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An investigation of the application of 3-aminophthalhydrazide to the forensic detection of blood stains

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An investigation of the application of 3-aminophthalhydrazide to the forensic detection of blood stains

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Content AN INVESTIGATION OF THE APPLICATION OF 3-AMINOPHTHALHYDRAZIDE TO THE FORENSIC DETECTION OF BLOOD STAINS •A T h e s is P r e s e n t e d t o The F a c u lt y o f The C h e m istry D ep artm en t U n i v e r s i ty o f S o u th e rn C a l i f o r n i a I n P a r t i a l F u l f i l l m e n t -of th e R eq u ire m e n ts f o r th e D egree M a ste r of S c ie n c e by .Norman J e f f e r i s Ho I t e r May, 1938 UMI Number: EP41494 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dlsf Rsbliilbteg UMI EP41494 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 This thesis, written by .....................H p M M .. J.!,..H0LTER under the direction of AJLs_ Faculty Com m ittee, and approved by all its members, has been presented to and accepted by the Council on Graduate Stu dy and Research in partial fulfill m ent of the requirements for the degree of MASTER OF S CIENCE Dean ' Secretary Date J.U N E .r ...1 9 3 a Faculty Committee Chairman (UJ (I 3 ? TABLE OF CONTENTS CHAPTER PAGE 1 INTRODUCTION . . . ♦ ........................................................... 4 E a r l y b lo o d t e s t s • • • • • • • • • « • • • • 4 V alue o f b lo o d t e s t s • • • • » • • • • • • • • 5 T e s ts now i n g e n e r a l u s e ............................. 8 S ta te m e n t o f th e p ro b lem • • • • • • • • • « • 18 O r g a n iz a tio n o f re m a in d e r o f t h e s i s . • . • . 19 I I THE REACTION BLOOD-HYDROGEN PEROXIDE-3 - AMINOPTHALHYDRAZIDE The ch em ilu m in e sc en c e o f 3 -a m in o p h th a lh y d ra z id e 20 The c o m p o s itio n o f b lo o d s t a i n s ............................. . 23 P o s t u la te d m echanism s • • • • • • • • .................... 26 I I I . THEORETICAL CONSIDERATIONS • .................................................. 28 P h o to g ra p h ic and p h o t o e l e c t r i c ra d io m e tr y . • 28 C hem ical K in e tic s » « • • • • • • • • • • • • 35 IV EXPERIMENTAL.............................................................. 41 D e n s ito m e te r c o n s t r u c t i o n • » • • * • • • • • • 41 M easurem ents o f c h em ilu m in escen ce * • • • • • • 46 Mechanism o f 3 -a m in o p h th a lh y d ra z id e o x i d a t i o n . 54 The r a t e o f d e c o m p o sitio n of p e ro x id e • • • • • 62 M is c e lla n e o u s e x p e rim e n ts ........................ 78 V CONCLUSIONS . ............................................................................... 83 BIBLIOGRAPHY................................................................................................88 LIST OF PLATES NUMBER PAGE I D e n s ito m e te r i n C r o s s - S e c tio n • . . . ♦ • • 42 I I D e n s ito m e te r View ...............................................43 I I I Forms o f S i l v e r D e p o s its 46 LIST OF TABLES I L in e a r D im ensions o f E r y th r o c y te s . . . . . 11 I I C h a r a c t e r i s t i c Curve D ata 48 I I I C hem ilum inescence I n t e n s i t y as a F u n c tio n o f H ydroxide C o n c e n tr a tio n , • • • • • • • • • 51 IV C hem ilum inescence I n t e n s i t y as a F u n c tio n o f H ydrogen P e ro x id e C o n c e n tr a tio n ......................... 52 V C hem ilum inescence I n t e n s i t y a s a F u n c tio n o f Time • ..................... • • • • • « • • • • « • • 54 VI The D e co m p o sitio n o f Hydrogen P e ro x id e by C a ta la s e * • • • • • * • • • « • • • • * « • 65 V II The D e co m p o sitio n o f P e ro x id e by B lood • . . 66 V I I I The D eco m p o sitio n o f P e ro x id e by B lood . . ♦ 67 IX S p e c i f i c R e a c tio n R a te s f o r Blood C a t a l y s i s 67 X The D e co m p o sitio n of P e ro x id e by H em oglobin 68 XI The D e co m p o sitio n o f P e ro x id e by H em oglobin 69 X II S p e c if i c R e a c tio n R a te s f o r H em oglobin C a ta l y s i s .................... 69 X I I I The D e co m p o sitio n o f P e ro x id e by H em oglobin 71 XIV The R a te o f P e ro x id e D e co m p o sitio n a s a F u n c tio n o f H em oglobin C o n c e n tr a tio n • . ♦ . 75 XV The R ate o f P e ro x id e D e co m p o sitio n as a F u n c tio n o f H em oglobin C o n c e n tr a tio n • • ♦ . 75 LIST OF CURVES 1 C hem ilum inescence I n t e n s i t y as a F u n c tio n o f ‘ Time • * • • • • • « • • • • ................................... 56 I I The R a te o f D e co m p o sitio n o f P e ro x id e by B l o o d ................................................. 67 I I I The R a te o f D eco m p o sitio n o f P e ro x id e by H e m o g lo b in ............................. 70 IV The R ate o f D e co m p o sitio n o f P e ro x id e by H em oblobin ............................. 72 V The R a te o f P e ro x id e D e co m p o sitio n as a F u n c tio n of H em oglobin C o n c e n tr a tio n • . • . 77 CHAPTER O N E INTRODUCTION EARLY BLOOD TESTS The v a lu e o f h lo o d c l a s s i f i c a t i o n and o f t e s t s f o r th e p re s e n c e o f b lo o d i s o b v io u s t o any p r e s e n t day c r i m i n o l o g i s t a lth o u g h t h i s h a s n o t alw ays b een th e c a s e . In s p i t e of th e f a c t t h a t m ic ro s c o p ic e x a m in a tio n o f b lo o d was made in th e m id d le of t h e n i n e t e e n t h c e n t u r y , l i t t l e im p o rta n c e was a t ta c h e d t o th e r e s u l t s , and e v id e n c e from su c h e x a m in a tio n s was n o t a d m itte d in c o u r t . As e a r l y as 1679, how ev er, th e te s tim o n y o f a laym an, who d e s c r ib e d a s t a i n as b e in g a ’’s o r t o f w h i t i s h b lo o d ” , was adm itted'*’ w i t h w hat e f f e c t on j u s t i c e we can o n ly c o n j e c t u r e . The d i f f e r e n c e in s i z e b etw een human and o t h e r a n im a l e r y t h r o c y t e s was known b e fo r e c h e m ic a l and b i o l o g i c a l t e s t s w ere d e v e lo p e d , w h ich was aro u n d 1850. When th e im p o rta n c e of in f o r m a tio n a b o u t b lo o d became a p p a re n t t o i n v e s t i g a t o r s o f t h a t p e r i o d , many f lim s y and a b s u rd t e s t s w ere d e v is e d . F o r ex am p le, i t was th o u g h t t h a t m ale b lo o d c o u ld be d i s t i n g u i s h e d fro m fem a le b lo o d by m ix in g i t w ith 2 s u l f u r i c a c i d and s m e llin g i t . ^ Henry R hodes, C lues & Crime (London; Jo h n M urray 1933) p . 134. 2 T a y lo r , P r i n c i p l e s & P r a c t i c e o f M edical J u r i s p r u d e n c e . (London: C h u r c h il l 1&28J *" 4 T estim o n y r e g a r d i n g s u s p e c te d b lo o d s t a i n s soon became more f r e q u e n t a lth o u g h th e v a l i d i t y o f th e c o n c lu s io n s seems r a t h e r d o u b t f u l . T h is was p r o g r e s s w h ic h , i n s p i t e o f i t s g ro p in g n a t u r e , was s o r e l y n eed ed as many c a s e s c o u ld be m a te r i a l l y c l a r i f i e d by th e m ost e le m e n ta ry know ledge o f th e c o n s t i t u t i o n o f th e b lo o d o f d i f f e r e n t a n im a ls . F o r e x am p le, one c a se c i t e d by T a y lo r^ c o n c e rn e d a m urder in A u s t r a l i a i n 1851 w h e re in a s h e e p h e rd e r was s u s p e c te d and fo u n d t o have a b lo o d y axe w i t h w h ich he c la im e d he had s l a u g h te r e d some s h e e p . E v i d en ce was g iv e n t h a t th e b lo o d on th e axe was human and t h i s c o n t r i b u t e d t o th e c o n v ic ti o n o f th e s u s p e c t . I d e n t i f i c a t i o n o f human b lo o d by e r y t h r o c y t e m easurem ent was a d m itte d i n 1857 i n th e N a tio n c a se i n E ngland a lth o u g h s e v e r a l u n j u s t i f i e d c o n c lu s io n s w ere a l s o m ade. One u n s c i e n t i f i c c la im t h a t was made was t h a t i t c o u ld be d e te rm in e d by c o rp u s c le m easurem ent w h e th e r a k n if e had b e e n p lu n g ed i n t o l i v i n g o r r e c e n t l y dead f l e s h . Not u n t i l 1895 was a s a t i s f a c t o r y means o f d i f f e r e n t i a t i n g v a r io u s ty p e s o f b lo o d d i s c o v e r e d . At t h a t tim e B o rd e t made th e im p o rta n t d is c o v e r y o f b lo o d seru m . T h is gave us th e p r e c i p i t i n t e s t w h ich i s b a s e d on th e f a c t t h a t when a r a b b i t , f o r e x a m p le , i s i n j e c t e d w i t h a f o r e i g n p r o t e i n , i t s serum w i l l form a p r e c i p i t a t e when m ixed w ith a s o l u t i o n o f t h i s f o r e i g n 3 I b i d . p . 102. 5 p r o te in * The m e d ic o - le g a l im p o rtan c e o f t h i s t e s t l i e s i n th e f a c t t h a t th e t e s t i s s p e c i f i c and a p r e c i p i t a t e i s form ed o n ly w i t h th e p r o t e i n w h ic h i s o f t h e same s p e c ie s as t h a t o r i g i n a l l y i n j e c t e d . T h is t e s t g iv e s much more p o s i t i v e r e s u l t s t h a n do l i n e a r m easu rem en ts, can he u se d w i t h o ld e r b lo o d , and i s g e n e r a l l y re c o g n iz e d i n a l l c o u r t s o f law . The B o rd e t r e a c t i o n was e x te n d e d t o a n sw e rin g th e q u e s t io n o f w h ich s u s p e c t s m ight have sh ed c e r t a i n b lo o d . I r r e f u t a b l e n e g a tiv e e v id e n c e can be o b ta in e d by t h i s t e s t and c o u ld have p re v e n te d th e e x e c u tio n o f an in n o c e n t man i n 1721 had i t b e en known and a p p li e d a t t h a t t im e .^ The c a se was one o f s u i c i d e i n w h ich a man had b e en s u s p e c te d o f m urder b e c a u se o f c e r t a i n s t a i n s on h i s c l o t h i n g , w h ic h he p r o t e s t e d had b e en c a u se d by an o ld wound w h ich had b ro k e n o p en . He was con v i c t e d and e x e c u te d , a f t e r w hich a s u i c i d e n o te was fo u n d w h ich e x o n e r a te d him . The im p o rta n c e o f b lo o d as a c lu e was e x te n d e d when med’i c i n e gave u s in f o r m a tio n a b o u t th e m orphology o f su c h b a c t e r i a as Treponema p a llid u m and o t h e r b lo o d d i s e a s e o r g a n is m s . O b v io u s ly , h e l p f u l in f o r m a tio n can be o b ta in e d by th e co m p ariso n o f t h e b lo o d o f a v i c t i m w i t h t h a t o f a s u s p e c t when one o r th e o t h e r h as b e en fo u n d t o be s i p h i l i t i c , f o r ex am p le. Such e v id e n c e as d i r e c t i o n o f f l i g h t , f i n g e r p r i n t s i n b lo o d , and im p re s s io n s made by c lo th - w e a v e s , e t c . , do n o t 4 Henry R hodes, Q£. c i t . , p . 135. 6 c o n c e rn us h e r e . THE VALUE OF BLOOD TESTS The q u e s tio n s w hich may a r i s e and w hich can o f t e n he answ ered by b lo o d e x a m in a tio n a re p r i n c i p a l l y : 1) Is t h e s t a i n b lo o d o r does i t c o n ta in b lo o d ? 2) Is i t human b lo o d o r t h a t o f a low er an im al? 3) What k in d o f a n im a l sh ed t h i s b loo d? 4) What s u s p e c ts c o u ld n o t have p o s s i b l y sh ed t h i s b lo o d ? 5) What p a r t of th e body d id t h i s b lo o d come from ? 6) When was th e b lo o d shed? The f i r s t q u e s tio n w i l l be d i s c u s s e d i n more d e t a i l b e lo w , as i t i s t h i s q u e s tio n w h ich i n s t i g a t e d th e p r e s e n t i n v e s t i g a t i o n . The seco n d and t h i r d q u e s tio n s c o u ld f o rm e r ly be a n sw ered o n ly on th e b a s i s o f l i n e a r m easurem ents o f c o r p u s c l e s , w h ich m ethod is n o t v e ry s a t i s f a c t o r y and can be u se d o n ly w i t h f r e s h b l o o d s t a i n s . The p r e s e n t method is t h a t em ploying th e p r e c i p i t i n r e a c t i o n a lr e a d y m e n tio n e d . I t g iv e s q u i t e a c c u r a t e in f o r m a tio n when p r o p e r ly a p p li e d and le a v e s d o u b t o n ly in th e c a se o f c l o s e l y r e l a t e d a n im a ls su c h as th e h o rs e and m u le, dog and f o x , h a re and r a b b i t , hen and p ig e o n , sh e e p and cow, e t c . 5 C a r e f u l te c h n iq u e w i l l d i f f e r e n t i a t e ty p e s i n t h e s e c a s e s , as w e l l as i n th e c a se o f chim panzee o r g o r i l l a and human b lo o d . The te c h n iq u e is b a se d on th e f a c t t h a t th e 5 Soderman and 0*C o n n e ll, Modern C rim in a l I n v e s t i g a t i o n (New Y ork: Funk & W agnalls Company, l9'35)' p . 230. 7 r e a c t i o n may n o t g iv e a p o s i t i v e r e s u l t i n as h ig h d i l u t i o n s w ith one ty p e as w i t h a n o th e r* T his r e a c t i o n may be used w ith b lo o d w hich i s to o o ld f o r c o rp u s c le m easu rem en t, b u t i t to o i s a f f e c t e d when th e b lo o d has p u t r e f i e d or b e en chem i c a l l y a l t e r e d . A c c u ra te n e g a t iv e e v id e n c e may be o b ta in e d in d e c id in g from w hich p e rs o n s a c e r t a i n s t a i n may have come# T h at i s , i f a group o f s u s p e c ts m a in ta in t h e i r in n o c e n c e , c e r t a i n o f them can be e lim in a te d on th e b a s i s o f t h e i r b lo o d t y p e , th e r e m ain d er s t i l l b e in g o n ly s u s p e c t s . B lood g ro u p in g i s a l s o of h e lp in th e d e te r m in a tio n o f n o n - p a t e r n it y # The phenomenon o f a g g l u t i n a t i o n upon w hich su ch ty p in g i s b a s e d may be f u r t h e r e x te n d e d by f u t u r e r e s e a r c h so a s t o e l i m i n a t e more s u s p e c ts from a g ro u p o r t o i n c r e a s e an in n o c e n t p e r s o n ’s ch an ces o f b e in g p ro v en in n o c e n t of p a t e r n i t y c h a rg e s# P o s i t i v e con n e c t i o n w ith a s u s p e c t may som etim es be o b ta in e d when a b lo o d s t a i n i s found to c o n ta in b a c t e r i a n o t p r e s e n t in norm al b lo o d . In c h e c k in g a s u s p e c t ’s a s s e r t i o n t h a t c e r t a i n s t a i n s came from a n o s e b le e d , r a z o r c u t , m e n s tr u a ti o n , e t c . , p h y s i c a l a n d /o r c h e m ic a l e x a m in a tio n may v e r i f y b u t can n o t u s u a l l y d i s p ro v e su c h an a s s e r t i o n # Such v e r i f i c a t i o n may come from p ro v in g th e p re s e n c e in b lo o d o f s h a v in g cre am , n o se h a i r s , g e n i t a l h a i r s , m ucous, sem en, v a g in a l e p i t h e l i a l c e l l s , e t c . The d e g re e o f c o a g u la tio n o r d r y in g may i n d i c a t e th e l e n g t h o f tim e w h ich has e la p s e d s in c e th e b lo o d was s h e d , a lth o u g h t h i s i s s u b j e c t t o many u n c e r t a i n t i e s and v a r i a t i o n s . A f te r b lo o d flo w s from th e body, i t s e p a r a t e s i n t o th e coagu- lum and th e b lo o d -s e ru m , t h i s p ro c e s s b e g in n in g in two o r t h r e e m in u te s and e n d in g w i t h in t e n m in u te s in most c a s e s . P a t h o lo g i c a l c o n d itio n s may a l t e r t h i s tim e as w i l l c o n d itio n s o f te m p e r a t u r e , s u r f a c e e x p o s u re , a g i t a t i o n , d i l u t i o n , e t c . D ry in g i s a s e p a r a t e p r o c e s s from t h a t o f c o a g u la tio n and th e tim e in v o lv e d depends on c o n d i t i o n s , one .or two h o u rs b e in g t y p i c a l f o r a s i n g l e drop.® F or s t a i n s w h ich a r e q u i t e o l d , th e age i s s t i l l more u n c e r t a i n , b u t may b e som etim es e s tim a te d by i n v e s t i g a t i n g th e s o l u b i l i t y o f th e s t a i n . The s o l u b i l i t y i s a f u n c t i o n of a g e , b e in g l e s s f o r o l d e r s t a i n s . T h is w i l l be d e a l t w i t h l a t e r i n d i s c u s s i n g 3 - a m in o p h th a lh y d ra z id e , w h ich r e a g e n t seems t o o f f e r some p ro m ise of a id i n g in th e d e t e r m i n a t i o n o f th e age o f s t a i n s i n s o f a r as age i s r e l a t e d t o s o l u b i l i t y . TESTS N O W IN GENERAL USE T h is r e s e a r c h i s c o n ce rn e d p r i n c i p a l l y w i t h th e f i r s t q u e s t i o n , n am ely , i s a s u s p e c te d s t a i n b lo o d , o r does i t con t a i n b lo o d of any k in d ? T here a re many t e s t s a v a i l a b l e f o r g i v in g su c h i n f o r m a tio n , e a c h w ith c e r t a i n a d v a n ta g e s and d i s a d v a n ta g e s . The more im p o rta n t o f th e s e t e s t s a r e th e f o l lo w in g : 0 W e b ste r, L eg a l M edicine and T o x ic o lo g y , ( P h i l a d e lp h i a : S au n d ers 1930) p . 161. 9 7 1) The P r o t e i n T e s t , T h is t e s t is n o t s p e c i f i c and can o n ly t e l l u s t h a t t h e s t a i n c o n ta in s a r e d c o lo r i n g m a t t e r p lu s p r o t e i n m a t t e r . B o th su c h th in g s e x i s t i n b lo o d and can c a u se b lo o d t o b e d i f f e r e n t i a t e d from f r u i t , v e g e ta b le o r f lo w e r e x t r a c t s , and d y e s . However, any o t h e r p r o t e i n m a t t e r su c h as m ilk , pus o r sputum can become c o n ta m in a te d w ith r e d c o l o r i n g m a tte r and g iv e a p o s i t i v e t e s t . The e x t r a c t o f th e s t a i n i s a c i d i f i e d w ith a c e t i c a c i d and b o i l e d u n t i l th e r e d d i s h c o l o r , i f an y , h as f a d e d . The s o l u t i o n w i l l become c lo u d y and a p r e c i p i t a t e may s e t t l e o u t due t o p r o t e i n c o a g u l a t i o n . A few d ro p s of sodium hy d r o x id e w i l l th e n d i s s o l v e t h e p r e c i p i t a t e and th e s o l u t i o n becom es d i c h r o i c . 2) The Teichm an T e s t . T h is is a l s o c a l l e d t h e hem in t e s t , as i t c o n s i s t s of p r e p a r i n g and i d e n t i f y i n g hem in c r y s t a l s m i c r o s c o p ic a l l y from th e b lo o d o f t h e s t a i n . The f o r m a tio n o f th e c h a r a c t e r i s t i c hem in c r y s t a l s i s i n d i s p u t a b l e p ro o f o f t h e p re s e n c e o f b lo o d . I t i s th u s v e ry v a lu a b le e v en tho ugh i t does n o t d i s t i n g u i s h a n im a l from human b lo o d . The s p e c i f i c i t y i s in doubt o n ly i f in d ig o is p r e s e n t . T h is t e s t g iv e s good r e s u l t s i f s t a i n s a r e n o t h e a t e d , w ashed, o r to o o l d . Lim e, sand and i r o n r u s t i n t e r f e r e i n t h i s t e s t . T here a r e s e v e r a l te c h n iq u e s f o r p e rfo rm in g t h i s t e s t , 7 I b i d . , p . 165. Q t h a t g iv e n by Hawk b e in g t y p i c a l . He p la c e s a s m a ll d ro p o f b lo o d on a s l i d e and adds a l i t t l e w a te r t o la k e th e b lo o d . A f r a c t i o n o f a d ro p o f .9$ sodium c h l o r i d e s o l u t i o n i s added and th e m ix tu re i s slo w ly d r i e d o v e r a f la m e . G l a c i a l a c e t i c a c i d i s ru n u n d e r a c o v e r - s l i p and g e n tl y h e a t e d . More a c id i s added and th e s l i d e c o o le d , a f t e r w h ic h hem in c r y s t a l s can be re c o g n iz e d u n d e r th e m ic ro s c o p e . The c o m b in a tio n o f t h e i r sh a p e and c o lo r i s e a s i l y r e c o g n i z a b le . A v a r i a t i o n o f t h i s 9 p ro c e d u re i s g iv e n by N ip pe. T h is t e s t r e q u i r e s p r a c t i c e and is n o t alw ays r e p r o d u c i b l e . The c o r r e c t amount o f h e a t i n g , a c i d , and sodium c h lo r i d e can be l e a r n e d from e x p e r ie n c e . 3) Beam and F r a n k ys T e s t . ^ T h is i s n o t r e a l l y a n o th e r t e s t , b u t i s a r a d i c a l m o d if i c a t io n o f T eichm anfs m ethod o f p r e p a r in g hem in. The m a t e r i a l t o b e t e s t e d is p la c e d in th e b o tto m o f a f l a t a r s e n i c s u b lim a tio n tu b e and a few d ro p s o f g l a c i a l a c e t i c a c i d c o n ta in in g .01 - .1 $ sodium c h lo r i d e s o l u t i o n a r e a d d e d . A f i n e c o tt o n t h r e a d i s a d j u s t e d so t h a t i t s u p p e r end i s n e a r th e to p o f th e tu b e and th e lo w er end d ip s i n t o th e l i q u i d . The th r e a d sh o u ld to u c h th e s id e o f th e tu b e a t a l l p o i n t s . Upon s ta n d in g a t room te m p e r a tu r e , 8 Hawk, P r a c t i c a l P h y s i o lo g i c a l C h e m istry ( P h i l a d e lp h i a B la k is to n s 1937) p . £>9§. 9 N ip p e , D e u t. Med. W ochschr, 3 8 , 222 2 , 1912. Beam & F ra n k , B io ch e m ic al J . 9 (1 915) p p . 1 6 1 -1 7 0 . 11 r e l a t i v e l y l a r g e hem in c r y s t a l s form on th e t h r e a d due t o th e slow e v a p o r a tio n o f th e l i q u i d . About one h o u r is enough tim e f o r th e c r y s t a l s t o be se e n w i t h a low power m ic ro s c o p e . T h is t e s t has th e a d v a n ta g e o v e r th e Toichman t e s t t h a t th e m ix tu re c a n n o t be o v e rh e a te d and th e e v a p o r a tio n can be more c l o s e l y c o n t r o l l e d . I t s d is a d v a n ta g e i s th e l e n g t h o f tim e n e c e s s a r y . 4) M ic ro sc o p ic I d e n t i f i c a t i o n o f E r y th r o c y te s : T h is has b een m en tio n ed b e f o r e as a means of d i f f e r e n t i a t i n g v a r io u s ty p e s o f b lo o d . However, more c e r t a i n r e s u l t s can be o b ta in e d from m ic ro s c o p ic e x a m in a tio n m ere ly f o r th e p re s e n c e o f e r y t h r o c y t e s . Good c o n f ir m a to r y e v id e n c e i s o b ta in e d i f e r y t h r o c y te s a r e fou nd t o be p r e s e n t i n a s u s p e c te d s t a i n . I f th e b lo o d i s f r e s h , th e e x a m in a tio n can be e x te n d e d t o th e m easurem ent o f c o rp u s c le s i z e as w e l l . The f o llo w in g t a b l e i s o f i n t e r e s t in show ing th e v a r i a t i o n s t o be e x p e c te d in d i f f e r e n t t y p e s . ^ TABLE I Anim al E r y th r o c y te D ia m e te r (u) E le p h a n t 9 .2 8 G u in e a -p ig 7 .8 8 Man 7 .8 2 Monkey 7 .5 1 Dog 7 .1 3 R at 6 .9 6 R a b b it 6 .9 6 Mouse 6 .7 9 L ion 6 .1 3 Ox 6 .0 2 H orse 5 .9 9 P ig 5 .9 5 Gat 5 .8 1 Sheep 5 .1 7 Goat 4 .1 0 M usk-deer 2 .0 6 W ormley, M icro C h em istry of P o iso n s (New Y ork: L ip p i n c o t t 1887) 5) The S trz y o w sk i T e s t ; T h is i s p e rfo rm ed s i m i l a r l y t o th e 1 p Teichm an t e s t , b u t w i t h th e f o llo w in g r e a g e n t : A lc o h o l l e a . , d i s t i l l e d w a te r l c c . , g l a c i a l a c e t i c a c i d 1 c c . , h y d r io d ic a c id 4 d r o p s . T his t e s t i s in c o n v e n ie n t s in c e th e r e a g e n t m ust be p r e p a r e d f r e s h j u s t b e f o r e u s e . 6) The H em ato p o rp h y rin T e s t : T h is compound i s form ed when h em o g lo b in i s d i g e s t e d w i t h c o n c e n tr a te d s u l f u r i c a c i d and can be re c o g n iz e d by i t s p r o p e r t y of f l u o r e s c i n g i n u l t r a - v i o l e t l i g h t . A d ro p o f b lo o d is d r i e d on a s l i d e a f t e r m ix in g w ith a d ro p o f w a t e r , and a d ro p o f s t r o n g s u l f u r i c a c id i s a d d ed . I f t h i s i s exam ined u n d e r f i l t e r e d u l t r a - v i o l e t l i g h t , a re d f l o u r e s c e n c e w i l l g r a d u a l ly be se e n t o d e v e lo p . H em atoporphy r i n can a l s o be r e c o g n iz e d s p e c t r o s c o p i c a l l y . 7) S p e c tr o s c o p ic T e s t s . B lood may be r e c o g n iz e d by th e c h a r a c t e r i s t i c a b s o r p t io n s p e c t r a o f v a r io u s h em o g lo b in d e r i v a t i v e s . Such e v id e n c e i s q u i t e e x a c t and may be o b ta in e d from v e ry s m a ll sam ples by u s in g a m ic r o s p e c tr o s c o p e . The b lo o d i n a s t a i n may be a m ix tu re o f s e v e r a l compounds o f h e m o g lo b in . The pigm ent fo u n d i n f r e s h b lo o d i s u s u a l l y t h a t o f oxyhemo g l o b in and i n o ld e r b lo o d may be one o r more o f th e f o llo w in g : oxy h em o g lo b in , h e m o g lo b in , m eth em o g lo b in , h e m a tin , o r o f c o u rs e i n c e r t a i n p o is o n in g c a s e s - n i t r i c o x id e h e m o g lo b in , c a rb o n m onoxide h e m o g lo b in , and su lp h -h e m o g lo b in . 12 v L o c a rd , Manuel de T ech n iq u e P o l i c i e r e , P a r i s , 1934. 13 When an e x t r a c t is made i t is exam ined and th e re g io n s o f a b s o r p t io n a r e m e a su re d . The a b s o r p t i o n o f th e above com pounds h as b e e n w e l l mapped o u t and may be b e s t se e n from su c h e x c e l l e n t c o lo r p l a t e s as th o s e in Hawk. ^ I f , f o r e x am p le, oxyhem oglobin is r e c o g n iz e d , th e s o l u t i o n is re d u c e d w ith a few drops o f ammonium s u l f i d e and exam ined f o r hemo g l o b i n . O b s e rv a tio n o f t h i s c o n v e rs io n o f s p e c t r a i s im por t a n t , as i t e x c lu d e s s u b s ta n c e s w h ic h g iv e s p e c t r a s i m i l a r t o t h a t o f ox yhem oglobin. I f on f i r s t e x a m in a tio n , m ethem oglobin a p p e a r s , as i t p ro b a b ly w i l l in c a se th e s t a i n i s o ld o r p a r t i a l l y decom posed, i t can be c o n v e rte d i n t o h em oglobin by ammonium s u l f i d e t r e a t m e n t . I f th e sp e c tru m i s t h a t o f hema- t i n , su ch t r e a tm e n t w i l l c o n v e rt i t to t h a t o f hemochromogen. T h is b r i e f l y i n d i c a t e s th e way i n w hich b lo o d i s e x am ined s p e c t r o s c o p i c a l l y and i t can r e a d i l y be se e n why t h i s i s a d e p e n d a b le m ethod w i t h so many c o n fir m a to r y p r o c e d u r e s . An e x c e l l e n t d i s c u s s i o n o f th e s e t e s t s i s g iv e n i n W e b s t e r . ^ 8.) The P a ra d ia m in o d ip h e n y l ( b e n z i d i n e )_T e s t : T h is and th e n e x t t h r e e t e s t 3 c an be c l a s s i f i e d t o g e t h e r as c h e m ic a l o x id a t i o n t e s t s a l l d e p e n d in g on th e same p r o p e r t y o f hem oglobin w h ich makes i t s o in d is p e n s a b le t o th e l i v i n g b o d y . Hem oglobin form s w ith oxygen a nl o o s e ” compound known a s o x y -h em o g lo b in . 13 Hawk, o j d. c i t . , F r o n t i s p i e c e . 14 W e b ste r, L e g a l M edicine & T o x ic o lo g y ( P h i l a d e lp h i a : S au n d ers 1930) p p . l ¥ l - l 7 F I 14 The r e a c t i o n i s r e a d i l y r e v e r s i b l e so t h a t hem oglobin can a c t a s an "oxygen c a r r i e r ” , t h i s p r o p e r t y e n a b lin g hemo g lo b in to be d e t e c t e d by th e u se o f c e r t a i n o x id iz a b le com pounds w h ich y i e l d c o lo r e d p r o d u c t s . One su c h compound w hich i s q u i t e s a t i s f a c t o r y i s b e n z i d in e , a d e r i v a t i v e o f b e n z e n e . I f b e n ze n e is n i t r a t e d and th e n re d u c e d t o h y d ra z o b e n z e n e , b e n z id in e can be o b ta in e d by re a rra n g e m e n t o f t h e h y d ra z o b e n zene by tr e a tm e n t w ith a s t r o n g a c i d . In th e p re s e n c e o f b e n z id in e and h y d ro g en p e r o x id e , h em oglobin p ro d u ce s a d eep b lu e c o l o r . The f o llo w in g p ro c e d u re is. recommended by Lucas D is s o lv e .2 gram o f d r y sodium p e r b o r a te i n one c . c . o f d i s t i l l e d w a te r and 10 c . c . o f g l a c i a l a c e t i c a c i d . To t h i s add 1 c . c . o f a 10 p e r - c e n t s o l u t i o n o f b e n z id in e in 2 c . c . o f g l a c i a l a c e t i c a c i d . T his r e a g e n t s h o u ld be p r e p a r e d f r e s h from p u re b e n z i d i n e . P la c e a p o r t i o n o f th e s t a i n o r a p ie c e o f m o is te n e d f i l t e r p a p e r w hich .has been p r e s s e d a g a i n s t th e s t a i n on a f i l t e r p a p e r and add a d ro p o f b e n z id in e r e a g e n t . I f b lo o d i s p r e s e n t , a b l u i s h o r b l u i s h - g r e e n c o lo r i s p r o duced im m e d ia te ly and r a d i a t e s i n s t r e a k s from th e s t a i n . I f th e s t a i n i s v e r y o ld and i n s o l u b l e , a d ro p o f p j^rid in e w i l l h a s te n t h e r e a c t i o n . T h is t e s t is p ro b a b ly th e b e s t i n u se to d a y and i s v e ry r e l i a b l e as a n e g a tiv e t e s t (b la n k s a re o f c o u rs e u se d t o ch eck 15 L u ca s, F o r e n s ic C h e m istry & S c i e n t i f i c C rim in a l I n v e s t i g a t i o n (New Y ork: Longmans G reen 7 & C o . 1^35) p . 3 0 . 15 16 th e r e a g e n t ) . A cco rd in g t o Lucas no s t a i n w h ich g iv e s a n e g a tiv e t e s t has e v e r b e en shown t o c o n ta in b lo o d . The t e s t i s n o t l i m i t e d by th e age o f th e s t a i n s in c e b lo o d th o u sa n d s of y e a r s o ld has g iv e n a p o s i t i v e t e s t . Lucas a l s o r e p o r t s t h a t M An in s ta n ta n e o u s r e a c t i o n i s g iv e n by b lo o d s t a i n s w h ich have b e en k e p t i n a s e m i - t r o p i c a l l a b o r a t o r y f o r e ig h te e n y e a r s . B lood b o i l e d f o r an h o u r, and k e p t f o r t h i r t e e n y e a r s r e a c t s r e a d i l y . Blood s t a i n s on r u s t y i r o n k e p t f o r f o u r y e a rs r e a c t e d w i t h i n f i v e s e c o n d s . I t i s a lm o st im p o s s ib le t o w ash o u t b lo o d from c l o t h so t h a t th e f a b r i c w i l l f a i l t o g iv e a b lu e c o lo u r w ith t h e r e a g e n t . ” A p o s i t i v e t e s t can be g iv e n more w e ig h t i f th e s o l u t i o n t o b e t e s t e d i s b o i l e d t o remove any o x id a s e s o r p e r o x i d a s e s t h a t may be p r e s e n t and w hich w ould g iv e a p o s i t i v e t e s t . 9)‘ The Guaiacum T e s t . T h is t e s t , som etim es known as van D eenfs , D ay’s o r S c h o n b e in ’s t e s t , i s l e s s s e n s i t i v e th a n th e b e n z id in e t e s t (1 t o 5 0 ,0 0 0 -1 0 0 ,0 0 0 ) and i s b a s e d on th e o x i d a t i o n o f g u a ia c r e s i n t o g u a ia c o n ic a c i d and t h e n t o g u a ia c b l u e . I t is p e rfo rm ed by a d d in g a few d ro p s o f f r e s h g u a ia c t i n c t u r e t o <the s t a i n e x t r a c t , w a i ti n g t o se e i f a b lu e c o lo r d e v e lo p s and th e n p u t t i n g I n a few d ro p s of h y d ro g en p e ro x id e . The b lu e c o lo r s h o u ld d e v e lo p a f t e r a d d in g th e p e ro x id e and n o t b e f o r e , t o i n d i c a t e a p o s i t i v e t e s t . 16 I b i d . , p . 3 1 . 16 T h is t e s t i s a l s o a p p l i c a b l e to q u i t e o ld s t a i n s b u t l i k e t h e b e n z id in e r e a c t i o n i s m ost r e l i a b l e as a n e g a tiv e i n d i c a t i o n . Some of th e s u b s ta n c e s w h ich i n t e r f e r e by g iv in g a p o s i t i v e t e s t a re p o ta s s iu m perm an g an ate and m an g an ate, m anganese p e r o x id e , le a d p e ro x id e , c h l o r i n e , brom ine, io d in e , n i t r i c and chrom ic a c i d s , f e r r i c c h l o r i d e , f e r r i c s u l p h a t e , i r o n alum , f e r r o - a n d f e r r i c y a n i d e o f p o ta s s iu m , c u p ric s u l p h a te and n i t r a t e , in d ig o , gum a c a c a i s , g l u t e n , m ilk , raw p o t a t o , p u s , s w e a t, and any l i v i n g c e l l o r i t s i n t r a c e l l u l a r 17 enzym e. The d is a d v a n ta g e s a re obvious* 10) The P h e n o lp h th a le in T e s t * T h is i s a n o th e r o x i d a tio n t e s t , i n w h ic h re d u c e d p h e n o lp h th a le in i s a g a in o x id iz e d t o p h e n o lp h - t h a l e i n i n a l k a l i n e s o l u t i o n w ith th e p r o d u c tio n of a r e d c o l o r . P h e n o lp h th a lin , th e re d u c e d compound, may be p re p a re d b y r e d u c t i o n o f p h e n o lp h th a le in w ith z in c in a l k a l i n e s o l u t i o n . I t i s c o l o r l e s s u n l e s s some p h e n o lp h th a le in is p r e s e n t w hich g iv e s 18 a r e d d i s h t i n g e t o th e a l k a l i n e s o l u t i o n . W ebster d i s s o l v e s .052 gram o f p h e n o lp h th a lin in 21 c . c . o f N/lO sodium h y d ro x id e and makes i t up to a volume o f 100 c . c . w ith w a t e r . Two p a r t s o f t h i s r e a g e n t a re added t o one p a r t o f aqueous s t a i n e x t r a c t a lo n g w ith a d ro p o f h y d ro g en p e r o x id e . The p re s e n c e o f b lo o d i s i n d i c a t e d by a p u r p le t o p in k c o l o r . A c o n t r o l s h o u ld be ru n on w a te r a lo n e s in c e th e t e s t i s e x tre m e ly d e l i c a t e and th e m e re s t t r a c e s o f b lo o d from p re v io u s t e s t s may g iv e an e rro n e o u s 17 W e b ste r, o £ . c i t . , p . 167. 18 I b i d . , p . 1 6 8 . 17 p o s i t i v e r e s u l t . T h is w ould seem t o be th e m ost d e l i c a t e t e s t now a v a i l a b le s i n c e one p a r t o f b lo o d i n 8 ,0 0 0 ,0 0 0 p a r t s o f w a te r can be d e t e c t e d , and K a s t l e - ^ h a s d e t e c t e d one p a r t i n 8 0 ,0 0 0 ,0 0 0 . The maximum s e n s i t i v i t y i s n o t o b ta in e d in th e p re s e n c e of v a r io u s t i s s u e s or body s e c r e t i o n s and t h i s t e s t , a lth o u g h a ls o v e ry c o n v in c in g in th e n e g a t i v e , i s n o t t h e most recommended. 11) The L e u c o -M a lc a c h ite T e s t . T h is is th e l a s t o f th e more common o x i d a t i o n t e s t s and i s more s p e c i f i c th a n th e o t h e r s , 20 and a l s o , a c c o r d in g to Soderm an, th e m ost s e n s i t i v e . The l i t e r a t u r e i s c o n f l i c t i n g on th e r e l a t i v e s e n s i t i v i t i e s of th e le u c o - m a la c h ite and th e p h e n o lp h th a lin t e s t s . 21 The t e s t is p erfo rm ed w ith th e f o llo w in g r e a g e n t : L e u c o -m a la c h ite g r e e n - lg m ., G l a c i a l a c e t i c a c i d - 100 c . c . , and d i s t i l l e d w a te r-1 5 0 c . c . I t k e ep s b e s t i n a d a rk b o t t l e w ith a p a r a f f i n e d c o v e r . 8 c . c . o f t h i s s o l u t i o n and 2 c . c . o f h y d ro g en p e ro x id e g iv e a g re e n t o b lu e c o lo r w i t h i n a m in u te i f a b lo o d s t a i n e x t r a c t i s m ixed w ith a l i t t l e r e a g e n t on a f i l t e r p a p e r . The c o lo r i s due t o th e o x i d a t i o n o f th e c o l o r l e s s le u c o - b a s e t o m a la c h ite g re e n . The mechanism o f th e o x id a tio n s in th e s e t e s t s i s n o t as s t r a i g h t f o r w a r d as i t m ight seem a t f i r s t t h o u g h t, b u t 10 The B r i t i s h M edical J o u r n a l 1926, p p . 6 5 0 -2 , 808. A l l e n ’s Com m ercial O rganic A n a ly s is 10 (1933) p p . 3 4 -5 . 20 Soderman & O’C o n n e ll, Modern C rim in a l I n v e s t i g a t i o n (New Y ork: Funk & W agnalls Co. 1935) p . 226. pi M edinger, Revue I n t e r n , de C r i m i n a l l s t i q u e No. 7 , 1931. 18 b r i e f l y th e t e s t s depend upon th e c a t a l y t i c d e c o m p o s itio n o f h y d ro g en p e ro x id e b y b lo o d , fo llo w e d by o x i d a t i o n o f th e t e s t r e a g e n t . The s t u d y o f t h i s m echanism c o n s t i t u t e s p a r t of th e f o llo w in g i n v e s t i g a t i o n s in c e i t i s in v o lv e d in t h e new t e s t t o be d e s c r i b e d . STATEMENT OF THE PROBLEM A b lo o d t e s t i s n eed ed w h ic h is more s p e c i f i c th a n th e above o x i d a t i o n t e s t s and w hich can be j u s t as sim p ly a p p l i e d . The more s p e c i f i c t e s t s now a v a i l a b l e su c h as th e v a r io u s hemin t e s t s r e q u i r e a c e r t a i n amount o f l a b o r a t o r y equipm ent and e x p e rie n c e n o t p o s s e s s e d by th o s e m ost l i k e l y t o make p r e l im i n a r y e x a m in a tio n s o f crim e c l u e s . The s p e c t r o s c o p i c t e s t s a r e s p e c i f i c b u t a r e , o f c o u r s e , n o t r e a d i l y u s a b l e by p o l ic e o f f i c e r s . The p ro b lem is b a s e d on th e use o f th e compound 3-am in o p h th a lh y d r a z id e f o r t h e d e t e c t i o n o f b lo o d in s u s p e c te d 22 s t a i n s . The use o f t h i s compound was f i r s t s u g g e s te d b y S pecht who made c e r t a i n q u a l i t a t i v e e x p e rim e n ts and fo u n d th e t e s t t o b e q u i t e s p e c i f i c . The t e s t b e lo n g s in th e c l a s s o f o x i d a t i o n t e s t s m en tio n ed a b o v e , th e h y d ra z in e compound lu m in e s c in g i n th e d a rk u n d e r t h e a c t i o n o f th e decom posing p e ro x id e i n s t e a d o f g iv in g a c o lo r r e a c t i o n upon o x i d a t i o n . T h is i n v e s t i g a t i o n i s made p r i n c i p a l l y t o exam ine th e m echanism o f th e b lo o d - p e r - o x id e -a m in o p h th a lh y d ra z id e r e a c t i o n from a p h y s ic o -c h e m ic a l 22 S p e c h t, Z e i t . f u r angew . Chem. 5 0 , 8 , 155, 1937. s t a n d p o in t and a p p ly su c h in f o r m a tio n t o d e v is in g th e m ost e f f i c i e n t r e a g e n t f o r f o r e n s i c use# The p u r e ly s c i e n t i f i c a s p e c ts of t h i s r e a c t i o n a re s t r e s s e d more t h a n a re th e p r a c t i c a l . ORGANIZATION OF THE REMAINDER OF THE THESIS The n e x t c h a p te r d i s c u s s e s th e c o m p o s itio n o f b lo o d and t h e p r o p e r t i e s of 3 -a m in o p h th a lh y d ra z id e i n s o f a r as th e y may be o f co nsequence i n th e b lo o d - p e r o x id e - h y d r a z id e r e a c t i o n . V a rio u s p o s s i b l e m echanism s a r e p o s t u l a t e d f o r t h e p r o d u c tio n o f ch em ilu m in e sc en c e by b lo o d . C h a p te r I I I d i s c u s s e s th o s e a s p e c ts o f th e th e o r y o f p h o t o e l e c t r i c and p h o to g ra p h ic m ea su re m ents o f r a d i a n t e n e rg y w h ich a re in v o lv e d in th e s u b s e q u e n t e x p e r im e n ta l c o m p ariso n s o f th e lu m in e sc e n c e s of v a r io u s m ix t u r e s . Some r e a c t i o n - k i n e t i c s th e o r y i s p r e s e n te d l a t e r in c o n n e c tio n w ith th e c a l c u l a t i o n s o f e x p e r im e n ta l r e s u l t s # C h a p te r IV d e s c r i b e s th e c o n s t r u c t i o n and o p e r a t io n o f th e a p p a ra tu s u se d i n m easurem ents o f c h e m ilu m in e sc e n c e , e x p e rim e n ts on r e a c t i o n s r a t e s , o x i d a t i o n m echanism s, d e c o m p o s itio n mech anism s and m is c e lla n e o u s e x p e rim e n ts . C h a p te r V p r e s e n t s con c lu s io n s and s u g g e s tio n s f o r f u t h e r w ork. 20 CHAPTER I I 3-AMINOPHTHALHYDRAZIDE, C ^ N g O g T h is compound has r e c e n t l y b e en s y n th e s iz e d by a Sim p'S p l i f i e d p ro c e d u re by H u n tr e s s , S ta n le y and P a r k e r . I t was f i r s t s y n th e s iz e d by a r a t h e r te d io u s, p r o c e s s from t r i e t h y l 24 25 h e m i m e l l i t a t e , f o llo w e d by v a r io u s a l t e r n a t i v e m eth o d s. The ch em ilu m in escen ce e x h i b i t e d by t h i s compound upon o x i- 26 d a t i o n was n o te d in 1928 by A lb re c h t who p r e p a r e d th e com pound by th e p a th 3 - n i t r o p h t h a l i c a c i d , i t s diammonium s a l t , 3 - n i t r o p h t h a l i m i d e , 3 -a m in o p h th a lim id e , 3 -a m in o p h th a lh y d ra - 23 z i d e . H u n tre ss r e p o r t s t h i s method t o be u n s a t i s f a c t o r y . The a u th o r h a s p r e p a r e d 3 -a m in o p h th a lh y d ra z id e by H u n tr e s s 1 i m ethod and found i t q u i t e s a t i s f a c t o r y , y i e l d i n g a good q u a n t i t y o f m a t e r i a l s u i t a b l e f o r many e x p e rim e n ts on chem i lu m in e s c e n c e . The m a t e r i a l u se d in t h i s r e s e a r c h was o b ta in e d i n somewhat p u r e r form from th e Eastm an Kodak Company ( P r e p a r a t i o n # 3 6 0 3 ). The m ethod of p r e p a r a t i o n g iv e n by H u n tre s s i s as f o l low s: 3 - N itr o p h th a lh y d r a z id e i s f i r s t p r e p a r e d . 23 J . Chem. Ed. 1 1 ' , 142, 1934, a l s o J .A .C .S . 5 6 , 2 4 1 ,1 9 3 4 . 24 S c h m itz , J . F r a k t . Chem. 91 4 6 , 97 (1 9 1 5 ). 23 C u r t i3 s , B e r ., 46 1162-71 (1913) A le x ra ,. Z. PhysTE. Chem., B8 382-94 (1930) B o g e r t, J . Am. Ctoem. Soc. 25 750 (1901) A lso G lev. J . P r a k t . Chem.“T 4 6 , 129, 1936. Z. P h y s lk . Chem., 136, 321-30 (1928) 21 " S o lid h y d ra z in e s u l f a t e and c r y s t a l l i z e d sodium a c e t a t e (272 g . o r 2 m o ls ) a r e d i s s o l v e d in 400 m l. o f h o t w a te r and t h e c l e a r s o l u t i o n i s added t o s o l i d 3 - n i t r o p h t h a l i c a c i d (211 g . o r 1 m ol) c o n ta in e d in an 8n p o r c e l a i n e v a p o r a tin g d i s h . T h is i s p la c e d upon a t r i p o d and e v a p o r a te d as r a p i d l y as p o s s i b l e o v e r a f r e e flam e w ith con s t a n t s t i r r i n g w i t h a f l a t p o r c e l a i n s p a t u l a to a v o id bumping o r d e c o m p o s itio n . T his o p e r a t io n r e q u i r e s a b o u t 1 .5 h o u rs and i s b e s t c o n d u c te d a t a hood i n o r d e r t o f a c i l i t a t e e v a p o r a tio n and t o rem ove th e a c e t i c a c i d v a p o r s . The r e s i d u a l d r y s o l i d i s r e moved from th e d i s h , g round t o a f i n e pow der, p la c e d i n a w ide-m outh ed f l a s k o r b e a k e r , and h e a te d f o r a t l e a s t t h r e e h o u rs a t a te m p e r a tu re o f 160 p lu s o r m inus 10 d e g . i n a s u i t a b l e o i l b a t h . D u rin g t h i s o p e r a t i o n th e s o l i d s h o u ld f r e q u e n t l y be s t i r r e d and i f t h e r e i s an y te n d e n c y to w ard c a k in g i n th e e a r l i e r p a r t o f th e b a k in g i t may b e n e c e s s a r y t o remove and powder th e m a t e r i a l b e f o r e c o n ti n u i n g . I t i s a d v i s a b l e t o m easure th e te m p e r a tu re i n th e h e a te d powder r a t h e r th a n e x t e r n a l l y and c a r e m ust be ta k e n n o t t o o v e r h e a t th e m ass. The e sc a p e o f steam may a l s o be f a c i l i t a t e d by p a s s in g a g e n t l e s tre a m o f a i r t h r u th e f l a s k . When th e h e a t i n g i s f i n i s h e d , rem ove th e s o l i d , pow der, and e x t r a c t tw ic e w ith 350 m l. o f h o t w a te r t o remove sodium s u l f a t e . The r e s i d u a l s o l i d i s th e n d r i e d a t 105 d e g . t o c o n s t a n t w e ig h t: y i e l d , 206 g . 3 - n i t r o p h t h a l h y d r a z i d e ♦ The c ru d e n i t r o com pound is th e n g r a d u a l l y added in s m a ll in c re m e n ts t o 1 l i t e r o f 6N ammonium s u l f i d e s o l u t i o n . V igorous sp o n ta n e o u s r e d u c t io n o c c u rs and th e f l a s k may r e q u ir e c o n s id e r a b le e x t e r n a l c o o li n g . A f te r a l l th e s o l i d n i t r o h y d r a z i d e h a s been a d d e d , th e r e s u l t a n t s o l u t i o n i s k e p t a t th e b o i l i n g p o in t f o r an h o u r w h ile a d d i t i o n a l h y d ro g en s u l f i d e i s p a s s e d i n t o th e m ix tu r e . D u rin g t h e r e d u c t io n th e o r i g i n a l n i t r o h y d ra z id e f i n a l l y c o m p le te ly d i s s o l v e s ; l a t e r , how e v e r , th e p r e c i p i t a t i o n o f th e r e s u l t a n t " lu m in o l” (T h is i s a s im p le r name s u g g e s te d by H u n t r e s s - a u t h . ) b e g in s , and th e s e two p r o c e s s e s may som etim es o v e r la p i n su c h a way t h a t t h e r e i s alw ay s some s o l i d p r e s e n t i n th e f l a s k . A f te r t h e d i s c o n tin u a n c e o f th e h y d ro g en s u l f i d e t r e a t m e n t , t h e s o l u t i o n i s b o i l e d f o r an h o u r more to c o m p lete th e r e a c t i o n and th e n a llo w e d t o s ta n d u n t i l c o ld . The y e llo w p r e c i p i t a t e o f m ixed s u l f u r and " lu m in o l1 1 i s f i l t e r e d w ith s u c t i o n , th o ro u g h ly w ashed w ith w a t e r , 22 and d r i e d . The f i l t r a t e from t h i s p rim a ry p r e c i p i t a t e is t h e n a c i d i f i e d w i t h a s l i g h t e x c e ss of g l a c i a l ■ - a c e t i c a c id and t h e r e s u l t a n t p r e c i p i t a t e i s f i l t e r e d w i t h s u c t i o n , w ashed w ith w a t e r , and d r i e d . In o r d e r to f r e e th e a m in o p h th a lh y d ra z id e from s u l f u r , th e c ru d e m ix tu re i s s t i r r e d up w ith t h a t amount of b% aqueous sodium h y d ro x id e w hich w ould j u s t c o rre sp o n d t o th e a ssu m p tio n t h a t th e d ry s o l i d c o n ta in e d no s u l f u r . A f te r s t i r r i n g and v e ry s l i g h t w arm ing, th e s o l u t i o n i s f i l t e r e d from u n d is s o lv e d s u l f u r , c o o le d to z e r o , s t i r r e d , and s c r a t c h e d . P r e s e n t l y p r e c i p i t a t i o n of th e mono sodium s a l t o f 3 - a m in o p h th a lh y d ra z id e b e g in s and in c r e a s e s f o r some tim e . F i n a l l y , t h e s o l i d i s f i l t e r e d w ith s u c t i o n , p r e s s e d as d ry a s p o s s i b l e on th e f i l t e r , th e n w ashed s p a r i n g l y w i t h d r y a l c o h o l o r e t h e r . I t may th e n be d r i e d i n th e a i r i f i t is t o be p r e s e r v e d as the sodium s a l t . To c o n v e rt t o ”lu m in o l,f i t s e l f th e o r i g i n a l s o l i d is n o t w ashed w ith a lc o h o l b u t is d i s s o l v e d i n w a te r and r e p r e c i p i t a t e d by a d d in g a s l i g h t e x c e ss o f g l a c i a l a c e t i c a c i d . The p r e c i p i t a t e i s a g a in f i l t e r e d w i t h s u c t i o n , washed f r e e from sodium a c e t a t e w i t h w a t e r , and d r i e d .* 1 C hem ilum inescence ta k e s p la c e o n ly in a l k a l i n e s o l u t i o n and i s b e s t s e e n by a d d in g h y d ro g en p e ro x id e and p o ta s s iu m f e r r i c y a n i d e i n v a rio u s ways* T h ere a r e many o t h e r c h e m ilu m in e sc e n t r e a c t i o n s known, p r i n c i p a l l y o x id a t i o n s , b u t none so s t r o n g as t h i s o n e . As w i l l be se e n l a t e r , n o t a l l s u b s ta n c e s w hich decompose p e ro x id e c au se th e a m in o p h th a lh y d ra z id e to lu m in e sce and i t i s t h i s f a c t w h ich g iv e s th e f o r e n s i c t e s t u s in g t h i s r e a g e n t a v a lu a b le s p e c i f i c i t y . The m ost r e c e n t w ork on th e c h em ilu m in e sc en c e of p h th a lh y d r a z id e d e r i v a t i v e s i s t h a t o f Z e l l n e r 27 who has 27 J . A. C. S. 59 2580, 1937 23 p r e p a r e d num erous d e r i v a t i v e s of p h th a lh y d r a z id e and com pared t h e i r lu m in e s c e n t p r o p e r t i e s by a p h o t o e l e c t r i c m ethod. H is r e s u l t s v e r i f y w hat was fo u n d i n th e e a r l y c o u rs e o f th e p r e s e n t i n v e s t i g a t i o n as r e g a rd s th e q u a l i t a t i v e r e l a t i o n b etw een l i g h t i n t e n s i t y and 3 -a m in o p h th a lh y d ra z id e c o n c e n tr a ti o n and l i g h t i n t e n s i t y and sodium h y d ro x id e c o n c e n t r a t i o n . H is m easurem ents w ere made d i r e c t l y by a P h o tr o n ic c e l l w hich was p o s s i b l e due t o th e h ig h lu m in e sc e n c e p r e s e n t when h y p o c h lo r i te i s u se d as o x i d a n t . In com paring v a r io u s i n t e n s i t i e s o f b lo o d r e a c t i o n s , th e a u th o r has fo u n d th e i n t e n s i t y to o low t o a llo w d i r e c t p h o t o e l e c t r i c m easu rem en t, and h a s u se d a c o m b in a tio n p h o t o g r a p h i c - p h o t o e l e c t r i c m ethod by w hich an i n t e g r a t e d e f f e c t is o b ta in e d s o t h a t w e ak e r i n t e n s i t i e s can be q u a n t i t a t i v e l y m e a su re d . THE COMPOSITION OF BLOOD STAINS I t w ould seem d e s i r a b l e t o t r y t o f i n d w hich b lo o d c o n s t i t u e n t i s t h e p r i n c i p a l r e a c t a n t in th e lu m in e s c e n t r e a c t i o n p ro d u ce d when b l o o d , p e r o x id e , and 3 -a m in o p h th a lh y d ra z id e * a r e b ro u g h t t o g e t h e r i n a l k a l i n e s o l u t i o n . F o r t h i s r e a s o n , t h e c o m p o s itio n o f b lo o d h as been s t u d i e d and r e a c t i o n s b etw een v a r io u s c o n s t i t u e n t s and h y d ro g en p e ro x id e have b e e n m e a su re d . Of im p o rta n c e from th e s t a n d p o in t of th e f o r e n s i c c h e m is t i s th e c o m p o s itio n o f b lo o d w h ic h i s o ld and i n s o l u b l e or p a r t i a l l y decom posed. I t i s d e s i r a b l e t o have a r e a g e n t T h is compound i s h e r e a f t e r r e f e r r e d to a s " h y d r a z id e 1 1 . 24 w hich w i l l w ork e q u a l ly w e ll w ith s t a i n s o f a l l ag es o r even one w hich would d i f f e r e n t i a t e s t a i n s o f v a rio u s a g e s . I t seems p o s s i b l e t h a t i f th e c o n s t i t u e n t s a r e s u f f i c i e n t l y d i f f e r e n t as d e c o m p o s itio n p r o g r e s s e s , t h a t th e d i f f e r e n c e c o u ld be made a p p a re n t b y v a r i a t i o n s in th e i n t e n s i t y o f ch em ilum i- n e s c e n c e . T h is phase of th e problem d e s e r v e s f u r t h e r i n v e s t i g a t i o n , as do a l s o s o l u b i l i t y d e te r m in a tio n s by e x t r a c t i n g s t a i n s and d e te rm in in g th e power o f th e e x t r a c t t o e x c i t e lu m in e s c e n c e . I t m ig h t a ls o be o f i n t e r e s t t o f i n d how th e b lo o d o f v a r i o u s p o is o n in g v ic tim s r e a c t s . H em oglobin, as i s w e ll known, i s th e r e d c o lo r i n g m at t e r of th e b lo o d a n d t h i s i s known t o decom pose h y d ro g en p e r ox id e# On s t a n d i n g , hem og lob in ch an g es i n t o m ethem oglobin i f ex p o sed to s u n l i g h t . I f th e c o n d itio n s of s ta n d in g a r e such t h a t th e b lo o d i s h e a te d o r k e p t damp, th e compound form ed is 28 more l i k e l y t o be h e m a tin . In o t h e r c a s e s , a s w here th e i r o n o f t h e h em oglobin i s rem oved by c h e m ic a l a c t i o n , hem ato- p o r p h y rin is form ed# Thus i t may be se e n t h a t a g iv e n s t a i n may c o n ta in many m o d if i c a t io n s o f h e m o g lo b in , w h ich may be i d e n t i f i e d and c o n v e rte d i n t o s t i l l o th e r m o d if i c a t io n s , as was d i s c u s s e d u n d e r th e s p e c t r o s c o p i c m ethod. Blood c o n s t i t u e n t s o t h e r th a n h em o glo bin and i t s d e r i v a t i v e s do n o t c o n c e rn us h e re and th e f o llo w in g d i s c u s s i o n 28 W e b ste r, L e g a l M edicine and T o x ic o lo g y ( P h i l a d e lp h i a : S au n d ers 1930) p . lVST 25 in v o lv e s o n ly th o se m o d if ic a tio n s o f hem oglobin w h ich w ould be m ost l i k e l y t o b e fo u n d in f o r e n s i c c h e m is tr y . Hem oglobin i s i t s e l f v e ry com plex a n d c o n s i s t s o f a p r o t e i n p a r t c a l l e d g lo - 29 b i n and an i r o n - p y r r o l compound known as h e m a tin o r heme. O xyhem oglobin may be b ro k e n up by a c i d o r a l k a l i i n t o a c i d o r a l k a l i n e hem atin* The a l k a l i n e compound may be re d u c e d t o hemochromogen w h ic h i s a compound o f re d u c e d heme and g lo b in . Hem i n , C^H^gN^Q^FeCL, i s h e m a tin h y d r o c h lo r id e w h ich can be p r e p a r e d fro m h e m a tin and h y d r o c h lo r ic a c i d . T h is has a ls o so b e e n s y n t h e s i z e d . I t can be made by t r e a t i n g h em oglobin w ith g l a c i a l a c e t i c a c id and a h a lo g e n c h l o r i d e as in th e 31 Teichm an t e s t . Heme o r h e m a tin form s a w hole s e r i e s o f compounds known as hem ochrom ogens. Members of t h i s group have th e same g e n e r a l a b s o r p t io n s p e c t r a , th e bands b e in g s l i g h t l y d i s p l a c e d f o r i n d i v i d u a l m em bers. H em atin i s p ro b a b l y th e p r i n c i p a l c o n s t i t u e n t o f o ld b lo o d s t a i n s . I t i s i n s o l u b l e i n w a t e r , d i l u t e a c i d s , a l c o h o l , e t h e r and c h lo r o fo r m . T h is a c c o u n ts f o r t h e slow r e a c t i o n of v e ry o ld s t a i n s w ith th e u s u a l t e s t s . I t d i s s o l v e s in d i l u t e a l k a l i , a c i d i f i e d a l c o h o l , o r g l a c i a l a c e t i c a c i d . When th e h e m a tin i s v e r y i n s o l u b l e , as i n s t a i n s w hich have b een h e a t e d , i t may be c o n v e rte d by s u l f u r i c a c i d i n t o h e m a to p o rp h y rin , w h ich i s th e n s o l u b l e i n a l c o h o l , a l k a l i e s and a c i d s . 29 B a n c r o f t, Hem oglobin (Cam bridge U n i v e r s i t y P re s s 1 9 2 8 ). 30 F i s c h e r & Z eile m , Ann. 4 6 8 , 9 8 , 1929. 31 B odansky, P h y s io lo g ic a l C h em istry (New York: W iley & Sons 1934) p 7 " 2 ? l. M ethem oglobin c o n ta in s th e same am ount o f oxygen and i s is o m e ric w ith oxyh em og lob in. POSTULATED REACTION MECHANISMS 1 Prom a c o n s i d e r a t i o n o f th e above p o s s i b l e r e a c t a n t s and t h e i r a c t i o n on h y d ro g en p e r o x id e , th e f o llo w in g r e a c t i o n s s u g g e s t th e m s e lv e s . When 3 -a m in o p h th a lh y d ra z id e i s d is s o l v e d i n a l k a l i n e s o l u t i o n and a s m a ll amount o f p e ro x id e i s added a lo n g w i t h a b lo o d s t a i n o r e x t r a c t , th e h y d r a z id e is known t o u n d erg o o x i d a t i o n by th e b l u i s h lu m in e sc e n c e w h ic h o c c u r s . S e v e ra l t h i n g s s u g g e s t th e m s e lv e s : 1) D eco m p o sitio n o f h y d ro g en p e ro x id e by h e m o g lo b in , m ethe m o g lo b in , oxyhem oglobin, o r h e m a tin , fo llo w e d by o x i d a t i o n o f th e h y d r a z id e by oxygen from th e p e ro x id e d e c o m p o s itio n . The o x i d a t i o n may o r may n o t be r e s t r i c t e d t o e i t h e r ato m ic or m o le c u la r oxygen. 2) D i r e c t o x i d a t i o n o f h y d ra z id e by h y drogen p e r o x id e . 3) D i r e c t o x i d a t i o n o f h y d r a z id e by n a t u r a l l y decom posing p e r o x id e . 4) O x id a tio n o f h y d ra z id e by d i s s o l v e d oxygen. 5) D ir e c t o x i d a t i o n o f h y d r a z id e by b lo o d . 6) A ll o f t h e above r e a c t i o n s a t o n c e. * 3 tQ S pecht say s i n th e t i t l e t o h i s a r t i c l e t h a t t h e 32 S p e c h t, Z e i t . f u r angew . Chem. 5 0 , 8 , 1 44 , 1937. 27 lu m in e sc e n c e i s due t o hem in and i n th e "body o f h i s p a p e r he sa y s t h a t i t i s c a u s e d by. h e m a tin . No q u a l i t a t i v e o r q u a n t i t a t i v e d a t a i s p r e s e n t e d i n c o n n e c tio n w ith e i t h e r c o n c lu s io n . P ro b a b ly th e m echanism i s a com plex one n o t due t o any one b lo o d c o n s t i t u e n t . T h is m a tte r i s d e a l t w ith in th e e x p e r i m e n ta l p a r t of t h i s p a p e r . I t a p p e a rs t h a t th e o x i d a t i o n o f th e h y d ra z id e may ta k e p la c e o n ly i f th e oxygen i s r e l e a s e d from th e p e ro x id e in th e a to m ic s t a t e . The e v id e n c e f o r t h i s seems t o be t h a t i r o n r u s t and o t h e r s u b s ta n c e s w hich i n t e r f e r e by decom posing th e p e ro x id e i n some o f t h e o x i d a tio n t e s t s do n o t i n t e r f e r e in t h e h y d ra z id e t e s t , as I s h a l l c a l l th e b lo o d - p e r o x id e - 3 - a m in o p h th a lh y d ra z id e r e a c t i o n . The k i n e t i c s o f p e ro x id e d e c o m p o s itio n by i r o n r u s t show t h a t m o le c u la r oxygen i s form ed w ith o u t th e in te r m e d i a t e s t e p o f a to m ic oxygen b e in g fo rm ed . T h e re fo re i t becomes l o g i c a l t o p o s t u l a t e t h a t f o r th e h y d ra - . z id e t o lu m in e s c e , th e o x i d a n t , i f i t i s oxygen, m ust be a to m ic . C o n c lu sio n s on t h i s p o in t a re l a t e r made on th e b a s i s o f e x p e r im e n ta l e v id e n c e p r e s e n te d i n C h a p te r IV. T h is m a t t e r c o u ld a lo n e form t h e b a s i s o f an e x te n s iv e r e s e a r c h in r e a c t i o n k i n e t i c s . CHAPTER I I I . THEORETICAL CONSIDERATIONS RADIOMETRY In o r d e r t o make a q u a n t i t a t i v e o r s e m i - q u a n t i t a t i v e s tu d y o f t h e phenomenon of ch em ilu m in e sc en c e accom panying th e o x i d a t i o n o f 3 -a m in o p h th a lh y d ra z id e i n a l k a l i n e s o l u t i o n , i t i s o b v io u s t h a t v i s u a l e s t i m a t i o n o r co m p ariso n a lo n e i s in a d e q u a te . The m ost l o g i c a l p ro c e d u re w ould seem to be in th e u s e o f th e p h o t o e l e c t r i c c e l l i n one of i t s s e v e r a l form s in c o n ju n c tio n w ith s u i t a b l e i n d i c a t i n g a p p a r a tu s and a m p lify in g equipm ent of one k in d o r a n o th e r , i f n e c e s s a r y . T here a r e many f a c t o r s i n f lu e n c i n g th e p ro p e r c h o ic e o f m e a su rin g e q u ip m e n t. T h ere a r e i n a d d i t i o n , v a r io u s a d v an ta g e s and d is a d v a n ta g e s f o r e a c h ty p e o f p h o t o c e l l . F o r e x am p le, i f one u s e s a vacuum p h o to e m is s iv e t y p e , th e r a t e o f e l e c t r o n e m is s io n , and t h e r e f o r e th e c u r r e n t , is so s m a ll i f th e l i g h t i n t e n s i t y i s s m a ll , t h a t a m p lify in g equ ipm ent i s u s u a l l y n e c e s s a r y . The u se o f a m p l i f i e r s u s u a l l y i n t r o du ces s e n s i t i v i t y a t a s a c r i f i c e o f th e s t a b i l i t y w h ic h i s d e s i r a b l e i n q u a n t i t a t i v e w ork. A s t r i c t l y l i n e a r a m p l i f i e r i s a l s o n e c e s s a r y . The b a r r i e r - l a y e r ty p e o f c e l l ( a l s o c a l l e d p h o to e . m . f . o r p h o t o v o l t a i c ) i s c h a r a c t e r i z e d by a r e l a t i v e l y h ig h o u tp u t w h ich i n a p r o p e r l y d e s ig n e d c i r c u i t i s n e a r l y p r o p o r t i o n a l t o t h e i n c i d e n t e n e rg y . The e x t e r n a l 29 r e s i s t a n c e m ust be k e p t below a c e r t a i n v a lu e i n o r d e r t o k eep th e c u r r e n t - i n c i d e n t e n e rg y c u rv e l i n e a r . A lth o u g h th e c u r r e n t s e n s i t i v i t y o f t h i s ty p e o f c e l l can be e q u a l le d in a good q u a l i t y gas f i l l e d p h o to tu b e , th e gas ty p e has th e d is a d v a n ta g e o f r e q u i r i n g an e x t e r n a l s o u rc e o f e . m . f . j u s t as in th e c a se o f th e l e s s s e n s i t i v e vacuum t y p e . The d i s a d v a n ta g e o f th e l i q u i d ty p e o f p h o t o v o l t a i c c e l l i s o b v io u s . The s p e c t r a l c h a r a c t e r i s t i c s o f th e l i g h t from th e c h e m ilu m in e sc e n t r e a c t i o n sh o u ld be c o n s id e r e d in any m ethod o f d i r e c t l y m e a su rin g i t s i n t e n s i t y p h o t o e l e c t r i c a l l y . The c o m p o s itio n o f th e l i g h t may v a ry w ith th e c o n c e n t r a t i o n , r a t e , te m p e ra tu re o r o t h e r f a c t o r s . Such v a r i a t i o n , i f p r e s e n t , i s to o s l i g h t t o w a r r a n t a s p e c t r a l a n a l y s i s o f th e l i g h t u n d e r v a r i o u s c o n d i t i o n s , a lth o u g h su c h a s tu d y sh o u ld b e m ost i n t e r e s t i n g . I t w ould, h o w ev er, be w e l l t o know w h e th e r any u l t r a - v i o l e t l i g h t i s e m itte d by th e s o l u t i o n , s i n c e t h i s w ould b e im p o r ta n t, p a r t i c u l a r l y i f p h o to g ra p h ic m ethods w ere em ployed. B r i e f e x p e rim e n ts in t h i s c o n n e c tio n a r e d e s c r ib e d in C h a p te r IV. L i n e a r i t y o f r e s p o n s e i s p ro b a b ly th e m ost d e s i r e d c h a r a c t e r i s t i c in a c e l l u se d f o r l i g h t i n t e n s i t y m easu rem en t, o th e r w is e th e c a l c u l a t i o n s a r e more in v o lv e d and n o t as a c c u r a t e . The a c t u a l p h o t o e l e c t r i c c u r r e n t i t s e l f , t h a t i s , t h e num ber o f e l e c t r o n s r e l e a s e d from th e anode i s d i r e c t l y p r o p o r t i o n a l t o th e i n c i d e n t f l u x o v e r a l l p o s s i b l e ra n g e s 30 o f v a l u e s . However, due t o v a r io u s c o m p lic a te d phenom ena, th e c u r r e n t o u tp u t of a vacuum o r gas tu b e i s n o t l i n e a r , th e l i n e a r i t y b e in g f o r one t h i n g a f u n c t i o n o f th e anode v o l t a g e . Where th e c h o is e of m e a su rin g tu b e is b e tw ee n the vacuum o r gas f i l l e d p h o to tu b e , th e vacuum ty p e i s more d e s i r a b l e f o r m easurem ent From a c o n s i d e r a t i o n o f th e above f a c t s and th e a v a i l a b le e q u ip m e n t, i t was d e c id e d to use th e b a r r i e r - l a y e r ty p e o f c e l l f o r m ea su rin g th e b r i l l i a n c y o f c h e m ilu m in e sc e n e e . T hat u se d was made by th e W eston In s tru m e n t Company u n d e r th e t r a d e name " P h o tro n ic C e l l ” . Such c e l l s a r e made w ith e i t h e r g l a s s o r q u a r tz windows f o r u se i n th e v i s i b l e o r u l t r a - v i o l e t r e g io n s of th e sp e c tru m . I t was fo u n d , as l a t e r d e s c r i b e d , t h a t t h e l i g h t i n t e n s i t y was to o s m a ll to a llo w d i r e c t u se o f th e c e l l so a d e n s ito m e te r was b u i l t f o r m ea su rin g th e d e p th o f s i l v e r d e p o s i t s in p h o to g ra p h ic f ilm s a f t e r e x p o su re to th e r e a c t i o n m ix tu re i n a s p e c i a l c e l l , ^he d e n s ito m e te r em bodies a p h o tr o n ic c e l l w ith a g l a s s window. Such an i n d i r e c t m ethod o f m e a su rin g th e lu m in e sc e n c e has c e r t a i n a d v a n ta g e s , among w hich a r e th e i n t e g r a t i n g a b i l i t y o f p h o to g ra p h ic m a t e r i a l s w hich a llo w s w eaker l i g h t t o be m e a su re d , th e e l i m i n a t i o n o f a m p lify in g equipm ent (w hich c a n n o t be u se d anyway w i t h low v o l ta g e d i r e c t c u r r e n t s ) , and th e 33 F o r s y th e , M easurem ent o f R a d ia n t E nergy (New Y ork: M cG raw -H ill 1937) p . "'230'. 31 a v o id a n c e of a " c r e e p i n g 1 1 o u tp u t r e s u l t i n g from a c o n s t a n t l y c h an g in g l i g h t i n t e n s i t y . W ith th e d e n s ito m e te r m ethod, c h an g in g i n t e n s i t y i s r e c o rd e d as th e t o t a l i n t e n s i t y o v e r a g iv e n p e rio d and t h i s t o t a l th e n does n o t change and may be m easured from th e s i l v e r d e p o s i t a t l e i s u r e . In a d d i t i o n , t h i s e n a b le s a v e ra g e v a lu e s t o be d i r e c t l y o b ta in e d . When m aking c o m p a ra tiv e m easu rem en ts, th e r a t i o of s i l v e r d e p o s i t d e n s i t i e s may be u se d to g iv e t h e d e s i r e d i n f o r m a tio n , b u t i f a b s o lu te m easurem ents a re to be m ade, o r i f th e c o m p a ra tiv e v a lu e s o f lu m in e sce n c e i n t e n s i t i e s a r e d e s i r e d , i t is n e c e s s a r y t o rev iew th e t h e o r y o f p h o to g ra p h ic r a d io m e try i n o r d e r to make th e p ro p e r c a l c u l a t i o n s . The f o llo w in g c o n s i d e r a ti o n s c o n s t i t u t e th e b a re fu n d a m e n ta ls of 34 su c h th e o r y : P h o to g ra p h ic m a t e r i a l s a re s e n s i t i v e r e c e i v e r s o f t h e s e l e c t i v e ty p e and f o r a b s o lu te m easurem ents i n s ta n d a r d u n i t s , m ust be c a l i b r a t e d by n o n - s e l e c t i v e r e c e i v e r s , su ch a s th e r m o p il e s . F o r th e p r e s e n t p u rp o se t h i s i s n o t n e c e s s a r y , s in c e i n t e n s i t i e s can be e x p re s s e d i n a r b i t r a r y u n i t s . Even t h i s is n o t n e c e s s a r y in e v e ry c a s e ,s i n c e g a lv a n o m e te r d e f l e c t i o n s , f o r ex am p le, can be u s e d as an e x p r e s s io n of l i g h t i n t e n s i t y . C om parisons by t h i s m ethod have b e en made 35 by Z e l l n e r who u se d a p h o t o e l e c t r i c c e l l and g a lv a n o m e te r 34 I b i d . , p p . 2 4 6 -2 8 2 . 35 J .A .C .S . 5 9 , 2580, 1937. 52 d i r e c t l y , e x p r e s s in g ch em ilu m in escen ce as cm. o f g a lv a n o m e te r d is p la c e m e n t. When u s in g l i g h t s e n s i t i v e e m u lsio n s as th e p rim a ry r e c e i v e r , th e d e f l e c t i o n s from a g a lv a n o m e te r, b e in g u se d to m easure t h e tr a n s m is s io n o f l i g h t t h r u th e s i l v e r d e p o s i t , w ould of c o u rs e be i n v e r s e l y p r o p o r t i o n a l t o th e i n t e n s i t y o f t h e lu m in e sc o n c e . The r e l a t i o n s e x i s t i n g b etw een l i g h t i n t e n s i t y and s i l v e r d e n s i t y (s tim u lu s and r e s p o n s e ) c o n s t i t u t e w hat i s known as th e c h a r a c t e r i s t i c cu rv e o f th e e m u lsio n u n d e r con s i d e r a t i o n * Such a c u rv e r e s u l t s when v a lu e s o f th e lo g a r ith m o f t h e e x p o su re a re p l o t t e d as a b s c i s s a and v a lu e s o f th e den s i t y as o r d i n a t e s . The m agnitude o f th e re s p o n s e i s a fu n c t i o n o f th e r a t e a t w h ich r a d i a n t e n erg y f a l l s on th e s u r f a c e and t h e l e n g t h o f tim e d u r in g w h ich i t d o es s o . E, th e e x p o s u r e , i s d e f i n e d as ( r a t e x tim e ) . In p h o to g ra p h ic p r a c t i c e , E i s e x p re s s e d i n l i g h t u n i t s , b u t i n r a d io m e tr y in v o lv in g r e g io n s of th e s p e c tru m i n s e n s i t i v e to t h e eye and t h e r e f o r e o f no co n seq u en ce i n p i c t u r e t a k i n g , i t i s e x p re s s e d in e n e rg y u n i t s . The u se o f l i g h t u n i t s i s a l l r i g h t h e re s in c e t h e so u rc e o f l i g h t i n th e d e n s ito m e te r i s sim p ly an in c a n d e s c e n t lamp* I f t h e c h em ilu m in escen ce w ere t o be r e c o r d e d d i r e c t l y t h r u a p h o t o c e l l , e n e rg y u n i t s w ould p r o b a b ly be b e t t e r in c a se t h e r e s h o u ld be any u l t r a - v i o l e t e m is s io n . The u n i t in th e fo rm e r c ase i s th e m e te r c a n d le so t h a t E = I x t m e te r- c a n d le -s e c o n d s w here I i s th e i l l u m i n a t i o n in m e te r c a n d le s 33 anS. t i s th e tim e in s e c o n d s . When u s in g u n i t s w h ic h ta k e i n t o c o n s i d e r a t i o n a l l p o s s i b l e s p e c t r a l c h a r a c t e r i s t i c s o f t h e l i g h t , E = I x t e r g - c m .- s e c s # w here I i s th e r a d i a n t f l u x d e n s i t y i n e rg s p e r s q u a re cm ., and t i s tim e in seco n d s# D, th e d e n s i t y , is an e x p r e s s io n of th e l i g h t - a b s o r b in g power of th e exposed and d e v e lo p e d p h o to g ra p h ic m a t e r i a l and e q u a ls th e lo g a r ith m to th e b a se 1 0 , o f 0 th e o p a c ity # The r e l a t i o n o f th e s e f a c t o r s t o th e s tim u lu s an d o t h e r f a c t o r s i s se e n from th e fo llo w in g : F0 = lum inous f l u x i n c i d e n t on th e s i l v e r d e p o s it# F^ = lum inous f l u x t r a n s m i t t e d by s i l v e r d e p o s i t . T = t r a n s m i s s io n f a c t o r . 0 = o p a c i t y . D = dens i t y . X = F i/P r,. l /T = 0 = Fq/ F ^ . D * log^QO# The c h a r a c t e r i s t i c c u rv e , a l s o c a l l e d th e D -lo g E c u rv e i s p r a c t i c a l l y l i n e a r o v e r p a r t o f i t s ra n g e f o r c e r t a i n m a t e r i a l s . One re a s o n why e r r o r s a r e o f t e n e n c o u n te re d i n p h o to g ra p h ic m ethods o f e n e rg y m ea su re m e n ts, i s t h a t th e form o f th e c h a r a c t e r i s t i c c u rv e i s n o t ta k e n i n t o c o n s i d e r a t i o n . N a t u r a l l y i t i s d e s i r a b l e to have th e d e n s i t y o f th e s i l v e r d e p o s i t d o u b le when lo g E i s d o u b le d so t h a t d e n s i t y r a t i o s w i l l t r u l y r e p r e s e n t e x p o su re r a t i o s . C h a r a c t e r i s t i c c u rv e s can be o b ta in e d fro m th e m a n u fa c tu re r o r can be d e te rm in e d e x p e r im e n ta lly and i t i s th e n o n ly n e c e s s a r y to w ork in t h a t ra n g e w here th e c u rv e i s l i n e a r i n o r d e r t o a v o id e r r o r . L a t it u d e i s th e te rm u se d t o e x p re s s th e l e n g t h o f th e l i n e a r 54 p a r t of th e D -lo g E c u r v e . When th e l i n e a r p o r t i o n i s p r o j e c t e d o nto th e lo g E a x i s , th e d i s t a n c e b etw een th e p o in ts on t h e a x is i s th e l a t i t u d e and may be e x p re s s e d in term s o f E o r o f lo g E. T h is L = lo g Eg - lo g E^ o r L = E 2 /E ^ . I t i s r e a d i l y s e e n t h a t th e more l a t i t u d e an e m u lsio n h a s , th e l e s s chance t h e r e is o f w o rk in g on th e n o n - l i n e a r p a r t of th e c u rv e , w i t h c o n se q u e n t e r r o r . L a titu d e i s a f u n c t i o n of d e v e lo p in g c o n d itio n s and s p e c t r a l q u a l i t y of th e s tim u lu s , b u t w ith o r d i n a r y c a r e one can a v o id w o rk in g a t to o low or to o h ig h ex p o s u r e . In o t h e r w o rd s, th e l i n e a r p o r t i o n of th e c u rv e is th e r e g i o n o f c o r r e c t e x p o su re and one need o n ly r e g u l a t e th e ex p o s u re tim e so t h a t t h e r e s u l t i n g d e n s i t y l i e s in t h i s r e g i o n . A lth o u g h i t w ould seem t h a t D w ould be th e same fu n c t i o n o f E r e g a r d l e s s o f th e v a lu e s o f I and t w h ich make up E, t h i s i s n o t alw ays s o . The d iv e rg e n c e i s known a s th e f a i l u r e o f the r e c i p r o c i t y law and n e c e s s i t a t e s a d i s t i n c t i o n as to w h e th e r th e c h a r a c t e r i s t i c c u rv e was c o n s tr u c te d on an i n t e n s i t y s c a l e or a tim e s c a l e . In th e f o r m e r , th e tim e i s con s t a n t and in th e l a t t e r th e i n t e n s i t y i s c o n s t a n t . Most c u rv e s r e p r e s e n t e x p o su re s i n w hich t h e tim e was v a r i e d and th e i n t e n s i t y k e p t c o n s t a n t . In th e w ork of t h i s i n v e s t i g a t i o n th e i n t e n s i t y o f ch em ilu m in escen ce i s p r a c t i c a l l y con s t a n t w h ile th e tim e o f ex p o su re t o th e e m u lsio n is v a r i e d . T h e re fo re th e e x p o su re s a r e made to f a l l on th e l i n e a r p o r t i p n o f a tim e - s c a l e c h a r a c t e r i s t i c c u rv e . The r e c i p r o c i t y - l a w 35 36 f a i l u r e has been s t u d i e d i n d e t a i l by many w o rk e rs . The d e n s i t y f l u x r e l a t i o n s d is c u s s e d above a r e c a l c u l a t e d from e x p e r im e n ta l r e s u l t s by d e te r m in in g Fq and F ^ , i n term s of g a lv a n o m e te r d e f l e c t i o n s , D = l o g 10 Fq/ F ! D = gT8 w here g i s th e d e - 8 i 89 f l e c t i o n c o rre s p o n d in g t o F q and G -1 i s t h a t c o rre s p o n d in g to F ^ , Gq i s th e d e f l e c t i o n f o r i n t e n s i t y e q u a l to z e r o , CHEMICAL KINETICS In d e te r m in in g th e m echanism o f r e a c t i o n s , th e o rd e r o f th e r e a c t i o n w ith r e s p e c t to c e r t a i n r e a c t a n t s is m ea su re d , and t h i s may be done in s e v e r a l w ay s. Among t h e s e a re a ) su b s t i t u t i n g e x p e r im e n ta l d a ta in t h e d i f f e r e n t i a l e q u a tio n s e x p r e s s i n g th e r a t e c o n s ta n t a s a f u n c t i o n o f i n i t i a l co n cen t r a t i o n an d c o n c e n tr a tio n s o f r e a c t a n t s a t tim e t , and f i n d i n g w hich e q u a tio n s g iv e s a " c o n s t a n t ” w hich i s a c t u a l l y c o n s t a n t , b ) O s tw a ld fs " i s o l a t i o n " m ethod by w hich e a c h r e a c t a n t i s ta k e n in t u r n (by t a k i n g su ch a l a r g e e x c e ss of th e o th e r r e a c t a n t s t h a t t h e i r c o n c e n tr a ti o n ch an g es a r e n e g l i g i b l e ) , th e r e l a t i o n b etw een r e a c t i o n v e l o c i t y and th e c o n c e n tr a ti o n o f t h i s m in o r ity r e a c t a n t th e n b e in g d e te rm in e d e x p e r i m e n t a l ly , ^ Abney, P h o t, J o u r , , 1 8 , 3 0 2 , 1893. S c h w a r z s c h ild , A s tr o p h y s . J o u r . 1 1 , 8 9 , 1900. 36 c) M easurem ent of th e tim e n e c e s s a r y t o co m p lete a c e r t a i n f r a c t i o n o f th e r e a c t i o n , d o u b lin g and t r e b l i n g th e co n cen t r a t i o n o f th e r e a c t a n t b e in g c o n s id e r e d and m e a su rin g th e tim e a g a in t o f i n d how th e tim e depends on i n i t i a l c o n c e n tr a t i o n , d ) P l o t t i n g v a r io u s f u n c t i o n s o f th e c o n c e n tr a ti o n a g a i n s t tim e t o d e te rm in e w h ich one g iv e s a s t r a i g h t l i n e . The b lo o d - p e r o x id e - h y d r a z id e r e a c t i o n comes u n d e r th e c l a s s i f i c a t i o n of com plex r e a c t i o n s p ro b a b ly in c lu d in g b o th s i d e - r e a c t i o n s , s im u lta n e o u s r e a c t i o n s , and c o n s e c u tiv e r e a c t i o n s . S in c e th e m easurem ent o f t h i s r e a c t i o n as a w hole i s p r a c t i c a l l y im p o s s ib le , the v a rio u s p o s t u l a t e d s t e p s o f th e r e a c t i o n a r e i n v e s t i g a t e d by one of th e above m e th o d s. Method a ) g iv e s e rro n e o u s r e s u l t s i f c o m p lic a tin g s im u lta n e o u s r e a c t i o n s a r e p r e s e n t so s h o u ld o n ly be u se d i n sim p le r e a c t i o n s . I f no s a t i s f a c t o r y c o n s ta n ts a re o b ta in e d 37 by any f o rm u la , th e r e a c t i o n i s n o t s im p le . Method b ) may c o n v e n ie n tly be a p p li e d to r e a c t i o n s even up t o f i f t h o r d e r . In m ethod c) th e c r i t e r i a f o r th e d e te r m i n a ti o n o f o rd e r a r e th e f o llo w in g : 1) F o r f i r s t o r d e r , th e tim e f o r a g iv e n f r a c t i o n t o be c o m p leted i s in d e p e n d e n t o f th e i n i t i a l c o n c e n tr a t i o n . 2) F o r se c o n d o r d e r , th e tim e f o r the same f r a c t i o n is i n v e r s e l y p r o p o r t i o n a l t o th e i n i t i a l c o n c e n tr a - 37 Getman, T h e o r e t i c a l C h em istry (New Y ork: W iley 1937) p . 324. 37 t i o n , e . g . , i f th e c o n c e n t r a t i o n o f th e r e a c t a n t i s d o u b le d , th e tim e i s h a lv e d . v e r s e l y p r o p o r t i o n a l to th e ( n - l ) t h pow er o f th e i n i t i a l c o n c e n t r a t i o n . v e r s u s tim e i s l i n e a r , w here a* i s th e i n i t i a l c o n c e n tr a ti o n o f th e r e a c t a n t and x i s th e c o n c e n t r a t i o n a t tim e t . 2) F o r b im o le c u la r r e a c t i o n s , l / ( a - x ) i s a l i n e a r f u n c t i o n o f th e t im e . l i n e a r f u n c t i o n o f t h e tim e . In b o th m ethods b ) and c ) , th e o r i g i n a l c o n c e n tr a ti o n s o f th e r e a c t a n t s m ust be e q u a l . The d i f f e r e n t i a l e q u a tio n s f o r th e v a r io u s r e a c t i o n o r d e r s a r e d e v e lo p e d a s f o l l o w s . In a l l o f th e s e c a s e s th e f o l low ing n o m e n c la tu re i s u s e d : 3) F o r an n t h o rd e r r e a c t i o n , th e tim e i s i n In m ethod d) th e c r i t e r i a a r e : 1) F o r u n im o le c u la r r e a c t i o n s , lo g ( a -x ) 3) F o r a te r m o le c u la r r e a c t i o n , l / ( a - x ) ^ i s a k = s p e c i f i c r e a c t i o n r a t e a * i n i t i a l c o n c e n t r a t i o n o f r e a c t a n t A k - li ti it .n c = ” V " C x = c o n c e n tr a ti o n o f p r o d u c ts a t tim e t U n im o le c u la r C ase- d x / d t = k ( a - x ) f k d t o 38 - I n / ( a - x ) - C -ln a ) = k t In a / a - x = k t k « * 2 .3 0 3 / t • lo g a / a - x I t i s n o t n e c e s s a r y t h a t th e o r i g i n a l c o n c e n tr a ti o n be known as t h i s form of th e e q u a tio n w ould i n d i c a t e . The f o llo w in g form a llo w s k t o be c a l c u l a t e d from a n a l y t i c a l r e s u l t s ob t a i n e d a t two tim e s a f t e r th e r e a c t i o n has s t a r t e d : k * 2 . 3 0 3 / t g - t ^ • lo g (a -x ^ J/C a -X g ) B im o le c u la r C ase- T here a re two c a s e s to be c o n s id e r e d h e r e , a ) th e two r e a c t a n t s a r e p r e s e n t a t th e same i n i t i a l co n cen t r a t i o n , b ) th e y a r e p r e s e n t a t d i f f e r e n t c o n c e n t r a t i o n s . Case a ) d x / d t = k ( a - x ) 2 x . r j d x / ( a - x ) ^ = ( k d t / o o l / ( a - x ) - l / a a k t x / a ( a - x ) = k t Case b ) d x / d t = k ( a - x ) ( b - x ) k d t ^ d x / ( a - x ) (b -x ) = j] k - 2 . 3 0 3 / t ( a - b ) • lo g b ( a - x ) / a ( b - x ) T h ere Is a s p e c i a l c a se in c o n n e c tio n w ith b im o le c u la r r e a c t i o n s w h e re in i f th e i n i t i a l c o n c e n tr a ti o n s o f th e r e a c t a n t s a r e n e a r l y e q u a l Case a ) becomes 39 k = l / t x / a ^ a - x ) 1 w here a* i s th e mean o f a and b and w here ( a - x ) 1 i s th e mean o f ( a - x ) and ( b - x ) . T e rm o le c u la r C ase- ( f o u r ty p e s ) a ) R e a c ta n ts a t same i n i t i a l c o n c e n tr a ti o n : d x / d t - k / a - x ) 3 | d x / ( a - x ) 3 = f k d t • * 0 O -j X l / a ( a - x ) 2 i = k t J O k = l / t x ( 2 a - x ) / 2 a 2 ( a - x ) 2 b) R e a c ta n ts a t u n e q u a l i n i t i a l c o n c e n tr a ti o n s : d x / d t = k ( a - x ) ( b - x ) ( c - x ) / d x / ( a - x ) (b -x ) (c - x ) = / k d t / O ''o k = 2 #303 lo g a - 4- ^ •5Q.5 lo g ft _ i~ ♦ 303 lo g a*c * t 1 6 a -x a 'b *t * b - x ^ b » c * t K c -x w here a* = ( a - b ) b* = (b - c ) c 1 = ( c - a ) c) S to ic h im o e tr y is o f form 2A ■ + * C = p r o d u c ts : d x / d t = k ( a - 2 x ) ( c - x ) f d x / ( a - 2 x ) ( c - x ) = f k d t /o 'o , 1 r(2c-a)2x ^ ^ ( (a-2x)c (a -2 c) [ a T O I j + 2*303 ® (c -x )a _ d ) Two r e a c t a n t s have same i n i t i a l c o n c e n tr a ti o n and t h i r d d o es not* These th e n a r e th e e q u a tio n s to be u s e d i n a p p ly in g m ethod a) f o r th e d e te r m in a tio n of th e o r d e r o f a r e a c t i o n w ith r e s p e c t to a c e r t a i n com ponent. They a r e u se d when i n v e s t i g a t i n g th e r e a c t i o n betw een h y d ro g en p e ro x id e and c e r t a i n b lo o d c o n s t i t u e n t s i n th e n e x t c h a p t e r . d x / d t = k ( a - x ) ^ ( c - x ) CHAPTER IV DENSITOMETER CONSTRUCTION A d e n s ito m e te r i s a n in s tr u m e n t f o r d e te r m in in g th e d e n s i t y of a p h o to g ra p h ic d e p o s i t , and th u s i n d i r e c t l y to m easure l i g h t i n t e n s i t y . I f th e a re a b e in g m easured i s r e s t r i c t e d t o t h a t c o v e re d by a s m a ll s l i t o r p i n h o le , th e i n s tru m e n t i s c a l l e d a m ic ro p h o to m e te r o r m ic r o d e n s ito m e te r The e s s e n t i a l f e a t u r e s o f a d e n s ito m e te r a r e a ) a s o u rc e o f l i g h t b ) a means o f d i r e c t i n g th e l i g h t t h r u th e d e s i r e d p o r t i o n o f th e e m u ls io n , and c) a means o f com paring th e b r i g h t n e s s a f t e r t r a n s m i s s io n w ith th e b r i g h t n e s s b e f o r e t r a n s m i s s i o n . Then th e d e n s i t y c a n be c a l c u l a t e d from th e fo rm u la D = lo g Fq/F-^. The s u b j e c t i v e ty p e o f in s tr u m e n t depends on t h e o b s e r v a tio n of an a d ju s tm e n t a t w h ic h two i n t e n s i t i e s a re e q u a l . The o b j e c t i v e ty p e i3 n o t o n ly more a c c u r a t e i n t h a t i t does away w ith depend ence on tbe o b se rv e r* e y e , b u t i s more c o n v e n ie n t. The in s tr u m e n t u se d i n t h i s i n v e s t i g a t i o n i s o f th e l a t t e r t y p e , u s in g a W eston P h o tro n ic c e l l and a lamp and s c a l e g a lv a n o m e te r f o r i n d i c a t i n g e q u ip m en t. The m ost a c c u r a te ty p e o f d e n s ito m e te r w ould be one w hich o p e r a te d as a n u l l i n s tr u m e n t, u s in g p h o t o e l e c t r i c c e l l s f o r d e te r m in in g th e b a la n c e c o n d i t i o n , b u t th e d e f l e c - < Z Q F o r s y th e , M easurem ent o f R a d ia n t E nergy (New Y ork; McGraw H i l l 1937) p p . 2&5-364T 42 t i o n ty p e o f in s tr u m e n t i s q u i te a c c u r a t e enough f o r th e p u rp o se h e r e . The component p a r t s w ere m ounted i n an o b lo n g wooden box made o f t h r e e - p l y wood. The box was p ro v id e d w i t h two p a r t i t i o n s i n w hich h o le s w ere c u t t o p r o p e r l y d i r e c t th e l i g h t beam from a lamp i n one end t h r u th e s i l v e r d e p o s i t to th e p h o t o e l e c t r i c c e l l . The box was 30 cm. lo n g , 14 cm. w id e , and 11 cm. h ig h p ro v id e d w ith a c o v er w hich k e p t th e i n t e r i o r a d e q u a te ly p r o t e c t e d from s t r a y o u t s i d e l i g h t . The c o n s t r u c t i o n , r e l a t i v e p o s i t i o n o f th e p a r t s , and d im e n s io n s , can be s e e n from th e c r o s s - s e c t i o n d iag ram o f P l a t e 1 , and th e to p view p h o to g ra p h ( P l a t e 2 ) . In t h i s s i d e view d raw in g th e m e te r i s n o t shown, as i t o b s t r u c t s th e v ie w . The lamp c i r c u i t was composed o f a 25 w a tt Mazda lam p, a 0 -250 a l t e r n a t i n g c u r r e n t m i l l i a m e t e r , a 2500 ohm w ir e - wound v a r i a b l e r e s i s t o r and a s w itc h , a l l c o n n e c te d i n s e r i e s . The lamp s o c k e t was m ounted on th e end o f th e box i n th e c en t e r and c o n n e c te d on one s i d e to th e r e s i s t o r and on th e o th e r t o th e m e te r . I n f r o n t o f th e lamp in th e c e n t e r o f th e f i r s t p a r t i t i o n was m ounted a n i r i s d iap h ra m and compur s h u t t e r . The d iap h ra m was u s e d , as w i l l be e x p la in e d l a t e r , t o r e g u l a t e th e l i g h t i n t e n s i t y t o th e p r o p e r v a lu e and th e s h u t t e r was u se d when m aking t e s t e x p o su re s f o r th e d e te r m i n a ti o n o f D -lo g E c u r v e s . The r e s i s t o r was u se d t o a d j u s t th e c u r r e n t flo w t h r u th e f ila m e n t o f th e lamp and th e m e te r was u se d to in s u r e pi-K W O dP C o y /r n 3//[ " P a h t i t v/A-ys.iv/r p.: :w/:::zz>. ;.-• ?; j rr T ^ lw /3 /./,/,/' ; / / y / ^ / / / // / / / /3 / ; .' /3 / / / / / / ' / ^ / / 7 V / / 3^373/ 7/ 37^3Z 3W ' r r ^3 / ; y/ - 4 5 0 v A > - 2 5 W 25 w L a m p & R O u n d G y a s s f LATE- i 1 1W 5 Diaphragm S vC W / V3///// 1 J ^ y L Y \3 /oco \\\v \\V > > • M S ^ - p a o n g- T u a e s o c K e r vV s / 1 5 T O N P H O T R O N I C C e L L [----------------------------------------------------------- P h ^ p E l e c t r i c D e n s i t o m e t e r C r o s s S e c t i o n Sr a l e ~ p O 2 D P P ''o x . K*------------ 10.00 0 o U ~ x s A /W W V I 450 w O / 25 w -vV V W A s w u , J/OVA.C. 43 d u p l i c a t e v a lu e s o f t h i s c u r r e n t . The p u rp o se o f su c h con t r o l may he s e e n from th e f o llo w in g c o n s i d e r a t i o n s . When th e d e n s i t y o f a d e p o s i t i s m easured by d e te rm in in g i t s power o f c u t t i n g down th e i n t e n s i t y o f a l i g h t beam p a s s in g t h r u i t , th e v a lu e o b ta in e d w i l l be p a r t l y a f u n c t i o n o f th e s p e c t r a l q u a l i t y o f th e l i g h t u s e d . When th e f ila m e n t c u r r e n t changes i n a lam p, th e te m p e ra tu re o f th e f i la m e n t changes c o r r e s p o n d in g ly . S in c e th e w a v e -le n g th -e n e rg y d i s t r i b u t i o n of a beam i s a f u n c t i o n of th e te m p e ra tu re o f th e s o u r c e , c u r r e n t c o n t r o l becomes n e c e s s a r y i n o r d e r to in s u r e th e same v a lu e f o r th e m easured d e n s i t y when m easured a t two d i f f e r e n t t im e s . The r e d u c t i o n in i n t e n s i t y v a r i e s w ith th e w a v e -le n g th so t h a t th e d e n s i t y as m easu red w ith b lu e l i g h t i s g r e a t e r th a n t h a t m easured w ith r e d . In a d d i t i o n t o t h i s , th e s p e c t r a l re s p o n s e o f th e p h o t o c e l l m ust be c o n s id e r e d . The c u r r e n t o u t p u t, w hich i s th e i n d i c a t i n g e n t i t y , w i l l n o t be th e same f o r two beams o f d i f f e r e n t c o l o r s , th o u g h th e y be o f th e same i n t e n s i t y . For th e s e r e a s o n s , th e te m p e ra tu re o f t h e lamp i s k e p t p r a c t i c a l l y c o n s ta n t d u r in g a m easu rem ent. I f th e l i g h t u se d w ere m onochrom atic su c h c o n t r o l w ould n o t be n e c e s s a r y , b u t i n m ost c a s e s w h ite l i g h t i s n e c e s s a r y i n o r d e r t o o b t a i n a s u f f i c i e n t l y i n t e n s e beam. F o r m ost m easu re m e n ts, th e c u r r e n t was r e g u l a t e d t o 150 m il l ia m p e r e s , th e s p e c t r a l q u a l i t y th u s s t a y i n g p r a c t i c a l l y c o n s t a n t ,a n d i f i t became n e c e s s a r y t o change th e i n t e n s i t y o f th e l i g h t t h i s 44 was done by means o f th e i r i s d ia p h ra m . The cham ber c o n t a i n in g th e lamp was a l s o p ro v id e d w ith two c o p p er tu b e s by means o f w hich a i r c o u ld be c i r c u l a t e d t h r u t h e c lo s e d com partm ent d u r in g e x te n d e d m easurem ents t o keep th e h e a t from a c c u m u la tin g , w i t h p o s s i b l e damage t o th e r e s i s t o r and e r r o r in th e m ea su re ment . A f te r th e beam goes t h r u th e i r i s d ia p h ra m , i t t r a v e r s es a ground g l a s s , th e n th e s i l v e r d e p o s i t to be m ea su re d , and e n te r s th e p h o t o c e l l window. The ground g la s s was f a s t e n e d to a h in g e and a c l i p p ro v id e d so t h a t i t c o u ld be swung away from th e p h o t o c e l l window t o a llo w th e p h o to g ra p h ic m a t e r i a l to be i n s e r t e d . The p h o to g ra p h shows th e g la s s open and a f i l m i n s e r t e d re a d y t o be r a i s e d and clam ped i n t o p o s i t i o n in f r o n t o f th e c e l l . The d e n s i t y as m easured by t h i s a rra n g em e n t i s p ro b a b ly c l o s e r to w hat i s known as d i f f u s e d e n s i t y th a n t o s p e c u la r d e n s i t y . The d i f f e r e n c e in th e two ty p e s i s in th e n a tu r e of th e l i g h t beam a f t e r t r a v e r s i n g th e f i l m . Looking a t th e fo rm u la D = lo g Fq/ F ^ , i t may be s e e n t h a t th e c a l c u l a t e d v a lu e f o r th e d e n s i t y w i l l v a ry i f th e l i g h t r e a c h in g th e p h o t o c e l l i s o n ly t h a t l i g h t i n th e c y l i n d r i c a l volume d e f i n e d by th e shape and s i z e o f th e i n c i d e n t beam. I f a l l of th e l i g h t s c a t t e r e d in any d i r e c t i o n w h a te v e r a f t e r t r a v e r s in g th e d e p o s i t i s m ea su re d , F has a n o th e r v a lu e . The d e n s i t y c o rre s p o n d in g t o th e fo rm e r ty p e o f m easurem ent i s c a l l e d 45 th e s p e c u la r d e n s i t y and t h a t t o th e l a t t e r c a se th e d i f f u s e d e n s ity # In th e d e n s ito m e te r d e s c r i b e d a b o v e , th e beam is d i f f u s e b o th b e f o r e and a f t e r t r a v e r s i n g th e f i l m , a lth o u g h n e a r l y a l l o f th e t r a n s m i t t e d l i g h t e n t e r s th e p h o t o c e l l w in dow* The e x a c t s i t u a t i o n n e ed n o t be known, s in c e c o n d itio n s can be a c c u r a t e l y d u p l i c a t e d in m aking m easurem ents and th e v a lu e s o f th e d e n s i t y a r e u se d o n ly f o r th e d e te r m i n a ti o n o f c o m p a ra tiv e d i f f e r e n c e s # - The p h o t o c e l l c i r c u i t was made up o f a W eston P h o tro n ic c e l l , a t e n - th o u s a n d ohm p o t e n t i o m e t e r , and a L eeds and N or- th r u p ty p e P w a l l g a lv a n o m e te r o f #00072 m ic ro amp•/mm. s e n s i t i v i t y , The p o te n tio m e te r was m ounted on a s i d e w a ll and c o n n e c te d to b in d in g p o s ts and th e p h o t o c e l l s o c k e t . I t was 40 c o n n e c te d a s a n A ry to n s h u n t , i n o r d e r t o d e c r e a s e th e s e n s i t i v i t y o f th e g a lv a n o m e te r when n e e d e d . I t s v a lu e e q u a le d t h a t recommended as a c r i t i c a l dam ping r e s i s t a n c e f o r th e g a lv a n o m e te r. The c i r c u i t f o r th e sh u n t i s shown a t th e b o tto m o f P l a t e 1 . By means o f t h i s c o n t r o l , th e c u r r e n t t o th e l i g h t s o u rc e need n o t be d e c r e a s e d w i t h a t t e n d a n t s p e c t r a l v a r i a t i o n s , w henever th e c u r r e n t o u tp u t i s to o g r e a t and th ro w s th e g a lv a n o m e te r o f f s c a l e . The g a lv a n o m e te r s e n s i t i v i t y a n d /o r 39 I b i d . , p . 284. 40 Sm ythe, Advanced E l e c t r i c a l M easurem ents (New Y ork: Van N o stra n d 1932) p , 4 l . 46 th e i r i s d iap h ra m may he r e g u l a t e d so t h a t f u l l - s c a l e o r o th e r c o n v e n ie n t d e f l e c t i o n r e s u l t s when a b la n k o r no f i l m i s in th e d e n s i to m e t e r . Then t h e f i l m i s i n s e r t e d and th e d e c r e a s e i n th e g a lv a n o m e te r d e f l e c t i o n g iv e s th e n e c e s s a r y d a t a f o r th e d e n s i t y c a l c u l a t i o n . By t r a c i n g th e s t e p s from th e o r i g i n a l r e a c t i o n to th e d e c r e a s e from f u l l - s c a l e d e f l e c t i o n w hich r e s u l t s by i n s e r t i n g th e s i l v e r d e p o s i t i n t h e l i g h t beam , i t i s s e e n t h a t th e amount o f d e c r e a s e i s a m easure o f t h e i n t e n s i t y o f th e o r i g i n a l lu m in e sc e n c e w h ich can be ex p r e s s e d i n a r b i t r a r y u n i t s as c e n t i m e t e r s . THE MEASUREMENT OP CHEMILUMINESCENCE INTENSITY * o r t h e p u rp o se o f e x p o s in g th e r e a c t i o n m ix tu re t o th e p h o to g ra p h ic f i l m , a s p e c i a l c e l l was c o n s t r u c t e d . When th e lu m in e s c in g s o l u t i o n was ex p o sed to a f i l m i n a d a rk room t h r u th e b o tto m o f a s m a ll b e a k e r by p l a c i n g th e b e a k e r on t h e f i l m w i t h a p ie c e o f b l a c k p a p e r betw een and th e n w ith d raw in g th e p a p e r , th e r e s u l t i n g p a t t e r n was t h a t shown i n a ) P l a t e I I I . The h e t e r o g e n e i t y o f th e d e p o s i t i s r e a d i l y r e c o g n i z a b l e . T h is i s o f c o u rs e u n d e s i r a b l e as th e c a l c u l a t e d d e n s i t y w ould v a r y w i t h th e p o s i t i o n o f t h e d e p o s i t i n th e l i g h t beam . I f th e d e p o s i t be hom ogeneous, th e f i l m need o n ly be i n s e r t e d so t h a t th e d e p o s i t c o v e rs th e h o le i n f r o n t o f th e p h o t o c e l l window. T h is h o le i s somewhat s m a lle r th a n t h e d e p o s i t r e p r e s e n t e d by b) P l a t e I I I , w h ich i s a homogen- PLATE I I I HETEROGENEOUS DEPOSIT HOMOGENEOUS DEPOSIT 47 eous one o b ta in e d by a s i m i l a r e x p o su re t h r u th e c e l l w hich was made a s f o llo w s : A p ie c e o f g l a s s tu b in g o f 4 cm* i n s i d e d ia m e te r was c u t t o a l e n g t h o f 3 cm. and cem ented i n th e c e n t e r o f a 4 .5 cm. g l a s s s q u a r e . T h is g l a s s was o n ly one m i l l i m e t e r t h i c k and th o u g h n o t o p t i c a l l y f l a t was q u i t e f l a t enough f o r th e p u r p o s e . The o u t s i d e o f th e tu b e was la c q u e r e d w i t h d u l l b la c k t o c u t down i n t e r n a l r e f l e c t i o n s . The r e f l e c t i o n s a t th e j u n c t i o n o f t h e tu b in g and th e p l a t e do n o t ex te n d f a r enough t o p r e v e n t a homogeneous d e p o s i t o f s u f f i c i e n t s i z e t o c o v e r th e p h o t o c e l l o p e n in g . The f o llo w in g e x p e rim e n ts w ere made: E x p erim en t 1 . D e fen d e r SS P a n c h ro m a tic c u t f i l m was o b ta in e d i n s i z e 9 x 12 cm. and e a c h s h e e t was c u t i n t o two e q u a l p i e c e s . T hese w ere th e n a c o n v e n ie n t and e co n o m ic a l s i z e f o r f i t t i n g u n d e r th e e x p o su re c e l l and i n th e d e n s i to m e t e r . The l i n e a r p o r t i o n o f th e c h a r a c t e r i s t i c c u rv e was t h e n d e te rm in e d a s f o llo w s : A s o l u t i o n o f .1 % h y d r a z i d e , 1 % sodium h y d r o x id e , and .1 % h y d ro g en p e ro x id e was made up i n a t e s t tu b e and 5 d ro p s o f a w a te r e x t r a c t o f a b lo o d s t a i n a d d ed . Such a s o l u t i o n , lu m in e sc e d f o r 10-15 m in u te s w ith no a p p a r e n t d im in u tio n i n i n t e n s i t y so i t was assum ed t h a t o v e r a p e r io d o f 30 se c o n d s o r so th e i n t e n s i t y w ould be c o n s t a n t enough f o r t h i s p u r p o s e . F iv e p o r t i o n s o f th e h y d r a z id e r e a g e n t w ere m easured i n t o t e s t tu b e s from a b u r e t t e and 5 d ro p s o f b lo o d e x t r a c t w ere added to e a c h one j u s t b e f o r e e x p o s u re . 48 Each s o l u t i o n a s soon as i t was th o r o u g h ly m ixed and was lu m in e s c in g s t r o n g l y , was p o u red i n t o a c le a n e x p o su re c e l l w h ich was s e t t i n g on a p ie c e of f i l m . The b la c k p a p e r was rem oved and th e e x p o su re was tim ed w ith a m etronom e. The f ilm s w ere a l l d e v e lo p e d t o g e t h e r i n D-76 d e v e lo p e r a t 21 d e g re e s C e n tig ra d e and f i x e d i n F - l hypo f o r m u la . A f te r th e f i lm s w ere d r y , th e d e n s i t y of th e s i l v e r d e p o s i t s w ere m easu red w ith th e d e n s i to m e t e r . The lamp c u r r e n t was s e t a t 150 m illia m p e r e s by th e r h e o s t a t and th e i r i s d iap h ram a d j u s t e d so t h a t 25 cm. d e f l e c t i o n r e s u l t e d w i t h no f i l m i n th e d e n s i t o m e t e r . T h is t h e n was th e v a lu e f o r g (s e e C h a p te r I I I R a d io m e try ). The r e s u l t s a r e p r e s e n t e d i n th e t a b l e b e low . The e x p o su re E, w h ic h i s th e p r o d u c t o f r a t e and tim e , was ta k e n a s a c o n s ta n t tim e s tim e so t h a t lo g t can be u se d f o r lo g E f o r th e p u rp o se o f d e te r m in in g l i n e a r i t y o f th e t i m e - s c a l e c h a r a c t e r i s t i c c u rv e . TABLE I I J L lo g E lo g Fq/ F j^ x 100 0 1 0 12.1 5 .7 0 6 3 .5 10 1.00 88.0 20 1 .3 0 1 1 6 .3 30 1 .4 7 1 2 6 .1 I t i s n o t s u r p r i s i n g t h a t th e D -lo g E c u rv e r e p r e s e n te d by t h e s e r e s u l t s i s l i n e a r w i t h i n th e l i m i t o f e x p e r im e n ta l e r r o r , s in c e th e f i lm u s e d was known t o have a w ide l a t i t u d e and th e e x p o su re s w ere tim e d t o g iv e what 49 e x p e rie n c e h a s shown t o be an a v e ra g e ( c e n t e r o f c o r r e c t e x p o su re r e g io n ) n e g a t i v e . These r e s u l t s i n d i c a t e d t h a t no e r r o r fro m n o n - l i n e a r i t y o f th e c h a r a c t e r i s t i c c u rv e w ould r e s u l t i n com paring th e i n t e n s i t i e s o f v a r io u s ch em ilu m i- n e s c e n t phenomena p ro v id e d th e s i l v e r d e p o s i ts o b ta in e d , a p p e a re d t o have a d e n s i t y somewhere betw een v e r y t h i n and v e ry h e a v y . E x p erim en t 2 . Though n o t an e x p e rim e n t on d e n s i t y m easu re m ent, t h i s e x p e rim e n t r e l a t e s t o th e d e n s ito m e te r i n t h a t i t was p e rfo rm ed w h ile th e d e n s ito m e te r was b e in g c o n s t r u c t ed i n o r d e r t o f i n d w hat k in d o f p h o t o c e ll window w ould be r e q u i r e d . Though n o t p ro b a b le t h a t th e r e a c t i o n produced any u l t r a - v i o l e t l i g h t , i t was th o u g h t n e c e s s a r y t o v e r i f y su ch a h y p o th e s is as f o l l o w s • A s o l u t i o n o f f l u o r e s c e i n i n a q u a r tz t e s t tu b e was ex p o sed t o th e l i g h t of a b r i l l i a n t l y lu m in e s c in g m ix tu re o f " lu m in o l1 1 and b lo o d . No f l u o r e s c e n c e , w hich i s known t o be p ro d u ce d in ev en th e s l i g h t e s t amount o f u l t r a - v i o l e t l i g h t , was n o te d . A ls o , when a t h i n g l a s s p l a t e was in tr o d u c e d i n t o th e sp a c e b etw een a b e a k e r of c h e m ilu m in e sc e n t m ix tu re and a P h o tro n ic c e l l w ith a q u a r tz window, no d im in u tio n o f c u r r e n t o u tp u t was n o te d a s w ould be th e c a se i f any u l t r a - v i o l e t l i g h t w ere b e in g e m itte d . E x p erim en t 3 . T h is e x p e rim e n t was b a se d on an u n e x p e c te d r e s u l t o f a p re v io u s e x p e rim e n t i n w hich a s to c k s o l u t i o n was p r e p a re d f o r u se i n c o r r e l a t i n g l i g h t i n t e n s i t y w ith 50 r a t e o f p e ro x id e d e c o m p o s itio n . The s t o c k s o l u t i o n was made up o f 2 gm. h y d r a z id e , 67 m l. o f 3$ h y d ro g en p e r o x id e , 90 m l. o f 1 % sodium h y d ro x id e , and th e w hole d i l u t e d to 2000 m l. w ith d i s t i l l e d w a t e r . The c o n c e n tr a ti o n s w ere ch o sen somewhat a t random , t h e r e b e in g j u s t enough ly e to e n su re s o l u t i o n o f t h e o r g a n ic compound and enough p e ro x id e t o f u r n i s h oxygen. When b lo o d was a d d e d , no lu m in e sce n c e was p ro d u c e d , i n con t r a s t t o w hat had alw ays o c c u rre d p r e v i o u s ly w ith any and a l l s o l u t i o n s made u p . Upon i n v e s t i g a t i o n th e th in g a b o u t t h i s s o l u t i o n t h a t was d i f f e r e n t th a n p r e v io u s ones was fo u n d t o be t h a t th e h y d ra z id e was p r e s e n t a t a much h ig h e r concen t r a t i o n , and a l s o t h a t th e sodium h y d ro x id e c o n c e n tr a ti o n as d e te rm in e d by t i t r a t i o n was lo w er th a n p r e v i o u s l y . The odd t h in g was t h a t lu m in e s c e n c e , a lth o u g h r e - e s t a b l i s h e d when th e b a se c o n c e n tr a ti o n was i n c r e a s e d , d id n o t a p p ro a c h i t s u s u a l i n t e n s i t y u n t i l th e h y d ra z id e c o n c e n tr a ti o n was c o n s id e r a b ly d e c r e a s e d . T h ere th u s e x i s t s a c r i t i c a l con c e n t r a t i o n o f h y d ro x id e and o f h y d ra z id e a t w hich th e lu m i n e sc e n c e i s th e m ost i n t e n s e . As th e h y d ro x id e c o n c e n tr a ti o n i s in c r e a s e d from a low v a lu e th e lu m in e sc e n c e p a s s e s t h r u a maximum and th e n d e c r e a s e s to z e r o , th e same t h in g h a p p e n in g i n th e c a se o f v a r y in g th e h y d r a z id e c o n c e n t r a t i o n . B oth o f t h e s e r e s u l t s have b e en r e c e n t l y v e r i f i e d by Z e l l n e r . 41 The 41 J . A. C. S. 5 9 , 25 81, 1937. 51 f o llo w in g d a ta was o b ta in e d by e x p o sin g s o l u t i o n s (*2% h y d ra z i d e , 1 % h y d ro g en p e r o x id e , p lu s 5 d ro p s b lo o d e x t r a c t ) o f v a r io u s h y d ro x id e c o n c e n t r a t i o n s , and n o tin g th e g a lv a n o m e te r d is p la c e m e n ts c o rre s p o n d in g t o th e l i g h t t r a n s m i t t e d by th e d e v e lo p e d f i l m s . In a l l w o rk, u n le s s o th e r w is e s p e c i f i e d , D e fen d e r b ra n d SS P a n c h ro m a tic f i l m was u s e d . TABLE I I I NaOH C o n c e n tr a tio n D e f l e c t i o n ( 2 5 - D e f l e c t i o n ) *005 N 1 4 .4 1 0 .6 .0 1 0 1 1 .0 1 4 .0 .020 6 .1 1 8 .9 .030 6 .0 1 9 .0 .050 7 .5 1 7 .5 .100 8 .5 1 6 .5 .200 1 2 .3 1 2 .7 .500 1 5 .7 1 1 .3 2 .0 0 0 2 3 .3 1 .7 The l a s t column e x p re s s e s th e d e f l e c t i o n s in a way so t h a t th e y a r e p r o p o r t i o n a l to th e i n t e n s i t y o f th e lu m i n e s c e n c e . From th e s e f i g u r e s , i t w ould seem t h a t th e optimum c o n c e n tr a ti o n o f h y d ro x id e f o r t h i s s o l u t i o n was b e tw ee n .02 and .0 3 N. E x p erim e n t 4 . The above s o l u t i o n ( . 2 % h y d r a z i d e , 1 % h y d ro x id e , 1 % p e ro x id e ) glowed v e ry v i v i d l y when b lo o d was p r e s e n t , b u t th e s t o c k s o l u t i o n o f h y d r a z id e , h y d r o x id e , and p e ro x id e i t s e l f glow ed t o a s m a ll e x t e n t w ith o u t b lo o d b e in g p r e s e n t due e i t h e r t o o x i d a t i o n by p e ro x id e o r t o oxygen from slo w ly d e com posing p e r o x id e . W ith t h i s in m ind, s e v e r a l s o l u t i o n s o f v a r i o u s p e ro x id e c o n c e n tr a ti o n s w ere t e s t e d t o se e i f th e glow 52 o f th e t e s t r e a g e n t was s tr o n g enough t o i n t e r f e r e by g iv in g an e rro n e o u s p o s i t i v e t e s t . To each:>of tw e lv e tu b e s was added 5 m l. o f th e s t o c k s o l u t i o n p lu s th e am ounts o f p e ro x id e i n d i c a t e d i n th e f o llo w in g t a b l e w hich a l s o p r e s e n t s th e c a lc u l a t e d d e n s i t i e s o f f i lm s exp osed in th e u s u a l way f o r e q u a l tim e i n t e r v a l s t o t h e s e s o l u t i o n s • TABLE IV Tube Number Amount o f P e ro x id e F ilm D e n s ity 1 5 m l. o f 5% 8 .4 2 3 m l. o f 5% 8 .3 3 1 m l. o f 5% 1 1 .0 4 .5 m l. o f 5% 2 0 .4 5 9 d ro p s o f 5% 4 3 .3 6 3 d ro p s o f 5% 7 7 .2 7 9 d ro p s o f .5% 9 8 .4 8 3 d ro p s o f .5% 9 6 .0 9 9 d ro p s o f .03 $ 4 2 .3 10 3 d ro p s o f .03$ 1 1 .0 11 1 d ro p o f .15$ 9 .5 12 none 0 .0 M ix tu re number 7 gave th e s t r o n g e s t lu m in e sc e n c e . To e a c h o f th e above t u b e s , a 3 m onth b lo o d s t a i n was added and th e l u m i n o s i t i e s , w h ich in c r e a s e d g r e a t l y , w ere com pared. I t c o u ld be r e a d i l y se e n t h a t th e s t o c k s o l u t i o n w hich gave th e b r i g h t e s t glow w ith o u t b lo o d a l s o f a r e x ce ed e d th e o t h e r m ix t u r e in b r i l l i a n c y when b lo o d was p r e s e n t . The c o n t r a s t b e tw een th e glow b e f o r e and a f t e r a d d in g b lo o d was so g r e a t t h a t no d o u b t f u l c o n c lu s io n s sh o u ld be o b ta in e d i f th e t e s t r e a g e n t u se d in t e s t i n g f o r b lo o d i s made up w ith th e v a r io u s optimum c o n c e n tr a ti o n s o f r e a c t a n t s d e te rm in e d from th e above e x p e r im e n ts . S p ech t q u e lle d th e weak lu m in e sc e n c e o f h i s 53 42 s to c k r e a g e n t w i t h in d o z o l - 4 - c a r b o n ic a c i d . The s to c k s o l u t i o n u se d th e n i n s u b s e q u e n t b lo o d s p e c i f i c i t y t e s t s , e t c . , was composed of .2 gm o f 3 -a m in o p h th a lh y d ra z id e and 1 gm o f sodium h y d ro x id e p e r l i t e r . Such a s o l u t i o n w i l l keep i n d e f i n i t e l y . A s i m i l a r s o l u t i o n k e p t i n th e a u t h o r ’s l a b o r a t o r y f o r f o u r y e a rs was made to lu m in e sc e s t r o n g l y when p e r o x id e and f e r r i c y a n i d e w ere a d d ed . The seco n d s to c k s o l u t i o n was one o f .3 # h y d ro g en p e ro x id e i n w a t e r . The t e s t s o l u t i o n was made up im m e d ia te ly b e f o r e u s in g by a d d in g 9 d ro p s o f .3 $ hy d ro g en p e ro x id e (o r e q u iv a le n t ) t o 5 m l. o f th e h y d ra z id e s t o c k s o l u t i o n . T h is s o l u t i o n glow ed f a i n t l y b u t when even a v e ry sm a ll sp e c k o f b lo o d s t a i n was a d d e d , i t glowed s u f f i c i e n t l y t o r e a d medium s i z e d p r i n t i n a d a rk room . E x p erim en t 5 . Among th e e v id e n c e p o i n ti n g t o th e f a c t t h a t th e r e a c t i o n i s much more c o m p lic a te d th a n a sim p le p e ro x id e d e c o m p o s itio n p lu s o x i d a t i o n by o x ygen, i s an e x p e rim e n t in w h ich th e s to c k s o l u t i o n w ith o u t an y p e ro x id e a d d ed , was made t o glow when p o ta s s iu m f e r r i c y a n i d e and b lo o d was a d d e d . T h is i s n o t u n u s u a l as th e f e r r i c y a n i d e c o u ld o x id iz e th e h y d ra z id e w ith o u t p e ro x id e b e in g p r e s e n t , b u t th e u n u s u a l f a c t i s t h a t when b lo o d i s a l s o p r e s e n t , th e lu m in e sc e n c e w hich o r d i n a r i l y d is a p p e a r s s h o r t l y a f t e r th e f e r r i c y a n i d e i s a d d ed , l a s t s q u i t e a b i t lo n g e r as th o u g h th e b lo o d w ere o x i d iz in g th e h y d r a z i d e . T h is m ix tu re i s i n v e s t i g a t e d w i t h th e d e n s ito m e te r t o d e te rm in e th e form o f th e r e l a t i o n betw een th e tim e w h ich 42 Z e i t . f u r angew . Chem. 5 0 , 8 , 155, 1937. 54 h a s e la p s e d from th e s t a r t o f t h e r e a c t i o n and t h e d e c r e a s e i n g a lv a n o m e te r d e f l e c t i o n when f i l m e x p o sed a t e q u a l i n t e r v a ls , i s p la c e d i n t h e d e n s i to m e t e r . The t e s t s o l u t i o n was p re p a r e d from th e s t o c k s o l u t i o n w ith o u t any p e ro x id e and a s m a ll c r y s t a l o f p o ta s s iu m f e r r i c y a n id e and a sp e c k o f 6 m o n th -o ld b lo o d s t a i n was a d d e d . The m ix tu re i n th e e x p o su re c e l l was e x p o sed in t u r n t o f o u r p ie c e s o f f i l m f o r p e r io d s o f one m in u te e a c h . D u rin g th e f o u r m in u te s of t o t a l e x p o s u r e , th e lu m in e sc e n c e s lo w ly d e c r e a s e d i n i n t e n s i t y , a lth o u g h much more s lo w ly th a n i f th e f e r r i c y a n i d e was p r e s e n t w ith o u t any b lo o d . The f o llo w in g e x p e r im e n ta l r e s u l t s a r e p r e s e n te d i n th e c u rv e (g ra p h 1 ) . TABLE V M inute d u r in g w h ich e x p o su re made d (2 5 -d ) 1 s t 3 .7 cm. 2 1 .3 cm. 2nd 9 .4 1 5 .6 3 rd 1 5 .3 9 .7 4 t h 2 1 .6 3 .4 I t was a l s o n o te d t h a t th e glow was i n t e n s i t i f e d by th e a d d i t i o n o f more b lo o d . The d e c o m p o s itio n o f h y d ro g en p e ro x id e was c o r r e l a t e d w ith h e m o g lo b in c o n c e n t r a t i o n in l a t e r e x p e rim e n ts b u t no p e ro x id e i s p r e s e n t in th e above e x p e rim e n t w h ich need s e l a b o r a t i o n . MECHANISM OF 3-AMINOPHTHALHYDRAZIDE OXIDATION E x p e rim e n ts a r e h e re d e s c r i b e d w h e re in i t was a tte m p e d t o f i n d w hat cau se d th e re m a rk a b le s p e c i f i c i t y o f 3 -am inoph - 55 t h a l h y d r a z i d e as a r e a g e n t f o r b lo o d t e s t i n g . Many s u b s ta n c e s i n t e r f e r e in th e o t h e r o x i d a t i o n t e s t s by decom posing th e p e ro x id e o f th e t e s t s o l u t i o n w hich r e s u l t s i n an e r r o n e o u s ly p o s i t i v e c o n c lu s io n . The fo llo w in g e x p e rim e n ts w ere made t o d e te rm in e w h e th e r 3 -a m in o p h th a lh y d ra z id e w ould be o x id iz e d when oxygen i n m o le c u la r form was th e o x i d iz i n g a g e n t, and when oxygen i n th e ato m ic form was th e o x i d iz i n g a g e n t . The l a t t e r form was p ro d u ce d i n v a rio u s ways t o ch eck th e r e s u l t s , th e s e ways b e in g by a) e l e c t r o l y s i s , b) by ozone d e c o m p o s itio n , and c) by u n im o le c u la r d e c o m p o sitio n o f hy d ro g en p e ro x id e . E x p erim en t 1 . I n t h i s e x p e rim e n t, th e e f f e c t o f m o le c u la r oxygen on an a l k a l i n e s o l u t i o n of h y d r a z id e was d e te rm in e d by b u b b lin g a i r t h r u a s o l u t i o n i n th e d a rk a f t e r th e eye was w e l l r e s t e d i n o r d e r t o d e t e c t th e f a i n t e s t p o s s ib l e lu m in e s c e n c e . No lu m in e sc e n c e was n o te d in t h i s c ase n o r when th e oxygen p a r t i a l p r e s s u r e was n e a r l y o n e . The l a t t e r c o n d i t i o n was o b ta in e d by g e n e r a t i n g oxygen from p o ta s s iu m c h l o r a t e and m anganese d i o x i d e . E x p erim en t 2 . T h is was p e rfo rm ed t o n o te th e r e s u l t o f e l e c - t r o l y z i n g an a l k a l i n e s o l u t i o n o f h y d r a z id e . When 6 v o l t s d i r e c t c u r r e n t was a p p li e d th ro u g h two c a rb o n e l e c t r o d e s i n th e d a r k , a b r i g h t glow was n o te d aro u n d th e p o s i t i v e e l e c t r o d e . T h is e n c o u ra g in g r e s u l t le d t o th e f o llo w in g e x te n s io n o f th e e x p e rim e n t. A p o te n tio m e te r o f t o t a l r e s i s t a n c e 5252 ohms was c o n n e c te d a c r o s s a 3 v o l t b a t t e r y and t o two C U R V E I 56 No. C 2 0 1 , University Book Store, Los Angeles 57 c arb o n e l e c t r o d e s i n su c h a m anner t h a t any v o l ta g e from z e ro t o 3 c o u ld be a p p li e d a c r o s s th e s o l u t i o n in a c o n t i n uous m anner as i t i s in th e c i r c u i t o f a p o la ro g ra p h # The e l e c t r o d e s o f th e above a p p a r a tu s w ere d ip p e d i n t o a d i l u t e (*4$) a l k a l i n e s o l u t i o n o f h y d r a z id e and th e l i g h t s t u r n e d out# A f te r a b o u t t e n m in u te s d u r in g w hich tim e th e eye r e a c h e d e q u i l i b r i u m , th e v o l ta g e a c r o s s th e e l e c t r o d e s was v e ry g r a d u a l ly in c r e a s e d by r o t a t i n g th e po t e n t i o m e t e r c o n tr o l# D u rin g t h i s tim e , th e eye was t r a i n e d c a r e f u l l y on th e e l e c t r o d e s w ith a le n s # As so on a s th e f i r s t t r a c e of lu m in e sc e n c e was n o te d a ro u n d th e p o s i t i v e p o l e , th e l i g h t s w ere tu r n e d on and th e r e s i s t a n c e o f t h a t p a r t o f th e p o te n tio m e te r w hich was a c r o s s th e e l e c t r o d e s was m easu red by a W h eatsto n e b rid g e # Prom th e v a lu e o f t h i s r e s i s t a n c e and th e p o t e n t i a l o f th e b a t t e r y , t h e p o t e n t i a l d i f f e r e n c e betw een th e e l e c t r o d e s , a t th e tim e t t e f i r s t lu m in e sc e n c e was n o te d , was c a lc u la te d # The v a lu e was found t o be f a i r l y r e p r o d u c i b le and o f c o u rs e dep ended somewhat on t h e c o n d it i o n o f th e e y e s . The mean o f s e v e r a l d e te r m in a tio n s o f t h e e .m .f# a t w hich lu m in e sc e n c e commenced was 1#72 v o l ts # The above v a lu e f o r th e ”c r i t i c a l v o l t a g e ” has b e en i n t e r p r e t e d i n th e l i g h t o f th e f o llo w in g t h e o r e t i c a l con s i d e r a t i o n s t o i n d i c a t e t h a t h y d ra z id e i s r e a d i l y o x id iz e d by oxygen i n th e a to m ic s t a t e # The d e c o m p o s itio n p o t e n t i a l o f many a c id s and b a s e s w ith p la tin u m e l e c t r o d e s i s i n th e 58 n e ig h b o rh o o d of 1 .7 v o l t s . A ll a c id s and b a s e s w hich decom pose a t 1 .7 0 v o l t s have b e en found to l i b e r a t e h y d ro g en and oxygen4 ^ so t h a t th e r e a s o n th e v a lu e i s th e same i s t h a t th e d e c o m p o s itio n p o t e n t i a l of w a te r i s b e in g m easured i n a l l su c h c a s e s . The f a c t t h a t th e lu m in e sc e n c e f i r s t o c c u r re d a t t h i s p o t e n t i a l i n d i c a t e s t h a t o x i d a t i o n ta k e s p la c e j u s t as soon as a to m ic oxygen i s form ed by th e e l e c t r o l y s i s . How ever, c a rb o n e l e c t r o d e s w ere u se d i n th e e x p e rim e n t and i t w ould have to be r e p e a t e d w ith p la tin u m and p e rh a p s w i t h an o b j e c t i v e m ethod o f n o tin g th e o n s e t o f ch em ilu m in escen ce b e f o r e p o s i t i v e c o n c lu s io n s c o u ld be made. These r e s u l t s s u g g e s t r e s e a r c h i n t o th e p o s s i b l e d e v e lo p m e n t, p ro b a b ly a f t e r o rg a n ic c h e m is try d is c o v e r s many more c h e m ilu m in e sc e n t r e a c t i o n s , o f w hat m ight be c a l l e d ’’o p t i c a l i n d i c a t o r s ” t o be u se d i n a n a l y t i c a l p o la r o g ra p h y . E x p erim en t 5 . Due t o th e i n s t a b i l i t y of o z o n e , p a r t i c u l a r l y i n a l k a l i n e s o l u t i o n , i t was th o u g h t t h a t i t c o u ld be u se d as a s o u rc e o f a to m ic oxygen. The m echanism o f i t s decompo s i t i o n i s n o t c e r t a i n , i n t h a t i t m ig ht decom pose u n im o le c u - l a r l y t o g iv e one m o le cu le of oxygen and one atom o f oxygen o r i t c o u ld decom pose b i m o l e c u l a r l y t o g iv e t h r e e m o le c u le s o f oxygen. W ith o u t i n v e s t i g a t i n g t h i s m echanism , ozone was p r e p a r e d and i t s e f f e c t on ’’lu m in o l” was n o t e d . I t was gen 43 Getman S c D a n i e l s , O u tlin e s o f T h e o r e t i c a l C h e m istry . (New York: W iley & S o n s, 1937)""p.' 476"----------------------------------------- 59 e r a t e d by a c ru d e p z o n iz e r made by w ra p p in g t i n f o i l i n s i d e and o u ts id e o f a c a rd b o a rd tu b e and th e n e n c l o s i n g t h i s "con d e n s e r ” in a l a r g e r tu b e w hich was c lo s e d a t b o th ends w ith th e e x c e p tio n o f h o le s t h r u w h ich g l a s s tu b e s c o u ld be u se d t o c i r c u l a t e a i r t h r u th e a p p a ra tu s * Leads from th e t i n f o i l l a y e r s w ere b ro u g h t o u t and c o n n e c te d t o a 3000 v o l t t r a n s form er* When th e s w itc h was c lo s e d and a i r f o r c e d i n t o th e a p p a r a tu s by means o f a r u b b e r b u l b , a v e r y s tr o n g o d o r o f ozone was d e t e c t e d in th e a i r coming o u t of th e e x i t h o le in th e t u b e , T h is ozone was l e d th ro u g h a r u b b e r tu b e below th e s u r f a c e o f a s o l u t i o n o f h y d ra z id e i n weak sodium h y d ro x id e* The ch em ilu m in e sc e was v e ry s t r o n g a t e v e ry o z o n e - liq u id i n t e r f a c e , d is a p p e a r in g im m e d ia te ly when th e ozone flo w was sto p p e d * T h is e x p e rim e n t i s d i f f i c u l t t o i n t e r p r e t in th e l i g h t o f th e c o n f l i c t i n g o p in io n s r e g a r d i n g th e m echanism o f ozone d e c o m p o s itio n . I t i s p ro b a b le t h a t a to m ic oxygen i s a t l e a s t m o m e n ta rily p r e s e n t and t h a t th e lu m in e sc e n c e i s f u r t h e r e v id e n c e o f th e p r e v i o u s l y p o s t u l a t e d r e a s o n f o r th e s p e c i f i c i t y o f th e h y d r a z id e - b lo o d t e s t (n am ely , o n ly th o s e s u b s ta n c e s w h ich decompose h y d ro g en p e ro x id e s u n im o le c u la r ly w ould i n t e r f e r e . Most d e c o m p o s itio n s o f p e ro x id e a re b i m o l e c u l a r ) . The p o s s i b i l i t y and p r o b a b i l i t y o f d i r e c t o x i d a t i o n o f h y d ra z id e by ozone m ust be k e p t i n m ind. F r ie n d 44 sa y s t h a t ozone i s 44 F r i e n d , In o rg a n i c & T h e o r e t i c a l C h e m istry (London: Longmans Green & C o ., 1922) V o l. V, p . 902. M onatsch, 1913, 3 4 , 6 65. 60 p ro b a b ly decom posed b i m o l e c u l a r l y . However, ozone i s con s i d e r e d a p a r t i c u l a r l y e f f e c t i v e o x i d iz in g a g e n t b e c a u se i t r e a d i l y l i b e r a t e s ’'n a s c e n t ” o x y g e n .45 He a l s o sa y s t h a t in o r d e r t o e x p la in c e r t a i n a s p e c ts o f i t s d e c o m p o sitio n i t i s n e c e s s a r y t o p o s t u l a t e th e p re s e n c e o f ato m ic o x y g e n .45 I t i s e s t a b l i s h e d t h a t ozone form s a to m ic oxygen when decom posed by m anganese s u lp h a te o r p o ta s siu m i o d i d e . F r ie n d a l s o men t i o n s a lu m in e sc e n c e w h ich accom pnies th e d e c o m p o sitio n o f ozone a t h ig h te m p e r a tu r e , b u t t h i s w ould n o t be c o n fu se d w ith th e glow o b ta in e d i n th e above e x p e r im e n t. E xperim ent- 4 . T h is was a n o th e r check o f th e h y p o th e s is o f o x i d a t i o n by ato m ic oxygen. The m ethod was to make up a t e s t r e a g e n t from th e s to c k s o l u t i o n s and th e n decompose th e p e r o x id e p r e s e n t by a m ethod known to form o n ly m o le c u la r oxygen. Such a r e a c t i o n i s t h a t b e tw e e n h y d ro g en p e ro x id e and p o ta s s iu m i o d i d e . The a c t u a l m echanism o f th e p ro c e s s has been w orked o u t so t h a t th e v a r io u s s te p s in v o lv e d i n th e s t o i c h i o m e t r i c a l 47 r e s u l t a re known. The s t e p s a re as f o llo w s : HgO gfcfs H20-*-lCT HgOgfclO — hgOg+Ogtl F i r s t , c o n c e n tr a te d p o ta s s iu m io d id e s o l u t i o n was added 45 M i t c h e l l , G e n era l P h y s io lo g y (New Y ork: M cG raw -H ill, 1932) p . 630. 46 I b i d . , p . 901. 47 Noyes & S h e r i l l , Advanced C hem ical P r i n c i p l e s (New Y ork: The M acM illan C o ., 19'3'oT p p . 140-1. 61 t o 3% h y d ro g en p e ro x id e and a r a p i d e v o lu t i o n o f oxygen was se e n and h e a rd when th e tu b e was c lo s e t o th e e a r . The t e s t r e a g e n t was th e n p la c e d in a tu b e and p o ta s s iu m io d id e ,w a s a d d e d . R apid d e c o m p o s itio n of th e p e ro x id e was n o te d b u t th e f a i n t lu m in e sc e n c e o f th e r e a g e n t was o n ly v e ry f a i n t l y e n h a n c e d . T h is i s th e b e s t e v id e n c e so f a r t h a t h y d ra z id e i s b e in g o x id iz e d by a to m ic oxygen i n th e b lo o d - h y d ra z id e t e s t . When a sp e ck of b lo o d s t a i n was added t o th e above m ix tu r e , i t im m e d ia te ly became b r i l l i a n t l y lu m in e sc e n t* T h is seems t o be c o n c lu s iv e enough so t h a t , i f i t c o u ld be shown t h a t ozone d id n o t o x id iz e h y d r a z i d e , th e above e x p e r im ent shows th e d e c o m p o s itio n o f ozone t o be u n im o le c u la r u n d e r t h e s e c o n d i t i o n s . E x p erim en t 5 . T h is e x p e rim e n t was made to check th e o t h e r p o s t u l a t e d m echanism s o f h y d ra z id e o x i d a t i o n d is c u s s e d in C h a p te r I I I . When th e t e s t r e a g e n t i s made up i t i s known t h a t i t can be se e n t o glow s l i g h t l y even w ith o u t b lo o d o r any o t h e r s u b s ta n c e toydecom pose th e h y d ro g en p e ro x id e * T h is can ta k e p la c e o n ly i f th e p e ro x id e i s d i r e c t l y o x i d i z in g th e h y d ra z id e o r i f th e p e ro x id e i s slo w ly decom posing and t h e r e s u l t i n g oxygen i s o x i d i z i n g i t . T his was ch eck ed by a n a l y s i s o f th e hyd ro g en p e ro x id e in su c h a s o l u t i o n w i t h o u t th e h y d r a z id e p r e s e n t . The s o l u t i o n was a n a ly z e d and a llo w e d t o s ta n d f o r f o u r w eek s. The c o n c e n tr a ti o n o f p e r o x id e was n o t fo u n d to have d im in is h e d , i n d i c a t i n g no decom 62 p o s i t i o n . By e l i m i n a t i o n th e n i t a p p e a rs t h a t d i r e c t o x id a t i o n by p e ro x id e i s one o f th e r e a c t i o n s ta k in g p la c e in th e b lo o d t e s t . I t was n o t th o u g h t t h a t b lo o d a lo n e would o x id iz e th e h y d r a z i d e , b u t th e fo llo w in g e x p e rim e n t was p e rfo rm ed t o ch eck t h i s p o i n t . The s t o c k s o l u t i o n o f h y d ra z id e in h y d ro x id e was ta k e n w ith o u t any p e ro x id e and a b lo o d s t a i n was p u t i n t o i t when th e ey es w ere w e ll a d j u s t e d t o th e d a rk . A v e ry f a i n t glow was d i s c e r n a b l e . Pure h e m o g lo b in p ro d u ced a s im i l a r r e a c tio n * Though a v e ry s l i g h t f r a c t i o n o f th e t o t a l l i g h t i s p ro d u ced by t h i s means in th e f i n a l t e s t , i t must be c o n s id e re d as one o f th e sim u lta n e o u s r e a c t i o n s t a k i n g p la c e i n th e m ix tu r e . D is s o lv e d oxygen i s n o t c o n s id e re d t o c o n t r i b u t e to th e e f f e c t s in c e i t h a s b e e n shown t h a t oxygen must be i n th e a to m ic s t a t e t o be e f f e c t i v e . THE RATE OF DECOMPOSITION OF HYDROGEN PEROXIDE I t a p p e a rs from th e r e s u l t s o f p r e v io u s e x p e rim e n ts t h a t th e d e c o m p o s itio n o f hydro gen p e ro x id e by b lo o d is th e m ost im p o rta n t r e a c t i o n o f th e s e v e r a l w h ich ta k e p la c e in th e r e a c t i o n m ix tu re o f sodium h y d ro x id e , h y d r a z id e , h y d ro gen p e r o x id e , and some c o n s t it u e n t o r c o n s t i t u e n t s o f b lo o d . An i n v e s t i g a t i o n o f v a r io u s a s p e c ts o f t h i s d e c o m p o sitio n seems d e s i r a b l e , p a r t i c u l a r l y i f i t can be shown t h a t th e 63 d e c o m p o s itio n by b lo o d i s u n im o le c u la r y i e l d i n g ato m ic oxy g e n . On th e b a s i s o f th e p re v io u s r e s u l t s , i t w ould seem t h a t th e d e c o m p o s itio n was u n im o le c u la r . A f u r t h e r s tu d y i s made a lo n g th e l i n e s d i s c u s s e d i n th e developm ent o f th e d i f f e r e n t i a l e q u a tio n s f o r th e v a r io u s o r d e rs o f r e a c t i o n . Among th e m ethods a v a i l a b l e f o r m e a su rin g r e a c t i o n 4ft r a t e s i n s o l u t i o n a r e : ° O b serv in g th e changes in volum e, d e n s i t y , r e f r a c t i v e in d e x , v i s c o s i t y , c o l l i g a t i v e p r o p e r t i e s , p r e s s u r e , c o l o r , o p t i c a l r o t a t i o n , c o n d u c t i v i t y , r e a c t a n t c o n c e n t r a t i o n , p ro d u c t c o n c e n t r a t i o n , e t c . The m ethods m ost c o n v e n ie n t in t h i s c a se a r e th o s e o f m e a su rin g th e change in c o n c e n tr a ti o n o f c e r t a i n r e a c t a n t s o r p r o d u c t s . Thus th e d e c r e a s e i n hyd ro g en p e ro x id e c o n c e n tr a ti o n o r th e in c r e a s e in oxygen c o n c e n tr a ti o n ( t h a t i s - r a t e of gas e v o lu tio n ) c o u ld be u se d e q u a lly w e l l to s tu d y th e b lo o d - p e r o x id e r e a c t i o n . These two m ethods have b e en u se d by i n v e s t i g a t o r s to d e t e r mine th e k i n e t i c s o f th e d e c o m p o s itio n of hydro gen p e ro x id e by h y d r o c h lo r ic a c i d . I t was fo u n d t h a t th e v a lu e o f th e 49 r a t e c o n s ta n t o b ta in e d by th e f i r s t m ethod was somewhat d i f f e r e n t th a n t h a t o b ta in e d by th e seco n d m ethod*^ u n d e r th e same c o n d i t i o n s . Such v a r i a t i o n s h o u ld n o t a f f e c t th e p r e s e n t i n v e s t i g a t i o n s in c e i t i s n o t g r e a t enough t o p r e v e n t th e R e illy - R a e , P h y sico C hem ical M easurem ents (London: M ethuen & Co. 1933) p p . 45*7-473. O s tw a Id -L u th e r, P h y slk o - Chemische Messungen (L e ip z ig : Akadem ische V e rla g l § 3 l ) p . *726. 49 L iv in g s to n & B ra y , J . A. C. S. 4 7 , 2069 (1 9 2 5 ). I b i d . , 4 6 , 2 9 0 , 1924. 64 o r d e r from b e in g d e te rm in e d by s u b s t i t u t i o n i n th e v a r io u s e q u a t io n s , a lth o u g h i t s h o u ld be c o n s id e r e d when p r e c i s e r a t e c o n s ta n ts a r e b e in g d e te r m in e d . C o n s id e rin g a g a in th e v a r io u s c o n s t i t u e n t s of b lo o d s t a i n s w hich w ould be in v o lv e d i n th e d e c o m p o s itio n (se e C h a p te r I I ) , i t h as b e e n shown t h a t h e m a tin i s n o t th e o n ly c o n s t i t u e n t r e s p o n s i b l e , i f i t i s r e s p o n s i b l e , s in c e f r e s h b lo o d has b e e n found t o g iv e a p o s i t i v e t e s t . A lth o u g h i t w ould be d e s i r a b l e t o i n v e s t i g a t e th e r e a c t i o n b etw een e ac h b lo o d c o n s t i t u e n t and h y d ro g en p e r o x id e , tim e d i d n o t a llo w t h i s and th e s tu d y was r e s t r i c t e d p r i n c i p a l l y t o h em o g lo b in , w hich i s r e a d i l y p r o c u r a b le i n p u re c o n d it i o n . Hem oglobin was shown t o g iv e a b r i l l i a n t l y p o s i t i v e t e s t w ith th e t e s t r e a g e n t . T here i s h e re m e n tio n e d , a p o in t on w hich th e l i t e r a t u r e does n o t seem t o a g re e i n c o n n e c tio n w ith th e decom posi t i o n o f p e ro x id e by c a t a l a s e , an enzyme p r e s e n t i n many a n i - 51 mal o rg a n s . I t i s s a i d t h a t c a t a l a s e e x h i b i t s no o x i d a t i v e e f f e c t s b e c a u se th e oxygen w hich i t l i b e r a t e s from h y d ro g en p e ro x id e i s in th e i n a c t i v e form o f m o le c u la r oxygen. How- 59 e v e r , e x p e r im e n ta l d a ta i s p r e s e n te d w hich can be u s e d to d e te rm in e th e o rd e r o f th e d e c o m p o sitio n by th e m ethod d i s - ^ M i t c h e l l , G e n e ra l P h y s lo lo g y (New York: M cGraw-Hill Book Co. 1932) p . 63 3 . 52 B e ll & G ro s s, P h y s ic a l C h em istry (New Y ork: Longmans, Green & Co. 1929) p . 254. 65 c u s s e d in C h a p te r I I I . T h is d a ta was o b ta in e d by m ix in g c a t a l a s e w ith h y d ro g en p e ro x id e and a n a ly z in g a l i q u o t e s a t i n t e r v a l s • TABLE VI Time P e ro x id e p r e s e n t P e ro x id e decom posed K 0 Min. 4 6 .1 0 .0 5 3 7 .1 9 .0 .0435 10 2 9 .8 1 6 .3 .0438 20 1 9 .6 2 6 .5 .0429 30 1 2 .3 3 3 .8 .0440 50 5 .0 4 1 .1 .0444 K was c a l c u l a t e d by s u b s t i t u t i n g th e d a ta i n th e equa t i o n K = l / t In a / a - x , d e r iv e d from c o n s i d e r a ti o n s o f a u n i m o le c u la r r e a c t i o n * The c o n s ta n c y o f K, as c a l c u l a t e d , shows th e c a t a l e s e - p e r o x i d e r e a c t i o n to be u n im o le c u la r and t h e r e f o r e th e k i n e t i c s p ro b a b ly t o be as f o llo w s : H202 = HgO + 0 0-t-0 = 0 2 I f th e s t o i c h i o m e t r i c a l e q u a t io n , SHgOg = 2H204-0g, e x p re s s e d th e a c t u a l m echanism o f th e r e a c t i o n , i t w ould be n e c e s s a r y f o r two m o le c u le s t o c o l l i d e w ith e a c h o th e r and th e number o f c o l l i s i o n s would be p r o p o r t i o n a l to th e s q u a re o f t h e p e ro x id e c o n c e n t r a t i o n . The r a t e w ould th e n be p ro p o r t i o n a l t o th e s q u a re o f th e c o n c e n tr a ti o n and n o t t o th e f i r s t pow er, b u t t h i s i s s e e n n o t t o be th e c a s e . The f o llo w in g e x p e rim e n ts were made t o d e te rm in e th e r a t e o f th e r e a c t i o n b etw een h y drogen p e ro x id e and b lo o d and b etw een hyd ro g en p e ro x id e and h e m o g lo b in . E x p erim en t 1 . T h is was a p r e l i m i n a r y e x p e r im e n t, i n w hich a s ix -m o n th o ld b lo o d s t a i n was e x t r a c t e d w ith a l i t t l e d i s t i l l e d w a te r and m ixed w i t h a c o n v e n ie n t s o l u t i o n o f p e ro x id e* One m l. of th e m ix tu re was rem oved a t i n t e r v a l s and added t o 250 m l. o f w a te r c o n ta in in g 20 m l. o f 7 w. s u l f u r i c a c i d t o ''q u en ch ” th e r e a c t i o n . The undecom posed p e ro x id e i n e a c h c a se was t i t r a t e d w ith .1 N p o ta s s iu m perm anganate* A b la n k composed o f b lo o d , s u l f u r i c a c i d and w a te r e x e r t e d no r e d u c in g a c t i o n on th e t i t r a n t so was c o n s id e r e d n o t t o i n t e r f e r e in th e a n a l y s i s . The f o llo w in g am ounts o f t i t r a n t w ere r e q u i r e d . These am ounts may be c o n s id e re d t o r e p r e s e n t th e p e ro x id e c o n c e n tr a ti o n i n a r b i t r a r y u n i t s . TABLE V II E la p s e d Time T i t r a n t R e q u ire d 0 s e c . 1 7 .1 9 m l. 1140 1 6 .5 8 2540 1 6 .5 6 The r e a c t i o n p ro c e e d e d f a s t e r th a n was a n t i c i p a t e d and was co m p lete d b e f o r e much p e ro x id e was decom posed. T here w ere th u s no e x p e r im e n ta l p o i n ts o b ta in e d betw een 0 and 1140 seco n d s a t w h ich tim e th e r e a c t i o n was p r a c t i c a l l y c o m p le te d . The form of th e c u rv e i s t h e r e f o r e unknown. E x p erim en t 2 . T h is was a r e p e t i t i o n o f th e f i r s t e x p e rim e n t w ith im proved te c h n iq u e and w eaker p e r o x id e . In th e f i r s t ex p e r im e n t, th e r e a c t i o n to o k p la c e i n a b u r e t t e w ith a w a te r No. 6201, University Book Store, Los Angeles j a c k e t aro u n d i t b u t i t was fo u n d t h a t th e e v o lv e d oxygen made th e m en iscu s d i f f i c u l t t o rea d * P i p e t t i n g from b e a k e rs was fo u n d t o b e more s a t i s f a c t o r y . 20 m l. o f Z % h y d ro g en p e r o x id e , 60 m l. of w a te r and an e x t r a c t o f a s ix -m o n th o ld s t a i n w ere m ixed and 5 m l. p o r t i o n s w ere removed f o r a n a l y s i s a t i n t e r v a l s . TABLE V I I I Time I n t e r v a l 0 Min. 2 2 2 18 16 20 Time E la p s e d T i t r a n t R e q u ire d 0 M in. 2 4 6 24 40 60 2 2 .1 1 Ml. 2 1 .6 0 2 1.44 2 1 .1 8 2 0 .0 9 1 9 .4 2 1 9 .2 0 The g r a p h i c a l r e s u l t s show t h a t n e i t h e r th e c o n c e n tr a t i o n n o r th e lo g a r ith m o f th e c o n c e n t r a t i o n o f p e ro x id e p l o t t e d v e rs u s th e tim e g iv e s a l i n e a r c u r v e . The fo rm er w ould be l i n e a r i f t h e r e a c t i o n w ere z e ro o r d e r w ith r e s p e c t to hy d ro g e n p e ro x id e and th e l a t t e r i f f i r s t o r d e r . The d a ta i s a l s o p u t i n to th e f o llo w in g form f o r e x a m in a tio n o f th e r e a c t i o n o r d e r by m ethod a ) , C h a p te r I I I . TABLE IX a a - x lo g a / a - x K .500 2 2 .1 1 2 1 .6 0 .0 1 0 1 .0117 .250 2 2 .1 1 2 1 .4 4 .1034 .0077 .167 2 2 .1 1 2 1 .1 8 .0187 .0073 .042 2 2 .1 1 2 0 .0 9 .0416 .0039 .025 2 2 .1 1 1 9 .4 2 .0563 .0032 .017 2 2 .1 1 1 9 .2 0 .0613 .0024 68 a i s p r o p o r t i o n a l t o th e o r i g i n a l c o n c e n t r a t i o n of p e r o x id e , x t o t h e p e ro x id e decom posed, and a -x t o th e p e r o x id e p r e s e n t a t any tim e t . U sing lo g a r ith m s to t h e b a se 1 0 , K ■ 2 * 3 0 3 /t lo g a / a - x . T hese r e s u l t s i n d i c a t e t h a t i f th e r e a c t i o n i s one sim p le o n e , w hich i t v e ry p ro b a b ly i s n o t , i t i s n o t f i r s t o r d e r n o r i s i t z e ro o r d e r w ith r e s p e c t t o p e r o x id e . A ls o , i f th e r e a c t i o n w ere a c t u a l l y a sim p le o n e , t h e r e s u l t s show t h a t i t i s n o t due t o c a t a l a s e i n w h ich c a s e , th e K 's w ould be c o n s ta n t and lo g C v e r s u s t w ould be l i n e a r * The r a t e as h e r e d e te rm in e d i s u n d o u b te d ly th e r e s u l t a n t o f many r e a c t i o n s o f v a r io u s o r d e r s and i t i s s u r p r i s i n g t h a t th e c u rv e i s a s sm ooth a s i t is * E x p erim en t 5 * T h is was s i m i l a r t o th e two p r e v io u s e x p e r i m ents b u t p u re hem o g lo b in was used* The f o l lo w in g d a ta was o b ta in e d by a n a ly z in g a l i q u o t s from a s u i t a b l e p e ro x id e -h e m o - g lo b in m ix tu re * TABLE X I n t e r v a l E la p s e d Time T i t r a n t R e q u ire d 0 M in. 0 Min* 5 .9 7 Ml. 1 1 5 .7 8 1 2 5 .6 5 1 3 5 .5 0 1 4 5 .3 9 T h is shows an a lm o st l i n e a r r e l a t i o n b etw een p e ro x id e c o n c e n tr a ti o n and tim e b u t may be th e f i r s t p a r t o f an ex p o n e n t i a l o r a l o g a r ith m ic c u rv e . 69 E x p erim en t 4 . • T his r e p e a t s E x p erim en t 3 w i t h more s u i t a b l e tim e i n t e r v a l s and c o n c e n t r a t i o n s , TABLE XI I n t e r v a l E la p s e d Time T i t r a n t R e q u ire d 0 M in. 1 0 1 0 1 0 1 0 1 0 0 M in. 10 20 30 40 50 1 1 .0 0 Ml. 10.10 9 .5 9 9 .3 2 9 .1 8 9 .1 0 The d a ta i s p l o t t e d on th e f o llo w in g page t o show th e r e l a t i o n s betw een p e ro x id e c o n c e n t r a t i o n and tim e and b e tw ee n th e lo g a r ith m o f th e c o n c e n tr a ti o n and tim e . The n o n - l i n e a r i t y i s i n t e r p r e t e d as b e f o r e i n th e c a s e o f b lo o d . R e a c tio n c o n s t a n t s c a l c u l a t e d as b e f o r e , u s in g th e f i r s t - o r d e r d i f f e r e n t i a l e q u a t io n , a l s o i n d i c a t e n o n - u n i m o l e c u l a r i ty . TABLE X II 2 .3 0 3 / t a a - x lo g a / a - x K .2303 1 1 .0 0 1 0 .1 0 .0371 .0085 .1151 1 1 .0 0 9 .5 9 .0596 .0069 .0768 1 1 .0 0 9 .3 2 .0720 .0055 .0756' 1 1 .0 0 9 .1 8 .0786 .0045 E x p erim en t 5* T h is was a re c h e c k on th e above r e s u l t s g a rd in g th e n o n - l i n e a r i t y o f th e v a r io u s f u n c t i o n s o f th e p e ro x id e c o n c e n t r a t i o n . 110 m l. o f .0 3 $ h y d ro g en p e ro x id e i n w a te r was m ixed w i t h 1 m l. o f 1$ h em o g lo b in i n w a te r and m a in ta in e d a t c o n s ta n t te m p e r a tu r e f o r 70 m in u te s . The f o llo w in g a n a l y t i c a l r e s u l t s a l s o show th e c a l c u l a t e d v a lu e s f o r th e lo g a r ith m s o f th e p e ro x id e c o n c e n t r a t i o n s . CURVE III No. 6201, University Bode Store, Los Angeles 71 TABLE X I I I I n t e r v a l Time E la p s e d T i t r a n t R e q u ire d Log C 0 M in. 0 Min. 1 0 .0 6 Ml. 1 .0 0 0 5 5 9 .2 9 .968 5 10 8 .7 0 .939 5 15 8 .3 0 .919 5 20 8 .0 5 .906 5 25 7 .9 6 .901 5 30 7 .8 0 .892 10 40 7 .6 9 .8 8 6 10 50 7 .6 0 .880 20 70 7 .5 0 .875 These r e s u l t s , i n g r a p h i c a l form on th e n e x t p a g e , s u b s t a n t i a t e th e p re v io u s e x p e rim e n ts on t h i s p o in t# E x p erim en t 6 . In t h i s e x p e rim e n t, m ethod c ) , C h a p te r I I I , was a p p li e d i n an a tte m p t to e v a lu a te th e e x p o n en t a o f th e f o llo w in g g e n e r a l e x p r e s s io n f o r th e p e ro x id e -h e m o g lo b in J lw v . w here C /^ c k r e p r e s e n t s th e c o n c e n tr a ti o n o f h y d ro g en p e ro x id e r a i s e d t o i t s p ro p e r (and unknown) power and C //< & * * * . r e p r e s e n t s t h a t o f h e m o g lo b in , a and b can be e v a lu a te d i n t u r n by d o u b lin g th e c o n c e n tr a ti o n o f th e r e a c t a n t i n q u e s t i o n and d e te r m in in g th e e f f e c t upon th e r a t e o f th e r e a c tio n * When th e p e ro x id e c o n c e n t r a t i o n , f o r ex am p le, i s d o u b le d , th e f o llo w in g r e l a t i o n s h o ld (53) from w hich M a w may be d e te rm in e d by f i n d i n g th e r a t i o o f th e r a t e s b e f o r e and a f t e r d o u b lin g . (Ax,') I {A* ^ K ^ C H xPy a C « 2 Getman & D a n i e l s , T h e o r e ti c a l C h em istry (New Y ork: W iley & S o ns, 1937) p . 326* No. 6201, University Book Store, Los Angeles 73 A p p ly in g t h i s m ethod t o p e r o x id e , 40 m l. o f .03$ and 40 m l. o f .015$ s o l u t i o n w ere p la c e d i n s e p a r a t e v e s s e l s and 10 m l. rem oved from e a c h f o r a n a l y s i s . 1 m l. o f 1$ h em oglobin was th e n added t o e a c h m ix tu re and a l i q u o t s w ere rem oved from e a c h a f t e r t e n m in u te s and a f t e r f i f t e e n m in u te s . D u rin g e ac h i n t e r v a l i t was fo u n d t h a t a p p ro x im a te ly ( w ith in 15$) th e same amount o f p e ro x id e was decom posed i n e a c h c a s e . I f one w ere j u s t i f i e d i n a tte m p tin g an i n t e r p r e t a t i o n , i t w ould be t h a t th e r e a c t i o n i s o f z e ro o r d e r w ith r e s p e c t to p e ro x id e s i n c e : R ate a f t e r d o u b lin g = i , i 5 = 2 a N e a re s t w hole No.= 1 R a te b e f o r e d o u b l i n g * Log 1 = a lo g 2 A = 0 I t i s n o t u n e x p e c te d t h a t th e o r d e r w ould be z e r o , i f one c o n s id e r s th e r e a c t i o n t o be a h e te ro g e n e o u s one t a k i n g p la c e on th e s u r f a c e o f a c o l l o i d . However, th e f a c t t h a t th e r a t i o o f th e r a t e s b e f o r e and a f t e r d o u b lin g d e v i a t e s fro m a w hole number by more th a n can be a c c o u n te d f o r on th e b a s i s o f e x p e r im e n ta l e r r o r s , i n d i c a t e s t h a t th e r e a c t i o n b e tw een hem oglobin and p e ro x id e i s n o t one r e a c t i o n b u t s e v e r a l . I f th e r e a c t i o n i s t r u l y one b etw een h em o g lo b in and p e ro x id e m o le c u le s , t h e r e a r e some s u r f a c e phenomena a t w ork w hich v i t i a t e th e m ethod b e in g u se d i n th e e x a m in a tio n o f th e r e a c t i o n . The p r e v io u s r e s u l t s on th e form o f th e c o n c e n tr a - t i o n - t i m e f u n c t i o n s may be i n t e r p r e t e d i n th e same l i g h t . E x p erim en t 7 . The above e x p e rim e n t was r e p e a t e d to se e how n e a r t h e r a t e s r a t i o was t o a w hole num ber. 200 m l. o f .03$ and 200 m l. o f .06$ h y d ro g e n p e ro x id e was u s e d i n e a c h c a s e . 74 5 ml* o f h em oglobin s o l u t i o n w ere added and th e f i r s t a n a ly s i s was made a f t e r two m in u te s o n ly , i n o r d e r t o be s u r e t h a t th e change i n c o m p o s itio n w ould be s l i g h t , w h ich c o n d i t i o n i s n e c e s s a r y so t h a t A X / a T w i l l n o t b e f a r from d x /d t* D uring th e f i r s t two m in u te s o f th e r e a c t i o n , th e s o l u t i o n o f g r e a t e r p e ro x id e c o n c e n t r a t i o n decom posed 1*50 tim e s as much a s d i d t h e w eak er m ix tu re* D u rin g th e n e x t f i v e m in u t e s , th e fo rm e r s o l u t i o n was decom posed .8 7 tim e s a s much as th e l a t t e r . I n o t h e r w o rd s , th e r a t i o h o v e re d aro u n d 1 a s th e n e a r e s t w hole num ber. The d e v i a t i o n from 1 i s to o g r e a t t o be a c c o u n te d f o r on th e b a s i s o f e x p e r im e n ta l e r r o r t h e c o n c lu s io n i s a g a in t h a t th e r e a c t i o n i s a r e s u l t a n t o f more th a n one r e a c t i o n . W hether th e p r o x im ity o f th e r a t i o t o 1 means t h a t th e r e a c t i o n i s p r e d o m in a te ly z e ro o r d e r , i s n o t im m e d ia te ly a p p a r e n t . E x p erim en t 8 . In t h i s e x p e rim e n t th e e f f e c t on th e r a t e , o f q u a d ru p lin g th e h y d ro g en p e ro x id e c o n c e n t r a t i o n , was d e te r m in e d . I t was fo u n d t h a t th e r a t e was i n c r e a s e d 1 .7 4 tim e s . By m ethod c ) , C h a p te r I I I , t h i s i s s e t e q u a l t o 4a whence a e q u a ls 1 .4 4 . C o n c lu sio n s from E x p erim e n ts 6 , 7 , and 8 a r e p r e s e n te d i n C h a p te r V. E x p erim en t 9 . T h is e x p e rim e n t was p e rfo rm e d i n th e same man n e r as th e l a s t o n e s , b u t was f o r th e p u rp o se o f e v a l u a t i n g f!b n e .g * t o d e te rm in e i f p o s s i b l e th e o r d e r w ith r e s p e c t t o 75 hem og lo bin by th e m ethod of d o u b lin g th e c o n c e n tr a ti o n and n o t in g th e e f f e c t on th e r a t e . A c o n c e n tr a te d w a te r s o l u t i o n o f hem oglobin was p r e p a re d and p la c e d i n e a c h o f f o u r b e a k e rs w ith 5 .ml. o f h y d ro gen p e r o x id e , th e c o n c e n tr a ti o n o f h em oglobin b e in g s u c c e s s i v e l y d o u b le d i n e a c h c a s e . A f te r a llo w in g e a c h m ix tu re t o s ta n d f o r f i f t e e n m in u te s , th e r e a c t i o n s w ere ’’q u en ch ed 1 * and . th e m ix tu re s a n a ly z e d . TABLE XIV P e ro x id e P e ro x id e S o lu tio n Hem oglobin Added P r e s e n t Decomposed #1 0 d ro p s 8 .8 1 0 .0 0 2 2 8 .7 8 .03 3 4 8 .7 2 .09 4 8 8 .5 9 .2 2 The r e s u l t s show th e amount o f p e ro x id e decom posed i n a g iv e n tim e t o be some f u n c t i o n o f th e hem oglobin con c e n t r a t i o n a lth o u g h u n d e r th e c o n d itio n s o f t h i s e x p e rim e n t, th e change i s to o s m a ll t o be a c c u r a t e . The r a t e i s s l i g h t l y more th a n d o u b le d when th e hem oglobin c o n c e n t r a t i o n i s d o u b le d . E x p erim en t 1 0 . T h is i s a r e p e t i t i o n o f th e l a s t e x p e rim e n t, u s in g h ig h e r r e a c t a n t c o n c e n t r a t i o n . D a ta: TABLE XV Ml. o f 1 % P e ro x id e P e ro x id e S o l u t i o n H em oglobin - P r e s e n t Decomposed #1 0 1 6 .6 7 0 2 .5 1 5 .8 9 .78 3 1 .0 1 5 .1 1 1 .5 6 4 2 .0 1 3 .4 8 3 .1 9 5 3 .0 1 1 .7 0 4 .9 7 6 3 .5 1 0 .9 2 5 .7 5 76 The r e s u l t s , p l o t t e d on th e n e x t p a g e , show a s t r i c t l y l i n e a r r e l a t i o n to e x i s t u n d e r th e c o n d itio n s o f t h i s e x p e r im e n t. The r a t i o o f th e r a t e a f t e r d o u b lin g th e h e m o g lo b in c o n c e n t r a t i o n to th e r a t e b e f o r e d o u b lin g th e h e m o g lo b in con c e n t r a t i o n h as th e v a lu e s 2 .0 0 , & 2 .0 4 , i n th e two c a s e s . The r a t i o a f t e r t r i p l i n g th e c o n c e n tr a ti o n o f h em o g lo b in i s 3 .1 8 . The e x p o n e n t o f th e h em oglobin c o n c e n tr a ti o n i n th e e x p r e s s io n o f page 71 i s th u s c a l c u l a t e d t o have th e v a lu e s 1 .0 0 , 1 .0 3 , and 1 .0 5 , th e a v e ra g e o f w h ich i s e q u a l t o th e w hole number 1, w i t h i n 2%• The o r d e r o f th e r e a c t i o n w i t h r e s p e c t t o hemo g lo b in i s t h e r e f o r e f i r s t . E x p e rim e n ts 11. 12 and 1 3 . These w ere a l l p e rfo rm e d t o check t h e r e s u l t o f e x p e rim e n t 10 w hich showed th e r e a c t i o n o r d e r w ith r e s p e c t t o h e m o g lo b in t o be f i r s t . V a rio u s c o n c e n tr a ti o n ra n g e s w ere u s e d i n o r d e r t o se e i f th e r e s u l t was c o n s i s t e n t . The v a lu e s o b ta in e d f o r th e ex p o n en t b w ere .7 5 , .6 6 , and .8 8 . The r e s u l t s , in s p i t e o f r i g i d e x p e r im e n ta l c o n t r o l , a r e n o t c o n s i s t e n t , show ing t h a t th e c o n c lu s io n from e x p e rim e n t 10 i s n o t g e n e r a l l y v a l i d . E v id e n tly th e s e e x p e rim e n ts s e r v e o n ly t o v e r i f y th e c o n c lu s io n re a c h e d from a s tu d y o f th e r e a c t i o n w i t h r e s p e c t t o h y d ro g en p e r o x id e , nam ely t h a t th e r e a c t i o n i s n o t one betw een p e ro x id e and hem oglobin as su ch b u t i s one b e tw een p e ro x id e and v a r io u s hem o glo bin c o n s t i t u e n t s . H em atin h a s b e en fo u n d n o t o n ly n o t to g iv e a p o s i t i v e r e a c t i o n by th e h y d r a z id e t e s t , b u t a c t u a l l y i n h i b i t s th e weak glow o f th e No. 6201, University Book Store, Los Angeles 78 t e s t r e a g e n t i n s t e a d o f g r e a t l y e n h a n c in g i t as do es b lo o d , h e m o g lo b in , o r hem in. 4 MISCELLANEOUS EXPERIMENTS The fo llo w in g a re b r i e f n o te s on v a r io u s m is c e lla n e o u s e x p e rim e n ts made d u r in g th e c o u rs e o f th e i n v e s t i g a t i o n . I f one a p p l i e s th e h y d ra z id e t e s t i n th e d a rk w ith o u t a llo w in g a m in u te o r so f o r th e eye t o become a d j u s t e d t o th e i l l u m i n a t i o n , th e ch em ilu m in escen ce h as th e a p p e a ra n c e o f b e in g slow i n s t a r t i n g i n c a se s w here th e lu m in e sce n c e i s w eak. The r e a c t i o n how ever b u i ld s up im m e d ia te ly t o i t s max imum lu m in e sc e n c e u n le s s a v e ry i n s o l u b le s t a i n i s b e in g t e s t e d . I f th e s o l u t i o n i s s p ra y e d on s t a i n s on c l o t h o r p a p e r , th e s t a i n a p p e a rs d a r k a g a i n s t th e l i g h t b a ck g ro u n d c a u se d by d i f f u s i o n o f t h e g lo w ing r e a g e n t . C are m ust be ta k e n n o t to to u c h d r o p p e r s , p i p e t t e t s , e t c . , t o s t a i n s b e in g t e s t e d . I f su ch g la s s w a re i s th e n d ip p e d i n to th e r e a g e n t , th e s t o c k s o l u t i o n w i l l glow s t r o n g l y due to th e c o n ta m in a tio n and s e n s i t i v i t y o f th e r e a c t i o n . The s e n s i t i v i t y i s a t l e a s t one p a r t i n s e v e r a l m i l l i o n a c c o rd in g t o b r i e f t e s t s w ith d i l u t e d b lo o d . S p e c i f i c i t y t e s t s i n d i c a t e t h a t a n y th in g w h ich n e u t r a l i z e s th e a l k a l i n e n a tu r e o f th e t e s t r e a g e n t , g iv e s a n e g a tiv e t e s t so t h a t s u b s ta n c e s sh o u ld be made a l k a l i n e 79 b e f o r e t e s t i n g . The f o llo w in g i s a b r i e f l i s t o f a few th in g s fo u n d to g iv e a d e f i n i t e l y p o s i t i v e t e s t w ith th e h y d ra z id e r e a g e n t : F r e s h b lo o d , b lo o d s t a i n s o f a g e s 1 m o ., 2 m o ,, 3 m o ,, 4 m o., 5 m o ., 6 m o ., 1 y r , , 2 y r s . , 2 .5 y r s . , 3 y r s . , 10 y r s . , 2 0 -1 0 0 ( ? ) y r s . , p u re h em o g lo b in , p u re h e m a tin h y d r o c h lo r id e (h e m in ), b o i l e d b lo o d s t a i n s , and s t a i n s h e a te d t o 110 d e g . w ith d ry h e a t . S p ech t fo u n d t h a t none o f th e fo llo w in g s u b s ta n c e s i n t e r f e r e d w ith h i s r e a g e n t s p e c i f i c i t y : i r o n r u s t , p u s , c o f f e e , s t a r c h , f u n g i , d y e s , l e a t h e r , e a r t h , wood, g r a s s . The a u th o r h a s found th e f o llo w in g s u b s ta n c e s t o r e a c t n e g a t i v e l y w ith th e m ix tu re d e v is e d b y him : Cowfs m ilk , g o a t ’s m ilk , human s k i n o i l , u r i n e , norm al f e c e s , s a l i v a , sem en, c a s t o r o i l , m ilk o f m a g n e sia , c o c a - c o l a , b e e r , b o urb on w h isk e y , s c o tc h w h isk e y , c a s c a r a , l i s t e r i n e , l a c t i c a c i d , g l y c e r i n e , b o r i c a c i d , a c e to n e , s u c r o s e , s u l f a n i l i m i d e . A ll o f th e o r d in a r y ru n o f l a b o r a t o r y c h e m ic a ls have b e e n found t o g iv e n e g a tiv e r e s u l t s w ith th e e x c e p tio n o f c e r t a i n o x i d iz i n g a g e n t s . Many o f th o s e g iv in g n e g a tiv e t e s t s w ere o f an a c i d n a tu r e and t h e i r a c t i o n may have b e en due t o th e n e u t r a l i z a t i o n o f th e t e s t r e a g e n t . I n d i v i d u a l c a s e s demand b la n k e x p e rim e n ts s u i t e d t o th e c o n d i t i o n s in v o lv e d . N a t u r a l ly i t i s e x tre m e ly u n l i k e l y t h a t su ch compounds as sodium h y p o c h lo r i te or p o ta s s iu m p erm anganate w i l l e v e r be fo u n d u n d e r c irc u m s ta n c e s w here th e y w i l l be con fu s e d w ith b lo o d . 54 S p e c h t, Z e i t . f u r angew. Chem. 5 0 , 8 , 155, 1937. 80 T e s ts w ere made on th e f o llo w in g m a t e r i a l s s u s p e c te d o f c o n ta in i n g b lo o d : Sm all p a r t i c l e s o f f l o o r s p l i n t e r s from th e s p o t w here a dynam ite bomb e x p lo d e d in th e hand o f a man who was c a r r y in g i t w ere p la c e d i n 10 ml* o f f r e s h l y p re p a re d t e s t s o l u t i o n i n th e dark-room * As e a c h p a r t i c l e moved a b o u t, i t l e f t an i l l u m i n a t e d t r a i l b e h in d i t i n th e s o l u t i o n and soon th e w hole tu b e was ag lo w . The t e s t was made a b o u t one y e a r a f t e r th e a c c id e n t to o k p l a c e . The f l o o r had b e e n w ashed i n th e mean tim e and no s t a i n s w ere v i s i b l e * F i n e l y s h a t t e r e d g l a s s was sw ept from th e f l o o r o f a l a b o r a t o r y a t th e s p o t w here a h y d ro g en e x p lo s io n i n a hood i n j u r e d a s t u d e n t . The g a th e r in g s gave im m ediate i n d i c a t i o n o f c o n ta in in g blood* D ust g a th e r e d s e v e r a l f e e t away a ls o gave a p o s i t i v e t e s t and s in c e a n e g a tiv e t e s t was g iv e n by a d u s t sam ple from a rem o te p a r t o f th e f l o o r , i t seems l i k e l y t h a t a f i n e b lo o d s p ra y r e s u l t e d from th e f o r c e o f th e e x p l o s i o n . S t a in s ta k e n from th e s h o e s , b e l t , and a u to m o b ile o f a man s h o t i n th e s h o u ld e r by a b a n d it gave d e f i n i t e p o s i t i v e r e s u l t s w ith th e h y d r a z id e t e s t * The s h o e s , w h ich w ere w h i t e , h ad b e e n c le a n e d w i t h w h ite p o l i s h i n th e m eantim e and a f r a c t i o n o f a m illig r a m o f s c r a p in g from th e seams gave a p o s i t i v e t e s t . The b e l t had some sm a ll s t a i n s w hich w ere h a rd t o d i s t i n g u i s h from th e l e a th e r * P i n - p o i n t sam ples 81 gave im m ediate p o s i t i v e r e s u l t s . T h is r e s u l t was v e r i f i e d by th e b e n z id in e t e s t . A few seco n d s en su e b e f o r e th e a p p e a ra n c e o f lu m in e s cence i f a sam ple i s many y e a r s o l d . P a r t i c l e s o f s u s p e c te d m a t e r i a l a r e b e s t added t o a few m l. o f r e a g e n t and c lo s e w a tc h m a in ta in e d w h ile th e y a re g e n t l y s w ir l e d a ro u n d . A ll s t a i n s up t o s i x m onths o f age have r e a c t e d so t h a t th e r e was no n o t i c e a b l e d e la y . I t seems l i k e l y t h a t an e x p e rie n c e d o b s e rv e r whose ey es a r e w e ll a d j u s t e d t o t h e d a r k , c o u ld ob t a i n t e n t a t i v e e v id e n c e as t o th e g e n e r a l age o f th e s t a i n . B r i l l i a n c y o f th e t e s t i s d e f i n i t e l y n o t i n h i b i t e d by q u i t e o ld s t a i n s . I f w eighed s t a i n s o f s u f f i c i e n t age w ere a v a i l a b l e , a p h o t o e l e c t r i c s tu d y o f th e lu m in e sc e n c e w ould p ro b a b ly answ er th e q u e s t io n o f th e e f f e c t o f age on th e a b i l i t y o f th e b lo o d t o e x c i t e lu m in e s c e n c e . As a lr e a d y m e n tio n e d , H em atein (E . K. C o.) f a i l s t o g iv e a p o s i t i v e t e s t w h ereas t h i s i s e x p e c te d t o b e p r e s e n t i n aged s t a i n s . F u r t h e r i n v e s t i g a t i o n seems w a r r a n te d . E q u al w e ig h ts o f b lo o d s t a i n s showed no d i f f e r e n c e i n i n t e n s i t y o f th e t e s t r e a c t i o n w h e th e r f r e s h o r 5 mo. o l d . Blood s t a i n s k e p t above 180 d e g . f o r 5 m in u te s f a i l e d to g iv e a p o s i t i v e t e s t w i t h th e h y d r a z id e r e a g e n t . Those b o i l e d f o r s e v e r a l h o u rs and a l s o th o s e m a in ta in e d a t d r y h e a t a t th e same te m p e ra tu re gave im m ediate p o s i t i v e r e s u l t s . I n te r m e d ia te te m p e r a tu r e s have n o t b e en i n v e s t i g a t e d . Oddly e n u f, a tu b e q f g lo w in g r e a g e n t d i d n o t d im in is h i n i n t e n s i t y 82 when f r o z e n s o lid * W ith f u r t h e r c o o lin g t o th e te m p e ra tu re o f l i q u i d a i r , th e glow fa d e d c o m p le te ly b u t r e t u r n e d when th e tu b e was warmed t o s l i g h t l y below t h e f r e e z i n g p o in t* 83 CHAPTER V CONCLUSIONS The c o n c lu s io n s as t o th e e x a c t n a tu r e of th e r e a c t i o n s in v o lv e d i n a p p ly in g th e h y d ra z id e t e s t t o th e d e t e c t i o n o f b lo o d i n s t a i n s o r o th e r m a t e r i a l , a re n o t a b s o l u t e l y c o n c lu s iv e i n any c a se b u t a r e to be r e g a r d e d a s p o s s ib l e e x p la n a tio n s f o r th e e f f e c t s o b se rv e d i n th e v a r io u s p h y s ic o c h e m ic a l p ro c e d u re s u t i l i z e d i n th e i n v e s t i g a t i o n . These e x p la n a tio n s a r e th e m ost p ro b a b le ones i n th e l i g h t o f th e know ledge a t hand and f o r th e phenomena a s o b se rv e d u n d e r th e c o n d itio n s h e r e p r e s e n t . A v e r y s a t i s f a c t o r y t e s t s o l u t i o n has b e e n fo u n d t o have th e f o llo w in g c o m p o s itio n - S o lu tio n # l - . 2 gms. 3- a m in o p h th a lh y d ra z id e and 1 gm. o f sodium h y d ro x id e made up t o 2000 m l. w ith d i s t i l l e d w a te r ; S o lu tio n # 2- . hydrogen p e ro x id e i n w a t e r . The r e a g e n t i s p r e p a r e d from th e s e s o l u t i o n s when i t i s w an ted by a d d in g 9 d ro p s o f .3 $ p e ro x id e o r i t s e q u iv a le n t to 5 m l. o f s t o c k s o l u t i o n # 1 . The t e c h n iq u e o f a p p ly in g th e t e s t can be v a r i e d a c c o rd in g t o i n d iv i d u a l re q u ire m e n ts b u t i t i s b e s t a t a l l tim e s t o add th e m a t e r i a l t o be t e s t e d , t o th e r e a g e n t and n o t v i c e - v e r s a * Such p ro c e d u re c a u se s th e w hole o f th e r e a g e n t t o glow and i t i s p ro b a b le t h a t w ith e x p e rie n c e one can judg e th e s o l u 84 b i l i t y o f th e s t a i n by o b s e rv in g th e lu m in e s c e n t s tr e a m e r s w hich c o n s t i t u t e th e t r a i l o f th e m a t e r i a l as i t s e t t l e s i n t h e r e a g e n t . The room i s o f c o u rs e d a rk e n e d as c o m p le te ly a s p o s s i b l e and th e ey es a d j u s t e d t o th e c o n d itio n s o f illu m in a tio n * As t o th e m echanism o f th e r e a c t i o n w ith b lo o d and th e h y d r a z id e r e a g e n t , i t h a s b e e n shown t h a t i f ch em ilu m in escen ce i s p r o p e r e v id e n c e o f th e o x i d a t i o n o f 3 - a m in o p h th a lh y d r a z id e , th e o x i d a t i o n i s c a u se d by a ) th e d e c o m p o s itio n o f h y d ro g en p e ro x id e by b lo o d and th e su b s e q u e n t o x i d a t i o n o f h y d ra z id e by th e p ro d u c ts o f th e d e c o m p o s itio n , b) d i r e c t o x i d a t i o n o f h y d ra z id e by some c o n s t i t u e n t o f b lo o d , and c) d i r e c t o x i d a t i o n o f h y d ra z id e by h y d ro g en p e r o x id e , a l l o f th e s e r e a c t i o n s o c c u r r in g s im u lta n e o u s ly . The slow o x i d a t i o n o f th e t e s t r e a g e n t i s n o t due t o d i s s o l v e d oxygen o r t o decom p o s in g p e r o x id e , b u t t o d i r e c t o x i d a t i o n by p e r o x id e . 3 -a m in o p h th a lh y d ra z id e i s n o t o x id iz e d a t any d e t e c t a b l e r a t e by m o le c u la r oxygen b u t v e ry r e a d i l y by a to m ic oxy gen and p ro b a b ly d i r e c t l y by o zo n e. The s t r o n g e s t e v id e n c e f o r t h i s i s t h a t no ch em ilu m in escen ce i s o b se rv e d when th e h y d ro g en p e ro x id e o f th e t e s t r e a g e n t i s decom posed b y a g e n ts known t o p ro d u ce m o le c u la r oxygen o n ly and no ato m ic oxygen a t any s ta g e o f th e r e a c t i o n . I t was a tte m p te d t o s u b s ta n t i a t e t h i s by show ing t h a t when h y d ro g en p e ro x id e decom posed i n a m anner su c h t h a t ch em ilu m in escen ce o ccu rred ;, th e decom- 85 p o s i t i o n was o f su c h a ty p e t h a t a to m ic oxygen was form ed d u r in g some s t a g e o f th e p ro c e s s * I n i n v e s t i g a t i n g t h i s m a t t e r , th e m ost l i k e l y c o n c lu s io n i s t h a t th e r e a c t i o n i s n o t a s i n g l e r e a c t i o n and t h e r e f o r e c an n o t be a c c u r a t e l y s t u d i e d by t h e m ethods o r d i n a r i l y u se d f o r d e te r m in in g th e o r d e r o f r e a c t i o n w i t h r e s p e c t to. c e r t a i n r e a c t a n t s w h ich a r e in v o lv e d i n one r e a c t i o n o n ly . The m ethod o f d e te r m in in g th e m a th e m a tic a l r e l a t i o n s betw een r e a c t a n t c o n c e n tr a ti o n s and tim e showed th e r e a c t i o n b e tw een h y d ro g e n p e ro x id e and h em o g lo b in t o be n e i t h e r z e ro n o r f i r s t o r d e r w i t h r e s p e c t t o p e ro x id e * The same c o n c lu s io n i s made on th e b a s i s o f th e m ethod o f d e te r m in in g th e change i n r a t e when r e a c t a n t c o n c e n tr a ti o n s a r e a l t e r e d . L ik ew ise b y .t h e l a t t e r m ethod, i t h a s b e en shown t h a t th e o r d e r w i t h r e s p e c t t o hem oglobin i s n e i t h e r z e ro n o r f i r s t o r d e r . S in ce t h i s m ethod showed th e o r d e r s w ith r e s p e c t t o h e m o g lo b in and w ith r e s p e c t t o p e ro x id e t o be n e i t h e r z e r o , f i r s t , s e c o n d , o r any o t h e r o r d e r , th e c o n c lu s io n i s re a c h e d t h a t th e decom p o s i t i o n o f h y d ro g en p e ro x id e by h e m o g lo b in i s i n r e a l i t y two o r more r e a c t i o n s p o s s i b l y b etw een heme and p e ro x id e and b e tw een some o t h e r m a t e r i a l and p e r o x id e . The r e s u l t s by th e m ethod o f r a t e s r a t i o s , a lth o u g h show ing th e p e ro x id e h em og lob in r e a c t i o n t o be com plex, may be r e s e r v e d l y i n t e r p r e t e d t o mean t i i a t th e p re d o m in a tin g r e a c t i o n i s one w h ich i s z e ro o r d e r w i t h r e s p e c t t o h y d ro g en 86 p e r o x id e , z e ro o r d e r w i t h r e s p e c t t o h em oglobin and t h e r e f o r e f i r s t o r d e r f o r t h e t o t a l r e a c tio n * T h is p o i n ts t o a m echanism o f d e c o m p o s itio n r e q u i r i n g t h a t ato m ic oxygen be a t l e a s t a t r a n s i e n t p h ase o f th e r e a c t i o n * Sum m arizing th e above d i s c u s s i o n , i t may be s a i d t h a t t h e m ost p ro b a b le r e a s o n t h a t th e h y d r a z id e t e s t i s a s s p e c i f i c as i t i s , i s b e c a u se v e ry few o t h e r s u b s ta n c e s p ro d u ce a to m ic oxygen from h y d ro g en p e ro x id e * A ll s u b s ta n c e s w h ich decompose p e ro x id e s h o u ld be t e n t a t i v e l y c o n s id e r e d a s i n t e r f e r i n g s u b s ta n c e s i n th e h y d r a z id e t e s t , b u t i f i n v e s t i g a t i o n shows a g iv e n s u b s ta n c e t o decom pose p e ro x id e b i m o l e c u l a r l y , i t w i l l n o t i n t e r f e r e and th e t e s t c an s t i l l be r e g a rd e d as s p e c i f i c i n th e p re s e n c e o f t h a t s u b s ta n c e * C hem ilum inescence i n t e n s i t y has b e e n shown to d e c r e a s e l i n e a r l y w ith tim e u n d e r th e c o n d itio n s o f th e t e s t r e a c t i o n . The t e s t i s a t l e a s t as good as o t h e r s a s r e g a r d s th e d e s t r u c t i v e a c t i o n o f h e a t on s u s p e c te d m a t e r i a l . T h is m at t e r r e q u i r e s e la b o r a tio n * The p h o t o g r a p h i c - p h o t o e l e c t r i c m ethod o f s tu d y in g th e . r e a c t i o n h a s b e en fo u n d t o be a c o n v e n ie n t one and co u ld be a p p li e d t o th e i n v e s t i g a t i o n o f lu m in e sc e n c e i n t e n s i t y a s a f u n c t i o n o f b l o o d - s t a i n a g e , e t c . A l o g i c a l l i n e o f a t t a c k a lo n g th e l i n e s o f f u r t h e r e l u c i d a t i o n of th e k i n e t i c s in v o lv e d , w ould be to exam ine 87 th e m ech an ics o f th e r e a c t i o n b e tw ee n h y d ro g en p e ro x id e and h e m a tin . I t c o u ld he r e a d i l y shown w h e th e r o r n o t t h a t r e a c t i o n was s i n g l e o r th e r e s u l t a n t o f two o r more as was fo u n d to be th e c a se w i t h h e m o g lo b in , Hemin i s v e ry a c t i v e i n decom posing p e ro x id e i n a m anner t o b r i n g a b o u t b r i l l i a n t lu m in e s c e n c e • No c o n c lu s io n s can be made a b o u t th e r o l e o f h e m a tin i n th e lu m in e sc e n c e b ro u g h t a b o u t by b lo o d s t a i n s , s in c e one sam ple o f h e m a tin was fo u n d n o t o n ly t o g iv e a n e g a tiv e t e s t b u t a c t u a l l y t o i n h i b i t th e s l i g h t glow o f th e f r e s h l y p r e p a re d t e s t r e a g e n t . BIBLlC>GRAJr A. BOOKS A lle n , Com m ercial O rganic A n a l y s i s . P h i l a d e l p h ia : P. B l a k i s t o n #s Son & Company, IncT B a n c r o f t, H em oglobin. Cam bridge: Cam bridge U n iv e r s i t y P r e s s , 1928. 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Creator Holter, N. J (author)

Core Title An investigation of the application of 3-aminophthalhydrazide to the forensic detection of blood stains

Degree Master of Science

Degree Program Chemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, analytical,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Brinton, Paul H.M.P. (committee chair), [illegible] (committee member), Vollrath, Richard E. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-788902

Unique identifier UC11347801

Identifier EP41494.pdf (filename),usctheses-c17-788902 (legacy record id)

Legacy Identifier EP41494.pdf

Dmrecord 788902

Document Type Thesis

Rights Holter, N. J.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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