Corrosion Behavior of 55mass%Al–1.6mass%Si–Zn Alloy in Wet ...
Dry and Wet Ashing Techniques in Analyses for Zn,CU,Mn and Fe in Hair 1986
Transcript of Dry and Wet Ashing Techniques in Analyses for Zn,CU,Mn and Fe in Hair 1986
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CLIN.CHEM. 32/5, 739-742 (1986)
CLIN ICA L CHEM IS TRY , V ol. 3 2, N o. 5, 1986 739
Dry - and We t-Ashing Techn iques Compared in Analy ses fo r Z in c, Copper,M an ganese, and Iro n in H airJames K. Friel and C hau D . N gyuenPreparation o f h air specimens for trace-m etal analyses isrout inelydone by we t- o r d ry -a sh ing. We t- ash ing is mo retim e consum ing than dry-ashing and can be dangerous. W ewished to determine if dry -ash ingwas a su i tab le a lte rna tiveto wet -ash ingwi th HCIO4:HNO3or HNO3 alone in prepar ingha ir for measurement o f z inc , copper, i ron ,and manganeseby atomic absorption spectroscopy. Concentrations of Zn ,Cu, an d M n w ere n ot d iffe ren tly a ffected in h air th at was dry-or w et-ashed. A nalytical recovery of th ese elem en ts ad ded tohair sam ples ranged from 102 to 108% ; day-to-day C Vs w ere18 MWcm
was obtained from t he B ar ns te ad p ur if ic at io n system (Barn-D e pa rtmen t o f Biochemistry, Memor ia l U n iv er si ty o f Newfound-l and , S t . J ohn s, N ew foundl and , Canada, A 1B 3X 9.Rece iv e d Oc to b er 2, 1985; a cc ep te d J an u ary 16 , 1986.
stead Co., Boston, MA 02132). W e washed the hair sam pleswith a 10 mLIL solution of a non-ionic detergent (Aca-tionox; American Scientific Products, JL 60085) in de-io niz ed w ate r. Fo r dry-ashing we used Suprapur-gradeH NO 3 (E . M erck , D ain stad t, F .R .G . ); f or wet-ashing w e u sedreagent-grade H NO 3 an d H C1O 4 ( Fis he r S cie ntific C o., F airL aw n, N J 07410).To avo id con tamina t ion , we soaked all glassware in dilute(50 m LIL ) HNO3 for two days, and then rinsed with de-io niz ed w ate r. F or a ll injections in to th e g ra ph ite fu rn ac e weused trace-elem ent-free disposable polypropylene pipettetip s (F ish er S cie ntific Co., no. 22 34 190-1).
Standards. Zn, Cu, Fe, and Mn standards were preparedby diluting 1 g /L stoc k solutions ( Fi sh er S cie nti fi c Co. ) withde-ionized w ater. W orking standards at two concent ra ti on sfor both Zn an d Cu were 1 an d 4 tg/m L, for Fe 0.5 and 5pg/mL, and for Mn 5 and 15 ng/mL.Procedures
S pe ctr op ho to me te r s ettin g. For f lam e p ro ce du re s, w e usedth e glass impact bead in the assays of Zn, Cu, an d Fe. TheZ n h ollow -cath od e lam p current w as 15 m A; wavelength,213.9 ma; slit w idth , 0.7 nm . The Cu hollow -cathode lampcm-rent was 15 mA; wavelength , 324.8 nm ; slit w idth , 0.7nm . The Fe hollow-cathode lamp (multi-elem ent lamp)current was 30 mA; w avelength, 248.3 nm slit width, 0. 2nm . For all f la me a na ly se s th e in tegration time was 2 a;results were calculated from th e average of three read in gs.Flameless analyses for M n were perform ed w ith deuteriuma rc b ack grou nd c orrec tion ; th e M n hollow-cathode lam pcurrent was 15 mA, wavelength , 279.5 nm ; slit width, 0. 2m a.
G ra ph ite fu rn ac e p ro gr am . The basic settings used fo r t hegraphite furnace were: Dry: 1 30 # {1 76 }C ,amp 20 a, hold 60 a;Char 1 07 0 # {1 76 }C ,ange 30s, hold 30s; A tom ize : 24 00 #{ 176 }C ,am p0 a, hold 5 s; C lean: 27 00 # {1 76} C,am p 1 s, hold 2 a. The argong as f lo w was 300 m L/m in du ring th e entire procedure exceptfor 4 s during atom ization , when it was 0 m lim in .
Collection of h air sam ples. For the control study, w ecollected hair from various areas of the head of one of us(J . K . F .), cut it with s ta in le ss -s te el s cis so rs , an d stored it inpolyethylene tubes. The entire fibers (not segm ents) w erein clu ded in th e sam ples.
Using s ta nd ard iz ed p ro ce du re s (12), we a ls o c ol le ct ed hairsamples from 20 women who ha d recently given birth toinfants with neural tube defects or to norm al, healthy full-term infants. F or b ot h groups of w om en, h air was cut aboveth e nape of the neck, as close to th e scalp as possible. Onlythe 1-2 cm of hair closest to the scalp was retained foranalysis.
Preparation o f h air samples. W e placed the hair samplef or t he control study in a 500-mL a cid -w as he d E rle nm ey erflask an d added 20 0 mL of th e A catio nox so lu tion . T he flaskw as covered with Parauilm an d agitated at room tempera-tare for 60 mmwith a mechanical sh ak er. W e th en filtered
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Mean (SD), L9/g dry wt
Percent r ec ov er y, m e an (SD)
Mean (SD), g /g dry w tZn Cu Mn
Certified value 852 (14) 63(3.5) 17.5 (1 .2) 195 (34)TreatmentDry 85 9 (1) 65 (0 .4) 18 (0 .8) 202 (7)HNO3 864 (2) 72 (3) 20 (1.6) 192 (4)HNO3/HCIO4 8 35 (1 00 ) 58 (8) 15.9 (0 .8) 218 (9)740 CLINICA LCHEMISTR Y,Vol .32,No.5, 1986
th e hair through an a cid -w ash ed B uc hn er f un ne l c on ne cte dto a vacuum aspirator and rinsed the sam ple several tim esw ith a to tal volume of 2 L of de-ionized water beforecovering it an d leaving it to dry overnight at 55 #{176}Cn adrying oven. F ou rte en p or tio ns of this con trol sa mp le o f h airwere transferred t o a ci d-washed 15-rnL porcelain crucibles,and 28 portions were placed i n a ci d -wa shed 250-m L P yrexbeakers. A ll sam ples weighed between 100 and 200 mg.
D ry -ashi ng . Ten of th e control hair samples were ashed ina m uffle furnace (Fisher Isotem p m uffle furnace, M odel184) for 12 h at 4 50 # {1 76 }C ,f te r o ve n- dr yin g at 250 #{176}Cor 2 h.After th ey h ad cooled for 1 h, w e removed the sam ples fromt he f ur na ce an d added five drops of Suprapur HNO3. Onehour later, w e returned th e samples to th e m uffle fu rn ace,where they were heated at 250 #{176}Cor 2 h, then ashed at4 50 # {1 76 }Cor an additional 8 h (11). W e again let the samplescool for 1 h, then added 1 mL of S uprapur 11N 03, in 4 mL ofwater, to each beaker. A fter 30 mm , we decanted th esamples into 25-m L volumetric flasks, washed th e b ea ke rswith de-ionized water, and used th e c omb in ed washings tobring th e sam ples to volum e.
Wet -ashing. W e placed 10 c on tr ol s am p le s of hair in 250-mL wide-mouthed Pyrex beakers, and added 10 mL ofreagent-grade H NO 3 to each beak er an d let these sit over-n ight. Ten other control samples of hair were placed insim ilar beakers and left to digest in 5 mL of HNO3 over-n ight. The follow ing morning we added 10 mL ofHC1O4:HNO3 (1:4 by vol) (4) to the latter 10 beakers. A llsamples were covered w ith watch glasses an d refluxed at120-140#{176}Cor about 4 h until the digests were clear and 2-3 m L of acid rem ained. A fter cooling for 1 h we decanted th edigested samples into 25-m L volum etric flasks an d broughtto volum e w ith added rinsings of d e- io niz ed w ate r.
Assessing accuracy and precision. Because no certifiedhair stan dard is currently availab le, w e used U .S . N at io na lBureau of Standards c er tifie d o ys te r t is su e (S ta nd ard R efe r-ence M aterial no. 1566) containing know n am ounts of Z n,Cu, Fe, and M n to assess th e a cc ur ac y of the methods. Fora na ly tic al -r ec ov er y e xp er im e nts , we added a k no wn a mo un to f e ac h metal to fou r portions of c on tr ol h air for each of thedry-ashing, HN O3-ashing, and H C1O4:H NO 3-ashing proce-dures. W e also added kn ow n am ounts o f e ac h metal to puresolutions, which were analyzed w ithout an y interveningashing step s.
M easuring m etals in h air s am ple s. For assay of Zn, Cu,an d Fe, w e aspirated wet- and d ry -d ige sted sam ples dire ctlyinto th e flame. For Mn det erm in a ti on s , we in je ct ed samplesmanually in to th e g ra ph ite tube w ith an E pp en dor f pipette.Sam ple size was 20 i.L , and each sample was analyzed intriplicate. W e repeated th e assays of 10 samples on differentdays to assess b etw ee n-r un v ar ia tio n. W e a ls o r ep ea te d th eassay of on e sample 10 t imes during th e same day to assessw ith in -ru n v ar ia tio n.
S ta tis ti ca l a na ly si s. D ifferences betw een hair concentra-tions in each group w ere assessed b y o ne -w ay analysis ofvariance (13).ResultsM easured concentrations of zinc, copper, and m anganese
(Table 1) in h air treated by dry- and wet-ashings did notdiffer. T he ir on c on ce nt ra tio n measured in hair that w asdry-ashed was lower than that in h air that was wet-ashed.Recovery of know n am ounts of metals added to puresolutions was: 99 1% for Zn, 103 3% for Cu, 96 10%
Table 1 . Concen tr atio n s o f Z In c, C o pp er, M a ng an es e,a nd Iro n In C on tro l H air Treated by Dry- a nd We t-AshingsZn Cu Mn
Dry 216 (4) 44 (1) 0.74 (0.06) 27 (9 )HNO3 216 (28) 42 (5) 0.82 (0.11) 38 (6)bHNO3:HCIO4 227 (31) 44 (6) 0 .77 (0.12) 41 (l2)LValues with differentsuperscrip ts are s ign i fi can t ly d i ffe ren t f rom eachother at p < 0.0 5. n = 10 .f or M n , an d 10 4 1% fo r F e. Recovery of known am ounts ofadded m etals to hair control sam ples w ere com parable fo rz in c, c o pp e r, an d iron in h air treated b y d ry - a n d we t- as hi ng .W e found high concentrations of manganese in h air tre ate dw it h HC 1O4:HNO3, com pared w ith the amount of manga-nese added (T able 2).Zinc, copper, iro n, and m anga nese con cen tratio ns o f o ys-te r tis su e tr ea te d by dry ashing w ere w ith in th e range ofNBS-certifled values (Table 3). The CVs for 10 co ntrol h air
s am ple s r e- an aly ze d on five different days were
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Zn Cu Mn17 7 (26) 32 (1) 2.07 (3.5)19 3 (30) 30 (21) 0 .88 (0 .85)
CLINICA LCHEMISTR Y,Vol .32, No.5, 1986 74 1
in determ ining if dry ashing of hair samples provides asatisfactory alternative to wet-ashing with eitherHC1O4:HNO3 or HNO3 alone.
Zinc. Concentrations of Zn in hair treated by threed if fe re nt a sh in g p ro ce du re s d id not differ (Table 1 ) , sugges t-ing that dry ashing is a suitable preparation for analysis forZn i n hair . V o la til iz at io n losses d ur in g a sh in g h ave n ot beenreported at the ashing temperature we used, but some Znm ay be retained o n c ru ci ble walls (20). W e di d not find anylosses of Zn after dry-ashing, because w e e va po ra te d al lsamples with HN O3, a method known to im pr ov e r ec ov er y(4). Recovery of added Zn (102 7% , Table 2) a nd a cc ur ac yof oyster tissue analysis (Table 3) supports th e su itability ofth e dry-ashing treatm ent prior to analysis of Zn in hair.
Copper. Concentrations of Cu in c on trol ha ir sam ples didnot differ am ong the three treatm ents (Table 1). Recovery(108 13%) a nd a cc ur ac y of Cu determinations after dry-ashing were com parable to those results obtained by wet-d igestion (Tables 2 an d 3) . Contamination with Cu fromporcelain crucibles has been reported previously (11) an dm ay explain the slightly higher analytical recovery f or d ry -ashed samples than was fo un d for w et-a sh ed s am ple s. Cu isnot volatile at the ashing temperature w e u sed (20); howev-er , there have b een re ports (4) of variable and increasedlosses of Cu as the temperature of d ry -ash in g in cre asesabove 4 00 # {1 76 }C .ry-ashing of hair sam ples is a reliablet reatment in preparation for C u analysis.
Manganese. Concentrations of M n in hair treated b y d ry -ashing did not differ significantly from t ho se r es ult s ob -tain ed after w et-a sh in g (T ab le 1 ). M n v olatilizatio n duringdry ashing is unlikely below 550 -6 00 #{ 17 6}C ,lthough som eretention of M n on crucible walls has b ee n re po rte d (21).Recovery of M n is improved with HNO3 (22), and therecovery from porcelain crucibles during dry-ashing in thisexp erim ent w as ac ceptable (108 5% , T ab le 2). Recovery ofMn after wet-digestion by H NO 3:H C1O 4 was high (120 10% , T able 2). This may be attributed to pipetting error orcontam ination, which is a risk because of the very lowcon cen tration s of M n in hair as compared w ith th ose of othermetals (Table 4). W e do not th ink that the slightly low ervalues for M n obtained during dry-ashing are ascribable toeither volatilization or retention of m anganese on crucib lewalls. O ther workers (4) have used ashing aid s for m an ga -nese analyses in tissues, but th is extra step would not beuseful, considering the low concentrations of manganese inhair a nd th e c or re sp on din gly increased p robability of con -tam ination . Recently , G uillard et al. (23) rep orted on m an-ganese analysis in hair after w et-d ig estion in T eflo n b om bs.T h e r ep or te d within-run CV (4% vs 8% ) an d r ec ov er y (1 04 9.2% vs 108 5% ) of M n w ere lower than ours. The slightlylower findings (23) may not justiir th e expense of the m in i-autoclave (current cost of th e P erkin -E lm er n o. 078486,$140.00 U .S.) in com parison with th e cost of porcelain orsilica cru cib les ($6 .00 U .S ., F ish er Scien tific C o.).
T ab le 4 . C on ce ntra tio ns o f Z in c, C op pe r, a ndManganese In the HaIrofMothersofInfantsithNeural Tube Defect or Normal Healthy ChIldren
Neur al t ub e d ef ec t ( n = 10 )No rmal (n = 10 )
Mean (SD) , pQ/g dr y w t
Iron. Measured concentrations of Fe in hair were signiiI-cantly low er in s am ple s tre ate d by dry-ashing as com paredwith w et-ashing (Table 2). Fe concentrations of oyster tissueanalyzed after preparation by the dry-ashing technique(Table 3) were w ith in the e xpe cted ran ge (202 7 tg/g vs19534 jtg/g, c ertified value). M atrix d ifferenc es m ay haveaccounted fo r lo we r measured Fe concentration in hair ascompared with the measured Fe concentration in oystertissue. Some Fe may volatilize during dry-ashing, butp re vi ou s r es ea rc he rs have reported no Fe losses at 50 0 or600#{176}C24). R etention losses for Fe have also b ee n r ep or te dd ur in g d ry -a sh in g (27), although our analytical recovery byth e d ry -a sh in g te ch niq ue (110 9% ) w as com parab le to thato f th e HNO3 digestion m eth od (114 14% ) and high er th anth e r ec ov er y by the HC1O4:HNO3 digestion technique (100 11 % , T a ble 2). Recovery of Fe a ft er d ry -a sh in g w as h ig he rthan that found for Zn, Cu, or M n. P re vio us r es ea rc he rsh ave also re porte d higher recovery for Fe as compared withthat found for Zn an d Cu in liver (19) an d hair (5). Dry-ashing m ay not be suitable prior to determining Fe in hair.Dry-ashing for F e h as p reviou sly b ee n r ec omm en de d onlyfor large and (or) fa tty s am ple s (20). W e found that treat-ment with HNO3 gave results comparable w ith those ob-tained by H C1O 4:H NO 3 d igestion (T ab le 1). Treatm ent w ithHNO3 has previously been reported to be suitable for Feanalysis in h air (25). It should be noted t ha t, i f t emper at ur esexceed 1 50 # {1 76 }Cu rin g w et-a sh in g, som e Fe may be lost (4).
W atting and Wordale (4 ) reported that concentrations ofm etals in fish tissu e a fter w et. or d ry-ashin g were compara-ble . They suggested that contam ination during w et ox id a-tio n m ay resu lt from th e ad dition of reagents to samples, th econtam ination frequently exceeding th e amounts in thes am ple its elf. W e used Suprapur-grade HN O3 on ly d urin gd ry -a sh in g, b ec au se each s am p le r eq uir ed only 1 m L. Supra-pur H C1O 4 is not available an d the large amount of acidrequired for wet digestion (10 m L per s am p le ) p re clu de s th eu se of S up rapu r HNO 3 b ecau se of th e e xp en se . Thereforeblank values obtained w ith reagen t-grad e acid during w et-ashing were higher than Suprapur blank values obtainedduring dry-ashing. B lank values from dry ashing of 18b la nk s, c al cu la te d in m icrogram s per gram of dry hair, w erelow for Zn (0.04), Cu (0 .04), and M n (0 .02), but were high forFe (0 .85 ) -about 5-8% o f th e usual F e con cen tration in hair.A fu rther d ifficulty d urin g w et oxidation is t he p re pa ra ti onof acid-matched sta nda rd s, b ecau se the am ount of acid thatis e vap orated differs fo r e ac h sample.W e suggest that dry-ashing is a suitable treatment fo rhair before analyses for Zn, Cu, and M n. Wet-digestion w ithHNO3 rather than H C1O 4:H NO 3 is acce pta ble before th eanalysis of Fe in hair. Z n, C u, and M n concentrations in hairanalyzed after dry-ashing are presented in Table 4 formothers who gave birth to infants w ith neural tu be defectand mothers of normal healthy children . The results suggesta d if fe re nc e in trace-m etal status of th ese two groups. Alarger stu dy of th ese tw o g ro up s is curre ntly in p ro gress.
W e thank D r. C lark Fraser a nd H il lo ry V a va so ur fo r collectingth e h air sam ples, Drs. Gene H erzberg and L A . W . Feltham fo rtheir helpful com ments, and C arol M urph y for p re pa rin g th ismanu sc ri pt . F in an ci al s uppor t w as receiv ed fro m th e PresidentsNSERC Fund.
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