics of avidin- and ovomucoid-bonded silica columns as proteinous chiral stationary phases for HPLC were investigated. The enantioselectivity was greatly affected by changing the mobile-phase composition, possibly because of induced alterations in the protein conformation. Hydrophobic and Coulombic interactions play major roles in the retention, and there are some similarities between the avidin column and the ovomucoid column. However, the stereoselectivity does not necessarily reflect the retention processes on these columns. No significant decrease in the retention or enantioselectivity was observed, even after the injection of 200 samples.

Use of Soluble Filters for Preconcentration of Trace Elements in Water

Abstract: Trace elements can be enriched by collecting them on a solvent-soluble filter, followed by dissolution of the filter in a small volume of suitable solvent. The filter serves not only as a sieve but also as a sorbent. For the element to be sorbed, it must be converted into an electrically neutral, hydrophobic species. Ionic species are poorly retained by the filter, but made collectable by adding counter ions with hydrophobic groups. In effect, this enrichment method resembles solvent extraction, but has many advantages. A number of papers have been published on its application since our proposal in 1984. This article summarizes applications of this method and presents a brief description of the mechanism of retention by the filter and some points which may help those who attempt to apply this method to their own field.

Crystal structure of bastnaesite (Ce, La, Nd, Sm, Gd)CO3F

Abstract: Recent development of high technology industries hay created a great demand for rare earths, which are used irr laser materials, high power magnetic materials, phosphors, ionic conductors, etc.. Bastnaesite CeCO3F i~ one of the most important resources of rare earths; other$ include monazaite (Ce, La, Nd)PO4 and xenotime YPO4 Despite the industrial importance of this mineral, the crystal structure of bastnaesite is not accurately known Donnay and Donnayl proposed a structural model of bastnaesite based on the X-ray photographic data ir. 1953, but ever since no structure analysis of bastnaesite has been reported. Here we report the first complete structure analysis of Bastnaesite. The sample is natural mineral from Madagascar. Quantitative analysis of the sample was carried out by electron microprobe analyzer for rare earths and by elemental analysis for C and H. The results gave La2O3 29.2, Ce2O3 35.9, Nd2O3 7.78, Sm2O3 5.71, Gd2O3 2.37, subtotal 81.0 wt%, CO2 19.9 and H, not detected, total 100.9 wt%. The absence of H indicates lack of OH substitution for the F site, which is often observed in natural minerals. Thus full occupancy of the F site by F ions is assumed and the empirical formula, normalized to C03=1.0, is (Ce0.484, La0.397, Nd0.102, Sm0.073, Gd0.029)1.08(C03)1.00F, which is close to its ideal formula CeCO3F. The average atomic number of the rare earths is 58.3, so the atomic scattering factor2 of Ce was used as that of the rare earth site in the analysis. A single crystal (ca. o.13XO.1OXO.15 mm3) of bastnaesite was used in the structure analysis. The crystal data and experimental conditions of intensity measurements are listed in Table 1. Intensities of 911 independent reflections were measured in the 29 range of 3° through 100° using graphite-monochromated Mo Ka radiation (A=0.71069 A). Lattice parameters were determined by a leastsquares treatment of the 28 values of 25 reflections (19° <20<28°). The intensity data were corrected for Lorentz and polarization factors; absorption correction was not made (,u=161.4). The final atomic parameters are shown in Table 2, which also gives the parameters obtained by Donnay and Donnayl for comparison. The selected interatomic distances and angles are given in Table 3. A view of the crystal structure projected along a* is

Continuous Separation Method with Liquid Particle Extractor: Enantioseparation of Mandelic Acid

Abstract: Continuous enantioseparation of (±)-mandelic acid was performed by dual-flow countercurrent extraction. The optimum extraction condition was calculated by a semi-empirical computational method. Use of the two-phase system composed of a 0.2mM copper(II) acetate aqueous solution and butanol containing 10mM N-dodecyl-L-proline copper(II) complex as a chiral separator allowed (±)-mandelic acid to be separated continuously. HPLC analysis showed that the (-)-form was extracted into the organic phase and the (+)-form was recovered from the aqueous extract with ee higher than 99.9%, at the stationary state.

Enantiomer Separation of Synthetic Pyrethroids by Subcritical and Supercritical Fluid Chromatography with Chiral Stationary Phases

Abstract: The separation of enantiomers of synthetic pyrethroids by subcritical and supercritical fluid chromatography was investigated using the Pirkle-type Chiral stationary phases developed for high-performance liquid chromatography. The enantioselectivities of these stationary phases were found to be sufficient in tests with reference chemicals. Using these stationary phases, enantiomers of compounds with one or two chiral centers in acid and alcohol moieties were resolved; attempts were made to improve their resolution. Significant improvement resulted with the use of lower temperature, and thermodynamic considerations suggested the effectiveness of subcritical fluid chromatography for chiral separation of pyrethroids. The elution order of enantiomers was the same as in high-performance liquid chromatography in the two cases studied. This technique is very promising, as it requires a shorter analysis time.

Mass Spectrometric Analysis for Distinction between Regular and Premium Motor Gasolines

Abstract: A method to estimate the octane number of motor gasoline by mass spectrometry (MS) has been studied, by first examining whether the octane number can be estimated from its mass spectrum (MS). The MS of 29 different regular gasolines and 32 premium gasolines, sampled in the market from spring to autumn, were measured. We studied whether it is possible to extract any available parameters for clearly distinguishing between regular and premium gasolines, by applying both feature-selection and pattern-recognition methods to MS. It was found that a clear distinction between regular and premium gasolines could be made using MS information.

Determination of Tributyltin and Triphenyltin Compounds in Fish by Gas Chromatography with Flame Photometric Detection

Abstract: A gas-chromatographic method is described for the determination of tributyltin and triphenyltin compounds in fish. Fish tissue was hydrolyzed with a potassium hydroxide/ethanol solution. Organotin compounds were extracted with toluene. The extract was purified with a series of cation- and anion-exchange resin cartridges. Organotin compounds were propylated and then determined by gas chromatography with flame photometric detection. Separation of the organotin compounds was carried out using a Silicone OV-1 packed column and an Ultra-1 capillary column. Organotin compounds were stable during hydrolysis for 90min at 60°C. The detection limits of the two organotin compounds in their chloride forms were 0.06ng (6ng/g for 5g of the sample) for tributyltin compounds and 0.1ng (10ng/g for 5g of sample) for triphenyltin compounds. Compounds yielding propyltributyltin and propyltriphenyltin were found in fish at levels ranging from none detected to 120ng/g, and 10 to 520ng/g in their chloride forms.

Determination of silver(I) by an association complex formation between silver-adenine and eosin

Abstract: A sensitive and selective spectrophotometric determination of silver(I) based on an association complex between silveradenine and eosin was established. The apparent molar absorptivity of the complex, without solvent extraction, was 1.1×105dm3 mol-1 cm-1 at 560nm. No metal ion interfered in an equimolar ratio of silver(I) among the metal ions examined. The method was applied to assays of silver in silver solder and silver protein: the results were in good agreement with those obtained by ICP-AES. The sensitivity could be further enhanced about 5-10 times over that of spectrophotometry with the third-derivative spectrophotometry and/or the fluorescence quenching method. Thermodynamic parameters (ΔG=-6.34kcal mol-1, ΔH=-28.17kcal mol-1 and ΔS=-72.73cal mol-1 K-1) and the binding parameters (n=0.98 and K=4.10×104M-1 at 27°C) suggest that colored complex formed in this reaction system is an association complex between silver-adenine and eosin.