Microchemical Journal 74(2003) 181–186

0026-265X/03/$ - see front matter� 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0026-265X(03)00024-9

Slurry sampling electrothermal atomic absorption spectrometricdetermination of copper in herbal medicine samples with a

molybdenum tube atomizer

Md. Nurul Amin *, Satoshi Kaneco , Tohru Suzuki , Kiyohisa Ohtaa, a b a

Department of Chemistry for Materials, Faculty of Engineering, Mie University, Tsu, Mie 514-8507, Japana

Environmental Preservation Center, Mie University, Tsu, Mie 514-8507, Japanb

Received 4 November 2002; received in revised form 22 December 2002; accepted 4 January 2003

Abstract

An ultrasonic slurry sampling electrothermal atomic absorption spectrometric method with a molybdenum tubeatomizer has been developed for the determination of copper in herbal medicine samples. Glycerol solution(10%)was used as the slurry medium. The optimum pyrolysis temperature was 7608C. The detection limit was 72 fg(3=SyN ratio). Matrix element interference was investigated and it was found that glycerol as a chemical modifiereliminated the interference. The amounts of copper in herbal medicines determined by the method proposed are ingood accordance with those measured in dissolved acid-digested samples. The method enables rapid calibration, andsimple and rapid analysis of copper in herbal medicine samples at low cost.� 2003 Elsevier Science B.V. All rights reserved.

Keywords: Copper; Slurry sampling; Herbal medicine; Electrothermal atomic absorption spectrometry; Metal tube atomizer

1. Introduction

Copper is an abundant trace element, which isconsidered as an essential micronutrient and as atoxic element, depending on its concentration levelw1x. As the borderline distinguishing between toxicand essential concentration is narrow and easy tocross, especially for such a common metal, itsanalytical determination in different matricesbecomes of great importance for the protection ofhuman health and the environment. Recently, ithas been reported by a number of researchersw2,3x

*Corresponding author. Tel.:q 81-59-231-9427; fax:q81-59-231-9442.

E-mail address: nurul34@hotmail.com(M.N. Amin).

that some diseases, which are believed to beclosely linked to the chronic intake of elementalcopper, even at low concentration, will possiblybecome a serious problem in the future. In theUSA, the estimated safe and adequate dietaryintake for copper is 1.5–3 mgyday for adults, 0.4–0.6 mgyday for infants, and 0.7–2 mgyday forchildren w4x.

From the viewpoint of public health, consuma-ble products such as foods and medicines shouldbe regularly monitored in order to reduce risks tohealth. Therefore, it is necessary to develop a rapidand simple method for the determination of copperat low levels in human consumable products. Thusfar, numerous studies on the determination of

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copper in these samples by graphite furnace atomicabsorption spectrometry(GFAAS) have beenreportedw5–7x. Recently, solid-sampling GFAAShas been developed for the determination of manytrace elements in a variety of materialsw8–39x.The solid sampling technique has many benefitsfor solid analysis, including decreased samplepreparation time, decreased likelihood of analyteloss owing to volatilization prior to analysis orretention by an insoluble residue, decreased use ofacids and a corresponding reduction in the produc-tion of waste; and decreased possibility of samplecontamination. A fairly comprehensive account ofrecent solid-sampling GFAAS research activitiescan be found in a review by Bendicho and Loos-Vollebregt w9x, which includes more than 250references and concludes that slurry sampling givesbetter analytical performance than direct solidsampling because of the possibility of changingthe slurry concentration and because slurry sampleintroduction can be fully automated. Preparationof a slurry using finely ground solid materialfacilitates the introduction of slurry samples intothe furnace, combining the advantages of directsolid sampling and liquid sampling. In addition,this technique provides a convenient means ofinserting mg–mg amounts of solid material intothe furnace for analysis using conventional liquid-sample handling devices, such as autosamplers andpipettes.Electrothermal atomization AAS(ETA-AAS)

with a metal tube atomizer, which is similar toGFAAS, is known to provide better sensitivity formost metal elements, and better reproducibility atrelatively low power and low costw40–42x.Despite these advantages, there is still little infor-mation on the combination of the metal tubeatomizer in ETA-AAS with slurry sampling forthe determination of copper in drugsw43–45x.

In the present work, we report the determinationof copper in herbal medicine samples by slurrysampling ETA-AAS with a molybdenum tubeatomizer.

2. Experimental

2.1. Instrumentation

A molybdenum tube atomizer(20 mm=1.8 mmi.d., wall thickness 0.05 mm) made from pure

molybdenum sheet(99.95% purity, Rembar Co)was used for slurry sampling ETA-AAS. Thesamples were injected by means of a glass micro-pipette through a 0.3-mm-diameter hole at themid-point of the tube.Copper determinations were performed with a

Nippon Jarrell-Ash 0.5-m Ebert-type monochro-mator atomic absorption spectrometer equippedwith a R928 photomultiplier tube(HamamatsuPhotonics Co), a fast-response amplifier, a storageoscilloscope(Kenwood CS-8010) and a micro-computer(PC 9801 FA, NEC Corporation). Alldeterminations were performed at the resonanceline of 324.75 nm (Cu hollow cathode lamp,Hamamatsu Photonics Co) and with a deuteriumlamp (Original Hanau D200F) to compensatebackground absorption, a step-down transformerand a transformer(Yamabishi volt slider, S-130-30, capacity 3 kVA) to supply electric power forheating the atomizer, and two pinhole apertures(1.0 mm in diameter) placed in front and at therear end of the atomizer to collimate the lightbeam and eliminate radiation from the atomizersurface. The absorption signal from the amplifierand the output signal from a photodiode for themeasurement of atomizer temperature were simul-taneously fed into a microprocessor. Calibration ofthe atomizer temperature using the photodiodevoltage was achieved by means of an opticalpyrometer(Minolta TR-630) and microcomputersoftware. The baseline was measured by heatingwithout the sample. The time constant was 4 ms.The atomic absorption signal was evaluated fromthe peak height.The mass of chemicals was determined using a

Mettler H20 semi-micro analytical balance with aprecision of"0.01 mg. Herbal medicine powdersamples were ground to different particle diametersusing an agate mortar and a filter(Nippon Rika-gaku Kikai, ISO 38). An optical microscope(Ken-ko, KL-1200) was used to determine the particlediameters of ground medicine samples. A Uni-sealdecomposition vessel was used for digestion ofmedicine samples. Ultrasonic washing apparatus(B-12, 40 W, Branson Cleaning Equipment Co,USA) was used to homogenize slurried medicinepowder immediately before sample injection.

183M.N. Amin et al. / Microchemical Journal 74 (2003) 181–186

Ultrapure water was prepared using an AdventecCW-102 ultrapure water system.

2.2. Sample and reagents

Herbal medicine samples(powdered ginseng,powdered pueraria root and powdered immatureorange) were purchased from different companies.A standard copper solution(1 mgyml in 0.1 molyL nitric acid solution) and pure glycerol used asslurry medium were obtained from Nacalai Tesque(Kyoto, Japan). Standard stock solutions of thematrix elements for interference study were pre-pared as nitrates or chlorides in 0.1–6 molyL nitricacid or hydrochloric acid. Working standard solu-tions of appropriate concentration were preparedby dilution of stock standard solutions with purewater immediately before use. All reagents andchemicals used were of analytical grade or spec-troscopic purity.

2.3. Procedures

A powdered herbal medicine sample was groundto a very fine mesh, filtered, and material with aparticle size range of 26–38mm was collected.An accurately weighed sample of the medicine(approx. 2 mg) was transferred into a 100-mlvolumetric flask and diluted to the desired volumewith 10% glycerol solution. Immediately beforeinjection, ultrasonic agitation for 5 min was per-formed to homogenize the slurries. An aliquot(1ml) of the slurry was injected into the Mo tubeatomizer by means of a glass pipet. The samplewas then dried at 1008C for 10 s, pyrolyzed at760 8C for 20 s, and then atomized at 21008C(using a heating rate of 3.48Cyms) for 3 s in Ar480 mlymin qH 20 mlymin purge gas. For the2

interference study, a 1-ml portion of sample solu-tion containing copper(5 ngyml) and interferent(50 mgyml) was pipeted into the molybdenumtube atomizer. The sample solution was measuredunder the same conditions used for the slurrysample.

3. Results and discussion

Ultrasonic agitation has been used as an effec-tive system of homogenizing slurries for GFAAS

w9x and can be used in combination with bothmanual and automated introduction of the slurry.This sample preparation is more profitable thanmagnetic agitation and vortex mixing. Hence, inthe present work, ultrasonic agitation was used forthe slurry preparation.Slurry preparation in aqueous solution is rarely

suitable because most powdered materials undergorapid sedimentation. This sedimentation of sus-pended material usually occurs after mixing theslurry and can be quantified using Stoke’s law.The sedimentation rate depends on the densities ofthe diluent and solid material, the viscosity of thediluent medium and the radius of the sampleparticles. The slurry can be stabilized using ahighly viscous liquid medium. Thus far, viscalexw20–23x, glycerol w8,24x, non-ionic surfactantsw25x and organic solvents of high viscosityw26xhave been used as slurry stabilizing agents. In thestudy of Hoening and Hoeyweghenw24x, testresults showed that the viscosity of glycerol keptdifferent types of particles in suspension for asufficient time. Furthermore, it has been stated thatthe optimum concentration of glycerol was 10%for the homogenization of slurry herbal medicinesamplesw45x. Therefore, in the present work, 10%glycerol solution was used as the slurry stabilizingagent. During the pyrolysis step, despite heavysmoke as a result of decomposition of the glycerol,no significant background signals were generatedin the atomization step.It has been reported in a previous paperw42x

that the optimal purge gas for ETA-AAS with amolybdenum tube atomizer was Ar 480 mlyminqH 20 mlymin and the optimal atomization2

temperature was 21008C in order to obtain maxi-mum copper absorbance and protect the metalatomizer from oxidation by residual traces ofoxygen in the atomizer chamber. The subsequentexperiments for slurry sampling ETA-AAS weretherefore performed under these experimentalconditions.

3.1. Slurry concentration

One of the important factors in the slurry tech-nique is the slurry concentration. Samples of highanalyte content can be analyzed more readily by

184 M.N. Amin et al. / Microchemical Journal 74 (2003) 181–186

Table 1Effect of the particle size of the herbal medicine sample(pow-dered ginseng) on copper absorbance and the R.S.D. of theabsorbance

Particle size(mm) Absorbance R.S.D.

Range Average(%)

)150 – 0.038"0.009 2375–150 112 0.043"0.007 1538–75 56 0.048"0.008 1626–38 32 0.133"0.021 15

-26 – 0.080"0.014 17

The number of replicate measurements was at least five.

the slurry technique than by direct solid sampling,as the slurry can be easily diluted. On the otherhand, when the analyte content of the originalsample is very low, the concentration of the slurrycan be increased accordingly, although pipetingefficiency can deteriorate if slurries are moreconcentrated. Lynch and Littlejohnw27x estab-lished an optimum slurry concentration range forthe analysis of food. The accuracy deterioratedwhen the slurry concentration was)5% wyv, asa result of the excess of matrix. Slurry concentra-tions )5% also resulted in inefficient depositionof the slurry. Because ETA-AAS with a molybde-num tube atomizer is highly sensitive for copper,a slurry concentration of 0.002% was sufficientfor evaluation of the copper content of the herbalmedicine samples. Therefore, this slurry concentra-tion was used for the subsequent experiments.

3.2. Effect of pyrolysis temperature

The effect of the pyrolysis temperature(650–10608C) on the atomic absorption signal of copperwas investigated with a slurried drug sample inorder to achieve sensitive absorption. The atomicabsorption signal of copper was constant for pyrol-ysis temperatures up to 8308C. At temperaturesabove 8308C, the signal decreased abruptly. Inthe atomization of copper with a molybdenum tubeatomizer and a graphite furnace, it was reportedthat the appearance temperature of the coppersignal was approximately 9708C w42x and 9808Cw46x. Thus, the rapid decrease in the copper signalfor pyrolysis temperatures above 8308C might beattributable to vaporization of copper. Consequent-ly, 760 8C was selected as the optimum pyrolysistemperature for the determination of copper inherbal medicine samples, in view of the back-ground absorption and reproducibility.

3.3. Effect of particle size

The particle size of the solid material used tomake a slurry can influence the stabilization, dep-osition and atomization efficiency of the slurry,which in turn can influence both accuracy andprecision w9x. In the studies of Nakamura et al.,smaller particle diameters for tooth samplesw28x,

silicate rocksw29x and quartzw30x demonstratedthe best reproducibility. The effect of five differentparticle sizes of powdered medicine was investi-gated. The results are presented in Table 1. Withthe variation in particle size, significant changesin absorption were observed. The absorption signalgradually increased with decreasing particle size,down to the range 26–38mm. These phenomenamight be due to the good dispersion and homo-geneity of relatively small suspended particles. Forparticle sizes-26 mm, however, absorption sig-nals were lower and R.S.D. higher than for the26–38-mm range. This might be due to coagulationof the particles. The particle size range 26–38mmwas therefore selected.

3.4. Effect of agitation time

The effect of ultrasonic agitation time of amedicine powder slurry sample with a particle sizein the range 26–38mm was investigated. Data forzero agitation time were observed from a slurrysample that was manually stirred for 2–3 s. Thepeak absorption increased for agitation times upto 5 min then reached a plateau. Based on theseresults, an agitation time of 5 min was recom-mended for the medicine powder samples.

3.5. Matrix interferences

Generally, herbal medicines contain largeamounts of Ca, Mg, Na and K. In a previous workw42x, it was observed that these elements severelyinterfered in the atomization of Cu from aqueoussolution. Using the molybdenum tube atomizer,

185M.N. Amin et al. / Microchemical Journal 74 (2003) 181–186

Table 2Results from determination of copper in herbal medicine samples

Sample Average Copperparticle

Added Found Recovery Acidsize(mgyg) (mgyg) (%) digestion(mm)

(mgyg)

Powdered ginseng 32 – 590"37 – 613"23250 852"68 105 –

Powdered pueraria root 32 – 90.9"12.9 – 107"16250 313"31 89 –

Powdered immature orange 32 – 235"29 – 260"35250 522"48 115 –

The number of replicate measurements was three.

the effects of Al, Ca, Fe, Mg, K, Na and Zn onthe copper absorption signal were investigated for10% glycerol solution. The change in Cu signalwas within less than 10% and so none of thematrix elements interfered significantly with thecopper absorption signal. It has been found thatglycerol acts as a chemical modifier in the deter-mination of leadw40x and manganesew45x whenusing a molybdenum tube atomizer. Glycerolmight, therefore, also serve as chemical modifierin copper atomization with the molybdenum tubeatomizer.

3.6. Detection limit, characteristic mass andreproducibility

The detection limit of copper by slurry samplingETA-AAS with the molybdenum tube atomizerwas 72 fg (3=SyN ratio, corresponding to 360ngyg in a 10-ml sample). The characteristic massof copper, defined as the mass of analyte giving apeak height absorbance of 0.0044, with the molyb-denum tube atomizer was 0.29 pg. These valueswere similar to or better than the detection limit(330 ngyg) w11x and characteristic mass(11 pg)w13x obtained using slurry sampling GFAAS forthe determination of copper in biological materials.The reproducibility for the copper atomic

absorption signal by slurry sampling ETA-AASwith the molybdenum tube atomizer was investi-gated. The R.S.D. with a herbal medicine samplewas 7% for seven measurements.

3.7. Determination of copper in herbal medicines

The proposed method of slurry sampling ETA-AAS with a molybdenum tube atomizer wasapplied to the determination of trace amounts ofcopper in herbal medicine samples. The dynamicrange of the calibration curve was up to 50 ngyml(2500 mgyg). When a deuterium lamp was usedas the light source, insignificant backgroundabsorption signals were observed for the herbalmedicine sample. The copper content of the herbalmedicine samples is shown in Table 2. Resultsfrom slurry sampling ETA-AAS are in good agree-ment with those obtained after acid digestion. TheR.S.D. for the determination of copper in herbalmedicine samples by the proposed method wasbetter than 15%. The recovery of spiked copperfrom herbal medicine samples was in the range of89-115%. This is indicative of excellent recoveryof copper.

4. Conclusions

Slurry sampling ETA-AAS with a molybdenumtube atomizer has been shown to be a feasiblemeans of determining copper in herbal medicinesamples. Use of the metal tube atomizer with anargon–hydrogen atmosphere has the benefit ofhigher sensitivity and longer metal tube lifetime(more than 5000 firings) w40–44x compared witha graphite atomizer(200–250 firings) w31x. Thus,the method recommended will be useful for relia-ble measurement of trace levels of copper, with

186 M.N. Amin et al. / Microchemical Journal 74 (2003) 181–186

high accuracy, in various types of herbal medicinesamples.

Acknowledgments

The present research was financially supportedby the Ministry of Education, Culture, Sports,Science and Technology of Japan. A part of thiswork was performed at the Mie University SatelliteVenture Business Laboratory(SVBL).

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