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Critical comparison of the quantative methods for determination of alcohol in brain

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Critical comparison of the quantative methods for determination of alcohol in brain

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Content CRITICAL COM PARISON O F THE QUANTITATIVE M ETH O D S FOR
DETERMINATION OF ALCOHOL IN BRAIN
G
A T h esis
P re se n te d to
th e F a c u lty o f th e D epartm ent o f C hem istry
U n iv e rs ity of S outhern C a lif o rn ia
In P a r t i a l F u lfillm e n t
o f th e R equirem ents f o r th e Degree
M aster o f S cience
W
Monroe G, Beyer
June 1936
U M I Number: EP41476
All rights reserved
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JU M T _
Ctesortation Pubfsh-.ng
UMI EP41476
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This thesis, w ritten under the direction of the
candidate's Faculty Com m ittee and approved by
all its members, has been presented to and ac
cepted by the Council on Graduate Study and
Research in partial fulfillment of the require
ments for the degree of
M ASTER OP SCIENCE
$secretary
Dean
Date....^.}TTt§..i2r. 9. 5. S .
Faculty Committee
A C K N O W L E D G M E N T
The w r ite r w ishes a t t h i s tim e to ex p ress h is
d e e p e st a p p re c ia tio n to D r. P a u l H. M .-P.
B rin to n whose guidance and p a tie n c e was a
d e c id in g f a c to r in th e com pleting of th is
w ork.
TABLE OP CONTENTS
SECTION PAGE
I . INTRODUCTION...................................................................... 1
I I . HISTORY OF RELATED STUDIES ...................................... 5
I I I . PURPOSE OF T H E S I S ........................................................ 25
IV. APPARATUS A N D MATERIALS IN GENERAL.................... 26
V. PROCEDURE . . . . . . ............................................... 27
Steam D i s t i l l a t i o n o f th e A lcohol . . . • 27
O x id atio n - G e ttle r and T ib er Method . . . 30
O x id atio n - G e ttle r and F r e i r e i c h ’s V a ria
tio n o f G e ttle r and T ib er Method . . . . 37
O x id atio n - O rig in a l V a ria tio n o f G e ttle r
and T ib er Method ....................... . . . . . . 38
O x id atio n - N icloux M e th o d ........................... 43
O x id atio n - O rig in a l V a ria tio n o f N icloux
Method . . . . . . . . 45
V I. SUMMARY..................................... 50
BIBLIOGRAPHY ..................................................................... 51
INTRODUCTION
1
”From alm o st ev e ry s ta n d p o in t,1 * says H unt, ne th y l
a lc o h o l must be re g a rd e d as th e most im p o rta n t p o iso n w ith
w hich m ed ical men and j u r i s t s have to d e a l; no o th e r p o iso n
causes so many d ea th s or e n a c ts to or i n t e n s i f i e s so many
d is e a s e s , b o th p h y s ic a l and m e n ta l, as does a lc o h o l in th e
v a rio u s form s in w hich i t i s ta k e n .” The d a ily e v e n ts con
s t a n t l y ta k in g p la ce in our p re s e n t s o c ie ty , e s p e c ia lly sin c e
th e r e p e a l o f p r o h ib itio n , have made th e t r u t h o f t h i s s t a t e
ment v ery e v id e n t. Judges and ju r ie s a re c o n tin u a lly c a lle d
upon to re n d e r judgment a s to w hether a d efe n d a n t was i n t o x i
ca te d a t th e tim e when an a c t was com m itted. C o m p lic atio n s,
a r is in g from s i t u a t i o n s , prove to ju d ic ia r y b oards t h a t th e y
cannot a c c e p t a s v a lid te stim o n y , s ta te m e n ts o f c h a ra c te r
w itn e sse s who, fo r exam ple, t e s t i f y t h a t an accused p a rty
was a confirm ed ch ro n ic a lc o h o lis t an d , th e r e f o r e , in f e r a
c o n d itio n o f in e b r ie ty when th e a c c id e n t o c c u rre d . M oreover,
such sta te m e n ts a re v a lu e le s s sin c e th e y do n o t prove con
c lu s iv e ly t h a t a man was in to x ic a te d when a f a t a l i t y took
p la c e . Does th e fin d in g of tr a c e s o f a lc o h o l in th e u rin e of
a su sp e c t by an in e x p e rie n c e d county c o n s ta b le d e f i n i t e l y
■^J. L egal Med. and T o x ., 2 : 216, 1923.
s
e s ta b lis h , a man as drunk? Like s itu a ti o n s a re c o n s ta n tly a p
p ea rin g b e fo re ju r ie s th ro u g h o u t th e c o u n try . This t h e s i s is
devoted to answ ering th e q u e s tio n : How can in to x ic a tio n be
proven?
T his q u e s tio n can n o t be answ ered d i r e c t l y by a ch em ist
a lo n e . There i s need f o r th e p h arm a co lo g ist and p h y s ic ia n .
However, th e r e s u l t o f th e c h e m is t's a n a ly s is i s th e b a s is
upon w hich th e d o cto r and p h arm aco lo g ist d e f i n i t e l y form
t h e i r su p p o rtin g o p in io n . N a tu r a lly , a lc o h o l a f f e c t s each
in d iv id u a l outw ardly in a d i f f e r e n t way, b u t i n t e r n a l l y , a l
co h o l i s tr e a te d th e same way by ev ery body mechanism. Thus,
from symptoms or p h y s ic a l sig n s produced by th e in g e s tio n o f
a lc o h o l, we cannot conclude th a t a p erso n i s in to x ic a te d .
E x p erim en tal work by many o b se rv e rs has shown th a t a lc o h o l
in g e s te d i s r a p id ly ab so rb ed u n a lte re d in to th e blood streamm
and i s d is t r ib u t e d u n ifo rm ly th ro u g h o u t th e e n t ir e body w ith
th e e x c e p tio n of th e f a t and th e bone s e c tio n s . I t i s ab
sorbed in v ary in g amounts in th e d i f f e r e n t tis s u e s o f th e
body. Why i t ta k e s so l i t t l e to a f f e c t some and much more to
a f f e c t o th e rs i s ex p lain e d in th e f a c t th a t th e r a t e o f a b
s o rp tio n o f th e a lc o h o l from th e blood stream to th e b r a in i s
v ery ra p id fo r th e e a s ily in to x ic a te d and r e l a t i v e l y slow er
f o r th e heavy d r in k e r . T h e re fo re , we cannot use th e amount
consumed a s an index o f in e b r ia tio n .
S
Em erson, in h is book on a lc o h o lis m , has graphs showing
2
Haven Emerson, A lcohol and Man.
3
th e e f f e c t s o f a lc o h o l re a c h a maximum ab o u t one and o n e -h a lf
hours a f t e r th e ta k in g o f th e d rin k . From t h a t tim e on,
th e re i s a ra p id d e c re a se in th e a f f e c tin g o f th e mechanism
u n t i l ab o u t th re e or fo u r hours l a t e r when th e s tim u la tio n s
have co m p letely d is a p p e a re d . T hereby, th e re i s a tim e i n t e r
v a l of fo u r to f iv e hours in w hich th e p resen ce of th e a lc o h o l
i s d e te c ta b le . T races o f a lc o h o l have been found in th e
u rin e as long as te n hours a f t e r a d rin k was f i r s t consumed.
Hence th e tim e coupled w ith th e a c tio n observed of th e su s
p e c t b e fo re and a f t e r th e a c c id e n t w i l l g iv e s u f f i c i e n t l i g h t
upon th e s u b je c t to en a b le th e c o u rt to d ecid e ro u g h ly w hether
th e re i s re a s o n fo r an a n a ly s is . But th e d e c is io n as to
w hether to conduct an a n a ly s is or n o t should n o t be dependent
upon w hether th e d efen d an t acknow ledges he i s a d rin k in g man
or n o t. U su ally q u a l i t a t i v e t e s t s re q u irin g b u t a few m inutes
to perform w i l l be th e most u s e f u l in e n a b lin g th e chem ist to
t e l l w hether to rim th e q u a n tita tiv e t e s t or n o t. I f he does
su sp e c t th e p resen ce o f a lc o h o l in a given sam ple, he should
i n t e r p r e t h is r e s u l t s in re fe re n c e to how much tim e has -elapsed
a f t e r d rin k in g up to - th e tim e o f th e a c c id e n t.
The n e x t p o in t i s what organs or f lu id s can be tak en
as le g a l sam ples, th e r e s u l t o f an a n a ly s is upon them being
s u b s ta n tia te d by th e c o u r ts . N a tu r a lly , only th o se su b stan ces
w hich v ary d i r e c t l y w ith th e in ta k e of a lc o h o l can be c o n sid
e re d . E xperim entors have found th a t th e b r a in , s p in a l f l u i d ,
4
u r in e , and blood a r e e x c e lle n t as sam ples. N a tu ra lly , we can
n o t use th e b r a in i f th e p erso n i s n o t d e a d . For a liv in g
perso n we can only use th e s p in a l f l u i d , u r in e , and b lo o d .
The e v a lu a tio n o f each of th e s e sam ples w i l l be given in th e
h is to r y o f r e la te d s tu d ie s under th e su p p o rtin g in v e s tig a to r s .
Im m ediately a f t e r an a c c id e n t, b o th s id e s or p a r tie s should
be re q u ire d by th e h o s p ita l a tte n d a n ts to g iv e a sample s u i t
a b le f o r a n a ly s is . These sam ples should be k e p t c o n s ta n tly
ic e d in -'o rd e r to p resev e them in t h e i r o r ig in a l s t a t e . S u f f i
c ie n t sam ple must be drawn th a t th e re w i l l be enough f o r sev
e r a l d e te rm in a tio n s by th e p ro se c u tin g ch em ists and as much
l e f t f o r th e d efen d an t to have an aly zed by h is own c h e m ists.
As to th e method o f a n a ly s is , t h i s th e s is i s d e d ic a te d
to th e com paring o f th e v a rio u s methods and e s ta b lis h in g one
as th e b e s t . As th e re a re only two m ethods f o r q u a n t ita tiv e
r e s u l t s , nam ely th e N icloux and th e G e ttle r and T ib e r, t h i s
th e s is i s devoted to a c r i t i c a l com parison o f th e se two
m ethods.
HISTORY O F RELATED STUDIES
In p a s t l i t e r a t u r e , many re fe re n c e s have been c o n t r i
b u ted by th e numerous w orkers in t h i s s p e c ia l f i e l d . S e v e ra l
methods have been developed and recommended by th e s e e x p e ri
m e n te rs. Follow ing i s a summary o f some o f th e most h ig h ly
recommended o f th e s e m ethods, and th e c r iti c is m s t h a t contem
p o rary ch em ists have o ffe re d a g a in s t them . Much o f t h i s
3
summary was com prised by G e ttle r and T ib e rj a good amount
o f t h i s m a te r ia l has been made use o f in t h i s s e c tio n o f the
t h e s i s .
By Means of P h y s ic a l P r o p e r tie s . These methods of
d eterm in in g th e a lc o h o lic c o n te n t depend upon th e steam d i s
t i l l i n g of a l l of th e a lc o h o l over in to a re c e iv in g f l a s k
and th e n d eterm in in g th e amount of a lc o h o l in th e d i s t i l l a t e
4
by c e r t a in p h y s ic a l p r o p e r tie s . 'Sidersky d eterm in ed th e a l
c o h o lic c o n te n t o f a g iv en s o lu tio n by m easuring th e in c r e a s
ed m is c ib i lity o f e th e r and w ater in th e p rese n ce o f a lc o h o l
and com paring t h i s r e s u l t w ith f ig u r e s o f known in c re a se d
m i s c i b i l i t y from a lc o h o lic s o lu tio n s of g iv en g rad u ated
s tr e n g th . A lthough S id e rs k y 1s method may be s u c c e s s fu lly ap -
^A rch. P a th , and la b . M ed., 3 :7 5 , 1927.
^ B u ll. Amer. Chem. S uer. D i s t . , 27:5 6 2 , 1909.
6
p lic a b le to h ig h e r c o n c e n tra tio n s o f a lc o h o l, i t was n o t a c
c u ra te enough w ith in th e ran g e of 0 .1 to 1 .0 p er c e n t.
5
Lyon’s method o f d eterm in in g o f c o e f f ic ie n t o f expansion
was a ls o n o t a p p lic a b le to th e ran g e o f t h i s work.
The s a ltin g o u t o f th e a lc o h o l from th e aqueous s o l-
u tio n by p o tassiu m f lu o r id e a s proposed by H aines and Madden
was o b je c te d to because o th e r su b stan c e s o f human tis s u e
w hich a re o f te n c a r r ie d over in th e d i s t i l l a t i o n m ight be
s a lte d o u t a n d , f u r t h e r , th e ac c u ra c y o f t h i s method in sm a ll
p ercen tag e a lc o h o lic s o lu tio n s i s y e t to be e s ta b lis h e d .
7
K o lth o ff’s method fo r d eterm in in g th e change in e l e c t r i c a l
c o n d u c tiv ity was d isc a rd e d because o f s im ila r o b je c tio n s .
Q
Lachman’s method o f r e l a t i v e s o l u b i l i t y o f a n i lin e in v a rio u s
s tre n g th s o f s o lu tio n s o f a lc o h o l was in a p p lic a b le to a ran g e
of such s l i g h t a lc o h o lity .
The s p e c if ic g r a v ity method f o r a lc o h o lic c o n te n t was
in v e s tig a te d q u a n t ita tiv e l y by G e ttle r and T ib e rf b u t was d i s
5J . Amer. Pharm. A s s ., :8 0 7 , 1916.
6J . In d . and Eng. Chem., 13: 230, 1929.
^R ec. T rav . C him ., 39: 126, 1929.
8J . In d . and Eng. Chem., 13: 2 3 0 , 1921.
9Z ts c h r. f . p h y s io l. Chem., 50: 2 1 , 1906.
7
carded because a t 15° C th e s p e c if ic g r a v ity o f s o lu tio n s
ra n g in g from .003 to 0 .3 2 per c e n t l i e s betw een 0.9999 and
0 .9 9 9 4 . Such a sm a ll d if f e r e n c e a s 0.0005 could n o t p o s s ib ly
be used to d eterm in e w hether a man should go to th e gallow s
or n o t, as th e e x p e rim e n ta l e r r o r would p ro b ab ly exceed t h i s
v a lu e . F u r th e r , th e b r a in c o n ta in s o th e r v o l a t i l e su b sta n c e s
because a lc o h o l and th e re i s no a s su ra n c e th a t the. same amount
o f th e se su b sta n c e s w i l l be d i s t i l l e d over each tim e , th e re b y
u n ifo rm ly a l t e r i n g th e s p e c if ic g r a v ity a d e f in i t e am ount.
I n v e s tig a tio n s a s to th e a d v i s a b i l i ty o f th e use o f th e r e
f r a c tio n method were a ls o c a r r ie d o u t e x te n s iv e ly w ith in th e
range o f th e problem by G e ttle r and T ib e r. An Abbe R e fra c to -
m eter k e p t a t 20° C and d i s t i l l a t e s o f known a lc o h o lity were
used b u t th e r e s u l t s were in c o n s is te n t. T able I shows t h e i r
v a r ia tio n . From t h i s ta b le i t i s seen t h a t no q u a n tita tiv e
method has been found a p p lic a b le by means o f th e p h y s ic a l
p r o p e r tie s .
By Means of Io d in e D e riv a tiv e s (G ra v im e tric ). S t r i t a r
co n v erted th e a lc o h o l to e th y l io d id e by b o ilin g th e s o lu tio n
w ith h y d rio d ic a c id . The p ro d u ct was d i s t i l l e d o f f , th e
h y d rio d ic b ein g h eld back by th e a d d itio n o f sodium c a rb o n a te ,
and th e f r e e io d in e formed being ab so rb ed by re d phosphorus.
The e th y l io d id e , a f t e r d i s t i l l a t i o n , p assed in to s i l v e r
n i t r a t e , whereupon s i l v e r io d id e was p r e c ip ita te d and w eighed.
G e ttle r and T ib er re p o rte d t h a t t h i s r e a c tio n does n o t ta k e
TABLE I
DETERMINATION OF ALCOHOL BY REFRACTIVE INDEX
Case A lco h o l C ontent R e fra c tio n Index
P ercen tag e
1 none 1.3312
2 none 1.3310
3 none 1.3313
4 0 .0 2 1.3310
5 0 .1 0 1.3314
6 0.15 1.3312
7 0 .1 5 1.3314
8 0.20 1.3315
9 0.25 1.3315
10 0 .3 0 1.3313
p la c e q u a n t i t a t i v e l y in d ilu t e aqueous s o lu tio n s . This r e
s u l t i s f u r th e r confirm ed by th e p re s e n t in v e s tig a tio n in
w hich r e s u l t s v a ry in g over 100 per c e n t were found on the
same sam ple.
The fo rm a tio n o f th e iodoform from th e a lc o h o l and
th e w eighing o f th e iodoform or d eterm in in g th e io d in e c o n te n t
10
was proposed by S c h o ttm u lle r. This t e s t was o r i g i n a l ly su g -
1 1
g e ste d as a q u a l i t a t i v e t e s t by L ieb en . W ebster c r i t i c a l l y
s t a t e s
This r e a c tio n i s a v ery d e lic a te one b u t n o t c h a r
a c t e r i s t i c o f e t h y l a lc o h o l as o th e r p rim ary a lc o h o ls ,
many secondary a lc o h o ls , a ld e h y d e s , a c e to n e , a c e tic
e th e r , and l a c t i c a c id g iv e t h is t e s t .
Many o f th e above m entioned su b sta n ce s a re p re s e n t in th e
b r a in in sm a ll q u a n titie s and th e re fo r e would be v o l a t il i z e d
over in to th e d i s t i l l a t e by th e steam d i s t i l l a t i o n . I t seem s,
th e r e f o r e , th a t a l l io d in e d e r iv a tiv e s used to d eterm in e a l
co h o l g r a v im e tr ic a lly w i l l have to be d is c a rd e d .
Bv Means o f Io d in e D e riv a tiv e s (V o lu m e tric ). A conven
i e n t m icro-m ethod fo r th e e s tim a tio n of a lc o h o l in blood and
u rin e has been advanced by S o u th g ate . The a lc o h o l i s o x id iz e d
•^ N e u ro l. C e n t r a l b i ., No. 16, 1912.
11
L egal M edicine and T oxicology, p . 739.
12Biochem. J . , 18: 101, 1925; 19: 737, 1926. Reviewed
by Sm ith and G la i s te r , R ecent Advances in F o re n sic M ed icin e.
p. 183.
10
to a c e tic a c id by a m ix tu re o f p o tassiu m b ich ro m ate and s u l
p h u ric a c i d . Excess o f th e bichrom ate m ix tu re i s p re s e n t
d u rin g th e r e a c tio n , and th e ex cess i s e s tim a te d by a llo w in g
i t to o x id iz e h y d rio d ic a c id (p otassium io d id e ) to io d in e ,
and t i t r a t i n g th e lib e r a te d io d in e w ith sodium th io s u lp h a te .
The e q u a tio n s a r e :
8KgCrs 07 + 8BgS04 + 3CgH 50H -*2C rs (S04 ) 3 + 3CHSC00H
4 - Hg0 + SKgS04
KgCrg 07 + 7HgS04 + 6KI ->4KgS04 + Crg (S04 ) 3 + 7Hg0 + 3 I g
SNagSgOg + • Ig NagS4 0g + - 2NaI
The a p p a ra tu s i s immersed in a beaker o f w ater a t about 80° C.
A co n tin u o u s a i r c u rre n t i s m ain ta in ed th ro u g h o u t th e e n t ir e
p ro c e d u re . The a lc o h o l v o l a t i l i z e s and i s c a r r ie d th ro u g h
th e o x id iz in g m ix tu re . The b ich ro m ate m ix tu re i s l a t e r made
up to ab o u t 500 c c . in volum e, and a f t e r th e p o tassiu m io d id e
i s ad d ed , th e m ix tu re i s t i t r a t e d w ith sodium th io s u lp h a te ,
u sin g s ta r c h in d ic a t o r . The r e s u l t s o f t h i s method a re of
q u a l i ta t i v e v a lu e o n ly . A t f i r s t i t was hoped th a t q u a n tita
ti v e r e s u l t s would be o b ta in e d , b u t v ery few in v e s tig a to r s
would use t h i s method because o f th e c o s tly a p p a ra tu s , th e
la rg e p o s s i b i l i t y o f e x p e rim e n ta l e r r o r s from re d u c in g m a tte r
in th e ru b b er sto p p e rs and im p u r itie s in th e r e a g e n ts , and
th e p o s s i b i l i t y o f su b stan ce s o th e r th a n a lc o h o l p re s e n t in
th e blood and u rin e would red u ce th e b ic h ro m a te.
11
By Means of N i t r i t e D e riv a tiv e s . The e th y l a lc o h o l is
co n v erted to e th y l n i t r i t e by sodium n i t r i t e and a c e tic a c id ,
th e e th y l n i t r i t e form ed b ein g c a rr ie d a t 40° C by means o f a
stream o f carbon d io x id e in to a s o lu tio n o f p o tassiu m io d id e .
A c id ify in g w ith h y d ro c h lo ric a c id forms n itr o u s a c id by hydro
l y s i s , w hich l i b e r a t e s f r e e io d in e w hich i s t i t r a t e d w ith
th io s u lp h a te . The e q u a tio n s a r e :
CgHgOH + CgH5N0g + HgO
W ° a + H s ° * C 2 E5 0ii -
SHNOg + 3 H I - * I g + 2 N 0 + 2HgQ
2NagSg 0g + Ig — >NagS4 06 + 2NaI
13
T his m ethod, f i r s t proposed by F isc h e r and S chm idt, was t r i e d
by G e ttle r and T ib er who re p o rte d th a t th e io d in e lib e r a te d
from th e b la n k sam ples, even u sin g a d ark room f o r th e p ro ce
d u re , exceeded th a t amount w hich would be li b e r a te d by th e
a lc o h o lic s o lu tio n s ran g in g from 0.05 to 0 .8 p er c e n t, the
ran g e in w hich alm o st a l l c a se s f o r a n a ly s is w i l l f a l l . How
e v e r, F is c h e r and Schm idt s p e c if ie d th a t d e te rm in a tio n s
should be made a f t e r a e r a tio n had tak en p la c e fo r two and o n e-
h a l f h o u rs. G e ttle r and T ib er in t h e i r r e p o r t ad m itte d t h a t ,
a lth o u g h th e y fo llo w ed d ir e c tio n s o f Schm idt and F isc h e r c a re
f u l l y , only one and o n e -h a lf hours were allo w ed f o r a e r a tio n .
"^B er. d . D eu tsch . Chem. G e s e lls c h ., 57: 695, 1924;
59: 679, 1926.
IS
However, r e s u l t s v a rie d by over 100 p er c e n t due to th e in
s t a b i l i t y o f th e f r e e io d in e , and th e y were n ev er a b le to
d u p lic a te th e r e s u l t s o f th e o r ig in a t o r s . T h e re fo re , t h i s
method in i t s p re s e n t s ta tu s would be im p r a c tic a l q u a n tita
t i v e l y .
By O x id atio n o f th e A lco h o l to th e A cetaldehvde (C olor
im e tr ic ) . The a lc o h o l was o x id iz ed to a c e ta ld e h y d e and r e
duced fu c h s in , th e r e s u l t a n t red c o lo r b ein g compared w ith a
s e r ie s of s ta n d a rd iz e d perm anganate s o lu tio n s by th e method
14
of A rgenson. As t h i s t e s t i s a p p lic a b le to o n ly a sm a ll
p o rtio n o f th e d i s t i l l a t e , an e r r o r in c o lo rim e tr ic e stim a
tio n would become m ag n ified many tim es and v e ry few c o u rts
could a c c e p t t h i s method a s a s a t i s f a c t o r y ev id en ce o f quan
t i t a t i v e r e s u l t s . F u r th e r , e r r o r s would a r i s e due to th e p re
sence o f a c e tic a c id , th e n e x t ste p ' fo llo w in g th e o x id a tio n
to th e a ld eh y d e.
By Form ation o f an A lco h o l D e r iv a tiv e . In th e p re s e n t
th e s is an a tte m p t was made to fin d a p o s s ib le d e r iv a tiv e of
th e a lc o h o l i t s e l f . U n su cc essfu l a tte m p ts to form e th y le n e
d ib ro m id e, e th y l e t h e r , and o th e r s im ila r p ro d u cts showed
th a t th e a lc o h o lic s o lu tio n s were much to o d i l u t e f o r th e se
m ethods.
14B u ll. Soc. Chim ., ’ 27: 1000, 1902
By Means o f O x id atio n o f A lcohol to A ce tic A c id .
These m ethods a re based on th e o x id a tio n o f th e a lc o h o l to
th e a c e tic a c id and th e t i t r a t i o n of th e l a t t e r . T his method
15
was f i r s t in tro d u c e d by V. S ubbotin who h eated th e a lc o
h o lic s o lu tio n w ith s u lp h u ric a c id - p o tassiu m d ich ro m ate
m ix tu re on a sand b a th f o r tw e n ty -fo u r h o u rs , th e n d i s t i l l e d
th e a c e ti c a c id over and t i t r a t e d i t a g a in s t sta n d ard b a s e .
The d isa d v a n ta g e s of t h i s method a re t h a t some o f th e a c e t i c
a c id i s c o n v e rted to carb o n d io x id e and w ater th e re b y b ein g
l o s t . The optimum amounts o f the. s u lp h u ric a c id and d ic h ro
mate to be used were n o t d e term in ed , and th e p ro cess o f tw en
ty - fo u r ho u rs o f h e a tin g was to o tim e consum ing.
■ j O
In 1927, G e ttle r and T iber p u b lish e d t h e i r method
o f q u a n t ita tiv e d e te rm in a tio n o f a lc o h o l in th e b r a in by th e
fo rm a tio n o f th e a c e ti c a c id from th e a lc o h o l. Up to t h i s
tim e th e re was no a c c u ra te method o f d eterm in in g a lc o h o l in
human t i s s u e ex c ep t by m ic ro -a n a ly s is or by th e N icloux m eth-
■ * S
od. As t h is method i s checked in g r e a t d e t a i l in t h i s t h e s i s ,
th e d ir e c tio n s a re given in d e t a i l : The t i s s u e , as soon as
i t i s removed from th e body, should be p laced in a t i g h t l y
c lo se d ja r and p la ced in a r e f r i g e r a t o r . When ic e c o ld , 500
1 ^
Z ts c h r. f . b i o l . Chem., 7: 361, 1871.
16A rch. P a th , and Lab. M ed., 3: 75, 218, 1927.
14
grams a re weighed o u t, q u ic k ly ground up and p la c ed in a 2
l i t e r f l a s k . To t h i s a r e added 600 c e . o f w a te r, 5 c c . of a
s a tu ra te d s o lu tio n o f t a r t a r i c a c i d , and 1 c c . o f w h ite min
e r a l o i l . This m ix tu re i s now d i s t i l l e d w ith steam . A lo n g ,
w e ll cooled condenser should he u se d , and th e d i s t i l l a t i o n
should he co n tin u ed u n t i l e x a c tly 800 c c . has been c o lle c te d .
The d i s t i l l a t e i s w e ll mixed and 300 c c . o f i t were p laced
in a 500 c c . d i s t i l l i n g f l a s k . Twenty grams o f p o tassiu m d i
chroma te and 40 c c . o f c o n c e n tra te d s u lp h u ric a c id were added.
The c o n te n ts were mixed w e ll and th e f la s k was th e n connected
to a long w e ll co o led condenser by means o f a Hopkins d i s t i l
lin g h ead , and th e d i s t i l l a t i o n was s t a r t e d . The h e a t must he
so re g u la te d th a t i t w i l l ta k e from f o r t y - f i v e to f i f t y min
u te s to c o l le c t e x a c tly 250 c c . of d i s t i l l a t e . A fte r m ixing
th o ro u g h ly , 50 c c . o f t h i s d i s t i l l a t e a re t i t r a t e d w ith 0.05
norm al sodium hydroxide s o lu tio n , u sin g p h e n o lp h th a le in a s arm
in d ic a t o r . From t h i s t i t r a t i o n f i g u r e , th e amount o f a lc o h o l
p re s e n t in 1 k ilo g ram o f b r a in tis s u e can he e a s i l y c a lc u
l a t e d .
Having developed t h e i r method to a q u a n t ita tiv e b a s is ,
G e ttle r and T ib er s e t o u t to fin d th e norm al a lc o h o lic con
te n t o f th e b r a in . A fte r an a ly z in g ab o u t 6,000 b r a in s o f
p erso n s n o t dead over 48 h o u rs, th e y found t h a t th e b ra in s of
p erso n s who had n o t had any a lc o h o lic b ev erag es f o r a p erio d
o f te n days b e fo re d e a th showed an a lc o h o lic c o n te n t o f from
15
.0005 to .0025 per c e n t. This amount does n o t mean th a t t h is
i s norm al f o r th e a lc o h o lic c o n te n t b u t sim ply th a t t h i s
amount o f v o l a t i l e m a te r ia l in b r a in t i s s u e y ie ld s upon d i s
t i l l a t i o n and o x id a tio n th a t a c i d i t y . T h e re fo re , th e y con
cluded th a t th e norm al b r a in n ev er co n ta in e d more th a n 0.0025
per c e n t a le o h o lity .
On th e o th e r hand, case h i s t o r i e s o f th e persons who
had ta k en b ev erag es a t th e tim e of d e a th were c a r e f u lly k e p t
and th e d eg ree of in to x ic a tio n a t th e e x a c t moment o f d e a th
was re c o rd e d . The b r a in s o f th e se deceased were an aly zed
and th e p er c e n t of a lc o h o l found was compared w ith th e d e
gree of in to x i c a tio n . In a l l cases in w hich th e a lc o h o lic
c o n te n t o f th e b r a in was below 0 .1 per c e n t, th e p a tie n ts
had been norm al in a l l t h e i r a c tio n s a t th e tim e of d e a th .
A lthough th e p a tie n ts whose a le o h o lity in th e b r a in was b e t
ween 0 .1 and 0 .2 5 p er c e n t showed some p h y sio lo g ic d i s t u r
b an ce, such a s a g g re s s iv e n e s s , a more or le s s lo s s of sense
o f c a re , e t c . , th e ev id en ce as re c e iv e d from th e a n a ly s is
was n o t s u f f i c i e n t to pronounce them in to x ic a te d .
Hovrever, th o se p erso n s whose b r a in upon a n a ly s is fo r
a lc o h o lic c o n te n t showed r e s u l t s above 0 .2 5 p er c e n t had,
b e fo re d e a th , d isp la y e d symptoms o f b ein g in to x ic a te d . De
g rees o f in to x ic a tio n v a ry in g from s l i g h t u n b alan cen ess and
s h o rt p e rio d s of b o is te ro u s n e s s to th o se o f com plete i n s t a b i l
i t y of e q u ilib riu m and deep coma were in d i r e c t c o r r e la tio n
w ith th e r e s u l t s of th e b r a in a n a ly s is a f t e r d e a th , th e f i g
u re s fo r a lc o h o l ran g in g from 0.25 to 0 .6 0 p er c e n t. As a
r e s u l t , th e y concluded th a t a p erso n could be le g a lly p ro
nounced in to x ic a te d i f , w ith in 48 hours a f t e r d e a th , an a n a ly
s is of h is b r a in showed an a lc o h o lic c o n te n t o f above 0.25
per c e n t.
So much fo r th e b r a in a n a ly s is , b u t now how can in e
b r i a t i o n be proven of a liv in g p erso n ? A g ain , th e s c i e n t i f i c
w orld i s in d e b te d to G e t t le r , who c o lla b o ra te d w ith F r e ir e i c h ,
and to g e th e r th e y worked o u t a q u a n tita tiv e method u sin g blo o d
and s p in a l f l u i d sam ples g a th e re d from liv i n g p erso n s. These
sam ples were an aly zed by th e G e ttle r and T ib er method b u t due
to th e f a c t t h a t sam ples e q u iv a le n t in w eig h t to th a t o f th e
b ra in were n a t u r a ll y n o t a v a ila b le , th e e n t i r e pro ced u re was
c a rr ie d o u t on a sm a lle r s c a le . F i r s t , th e y worked w ith sam
p le s from n o n -a lc o h o lic in d iv id u a ls to d eterm in e th e norm al
a lc o h o lic e q u iv a le n t of each o f th e f l u i d s . The r e s u l t s of
th e s p in a l f l u i d av erag ed 0.0071 p er c e n t w h ile th o se o f th e
blood were 0.0120 p er c e n t. T his s ig n if ie d th a t in norm al
sam ples o f blood and s p in a l f l u i d th e re a re p re s e n t v o l a t i l e
s u b s ta n c e s , n o t n e c e s s a r ily a lc o h o l, th a t pass in to th e d i s
t i l l a t e when th e sam ple is su b je c te d to steam d i s t i l l a t i o n
and a re o x id iz ed by th e s u lp h u ric a c id - d iehrom ate m ix tu re
17J . B io l. Chem., 92: 199, 1931.
17
whereby y ie ld in g an a c i d i t y o f 1 .3 3 c c . o f 0 .0 2 N e q u iv a le n t
to 0.012 p er c e n t of e th y l a lc o h o l or 1 .3 mg. o f e th y l a lc o
h o l per 10 c c . sample o f norm al b lo o d . In o rd e r to d eterm in e
th e optimum amounts of r e a g e n ts , v ary in g am ounts in th e r a t i o
o f 1 gram o f diehrom ate to 2 c c . o f c o n c e n tra te d a c id were
added to 100 c c . o f a p rep ared a lc o h o lic s o lu tio n having 0 .5
per c e n t a lc o h o l. A fte r d i s t i l l a t i o n , th e a c e ti c a c id was
t i t r a t e d and th e re c o v e ry o f a lc o h o l f ig u r e d . T able I I
shows t h e i r r e s u l t s . The optimum o f re a g e n ts re q u ire d was
found to be 7 .5 grams o f d ieh ro m ate to 15 c c . o f su lp h u ric
a c id . F u rth e r, th e y found th a t th e e m p iric a l f a c t o r of
w hich G e ttle r and T ib er e s ta b lis h e d from t h e i r b r a in a n a ly s is
h eld tr u e f o r th e o x id a tio n of th e d i s t i l l a t e from th e blood
and s p in a l f l u i d , 100 c c . o f d i s t i l l a t e b ein g ta k en and 80
c c . p o rtio n s c o lle c te d . In T able I I I th e s e r e s u l t s a re l i s t e d .
N ext, sam ples were drawn from p a t i e n t s , some in to x ic a te d
and o th e rs n o t, to see i f th e r e s u l t s o f th e a n a ly s is would
prove u s e f u l or n o t. G e ttle r and F r e ir e ic h concluded t h a t th e
s p in a l f l u i d was v ery v a lu a b le a s an in d ex o f in to x ic a tio n ,
a s a l l c a se s showing 0.272 per c e n t or more a lc o h o l were d e f
i n i t e l y in to x ic a te d , th e d eg ree o f w hich b ein g in d i r e c t c o r
r e l a t i o n w ith th e p er c e n t of a lc o h o l reco v ered ra n g in g from
0.272 a s m ild ly in to x ic a te d to 0.674 a s dead d ru n k .
The r e s u l t s from th e a n a ly s is o f th e blood v a r ie d to o
much to be used a s any index of in to x ic a tio n in th e q u a n tita -
TABLE I I
DETERMINATION OF OPTIM UM A M O U N TS OF REAGENTS
KgCrgO^ i n o x id
HgSO^ in o x id 0 .0 5 N a c e ti c CgH50H
iz in g m ix tu re iz in g m ix tu re a c id in d i s . re co v e re d
4
5 ,5 grams 11 c c . 18.95 c c .
p
4 3 .6 mg. 8 7 .2
6 .5 13 1 9.00 4 3 .7 8 7 .4
7 .5 15 19.80 4 5 .5 91.0
8 .5 17 19.25 4 4 .2 8 8 .4
10.5 20 18.90 4 3 .4 8 6 .8
TABLE I I I
RECOVERY OF ALCOHOL FRO M SPIRAL FLUID A N D BLO O D
50 mg. of a lc o h o l were added to each specim en.
Sample No. Specimen 0.05 N a c i d it y 0.05 N a c i d i t y A lcohol
produced due to a lc o h o l re c o v e r
added ed
c c . c c . per cen t
1 Blood 19.67 19.25 88.45
2
it
19.57 19.15 88.08
3
a
18.89 18.47 84.96
4
ii
18.84 18.42 84.74
5 S p in a l f lu i d 18.77 18.35 84.40
6
« a
18.34 17.92 82.44
7
a n
18.40 17.98 82.70
8
it ii
18.93 18.51 85.14
Average recc >very .................... 85.12
20
tiv e work b u t i s u s e f u l as a q u a l i t a t i v e m easure. This i s con
t r a r y to Widmark, who re p o rte d t h a t w h ile he was s t i l l e x p e ri
m enting w ith blood a s a q u a n tita tiv e m easure o f in to x ic a tio n
by methods o f m ic ro -a n a ly s is , he was d e f i n i t e l y su re o f i t s
u s e fu ln e s s in fo re n s ic c h e m istry . W hile due c r e d i t m ust be
g iv en to h is p a tie n c e i n c a rry in g o u t te d io u s work o f t h i s
n a tu r e , v e ry few la b o r a to r ie s in t h i s c o u n try a re o u t f i t t e d
w ith m icro a p p a ra tu s , and th e p r a c t i c a b i l i t y o f t h i s method
w i l l have to be answ ered by th e c rim in o lo g ic a l ch em ist o f th e
f u tu r e . R e c e n tly , he advanced a more p r a c t i c a l method in
w hich sam ples of blood a re drawn from th e e a r o f th e su sp e c t
and o x id iz e d d i r e c t l y by th e G e ttle r ana T ib er method w ith o u t
em ploying th e use o f steam d i s t i l l a t i o n . Along w ith t h i s
o x id a tio n , he runs a b la n k sample i n w hich only th e re a g e n ts
a re u se d , and from th e d if fe re n c e s in amount o f 0 .0 2 N base
re q u ire d to n e u tr a liz e th e a c id form ed, he d eterm in es w hether
a p erso n i s in to x ic a te d or n o t. W hile t h i s method i s f u r th e r
confirm ed by th e I n s t i t u t e fo r L egal and S o c ia l M edicine a t
B e r lin , Germany, th e re i s s t i l l a c o n tro v e rsy as to w hether
th e blood in c re a s e s d i r e c t l y in a le o h o lity as th e p e rso n b e
comes more in e b ria te d and a ls o how long a f t e r in to x ic a tio n
th e a lc o h o l w i l l rem ain in th e b lo o d . As i t was im p o ssib le
fo r th e p u rp o ses o f t h i s th e s is to draw sam ples of blood or
s p in a l f l u i d from p a t i e n t s , d i s t i l l a t e s were made up c o r r e s
ponding to th o se th a t would r e s u l t from th e steam d i s t i l l a
21
tio n and th e s e p rep ared s o lu tio n s were s u b je c te d to o x id a
tio n . T h e re fo re , th e c o n tro v e rsy w hich e x i s t s over th e a lc o
h o l c o n c e n tra tio n in th e blood w i l l have to be so lv ed by some
o th e r w orker in t h i s f i e l d .
By Means o f T itr a t io n o f th e A lco h o l w ith S tandard
18
S o lu tio n o f D ichrom ate S u lp h u ric A cid. B odlander was th e
f i r s t to in tro d u c e t h i s m ethod. He t i t r a t e d a d e f i n i t e
amount o f a sta n d a rd s o lu tio n o f d ich ro m ate - su lp h u ric a c id
w ith a s o lu tio n of a lc o h o l u n t i l a g reen c o lo r was o b ta in e d .
He c a rr ie d out h is t i t r a t i o n s w h ile h o t.
19
S trassm an extended t h i s method by making c o lo rim e t
r i c com parison u sin g a lc o h o lic s o lu tio n o f d e f in it e s tr e n g th
and g e ttin g c o lo r changes from orange to g re e n .
The use o f an ex cess o f d ich ro m ate - s u lp h u ric a c id
m ixture and th e d e te rm in a tio n of th a t e x c e s s , th e a lc o h o l
having been co n v erted to th e aldehyde by red u cin g i t w ith a
known ex cess of sta n d a rd s o lu tio n o f fe r ro u s ammonium s u l-
20
p h ate was in tro d u c e d by B en ed ict and N o rr is . The amount
o f fe rro u s ammonium su lp h a te in ex cess was d eterm in ed by
t i t r a t i n g w ith a sta n d a rd s o lu tio n o f p o tassiu m perm anganate,
u sin g a change o f c o lo r to pink as th e end p o in t. This method
^8P f lu g e r ’s A rch. f . d . g e s. P h y s io l., 32: 398, 1883.
19
P f lu g e r ’s A rch. f . d . g e s. P h y s io l., 49: 315, 1891.
^ J . Amer. Chem. S o c ., 20: 293, 1898.
22
i s c r i t i c i z e d by most w orkers a s b ein g to o cumbersome due to
i t s many s te p s and th e d i f f i c u l t y o f keeping a l l th r e e r e
a g e n ts s ta n d a rd iz e d .
21
C o tte d eterm in ed th e a lc o h o lic c o n te n t by adding a
known ex c ess of sta n d a rd chrom ic - s u lp h u ric a c id s o lu tio n
and t i t r a t i n g th e ex cess by u sin g sta n d a rd fe rro u s ammonium
s u lp h a te , u sin g potassium f e r r ic y a n id e as an o u ts id e in d ic a
t o r .
9 9
B au d rex el d i s t i l l e d th e a lc o h o l in to a m easured
amount of sta n d a rd chrom ic - s u lp h u ric a c id s o lu tio n and th e n
t i t r a t e d th e ex cess o f chrom ic a c id w ith a sta n d ard s o lu tio n
o f a lc o h o l. O b jec tio n i s r a is e d to t h i s method due to th e
lo s s in d i s t i l l a t i o n , o x id a tio n , and t i t r a t i o n w ith sta n d a rd
a lc o h o l.
23
B aren d rech t o x id iz e d th e a lc o h o l w ith an ex cess of
sta n d a rd s o lu tio n o f a l k a li n e perm anganate; th e n he reduced
th e ex cess perm anganate w ith an ex cess o f o x a lic a c id , f i n a l l y
t i t r a t i n g t h i s excess w ith a sta n d a rd s o lu tio n o f perm angan
a t e . However, th e same o b je c tio n s as fo r o th e r methods of
t h i s type s t i l l hold tr u e .
*^Rep. de P harm ., 9: 4 3 8 , 1852.
SSwchnschr. B ra u e re i, 2 8 : 2 1 , 1912.
23
Z ts c h r. f . a n a l. Chem., 52: 167, 1913.
23
24
Widmark used t h i s same method ex c e p t on a sm a lle r
s c a le , and fo r a n a ly s is o f blood e s p e c ia lly .
P robably th e b e s t method in t h i s p ro ced u re i s t h a t as
25
advanced by N icloux who f i r s t compared th e c o lo r w hich he
o b ta in e d on adding a lc o h o l s o lu tio n to a d e f i n i t e s o lu tio n of
chrom ic - s u lp h u ric a c id , w ith th e c o lo rs produced by a s e r ie s
o f known s tr e n g th s o lu tio n s o f a lc o h o l tr e a te d in th e same
way. L a te r , he im proved t h i s procedure by t i t r a t i n g th e a lc o
h o l s o lu tio n d i r e c t l y w ith a sta n d a rd s o lu tio n o f chrom ic -
su lp h u ric a c id u n t i l th e g reen f i r s t o b ta in e d changed to a
brow n-green. G e ttle r and T ib e r ’s c r iti c is m t h a t th e sta n d a rd
r e a g e n ts , e s p e c ia lly th a t o f th e chrom ic - s u lp h u ric a c id ,
d e t e r io r a te r a p id ly i s y e t to be proven, a s th e p re s e n t i n
v e s tig a tio n in d ic a te s th e c o n tra ry . This w i l l be f u r th e r d i s
cussed in d e t a i l l a t e r . They a ls o claim t h a t th e re i s some
lo s s of ald eh y d e due to v a p o riz a tio n d u rin g t i t r a t i o n . This
has been m inim ized by th e work re p o rte d in t h i s t h e s i s . They
a ls o claim t h a t b e s id e s th e a ld e h y d e , a c e ti c a c id and carbon
d io x id e a re a l s o p ro d u c ts o f t h i s o x id a tio n . L a s tly , th ey
have tro u b le in th e re c o g n itio n o f th e end p o in t, w ith e r r o r s
^4 S kandinav. A rch. f . P h y s io l., 35: 125, 1917.
^C o m p t. re n d . Soc. de B i o l ., 3: 841, 1906 and
D eutsches A rch. f . k l i n . M ed., 109: 272, 1913.
ran g in g from 10 to 20 d ro p s. T his method w i l l he d isc u sse d
in d e t a i l in th e th e s is and most o f th e s e f a u l t s w i l l be
overcome.
PURPOSE OF THESIS
The purpose of th is th e s i s i s to answ er th e q u e s tio n :
How can in to x ic a tio n be proven? As t h i s in v e s tig a tio n i s
c a r r ie d on w ith th e i n t e r e s t o f o b ta in in g q u a n tita tiv e r e
s u l t s o n ly , th e main p ro ced u re c e n te rs around th e stu d y of
th e two b e s t q u a n tita tiv e m ethods, nam ely, th e G e ttle r and
T ib er P rocedure and th e N iclo u x C la s s ic a l M ethod. The ex
p e rim e n ta l work in t h i s th e s is i s d iv id e d in to f iv e s e c tio n s .
The f i r s t i s th e checking of th e G e ttle r and T ib er P ro cess
o f O x id a tio n , in v o lv in g th e u se o f an e m p iric a l f a c t o r . The
s l i g h t v a r ia tio n as to th e optimum amount o f re a g e n ts to be
used as proposed by G e ttle r and T ib er w i l l be d e a lt w ith in
th e second s e c tio n . R e su lts o f v a r ia tio n s w hich t h i s in v e s
t i g a t o r employed w i l l be b ro u g h t o u t in th e t h i r d p a r t . The
c r i t i c a l stu d y o f th e N icloux method of o x id a tio n w i l l com
p r is e th e f o u r th s e c tio n . The f i n a l p o rtio n o f th e e x p e ri
m en tal work w i l l be devoted to th e improvement o f t h i s
N icloux method and th e r e s u l t s fo rth co m in g .
APPARATUS A M D MATERIALS IN GENERAL
A ll m a te r ia ls used in t h i s th e s is w ere ch em ically
p u re . The a b s o lu te a lc o h o l was a s s u re d by adding anhydrous
copper s u lf a te w ith no changing in th e c o lo r o f th e s u l f a t e .
The dichrom ate was checked by f i l t e r i n g a h o t s a tu r a te d s o lu
t i o n and c a tc h in g th e f i l t r a t e in a sm a ll e v a p o ra tin g d is h
w hich f lo a te d on th e s u rfa c e o f a co ld w a te r b a th . This
caused sm a ll c r y s ta ls o f th e d ich ro m ate to be form ed. These
were d r ie d in a vacuum d e s ic a to r over anhydrous s u lf u r ic
a c id . The f r e s h ly p rep a re d c r y s ta ls were p laced in one
f l a s k and c r y s ta ls from a b o t t l e la b e le d C .P. were p la ced in
a n o th e r f l a s k and b o th were su b je c te d to th e same p ro ced u re
o f o x id a tio n u sin g th e same amount o f s u lf u r i c a c id and th e
same s tr e n g th and amount o f a lc o h o lic s o lu tio n . Upon t i t r a
t i o n , th e r e s u l t s agreed w ith each o th e r to w ith in 5 p a rts
in te n th o u s a n d $ th e re fo r e i t was sa fe to conclude t h a t th e
C .P. d ich ro m ate was p u re . The d i s t i l l e d w ate r was assumed
to be p u re .
A ll a p p a ra tu s w i l l be d e s ig n a te d as used in th e main
body o f th e t h e s i s . A ll work was c a r r ie d o u t and c o rre c te d
to 20° C. P ip e ts and b u re ts were c a r e f u lly c a lib r a te d and
c o rre c te d f o r t h i s te m p e ra tu re .
PROCEDURE
Steam D i s t i l l a t i o n o f th e A lco h o l. The f i r s t s te p
a f t e r th e sam ple o f b r a in has been re c e iv e d f o r a n a ly s is i s
to d i s t i l over th e v o l a t i l e a lc o h o l. To acco m p lish t h i s ,
steam d i s t i l l a t i o n i s em ployed. The b r a in tis s u e i s immedia
t e l y fro z e n upon re c e iv in g i t in o rd er to m inim ize th e lo s s
o f a lc o h o l and o th e r su b sta n c e s th ro u g h v o l a t i l i z a t i o n . The
fro z e n tis s u e i s ta k en from th e chamber and 500 grams w eigh
ed out and ground up. As no human b r a in was a v a ila b le ,
lamb and b e e f b r a in s were pu rch ased and w h ile f r e s h , were
ground u p , w eighed and in o c u la te d w ith a d e f i n i t e amount o f
a lc o h o l. Im m ediately a f t e r th e in o c u la tio n , th e d i s t i l l a t i o n
was s t a r t e d and hence th e re was no lo s s due to v o l a t i l i z a
ti o n . By u sin g f r e s h ground b r a in , th e f re e z in g p ro cess was
n o t n e c e s s a ry . The ground b r a in was p la ced in a two l i t e r
round bottom f l a s k , 600 c c . o f d i s t i l l e d w a te r, 1 c c . of
N u jo l, 5 c c . o f a s a tu ra te d s o lu tio n of t a r t a r i c a c id and
th e a lc o h o l w ere th e n added. The d i s t i l l e d w ater was added
to g iv e th e b r a in enough f l u i d in th e s t a r t o f th e d i s t i l l a
t i o n , o th e rw ise c h a rrin g o f th e o rg a n ic m a tte r o f th e b r a in
would o c c u r. The N u jo l was added to p re v e n t f r o th in g , b ein g
found to be b e t t e r th a n th e p a r a f f in th a t F r e ir e ic h and G et
t l e r recommended f o r th e d i s t i l l a t i o n o f th e blood and s p in a l
f l u i d . The t a r t a r i c a c id was added to hold back many o f th e
o rg a n ic su b stan ce s from b ein g d i s t i l l e d o v e r. The f la s k
was im m ediately s to p p e re d , ru b b er s to p p e rs b ein g used
th ro u g h o u t th e e n t i r e a p p a ra tu s in o rd e r to have a i r t i g h t
c o n n e c tio n s . The steam g e n e ra to r c o n s is te d o f a one l i t e r
d i s t i l l i n g f l a s k , w ith a g la s s tu b e ex ten d in g from b e n e a th
th e s u rfa c e o f th e d i s t i l l e d w ater to a f o o t or more above
th e f l a s k , th u s a c tin g as a s a fe ty v a lv e in case th e steam
g e n e ra te d should b u ild up to o much p re s s u re a t any given
moment. T his was co n n ected to one o f th e two o u tl e t s in th e
ru b b e r sto p p e r o f th e d i s t i l l a t i o n f l a s k . The tu b e by means
o f w hich th e steam e n te re d th e f l a s k c o n ta in in g th e b r a in
m a te r ia l, extended alm o st to th e bottom o f th e f l a s k so th a t
th e steam would n o t escape from th e b o ilin g w ater v ery r a p id
ly and would s t i r th e m ix tu re around cau sin g a l l o f th e p a r t i
c le s o f a lc o h o l to be c a r r ie d over w ith th e steam . The o th e r
o u t l e t c o n tain e d a Hopkins d i s t i l l i n g head w hich k e p t most of
th e s o lid p a r t i c l e s and f a t t y tis s u e from being c a r r ie d o v e r.
T his was connected to a long condenser w hich was co o led by a
w ater ja c k e t. From th e co n d en ser, th e drops o f th e d i s t i l l a t e
flow ed th ro u g h a curved a d a p te r w ith a se a le d -o n e x te n sio n of
g la s s tu b in g . The end o f th e tu b in g extended to th e bottom
of a one l i t e r Erlenm eyer f l a s k w hich was surrounded by an
ic e and w ater b a th . T his f la s k was marked w ith re d crayon
a t a le v e l s ig n ify in g 800 m l. A g r a n ite pan was p la c e d be
n e a th th e f la s k c o n ta in in g th e b ra in s and used as a w ater
B 9
b a th . On s t a r t i n g th e d i s t i l l a t i o n , th e w ate r b a th was f i r s t
h eated and a f t e r th e w ater had b o ile d f o r a b o u t f i f t e e n m in u te s,
th e re b y h e a tin g th e m ix tu re in s id e th e f l a s k , th e steam g e n e r
a to r was s t a r t e d . I f t h i s was n o t done, th e amount o f steam
n e c e ssa ry to b o i l th e liq u i d in s id e th e b r a in f la s k i s v ery
la rg e and th e volume o f th e r e s u l t a n t m ix tu re o fte n exceeds
th e two l i t e r f l a s k , and much w ater and b r a in p a r t i c l e s a re
^bumped” over in to th e d i s t i l l a t e . I t u s u a lly re q u ire d ab o u t
o n e -h a lf to th r e e - q u a r te r s o f an hour to e la p s e b e fo re th e
f i r s t drop o f d i s t i l l a t e a p p e a rs . One sh u t do?m o f th e steam
g e n e ra to r was n e c e ss a ry to add more d i s t i l l e d w ater b u t th e
main f la s k was k e p t h eated by th e w ater b a th . - The e f f ic ie n c y
o f th e c o o lin g ja c k e t can be d eterm in ed by w hether th e
a d a p to r was h ea te d or n o t. The r a t e o f d i s t i l l a t i o n was
determ ined by th e maximum amount o f steam g e n e ra te d w ith o u t
having r e f lu x in g ta k e p la c e in th e stem o f th e Hopkins b u lb .
The tim e re q u ire d fo r t h i s d i s t i l l a t i o n av erag ed about fo u r
h o u rs. G e ttle r and T ib er d e f i n i t e l y showed th a t th e f i r s t
600 c c . o f th e d i s t i l l a t e c o n ta in e d p r a c t i c a l l y a l l o f th e
a lc o h o l b u t a d v ise d t h a t 800 c c . be c o lle c te d to a s s u re com
p le te d i s t i l l a t i o n o f th e a lc o h o l. They a r r iv e d a t t h i s con
c lu s io n by t r e a t i n g p o rtio n s o f th e d i s t i l l a t e w ith h o t cop
per w ire th e re b y o x id iz in g any a lc o h o l t h a t m ight have been
p re s e n t to th e aldehyde and proving th e p resen ce of t h i s
aldehyde by th e re d c o lo r when reduced fu c h s in was added.
30
The volume of th e d i s t i l l a t e was reco rd ed and th e n th e
f la s k c o n ta in in g i t was sto p p e re d u n t i l o x id a tio n sam ples
were to be draw n. There was a sm a ll amount o f f a t t y sub
sta n c e s c a r r ie d over b u t n o t enough to cause any tr o u b le .
O x id atio n - G e ttie r and T ib er M ethod. In t h i s method
th e a lc o h o l re c e iv e d in th e d i s t i l l a t e from th e steam d i s t i l
l a t i o n was o x id iz ed to th e a c e ti c a c id by a d ic h ro m a te -s u l
f u r ic a c id m ix tu re . Follow ing i s th e e q u a tio n o f th e r e a c
tio n :
3CgH50H + SKgCr2 07 + 8HgS04 —= > 3CHSC00H + 2Crg(S04 ) 3
4 2Kg S04 + llHgO
G e ttle r and T ib er claim ed th a t t h e i r e m p iric a l f a c to r based
on 85.67 p er c e n t re c o v e ry h e ld tr u e when 20 grams o f p o ta s
sium d ich ro m ate and 40 c c . o f c o n c e n tra te d s u lf u r ic a c id
was used to o x id iz e 300 c c . o f th e d i s t i l l a t e . C o n sid erin g
a 0 .5 per c e n t s o lu tio n , by in s p e c tio n o f th e e q u a tio n
above, i t i s e v id e n t t h a t only 6 .3 9 grams o f p o tassiu m d i
chrom ate and 13.54 grams o f s u lf u r ic a c id a re n eed ed . Thus,
in o rd e r to a s s u re a good re c o v e ry th e re a g e n ts w ere used
in ex c ess o f th r e e tim es t h e i r p re s c rib e d am ounts. A f i r s t
s te p in t h i s work would be th e checking o f th e re c o v e ry by
u sin g sta n d a rd p rep ared a lc o h o lic s o lu tio n s . These were
made up to r e p r e s e n t d i s t i l l a t e s i n d e f i n i t e p e rc e n ta g e s of
a lc o h o l by w eig h t. That m eant, n a t u r a l l y , th a t in a 1 .0 per
c e n t s o lu tio n o f a lc o h o l th e re was one gram o f a lc o h o l to
9 9 .0 grams o f w a te r. Using 0.7890 as th e ac cep ted s p e c if ic
g r a v ity o f a b s o lu te a lc o h o l a t 20° C, th e r e l a t i v e volumes
of th e s o lu tio n s were fig u re d o u t f o r b o th th e 8,1cohol and
th e w a te r. From a c a lib r a te d b u re t th e p re s c rib e d volumes
o f each liq u id were d e liv e re d in to g la s s sto p p e re d b o t t l e s ,
sh ak en , and s e a le d . Follow ing i s a ta b le showing th e con
te n ts o f th e v a rio u s s o lu tio n s :
S o lu tio n
number 1 2 3 4 5 6
Water
added (cc) 989.07 991.6 994.14 996.67 999.21 1000.47
A lco h o l
added (cc) 12.67 10.14 7.60 5 .0 7 2 .5 3 1 .2 7
Per c e n t 1.00 0 .8 0 0.60 0 .4 0 0 .2 0 0 .1 0
In th e checking o f th e f a c t o r , th e fo llo w in g p ro ced u re
was u sed : 300 c c . o f th e p rep ared s o lu tio n were p ip e tte d in to
a 500 c c . round bottom f l a s k , th e te m p eratu re reco rd ed and
th e volume c o rre c te d by b o th th e te m p e ra tu re and th e c a l i b r a
t i o n .
The w eighed o u t sam ples o f th e d ich ro m ate were added
slo w ly to th e f l a s k , c a re b ein g tak en n o t to lo s e any o f th e
d i s t i l l a t e by s p la s h in g . Two sm a ll beads were p la ced in th e
f la s k in o rd e r to p re v e n t bumping w hich o cc u rred f re q u e n tly
b e fo re t h i s improvem ent was u se d . The amount o f s u lf u r ic
a c id was d e liv e re d by p ip e ts . The a c id was allow ed to flow
down th e w a lls o f th e f l a s k and d id n o t s p la s h , and so th e
lo s s of any d i s t i l l a t e was p re v e n te d . F u r th e r , by adding th e
32
a c id in t h i s m anner, th e r e a c tio n was k e p t from s t a r t i n g
b e fo re th e f l a s k was connected to th e r e s t o f th e a p p a ra tu s .
A ll o f th e a c id having been d e liv e r e d , th e f la s k was co n n e c t
ed to a Hopkins d i s t i l l i n g head by a ru b b e r s to p p e r. A t h e r
mometer was in s e r te d th ro u g h th e sto p p e r in to th e f l a s k in
o rd e r to re c o rd th e te m p eratu re o f th e v a p o r. The Hopkins
b u lb was connected to a long w a te r-c o o le d condenser and t h i s
was a tta c h e d by an a d a p te r w hich c a r r ie d th e liq u i d in to a
250 c c . v o lu m e tric f l a s k . Table IV shows th e r e s u l t s r e
ceiv ed from p rep ared s o lu tio n s . As th e d i s t i l l a t e from th e
b r a in n ev er exceeds 0 ,6 0 p er c e n t in a lc o h o li ty , th e f i r s t
two r e s u l t s may be d isc a rd e d and th e o th e r fo u r conform
c lo s e ly w ith th e 85.67 per c e n t y ie ld re p o rte d by G e ttle r
and T ib e r.
The n e x t s te p was th e a p p ly in g o f th e se optimum
am ounts to a c tu a l d i s t i l l a t e s from th e b r a in . The same p ro
ced u re was fo llo w ed a s in th e o x id a tio n w ith th e p rep ared
s o lu tio n s . The b a se used in th e t i t r a t i o n o f th e d i s t i l l e d
a c e tic a c id was sta n d a rd iz e d a t f re q u e n t in t e r v a ls to p re
v e n t any e r r o r from th e changing o f th e s tr e n g th . I t was fo
found more advantageous to use 25 c c . p o rtio n s o f d i s t i l l a t e
r a t h e r .t h a n 50 c c . p o r tio n s . Three p o rtio n s o f th e
th o ro u g h ly mixed d i s t i l l a t e was draw n, th e f i r s t b ein g used
J
to f in d th e approxim ate end p o in t and th e o th e r two to check
each o th e r a t th e e x a c t end p o in t.
TABLE IV
PER CENT RECOVERY O F ALCOHOL FRO M PREPARED SOLUTIONS
S o lu tio n A lc o h o lic S tre n g th D ichrom ate A cid Per c e n t
n o . p er c e n t used used Recovery
1 1.00 8 0 .0 gm 40 cc 92.35
8 0.08 80.0 40 90.41
3 0.06 80.0 40 87.54
4 0 .0 4 20.0 40 85.87
5 0.08 2 0 .0 40 85.43
6 0.01 2 0 .0 40 85.64
T A B L E V
PEE CENT RECOVERY OF ALCOHOL OF GETTLER AND TIBER OXIDATION METHOD
P e r c e n t Sam ple I Sam ple I I
A lc o h o l T r i a l I T r i a l I I T r i a l I T r i a l I I A v erag e X F a c to r E r r o r
% % % % % % %
0 .6 0 3 8 9 1 .6 1 9 1 .3 8 9 0 .9 8 9 1 .3 6 9 1 .3 3 1 0 6 .6 0 6 .6 0
0 .5 4 9 3 8 8 .4 7 8 8 .5 2 8 8 .0 1 8 8 .2 0 8 8 .3 0 1 0 5 .0 5 3 .0 5
0 .5 0 4 9 8 6 .5 9 8 6 .6 7 8 6 .6 2 8 6 .6 4 8 6 .6 2 1 0 1 .1 1 1 .1 1
0 .4 4 1 8 8 7 .5 6 8 7 .5 4 8 7 .6 0 8 7 .5 4 8 7 .5 6 1 0 2 .2 3 2 .2 3
0 .3 9 9 2 8 2 .7 3 8 2 .6 8 8 2 .6 9 8 2 .7 0 8 2 .7 1 9 6 .5 4 3 .4 6
0 .3 7 8 7 8 6 .2 1 8 5 .9 7 8 5 .8 0 8 5 .9 6 8 5 .9 8 1 0 0 .3 6 ' 0 .3 6
0 .3 1 7 2 8 6 .8 5 8 5 .6 2 8 5 ,5 6 8 6 .9 1 8 5 .7 3 1 0 0 .0 7 0 .0 7
0 .3 1 2 4 8 6 .5 8 8 6 .4 1 8 6 .6 8 8 6 .4 7 8 6 .5 4 1 0 1 .0 2 1 .0 2
0 .2 8 7 1 8 5 .9 0 8 6 .0 0 8 6 .2 8 8 5 .9 7 8 6 .0 5 1 0 0 .4 4 0 .4 4
0 .2 2 3 4 8 7 .1 1 8 8 .0 1 8 7 .0 3 8 7 .0 9 8 7 .3 1 1 0 1 .9 2 1 .9 2
0 .1 5 7 8 9 4 .4 7 9 4 .3 6 9 2 .9 2 9 4 .0 5 9 3 .9 5 1 0 7 .1 4 7 .1 4
35
C a lc u la tio n s :
1 . The amount o f base re q u ire d in th e t i t r a t i o n was c o r r e c t
ed f o r te m p eratu re and c a lib r a ti o n and 1 .3 2 d educted as
th e c o r r e c tio n f o r th e norm al a lc o h o lic c o n te n t.
2 . (R e s u lta n t c c . o f b a se )(N o rm a lity o f b ase) _ N o rm ality of
(c c . of a c id used) ” a c id
3 . (N orm ality o f a c id ) x (Volume o f d i s t i l l a t e re c e iv e d ) _
(Volume o f d i s t i l l a t e used)
N o rm ality o f a c id in r e s p e c t to e n t i r e d i s t i l l a t e .
4 . (N orm ality o f e n t i r e a c id )(M o le c u la r w eight o f a c e ti c a c id )
4
Grams o f a c e ti c a c id re c o v e re d .
5 . Grams o f a c id reco v e re d x M olecular w eig h t of a l c o h o l _
M olecular w eight o f a c id
Grams o f a lc o h o l re c o v e re d .
6 . Grams o f a lc o h o l reco v e re d
Grams o f a lc o h o l in s e r te d x 100 Per c e n t R ecovery.
N um erical exam ple:
1 . 26.017 - .6 6 = 25.357 c c . o f b ase
2 . (2 5 .3 5 7 )(.0 4 6 3 2 )
(24.869) (0.04723)
3 . (,04723)1893^1)
(300.44) (0.1404)
4 . (0 .1 4 0 4 )(6 0 .0 3 )
4 2 .1 0 8 grams o f a c e tic a c id
5 . 2 .1 0 8 x 4 6 .0 2
60.03 1.616 grams o f a lc o h o l
6. 1.616
1.893 x 100= 85.38 per c e n t re c o v e ry
T h e re fo re , i t can he concluded th a t o u ts id e o f th e
extrem e c a ses in w hich th e a lc o h o lic c o n te n t re a c h e s a s h ig h
as 0 .6 0 p er c e n t, th e method i s v e ry a c c u ra te and checks
w ith in th e e x p e rim e n ta l e r r o r a llo w a b le . The 7 .4 per c e n t
e r r o r in th e 0.1578 d i s t i l l a t e i s n o t s e rio u s because th e
a lc o h o lic c o n te n t must be over 0.0500 per c e n t b e fo re th e
p erso n i s pronounced in to x ic a te d . As amounts le s s th a n 3.00
c c . o f a b s o lu te a lc o h o l were added to th e b r a in , th e 85 per
c e n t reco v e ry i s v ery good, due to th e change o f lo s s coming
from steam d i s t i l l a t i o n , in co m p lete o x id a tio n , e r r o r s in c a l i
b r a tio n , e r r o r s in te m p eratu re c o r r e c tio n s , and e r r o r s in th e
t i t r a t i o n . The d if fe re n c e o f a drop o f b ase in th e t i t r a t i n g
w i l l e f f e c t th e re co v e ry more th a n 0.30 o f a p e r c e n t. Cor
r e c t io n was made f o r th e v o l a t i l e su b stan ces in th e b ra in
w hich would upon d i s t i l l a t i o n and o x id a tio n , produce a c i d i t y .
1 .3 2 c c . were s u b tra c te d from th e t i t r a t i o n f ig u r e s to a c
count fo r t h i s phenomena. T his a l s o would c o n trib u te to th e
e x p e rim e n ta l e r r o r s , as an im al b r a in was used in s te a d of
human b r a in . However, p ro b ab ly some o f th e s e e r r o r s c a n c e lle d
each o th e r . G e ttle r and T ib er re p o rte d a v a r i a t i o n o f 1.02
per c e n t in e i t h e r d ir e c tio n when th e y d e v ise d t h e i r c o rre c
tio n f a c t o r , ?/hereupon, i t i s s a fe to conclude th a t t h is
method can be le g a ll y used w ith o u t f e a r t h a t th e su sp e c t
w i l l n o t be g iv e n a f a i r judgm ent.
37
O x id atio n - G e ttle r and F r e ir e i c h T s V a ria tio n of '
G e ttle r and T ib er M ethod. In working w ith s p in a l f l u i d and
b lo o d , th e method was used on a sm a lle r s c a le . A sm a lle r
amount of d i s t i l l a t e was c o lle c te d from th e steam d i s t i l l a
t i o n , only 10 c c . o f o r i g i n a l sample b ein g u se d . 100 c c .
p o rtio n s o f th e d i s t i l l a t e were su b je c te d to th e same o x id a
tio n p ro c e s s . However, th e y found t h a t th e optimum amount
o f p o tassiu m d ich ro m ate and s u lf u r ic a c id was 7 .5 grams and
15 c c . r e s p e c tiv e ly . In th e checking o f t h e i r w ork, th e p re
pared s o lu tio n s , 0 .6 p er c e n t and 0 .4 p er c e n t, used to
check th e G e ttle r and T ib er Method were a g a in u se d . The p ro
cedure i s th e same as t h a t m entioned in th e p rev io u s s e c tio n
ex c ep t t h a t 100 c c . o f d i s t i l l a t e were used and 7 .5 grams o f
dichrom ate and 15 c c . o f c o n c e n tra te d a c id . The tim e re q u ire d
f o r d i s t i l l a t i o n to b e g in was th e same, b u t as th e o th e r p ro
cedure to o k n e a rly one hour to c o l le c t 250 c c . , t h i s one to o k
f o r t y m inutes to c o l l e c t 80 c c . As th e re was no 80 c c . v o lu
m e tric f l a s k a v a ila b le , th e volume was made up to 100 c c .,
and sam ples drawn f o r t i t r a t i o n . The r e s u l t s re c e iv e d c o r
r e la te d w ith th o se o f G e ttle r and F r e ir e i c h , t h e i r r e s u l t s
b ein g p re v io u s ly m entioned on page 19 under th e d is c u s s io n
o f H isto ry o f R e lated S tu d ie s . I t was confirm ed th a t fo r
100 c c . p o rtio n s o f d i s t i l l a t e th e optimum amounts o f th e
p o tassiu m d ich ro m ate and s u lf u r ic a c id were 7 .5 grams and
15 c c . r e s p e c tiv e ly .
The q u e s tio n n e x t to be answ ered was w hether th e se
amounts were optimum when a p p lie d to 300 c c . o f th e d i s t i l
l a t e , The r e s p e c tiv e amount o f th e p o tassiu m dichrom ate b e
came 22.5 grams and th e c o n c e n tra te d s u lf u r ic ac id 45 c c .
The pro ced u re was th e same a s th a t m entioned in th e G e ttle r
and T ib er o x id a tio n . The method o f c a lc u la tio n was a ls o th e
same. T able VI shows th e r e s u l t s o f t h i s v a r i a t i o n .
A lthough th e r e s u l t s sho?/ e r r o r s ran g in g a s h ig h as
10.42 per c e n t, th e re a re sm a ll e r r o r s w ith in th e range in
w hich th e d e c is io n o f in to x ic a tio n i s to be made. Whereupon
t h i s method i s p r a c t i c a l . Upon com parison w ith th e r e s u l t s
o f G e ttle r and T ib e r, i t can be concluded th a t u sin g 300 c c .
o f b r a in d i s t i l l a t e , th e optimum am ounts o f re a g e n ts recom
mended by them i s b e t t e r th a n th o se am ounts p re s c rib e d by
G e ttle r and F r e i r e ie h .
O x id atio n - O rig in a l V a ria tio n o f G e ttle r and T iber
M ethod. In th e checking o f th e optimum amounts o f th e d i -
c h ro m a te -s u lfu ric a c id m ix tu re f o r th e s ta n d a rd G e ttle r and
T ib er o x id a tio n , a c e ta te io n was found in th e re s id u e a f t e r
th e d i s t i l l a t i o n had been co m p leted . W hereupon, i t was con
cluded t h a t in c o lle c tin g th e p re s c rib e d amount o f d i s t i l l a t e
t h a t th e re c o v e ry would be 85 p er c e n t, th e b a lan ce o f th e
a c e t i c a c id , th e b alan ce o f th e a c e tic a c id formed being l e f t
in th e f l a s k . 'With th e hope o f re c e iv in g a re c o v e ry b e t t e r
T A B L E VI
PER CEET RECOVERY OF ALCOHOL OF GETTLER AND FREIKEICH OXIDATION METHOD
P e r c e n t Sam ple I
A lc o h o l T r i a l I T r i a l I I
Sam ple I I
T r i a l I T r i a l I I A v erag e X F a c to r E r r o r
6058 9 0 .2 2 9 0 .1 4 9 0 .0 1 9 0 .3 1 9 0 .1 7 1 0 5 .2 2 - 5 .2 2
5495 8 7 .5 0 8 7 .6 1 8 6 .9 5 8 7 .1 0 8 7 .2 9 1 0 1 .9 1 - 1 .9 1
5049 8 5 .7 8 8 5 .8 2 8 6 .0 0 8 5 .9 1 8 5 .8 7 1 0 0 .2 3 - 0 .2 3
4418 8 7 .9 3 8 7 .9 8 8 8 .4 0 8 8 .2 1 8 7 .8 8 1 0 2 .5 6 - 2 .5 6
3992 7 9 .4 2 8 0 .9 6 8 3 .1 4 8 1 .0 4 8 0 .9 5 9 4 .4 7 + 5 .5 3
3787 8 5 .3 8 8 5 .3 8 8 5 .8 1 8 5 .4 3 9 9 .7 2 9 9 .7 2 + 0 .2 8
3172 8 7 .5 1 8 7 .4 9 8 7 .2 9 8 7 .3 1 8 7 .3 9 1 0 2 .0 - 2 .0 1
3124 8 7 .5 3 8 7 .2 9 8 7 .5 0 8 7 .7 8 8 7 .5 2 1 0 2 .1 7 - 2 .1 7
2871 8 7 .2 3 8 7 .7 7 8 7 .6 9 8 7 .1 5 8 7 .4 6 1 0 2 .0 8 - 2 .0 8
2234 8 8 .6 8 8 9 .0 1 8 8 .7 1 8 8 ,7 6 8 8 .7 9 1 0 3 .6 4 - 3 .6 4
1578 9 3 .3 7 9 5 .6 1 9 4 .1 7 9 4 .5 9 9 4 .5 9 1 1 0 .4 2 - 1 0 .4 2
40
th a n 85 p er c e n t, th e fo llo w in g pro ced u re was u sed :
One hundred c c . o f a p rep ared a lc o h o lic s o lu tio n ,
w ith in th e range o f .th a t w hich m ight r e s u lt.f r o m a steam d i s
t i l l a t i o n , was p ip e tte d in to a 500 c c . round bottom f l a s k .
7 .5 grams o f dichrom ate and 15 cc . o f s u lf u r ic a c id were
th e n added. The f la s k was connected to a w a te r-c o o le d con
d en ser by a Hopkins d i s t i l l i n g head, co n n ectio n s being made
by means o f ru b b er s to p p e rs . A therm om eter"w as in s e r te d in
to th e f l a s k and th e d i s t i l l a t i o n s t a r t e d . I t took f u l l y
f iv e m inutes b e fo re th e f i r s t drop reach ed th e re c e iv in g
f l a s k . The re c e iv in g f l a s k co n tain e d 100 c c . o f d i s t i l l e d
w ater or sta n d a rd b a s e . I t was surrounded by an ic e -w a te r
b a th ; an ic e s a l t b a th was d isc a rd e d as no b e t t e r re c o v e rie s
had r e s u l t e d . The condenser vms connected to th e re c e iv in g
f la s k by an a d a p te r w ith a g la s s e x te n s io n se a le d on. The
g la s s tu b in g was used to have th e end under th e liq u id in th e
f la s k a t a l l tim e s . The d i s t i l l a t i o n was co n tin u ed a t a
r a t e o f 35 drops a m inute w ith th e te m p e ra tu re rem aining a t
100° C u n t i l n ea r th e end o f th e p ro ce ss when th e m ercury
ro s e to 125° C. The d i s t i l l a t i o n was co n tin u ed u n t i l s o lid
p a r t i c l e s o f th e d ich ro m ate condensed on th e s u rfa c e o f th e
rem aining liq u i d o f th e r e s id u e . The a p p a ra tu s was co o led
and 100 c c . o f d i s t i l l e d w ater added. The d i s t i l l a t i o n was
re p e a te d and th e t o t a l d i s t i l l a t e made up to 500 c c . F if t y
c c . p o rtio n s were p ip e tte d o u t and t i t r a t e d w ith .20 N b ase
41
u sin g p h e n o lp h th a le in a s an i n d i c a t o r . Each s e p a ra te d i s t i l
l a t i o n took ab o u t one and o n e -h a lf h o u rs. By t h i s second d i s
t i l l a t i o n , th e re c o v e ry was in c re a s e d ab o u t 1 .5 p e r c e n t. At
f i r s t , th e r e s u l t s in d ic a te d th a t th e p ro c ess o f c o lle c tin g
th e sam ple in base was s u p e rio r b u t l a t e r e r r o r s from th e
changing n o rm a lity and d iffe re n c e in te m p e ra tu re s caused
t h i s method to be abandoned. Blank sam ples were ru n and, a s
th e r e s u l t s proved n e g a tiv e , th e h e a tin g to 125° C d id n o t
cause an y th in g o th e r th a n th e a c e ti c a c id to be c a r r ie d o v er.
C a lc u la tio n s :
For a 0 .6 p er c e n t a lc o h o lic s o lu tio n , th e d i s t i l l a t e
o f w hich was c o lle c te d in w a te r, th e r e a re .6 grams in 99.4
grams of w a te r. In 100.147 c c . o f s o lu tio n th e re a re .5996
grams of a lc o h o l or 0.01302 H or I a lc o h o lic s o lu tio n . This
a lc o h o l upon b ein g o x id iz e d y ie ld s 0.01302 M or N a c e tic
a c id . However, a s th e d i s t i l l a t e was o n ly made up to 500 c c .
th e n o rm a lity p re s e n t would be doubled o r 0.02604.
(c c . o f b ase ~used in t i t r a t i o n ) ( n o r m a l i t y o f b a s e ) _ N orm ality
(c c . o f a c id d e liv e re d by p ip e t) “ o f a c id
N orm ality o f a c id
T h e o r e tic a l n o rm a lity o f a c id x 100= % re co v e ry
N um erical exam ple:
(2 2 .3 8 )(0 .0 5 5 5 5 )
(50.0736) = 0.02389 N
0.02589
0.02604 x 100 = 95.04$
D i s t i l l a t e C o lle c te d in Base
N orm ality o f b ase added 0.01160
T h e o re tic a l amount o f a c id 0.02604 - 0 .0 1 0 6 0
C a lc u la te d amount 0.01518
% reco v e ry 96.26$
TABLE V II
PER CENT RECOVERY OF ALCOHOL FRO M ORIGINAL VARIATION M E T H O D
A lco h o lic s o lu tio n Per
%
0 .1 97
0 .2 95
0 .4 94
0 .6 93
0 .8 92
1 .0 93
c e n t re c o v e ry Average
%
29 95.45 96.37
38 94.37 94.87
96 96.03 95.49
09 91.94 92.50
27 94.07 93.17
41 94.64 94.03
Average R ecovery f o r E n tire Range 94.60
T able Y II shows th e r e s u l t s re c e iv e d by u sin g t h i s
m ethod.
Thereupon, in s te a d o f c o rre c tin g f o r a 85.67 per c e n t
re c o v e ry , i t would be recommended to d i s t i l l u n t i l alm ost
d ry n ess and c o r r e c t f o r a 94.60 per c e n t re v o c e ry . This c o r
r e c t io n f a c t o r would be 1 .0 5 7 1 .
O x id atio n - N icloux Method. This method i s based on
th e o x id a tio n o f th e a lc o h o l to th e ald eh y d e in a c o n c e n tra te d
s u lf u r ic a c id s o lu tio n . The e q u a tio n f o r t h i s r e a c tio n i s :
3CgH 5Q B + KgCrs 07 + 4HgS04 3CHgCH0 + Crg (S04 )
+ 7HgO * Kg S04
Nicloux^® advanced t h i s method in 1900 and sin c e t h a t tim e ,
many ch em ists have su p p o rte d i t , even to th e e x te n t o f p re
f e r r in g i t to th e r e c e n tly advanced G e ttle r and T ib er m ethod.
As t h i s method i s q u a n tita tiv e l y a p p lia b le , N icloux su g g ested
th e fo llo w in g p ro ced u re: 5 c c . o f d i s t i l l a t e from th e steam
d i s t i l l a t i o n p ro c e ss were p ip e tte d in to a .30 c c . Erlenm eyer
f l a s k , 3 c c . o f c o n c e n tra te d s u lf u r ic a c id were th e n added.
A dichrom ate s o lu tio n c o n ta in in g 19 grams p er l i t e r of p o ta s
sium dichrom ate was added dropw ise from a c a lib r a te d 10 c c .
b u r e t. A few drops o f th e d ich ro m ate were added a t a tim e .
A fte r each a d d itio n , th e f l a s k was sto p p e red and shaken u n t i l
pfi
N iclo u x , These de P a r is , 1900.
44
a c o lo r change from b lu e g reen to y ello w g reen was n o te d .
The f l a s k was th e n p la ced in b o ilin g w ater and a f t e r two min
u te s o f such tre a tm e n t coupled w ith o c c a s io n a l shaking i f th e
c o lo r changed back to b lu e g re e n , more dichrom ate was added
u n t i l th e y ello w g reen c o lo r rem ained p erm an en tly . The re a d
in g o f th e b u re t was reco rd e d and th e p rocedure was re p e a te d
on a n o th e r sample from th e same d i s t i l l a t e u sin g a few le s s
drops o f dichrom ate u n t i l th e end p o in t was re a c h e d .
T his p ro cess was c a r e f u lly c a r r ie d o u t in t h i s i n v e s t i
g a tio n , b u t a wide ran g e o f end p o in ts r e s u lte d from th e same
s o lu tio n . P rep ared s o lu tio n s m entioned in th e G e ttle r and
T ib er s e c tio n were used h e re . However, th e volume o f th e l i
quid b ein g t i t r a t e d became d ark due to i t s being in an B rle n -
meyer f l a s k and caused th e end p o in t to be re co rd ed b e fo re i t
was r e a l l y p r e s e n t. F u rth e r, as th e t h e o r e t i c a l end p o in t of
th e s o lu tio n being t i t r a t e d was known, p re ju d ic e was v ery
h ard to overcome when working w ith known s o lu tio n s . When work
was conducted w ith unknown s o lu tio n s , end p o in t v a r ia tio n s o f
over 20 per c e n t o f th e t h e o r e t i c a l was -re co rd ed . In th e c a l
c u la tio n o f th e t h e o r e t i c a l end p o in t, W ebster claim s t h a t 1
c c . o f dichrom ate s o lu tio n c o n ta in in g 19 grams p er l i t e r i s
e q u iv a le n t to .005 c c . o f a b s o lu te a lc o h o l and 1 c c . o f d i -
chrornate i s e q u iv a le n t to 0 .1 per c e n t a lc o h o l in d i s t i l l a t e .
T his c a lc u la tio n seems to be wrong and fo llo w in g i s a c o r r e c t
ed c a lc u la tio n :
In a 0 .1 p er c e n t s o lu tio n , 1.267 c c . o f a lc o h o l were
added to 1000.47 c c . o f w a te r.
1.2 6 7 c c . o f CgH50 H = .9 9 9 7 grams o f a lc o h o l.
In a 10 c c . p ip e t th e re a r e 9.9513 x .9997 = • .009943 grams
3.000.47
o f a lc o h o l.
From th e e q u a tio n 5(46.05') 294.22
.009943 ' X
X - .02118 grains of p o tassiu m dichrom ate n e c e s s a ry to c o n v e rt
0.009943 grams o f a lc o h o l to ald e h y d e .
There a r e 19.0072 grains o f KgCr^O^ in a 1000 c c . s o lu tio n ,
th e r e f o r e 0.0190072 grams in 1 c c .
The number o f c c . o f d ich ro m ate .re q u ire d to o x id iz e th e a lc o h o l
to ald eh y d e .02118 = 1.122 c c . w hich r e p r e s e n ts th e t h e o r e t i -
.0190072
c a l end p o in t.
However, t h i s method a s g iv en by N icloux i s n o t co n c lu
s iv e enough as even a tr a in e d o b se rv e r made e r r o r s in t i t r a t i n g
am ounting to over 20 p er c e n t. T h e re fo re , i t i s recommended
t h a t t h i s method be abandoned as th e r e s u l t s co u ld n o t be used-
to send a man to th e g allo w s.
O x id atio n - O rig in a l V a ria tio n of th e N icloux M ethod.
D e sirin g to overcome th e d isa d v a n ta g e s o f th e N icloux Method,
in v e s tig a tio n s were c a rr ie d o u t in w hich v a r ia tio n s in p ro
ced u re were c a r r ie d o u t. A e a lo r im e tr ic method o f com parison
proved s u c c e s s fu l in th e d e te rm in a tio n o f th e a lc o h o lic con
t e n t o f s o lu tio n s .
N e ssle r tu b e s h o ld in g 50 c c . o f s o lu tio n were used
and th e b e s t r e s u l t s were had by u sin g 10 c c . p o rtio n s o f th e
d i s t i l l a t e . F in a ll y , th e fo llo w in g p rocedure was ad o p ted :
A 10 c c . p o rtio n o f th e d i s t i l l a t e was p ip e tte d in to a 50 cc.
N e ss le r tu b e . 6 c c . o f ■ c o n c e n tra te d s u lf u r ic a c id were added
from a b u r e t, b ein g allo w ed to flow down th e w a ll o f th e
tu b e . The tu b e was s t i r r e d a s l i t t l e as p o s s ib le to p rev en t
th e r e a c tio n from s t a r t i n g . A c e r t a i n amount o f dichrom ate
was added r a p id ly from a 10 c c . b u r e t. The tu b e was sto p p e re d ,
sh ak en , and p la ced in b o ilin g w ater f o r f iv e to te n m in u te s,
b ein g o c c a s io n a lly shaken d u rin g th a t tim e . l a t e r th e tube
was p laced in a K e ss le r Tube Hack. A w h ite p la te o f p o rce
l a i n , d ia g n n a lly s i t u a t e d , a d m itte d w h ite l i g h t up through
th e bottom o f th e tu b e , and th e co rk having been rem oved, th e
c o lo r was observed by lo o k in g downward th ro u g h th e liq u i d .
I f th e c o lo r appeared b lu e , th e n th e p ro ced u re was
re p e a te d w ith a n o th e r tube u sin g one more c c . o f d ic h ro m a te.
Were th e c o lo r o f th e second tu b e to be o liv e g re e n , th en th e
ran g e in w hich th e end p o in t la y was known and i t rem ained
o n ly to re p e a t th e p ro ced u re v ary in g th e amount o f dichrom ate
s o lu tio n o n e -te n th of a cu b ic c e n tim e te r each tim e and p la c
in g th e tube in th e ra c k a c co rd in g to i t s r e l a t i v e amount o f
d ic h ro m a te; th e c o lo r was compared to th a t o f th e a d ja c e n t
tu b e u n t i l th e end p o in t was firm ly d eterm in ed . O ften the
end p o in t passed th ro u g h a t r a n s i t i o n sta g e and th e d e f in i t e
end p o in t was d i f f i c u l t to d e te rm in e . By u sin g th e av erag e
o f th e dichrom ate am ounts, th e t r a n s i t i o n s ta g e , th e r e s u l t s
ag reed v ery w e ll w ith th e t h e o r e t i c a l am ount. T able V III
shows th e r e s u l t s o f t h i s m ethod.
T A B L E VIII
P e r c e n t
A lc o h o l
E x p e rim en
t a l End
P o i n t
T h e o r e t i c a l
End P o i n t
P e r c e n t
D i f f e r e n c e
RESULTS USING GALORIMETRIC METHOD
0 .1 0 0 $ 0 .2 0 0 $ 0 .5 1 2 4 $ 0 .5 1 7 2 $ 0 .5 7 8 7 $ 0 .5 9 9 2 $ 0 .4 4 1 8 $
a d d e d
C o lo r KgCrgO?
a d d ed
C o lo r
K2C r2 °7
a d d e d
C o lo r KgCrgOy
ad d e d
C o lo r K gC r§07 C o lo r KoCr?0
a d d e d a d d e d
< j C o lo r KgCrgOi
a d d e d
■ j C o lo r
1 .5 0 B lu e 2 .7 5 ' B lu e 5 .5 0 B lu e 4 .0 0 B lu e 4 .0 0 B lu e 5 .5 0 B lu e 4 .5 0 B lu e
1 .7 5 B lu e 4 .0 0 B lu e 5 .7 5 B lu e 5 .7 5 B lu e 4 .2 5 B lu e 4 .0 0 B lu e 4 .7 5 B lu e
2 .0 0 B lu e 4 .1 0 B lu e 4 .0 0 B lu e 4 .0 0 B lu e 4 .5 0 B lu e 4 .2 5 B lu e 5 .0 0 B lu e
2 .1 0 B lu e 4 .2 5 B lu e 4 .1 0 B lu e 4 .1 0 G reen 4 .6 0 B lu e
G reen
4 .5 0 B lu e
G reen
5 .1 5 B lu e
G reen
2 .2 0 G reen 4 .5 0 G reen 4 .2 5 G reen 4 .2 5 G reen 4 .7 5 G reen 4 .7 5 G reen 5 .2 5 G reen
2 .5 0 G reen 4 .7 5 G reen 4 .5 0 G reen 4 .5 0 G reen 5 .0 0 O liv e
G reen
5 .0 0 O liv e
G reen
5 .5 0 G reen
2 .1 5 0 4 .5 7 5 4 .0 5 4 .0 5 4 .5 5 0 4 .5 0 0 5 .1 2 5
1.122 2 .2 4 4 5 .5 0 0 5 .5 5 9 4 .2 4 9 4 .4 7 5 4 .9 5 7
4 7 .8 9 6 1 .2 1 8 4 .2 8 8 5 .8 1 9 2 .9 4 9 9 .9 5 9 6 .6 7
T A B L E VIII
P e r c e n t
A lc o h o l
E x p e rim en
t a l End
P o i n t
T h e r o r e t i -
c a l End
P o i n t
P e r c e n t
D i f f e r e n c e
RESULTS USING CALORIMETRIC METHOD
0.5049Jg 0 .5 4 9 3 0.60385?
:gC r20 7 C o lo r K 2Gr2°7 C o lo r KgCrgO? C o lo r
a d d e d a d d ed a d d ed
5 .5 0 B lu e 6 .0 0 B lu e 6 .5 0 B lu e
6 .0 0 B lu e 6 .2 5 B lu e 7 .0 0 B lu e
6 .1 0 B lu e 6 .3 5 B lu e 7 .1 0 B lu e
G reen
6 .1 5 B lu e 6 .4 0 G reen 7 .2 0 B lu e
G reen G reen
6 .2 5 G reen 6 .5 0 G reen 7 .5 0 G reen
6 .5 0 O liv e 7 .0 0 O liv e 7 .5 0 D ark
G reen G reen G reen
6 .1 2 5 6 .3 7 5 7 .2 0 0
5 .6 6 5 6 .1 6 3 6 .7 7 4
9 1 .8 6 9 6 .5 6 9 3 .7 2
This p ro c e ss was found to hold only in th e range of
0.3000 to 0.6000 p er c e n t a lc o h o lic s o lu tio n s . S o lu tio n s
c o n ta in in g le s s th a n 0.3000 p er c e n t a lc o h o l gave v ary in g
r e s u l t s . R e s u lts o f 0.100 p er c e n t and 0 .200 per c e n t s o lu
tio n s showed r e s u l t s alm o st tw ice th a t o f th e th e o r e ti c a l end
27
p o in t. K ohn-A brest in h is book on to x ic o lo g y g iv e s the f o l
low ing e q u a tio n f o r t h i s r e a c tio n :
6Cs H 50H + KgCr207 + 4HgS04 Crg (S04 ) 3 4 - K g S04
+ 6CBLCH0 + 7HQ 0
3 2
T his e q u a tio n may hold tr u e f o r s o lu tio n s up to 0.3000 per
c e n t a lc o h o lity b u t th e e q u a tio n g iv en in th e d is c u s s io n o f
th e R icloux method h o ld s tr u e f o r s o lu tio n s c o n ta in in g
0.3000 to 0.6000 p er c e n t a lc o h o l.
However, t h i s method i s recommended a s a c o n firm a tio n
o f th e r e s u l t s re c e iv e d from a G e ttle r and T ib er o x id a tio n
p ro cess
27
E. K ohn-A brest, P re c is de T o x ic o lo g ie . p . 99.
SU M M A R Y
In th e answ ering o f th e q u e s tio n how can in to x ic a tio n
he p ro v en , a c r i t i c a l com parison o f th e q u a n t ita tiv e methods
f o r th e d e te rm in a tio n o f a lc o h o lic c o n te n t in th e b r a in was
co n d u cted .
The method o f G e ttle r and T ib er was d e f i n i t e l y e s t a
b lis h e d a s th e b e s t m ethod. T h eir e m p iric a l f a c t o r depended
upon a 85.67 p e r c e n t y ie ld and was confirm ed in t h i s stu d y .
The G e ttle r and F r e ir e ic h v a r ia tio n o f th e G e ttle r and
T ib er method was found to hold f o r sm a ll volumes o f d i s t i l l a t e
as th o se r e s u lt in g from th e steam d i s t i l l a t i o n o f s p in a l
f l u i d and b lo o d . However, t h e i r optimum amount of re a g e n ts
was n o t as a c c u ra te when a p p lie d to la r g e r d i s t i l l a t e s as
th o se optimum amounts p re s c rib e d by G e ttle r and T ib e r.
An o r i g in a l v a r ia tio n was recommended as a h ig h e r p er
c e n t re co v e ry o f a lc o h o l r e s u lt e d . This was n o t e s ta b lis h e d
to hold f o r d i s t i l l a t e s as only p rep ared s o lu tio n s were u se d .
The r e s u l t a n t e m p iric a l f a c to r was 1 .0 5 7 1 .
The N icloux method was found to be im p r a c tic a l as th e
t i t r a t i o n end p o in t was d i f f i c u l t to o b ta in a c c u r a te ly .
The v a r ia tio n o f th e N icloux method f o r d eterm in in g
th e end p o in t by means o f a c a lo r im e tric com parison w ith v a ry
in g amounts o f d ichrom ate s o lu tio n was found to be a p p lic a b le
o n ly w ith in th e ran g e o f 0.300 to 0 .600 p er c e n t a lc o h o lic
s o lu tio n s . I t can be recommended as a c o n firm a tio n t e s t f o r
th e r e s u l t s re c e iv e d from th e G e ttle r and T ib er o x id a tio n
p ro c e s s .
BIBLIOGRAPHY
BOOKS
A u te n r e ith , W., L ab o rato ry Manual f o r th e D e te c tio n o f P o i
sons and P ow erful D ru g s, tr a n s l a te d by W . H. W arren.
P h ila d e lp h ia : P. B la k is to n ’s Son and Company, 1928.
Em erson, Haven, A lco h o l and Man. New York: The M acm illan
Company, 1933.
K ohn-A brest, E ., P re c is de T o x ic o lo g ie . P a r is : G. Doin e t
C ie ., 1934.
W ebster, R. W., L egal M edicine and T o x ico lo g y . P h ila d e lp h ia :
W . B. Saunders Company, 1930.
PERIODICALS
A b ern eth y , R. J . , R u s s e ll, E. R ., and T h ien es, C. H ., ”The
e s tim a tio n o f e th y l a lc o h o l in b r a i n .1 1 J o u rn a l o f Labora
to r y and C lin ic a l M ed icin e. 19: 1014-18, 1934.
G e t t l e r , A. 0 . , and F r e ir e ie h , A. W., ’ ’D ete rm in atio n o f A lco
h o lic I n to x ic a tio n d u rin g L ife by S p in a l F lu id A n a ly s is .”
Jo u rn a l o f B io lo g ic a l C h em istry . 92: 199-209, 1931.
G e tt le r , A. 0 . , N ie r d e r l, J . B. , and B e n e d e tti- P ic h le r , A. A .,
"The i s o l a t i o n of pure anhydrous e th y l a lc o h o l from non
a lc o h o lic human and anim al t i s s u e . ” J o u rn a l o f American
Chem ical S o c ie ty . 54: 1476-86, 1932.
G e t t l e r , A. 0 . , and T ib e r, A ., ”Q u a n tita tiv e d e te rm in a tio n o f
e th y l a lc o h o l in human t i s s u e . ” A rchive o f P athology and
L ab o rato ry M edicine. 3: 7 5 -8 4 , 1927.
G e tt le r , A. 0 . , and T ib e r, A ., ’ ’The a lc o h o lic c o n te n t of th e
human b r a in - I t s r e l a t i o n to i n t o x i c a t i o n .” A rchives of
P athology and L ab o rato ry M edicine. 3, 218-227, 1927.

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Creator Beyer, M. G (author)

Core Title Critical comparison of the quantative methods for determination of alcohol in brain

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

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Advisor Brinton, Paul H.M.P. (committee chair), Harrison, Bruce M. (committee member), Roberts, L.D. (committee member)

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