iron and zinc by aas

8/14/2019 Iron and Zinc by AAS

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Mikrochimica Ac ta [Wien] 1974, 65--729 by Springer-Verlag 1974

Clinical Biochemistry La boratories, G overn me nt D epartm ent of Patholog y,Sepoy Lines, Singapore, 3D e te r mi na t i o n o f Se r um Iro n a nd Z i nc by to m i c

bso r pt i o n Spe c tr o sc o pyB y

Kum -Tatt Lee and Edward JacobReceived September 5, 1972)

IntroductionT h e u s e o f a t o m i c a b s o r p t i o n s p e c t r o s c o p y ( A A S ) a s a n a n a -

l y t i c a l t o o l i n t h e c l i n i c a l l a b o r a t o r y h a s i n t r o d u c e d p r e c i s i o n a n dr e l i a b i l i t y i n t h e d e t e r m i n a t i o n o f m e t a l s i n b i o l o g i c a l f l u i d s a n dt i ssues . The method i s en t i r e ly spec i f i c fo r a g iven e l emen t . I t hast h e d i s t i n c t a d v a n t a g e o v e r e x i s t i n g m e t h o d s i n t h a t i n t e r f e r e n c ef r o m o t h e r m e t a l s i s a l m o s t c o m p l e t e l y n e g a t e d , a s n o t w o m e t a l sa r e k n o w n , w h i c h h a v e t h e s a m e r e s o n a n c e lin e s. W h i l e s u c c es s h a sb e e n a c h i e v e d i n t h i s f i e l d w i t h s e r u m c a l c i u m a n d m a g n e s i u md e t e r m i n a t i o n s , t h e a p p l i c a t i o n o f t h is te c h n i q u e t o s e r u m i r o n h a ss h o w n t h a t t h e d i re c t a s p i r a ti o n o f s e r u m i n to t h e f la m e a s d e s c ri b e db y R o d g e r s o n a n d H e l f e r 1 a n d b y S p r a g u e a n d S la v in ~ is n o t a se f f e c t iv e a s t h e c h e l a t i o n o f i r o n a n d su b se q u e n t e x t r a c t i o n o f th ec o m p l e x i n t o a n o r g a n i c so l v e n t , w h i c h is t h e n a n a l y se d b y A A S .I n th e f o r m e r c a se , t h a t o f u s i n g d i l u t e d s e r u m o r p la sm a , p o s s i b l ec o n t a m i n a t i o n d u e t o th e p re s e n c e o f h a e m o g l o b i n i r o n c a n c o n -t r i b u t e s i g n i f i c a n t e r r o r t o t h e i r o n v a l u e e x h i b i t e d i n n o r m a l s e r u m .F u r t h e r m o r e , t h e h i g h p r o t e i n c o n t e n t t e n d s t o c l o g t h e b u r n e r h e a d ,r e su l ti n g i n u n s t a b l e r e a d i n g s . Z e t t n e r 3 f o u n d t h a t t h e d i r e c t a sp i r a -t i o n o f s e r u m i n t o t h e f l a m e g a v e su f f i c i e n t l y a c c u r a t e d a t a i n t h er a n g e a b o v e 2 0 0 / ~ g i r o n p e r 1 0 0 m l s e r u m , b u t u n r e l ia b l e r e s u lt sw e r e e n c o u n t e r e d i n t h e l o w e r r a n g e s b e c a u s e o f m a t r i x i n t e r f e r e n c e

~" Singapore Institute of Standards an d Industrial Rese arch, 179 R ive rValley Road, Singapore-6, Republic of Singapore.Mikr ochi m Acta 1974/1 5


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D e t e r m i n a t i o n o f S e r u m I r o n a n d Z i n c 6 7

Assay ProcedureI n t o g l as s s t o p p e r e d c e n t r i f u g e t u b e s (1 5 m l ) a r e t r a n s f e r r e d

1 .0 m l o f s e r u m ; 1 .0 m l w a t e r ( b l a n k s a m p l e ) ; a n d 1 .0 m l o fw o r k i n g i r o n a n d z i n c s t a n d a r d so l u t i o n ( 0.2, 0 .5 , 1 .0, 1 .5 , 2 .0 # go f m e t a l p e r m l ) . 4 m l o f w a t e r , 1 .0 m l o f 1 .0 N h y d r o c h l o r i c a c i d ,2 .0 m l o f 0 .1 M so d i u m su l p h i t e so l u t i o n a n d 2 .0 m l 1 0 ~ t ri -c h l o r o a c e t i c a c i d a r e a d d e d t o e a c h t u b e . T h e c o n t e n t s a r e m i x e da n d t h e t u b e s p l a c e d i n a b o i l i n g w a t e r b a t h f o r 5 m i n u t e s . T h ec o n t e n t s o f e a c h t u b e a r e t h e n s t i r r e d w i t h a f i n e g l a s s r o d , a n da f t e r c o o l i n g , t h e t u b e s a r e c e n t r if u g e d a n d 8 .0 m l o f t h e c l e a rs u p e r n a n t a n t f r o m e a c h t u b e a r e t r a n s f e r r e d t o 1 5 m l g l a s s s t o p p e r e dt e s t t u b e s . O n e m l o f P A P H Y s o l u t i o n i s a d d e d t o e a c h t u b e , t h ec o n t e n t s m i x e d a n d a l l o w e d t o s t a n d f o r 5 m i n u t e s . T h i s i s f o l l o w e db y th e a d d i t io n o f 1 .0 m l o f 4 0 ~ p o t a s s i u m h y d r o x i d e a n d 1 .0 m lo f a m y l a l c o h o l . T h e t u b e s a r e s t o p p e r e d a n d t h e c o n t e n t s m i x e dv i g o r o u s l y f o r a f e w s e c o n d s . T h e t w o l a y er s a r e a l l o w e d t o s e p a -r a t e a n d t h e o r g a n i c l a y e r i s t h e n a sp i r a t e d i n t o t h e a i r - a c e t y l e n ef l a m e .

D eterm inat io n o[ to ta l i ron b ind ing capac ity. T h e m e t h o d o fP e t e rs a n d c o w o r k e r s 7 w a s a d o p t e d w i t h s li gh t m o d i f ic a t io n s . 0 .0 5m l o f t h e f er ri c a m m o n i u m c i tr a te s o l u t io n is a d d e d t o 0 . 5 m l o fse r u m i n a t e s t t u b e , a n d a l l o w e d t o r e a c t f o r 5 m i n u t e s . 0 . 2 m l o ft h e r e s i n i s t h e n a d d e d a n d t h e c o n t e n t s o f t h e t u b e m i x e d c o n -t i n u o u s l y f o r 5 m i n u t e s . B u f f e r (2 .5 m l ) is a d d e d a n d a f t e r m i x i n gw e l l , t h e t u b e i s c e n t r i fu g e d . T o 2 .0 m l o f t h e c l e a r su p e r n a t a n t i sa d d e d 3 .0 m l o f w a t e r a n d t h e 5 .0 m l a l i q u o t i s t h e n t r e a t e d a sd e sc r i b e d f o r s e r u m i r o n .

In s t rum en ta t ion . A U n i c a m S P 9 0 a t o m i c a b s o r p t i o n s p e c t r o -p h o t o m e t e r , f i t t e d w i t h c a t h o d e l a m p s o f i r o n a n d z i n c , w a s u s e d .T a b l e I . I n s t r u m e n t a l P a r a m e t e r s f o r M e a s u r e m e n t s o f I ro n a n d Z i n c b y A A SP a r a m e t e r M e a s u r e m e n t

I r o nL a m p C u r r e n t . . . . . . . . . . . . . . . . . . . . 1 5 m A m pA b s o r p t i o n W a v e l e n g t h . . . . . . . . . . . . 2 4 8 .3 n mE n t r a n c e s li t . . . . . . . . . . . . . . . . . . . . . 2 / ~ mA i r p r e s s u r e . . . . . . . . . . . . . . . . . . . . . . 4 0 p s iA i r f l o w ' : . . . . . . . . . . . . . . . . . . . . . . . . 5 . 5 1 p e r m i n .A c e t y l e n e p r e s s u r e . . . . . . . . . . . . . . . . 8 .0 p s iA c e t y l e n e f l o w * . . . . . . . . . . . . . . . . . . . 1 .2 1 p e r m i n .S a m p l e a s p i r a t i o n r a t e . . . . . . . . . . . . . 2 .5 m l p e r m i n .

Z i n c1 2 m A m p2 1 3 . 9 n m2 m4 0 p s i

5 . 5 1 p e r r a i n .8 . 0 p s i1 .3 1 p e r m i n .3 . 0 m l p e r m i n .* M e a s u r e m e n t s o f a i r a n d a c e t y l e n e f lo w a r e e x p r e s s e d i n a r b i t r a r y u n i t si n d i c a t e d o n t h e f lo w m e t e r s o f th e g a s r e g u l a t o r u n i t s o f t h e U n i c a m S P 9 0 a t o m i ca b s o r p t i o n s p e c t r o p h o t o m e t e r .

5*


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68 K .-T. Lee and E. Jacob:T h e a b s o rp t io n l in e o f 2 48 .3 n m a n d 2 1 3 .9 n m fo r i r o n a n d z in cr e sp e c ti v e ly , w e r e u s e d t h r o u g h o u t t h e s t u d y .

S t a b i l i t y o f t h e b u rn e r h e a d , o p t i c a l a n d e l e c t ro n i c s y s t e ms i sv e r i f i e d b y m o n i t o r i n g t h e z e r o - p o i n t f o r s e v e r a l m i n u t e s w h i l ea my l a l c o h o l i s c o n t in u o u s ly a s p i r a t e d i n to t h e f l a me . Th e i n s t ru -me n t i s a d ju s t e d t o t h e o p e ra t i o n c o n d i t i o n s a s l i s t e d i n Ta b le I .M e a s u r e m e n t s o f a t o m i c a b s o r p t i o n T h e b l a n k , s t a n d a r d s a n dt h e u n k n o w n s a m p l e s a r e a s p i r a t e d i n t o t h e f l a m e , a n d t h e a b -s o rp t io n o f t h e s a mp le s i s d i r e c t l y r e a d o n t h e s c a l e o f t h e i n s t ru -m e n t . T h e c o n c e n t r a t i o n o f i r o n a n d z i n c i n t h e u n k n o w n i s t h e n

c o m p u t e d f r o m t h e c a l i b r a t i o n c u r v e s .

R e su l ts a n d i s c u s s i o nM o s t p u b l i s h e d m e t h o d s f o r t h e d e t e r m i n a t i o n o f s e r u m i r o nu t i l i z e a n i n i t i a l s p l i t t i n g o f t h e i r o n f ro m i t s p ro t e in c o mb in a t io nb y e x p o s u r e t o a c i d f o l l o w e d b y p r e c i p i t a t i o n o f t h e s e r u m p r o t e i n .B o t h w e l l a n d M a l l e t t s a n d P e t er s a n d c o - w o r k e r s~ h a v e s h o w n t h a tt h e u s e o f s t r o n g 2 t o 6 N) h y d ro c h lo r i c a c id g iv e s c o m p le t e e x -

t r a c t io n o f t h e i r o n f r o m p r o t e in . B a r k a n 9 i n t r o d u c e d t h e u s e o fd i l u t e 0 .2 t o 0 .3 N) h y d ro c h lo r i c a c id, w h i l e P e t e r s a n d c o -w o rk e r svc o m b i n e d d i lu t e h y d r o c h l o r i c a c i d w i t h a re d u c i n g a g e n t t hi o -g ly c o li c ac id ) a n d o b t a in e d s a t i s f a c to ry i r o n e x t r a c t i o n . R a m s a y 1~u s e d s o d i u m s u l p h i t e t o r e l e a s e t h e i r o n f r o m i t s c o m b i n a t i o n . W eh a v e c o m b i n e d s u c c es s fu l ly t h e u s e o f d i lu t e h y d r o c h l o r i c a ci d a n ds o d iu m s u lp h i t e . Du r in g t h e i n i t i a l d e p ro t e in i s a t i o n w i th t r i c h lo ro -a c e t i c a c id , r e l e a s e o f t h e i r o n a n d z in c f ro m p ro t e in i s f a c i l i t a t e db y b o i l i n g i n t h e p r e s e n c e o f h y d ro c h lo r i c a c id . Th e i r o n i s s imu l -t a n e o u s l y r e d u c e d b y t h e a c i d a n d s o d i u m s u l p h i t e . I n o r d e r t oa v o id a n y t r a p p in g o f t h e me ta l l i c i o n s i n t h e i n t e r s t i c e s o f t h ep re c ip i t a t e , i t wa s n e c e s s a ry t o s t ir t h e m ix tu r e we l l w i th a f i n eg la ss r o d . T h e i r o n a n d z i n c a r e e v e n l y d i s t ri b u t e d t h r o u g h o u t t h ea q u e o u s p h a s e . T h i s i s b o r n e o u t b y t h e r e s u l t s o f t h e r e c o v e r ye x p e r ime n t s , wh ic h i n d i c a t e t h a t n e i t h e r me ta l i s l o s t i n t h e p ro t e inprec ip i ta te .

A d i s t i n c t a d v a n t a g e o f t h i s a t o m i c a b s o r p t i o n m e t h o d i s t h a tt h e c o n c e n t r a t i o n o f i r o n a n d z i n c i n t h e a m y l a l c o h o l l a y e r w h i c his a s p i r a t e d i n to t h e f l a m e fo r a n a ly si s , is id e n t i c a l t o t h a t i n t h eo r ig in a l s e ru m. I f i t i s d e s i r e d , a s ma l l e r v o lu me o f o rg a n i c s o lv e n tc a n b e e m p l o y e d i n t h e fi n a l e x t ra c t i o n . T h i s w o u l d r e s u lt i n ac o n c e n t r a t i o n o f t h e s e ru m c o n s t i t u t e n t s b e in g i n v e s t i g a t e d , a n d i sa g r e a t a d v a n t a g e w h e n l o w v a l u e s a r e e n c o u n t e r e d . W i t h m o s t


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D e t e r m i n a t i o n o f S e r um I r o n a n d Z i n c 6 9c o t o r i m e t r i c p r o c e d u r e s , t h e s e r u m c o n s t i t u e n t s u n d e r g o a d i l u t i o nb e f o r e t h e f i n a l a n a l y s i s is c a r r i e d o u t .

R e p r o d u c i b i l i t y T h e r e s u l t s o f 10 a n a l y s e s f o r s e r u m i r o n a n dz i n c o n e a c h o f 2 p o o l e d s e r a b y t h e p r o c e d u r e d e s c r i b e d a r e s h o w ni n T a b l e I I. T h e p r e c i s i o n o f t h e m e t h o d f o r s e r u m i r o n i n t h e r a n g e

T a b l e I I . P r e c is i o n o f R e p e a t e d A n a l y s e sI ro n /~g % Zi nc /~g %

M e a nS . D .C . V .

S e r u m A S e r u m B S e r u m A S e r u m B24.0 54.0 80.0 143.025.0 53 5 82.0 145.026.5 57.0 81.5 143.024.5 55 5 83.0 145.526.0 55 5 82.5 145.024.0 54.0 81.0 144.025 5 53 5 80.5 144.526.5 55 5 83.5 145.024.5 55 5 80.5 143.525.0 54 0 81.5 144.525.2 54 8 81.6 144.3 0.94 _+ 1.16 1.15 0.883.75 2.12 1.41 0.62

b e l o w 6 0 g / 1 0 0 m l , t h e c l i n i c a l ly i m p o r t a n t r a n g e , is s h o w n t o b eg o o d , a s , w i t h t h e s e r u m p o o l s o f l o w i r o n c o n t e n t , 1 0 r e p e a t e da n a l y s e s o n t h e t w o s a m p l e s g a v e m e a n s o f 2 5 . 2 0 . 9 4 (S . D . ) a n d5 4 . 8 + 1 . 1 6 (S . D . ) . T h e c o e f f i c i e n t s o f v a r i a t i o n w e r e 3 7 5 ~ a n d2 . 1 2 ~ r e s p e c t i v e l y .

R e c o v e r y e x p e r i m e n t s T h e r e s u l ts o f a s e r i e s o f r e c o v e r y e x p e r i -m e n t s a r e s h o w n i n T a b l e I II . T h e s e w e r e c a r r i e d o u t b y t h e a d -

T a b l e I I I . R e c o v e r y o f I r o n a n d Z i n c A d d e d t o S e r u mS e r u m I r o n S e r u m Z i n c

I r o n C o n t e n t i n g / 1 0 0 m l Z i n c C o n t e n t i n /~ g / 1 0 0 m lA d d e d F o u n d E x p e c t ed S e ru m A d d e d F o u n d E x p e c t e dV a l u e

50 76 78 80 50 130 13050 82 83 87 50 135 137100 167 160 109 50 161 15950 136 135 125 100 223 22550 160 158 127 100 224 22750 280 280 153 50 201 203

S e r u mV a l u e28336085108230

d i t i o n o f 2 0 /~ 1 o f a d i l u t e d s t a n d a r d s o l u t i o n o f i r o n a n d z i n c t o1 m l o f s e r u m , t o g i v e f i n a l a d d e d i r o n a n d z i n c c o n c e n t r a t i o n s o f


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70 K.-T. Lee and E. Jacob:50 ,ug or 100/~g per 100 ml of serum. The recoveries were calcu-lated by comparing the results obtained before and after theaddition of the standard solutions. The sera chosen for the recoveryexperiments had a wide concentration range of iron and zinc, andthe observed consistent recoveries of added metal demonstrates theprecision of the method over a wide range.

C o mp a r i so n w i th a c o lo r ime t r i c p ro c e d u re Iron analyses werecarried out on the same serum samples by the ASS procedure andRams ay's i~ method. The results obtained are shown in Table IV.

Table IV. Comparison of Iron Values by Colorimetry and AASSample No. Serum Iron,/*g per 100 ml T.I.B.C., ~g per 100 mlColorimetry AAS Colorimetry AAS

1 62 62 388 3982 72 69 241 2333 35 25 316 3274 60 59 292 2705 80 71 270 2516 75 69 241 2327 70 71 324 3128 72 66 238 2299 250 240 392 38610 117 109 327 319

There is reasonable agreement between the methods. Colorimetricprocedures in general, are exposed to interference from pigments,other serum constituents and turbidity of the final coloured solutions.These drawbacks are of no consequence to atomic absorptionmethods, which are specific for the metal being investigated. Inter-ference from turbidity might explain the higher values obtained byRamsay's procedure.

Table V. Normal Values with New ProcedureNo. of Contents in /~g per 100 ml serumpersons Range Mean S.D.

Serum Iron . . . . . . . . . . . . . . . . 57Total Iron-binding Capacity 57Serum Zinc . . . . . . . . . . . . . . 57* Observed range.

76 --181 128.0 _+26.0230 --410 326.0 - -99 --146 122.0 + 24.0

N o r m a l v a l u e s The analyses of 57 morning samples fromhealthy normal individuals for serum iron, the total iron-bindingcapacity and serum zinc levels yielded the results shown in Table V.


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Determination of Sermn Iron and Zinc 71The mean value for serum iron is 128/~g per 100 ml, with a rangeof 76 to 181 g per 100 ml, while the mean for TIBC is 326 g per100 ml, with a range of 230 to 410/~g per 100 ml. The range ob-tained for serum zinc is 99 to 146/~g per 100 ml, with a mean valueof 122 g per 100 ml. The skewness of the distr ibution of the valuessuggest that a non-normal distribution may be characteristic of agroup of subjects who are heterogenous with respect to race, age,dietary habits and other characteristics. Table V summarizes thenormal values obtained with the present procedure.

ummaryDeterminat ion o[ Serum Iron and Zinc by Atomic Absorpt ionSpectroscopy

This paper presents a rapid sensitive atomic absorption spectro-scopic method for the simultaneous determination of serum ironand zinc levels. It is also applicable to the detection of these metalsin urine. The technique is based on the ability of pyridine-2-alde-hyde-2-pyridyl hydrazone (PAPHY) to form stable cationic bis com-plexes with iron or zinc, in the +2 oxidation state, while in anaqueous medium. When the pH of the medium is shifted towardsthe alkaline side, the charged complexes attain neutrality and arequantitatively extractable into an organic solvent. Thus, the metalsare free of the interfering effects of serum constituents, and areeasily estimated by atomic absorption spectroscopy.

ZusammenfassungEine rasche und empfindliche Methode zur gleichzeitigen Bestimmungvon Eisen und Zink im Serum durch Messung der Atomarabsorption wurdeangegeben. Sie eignet sich auch zum Nachweis der beiden Metalle im Ham.Sie beruht auf der Bildung stabiler kationischer Bis-Komplexe des zweiwer-tigen Eisens oder Zinks in w~if~riger Phase mit Pyridin-2-aldehyd-2-pyridyl-hydrazon (PAPHY). Wird das pH nach der alkalischen Seite verschoben, soverlieren die Komplexe ihre Ladung und werden mit einem organischenL6sungsmittel extrahierbar. Sonstige Serumbestandteile st6ren nicht.

References1 D. O. Rodgerson and R. E. Helfer, Clin. Chemistry 12, 338 1966).s S. Sprague and W. Slavin, Atomic Absorption Newsletter 4, 228 1965)Perkin Elmer Corp. Norwalk Conn.


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7 2 K . - T . L e e a n d E . J a c o b : D e t e r m i n a t i o n o f S e r u m I r o n a n d Z i n c3 A . Z e t t n e r , L . C . S y l v ia , a n d L . C a p a c h o - D e l g a d o , A m e r . J. C l in . P a th .45, 533 (1966) .4 j . E . A l l a n , S p e c t r o c h i m . A c t a 1 7 , 4 7 6 ( 1 9 6 1 ).9 5 F . L i o n s a n d K . V . M a r t i n , J . A m e r . C h e m . S o c . 8 0 , 3 8 5 8 (1 95 8).6 j . F . G e l d a r d a n d F . L i o n s , J . A m e r . C h e m . S o c . 8 4 , 2 2 6 2 ( 1 9 6 2 ) .7 T . P e t er s , T . G i o v a n n i e l l o , L . A p t , a n d J . F. R o s s , J . L a b . C l in . M e d .48 , 274 (1956) .8 T . H . R o t h w e l l a n d B . M a l l e t t , C l i n . S c i . 1 4 , 2 3 5 1955).9 G . B a r k a n , K l i n . W s c h r . 6 , 1 6 1 5 ( 1 9 2 7 ) .

1 0 W . N . M . R a m s a y , C l i n . C h i m . A c t a 2 , 2 1 4 ( 1 9 5 7 ) .

iron and zinc by aas

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