hu3i0 approved by
TRANSCRIPT
. ?HU3I0 APPROVED
By
V a jo r A d v iso r
Dean
/a stutjt o p she HB iC Tios op assim ohy
TDICHLOKIDS 71 ?H VKfAHUf A, CAROTENE,
AND ASSOCIATED SUBSTANCES,
BY
AIPE82) CHAKLEY ABBESSES
A THESIS
tthm ittod to the ? a c u ity o f The C ro lg h to n U n iv o r a ity In P a r t i a l F u l f 1 ll ia o n t o f th e R equirem ents
f o r th o D egree o f R anter o f S c ie n ce in th o Department o fB io lo g ic a l Chemistry
OTiAHA, 1935
1
C0II?E2FT3
C h ap ter Page
I BARLY 3ftTPXS8............................................................ 1I I 5H3 p b o b k :m • . . . ............... . .................... . . . . . . . 11i n m m x m m A i, m m .................................................... 12
Rea#*ents and S o lu t io n s . . . . . . . . . . . . . 12
P roced u re •••••*•••••.............. ........... .. 14iv D iscussion ........................................................ 3 1
v su im irr ............................................................ 3 9
BIBLIOGRAPHY ............................................................... 42
88226
i i
TABLES
Wwaber Page
I COLORS PRODUCE THROUGH THE REACTIONOP tJNSATURATCD FATTY ACIDS AND GLYCERYL ESTERS OP FATTY ACIDS OR RELATED SUBSTANCES WITH ANTIMONY TRICHLORIDE ....................................................... 16
I I CO^ARISOH OF THE COLORS PRODUCED BYOLEIC ACID AND BY VITAMIN A ITH ANTIMONY TRICHLORIDE IN THE PRESENCE OF AND IN THE ABS NCE OF RYnCOATRCHXH ................................................ 18
I I I
IV
V
VI
VII
RESULTS OF AN BT'GRT TO DETERMINE THE SMALLEST QUANTITY OF OLEIC ACID THAT WILL PRODUCE A PERCEPTIBLE RED COLOR ITH ANTIMONY TRICHLORIDE......................... 19
COMPARISON OF THE COLOR DEVELOPED BY A NATURAL SOURCE OF VITAMIN A WITH THAT DEVELOPED BY AN OIL DILUTION OF THE CONCENTRATE THEN SUBJECTED TO THEco?n :m oi?r of the t e s t ............................... 20
COMPARISON OF THE COLORS DEVELOPED BY SOLUTIONS OF OLEIC ACID, OLEIN. AND VITAMIN A NEAR THE LIMIT OF SENSITIVITY OF TITS TEST USING ANTIMONY TRICHLORIDE WITH AND ITIOUT FYROCATFCMIN................ 22
COMPARISON, USING A KLBTT COLORIMETER,OF THE RATE OF FADING OF THE COLORSPRODUCED BY TREATMENT OF VITAMIN A WITH ANTIMONY TRICHLORIDE IN THE PRESENCE OF .AND IN THE ABSENCE OF A POLYPHENOL................... 24
COLORS PRODUCED WITH VITAMIN A ANDANTIMONY TRICHLORIDE IN THE PRESENCE OF AND IN THE ABSENCE OF FTROCATECHIH INAN EFFORT TO DETERMINE THE SENSITIVITY OP TTU*: TE ST................... 27
i i i
iiiupbor
T i l l COLORS LEVBIDPED 3Y CHLOROFORM SOLUTION OF CHOLESTEROL UrOH SRBAttRS* WITH ANTIMOIIY tsrichlcrxdi! Hi TBS PR12SSHCI3 OF AND Hi TRS ABSENCE OF GUAIACG1 ............................... .
Page
30
iv
Em ber
I
FIGURE
GRAPHIC RECORD OF THE BATE 0? FADING OF THE COLORS FRO £0089 BY TREAT!!; HT OF VITAMX8 A WITH AJTTIM33T TRICHLORIDE IB THE FRESISICE OF AltjD IE THE x\B3®C! OF A POLY?HI.!!0L ............................ ..
Page
25
CHAPTER I
EARLY STUDIES
The use o f antimony t r ic h lo r id e as a reagent
fo r the d e te c tion and estim ation o f vitam in A and fo r
the d if fe r e n t ia t io n o f the vitam in and carotene i s
the r e s u lt o f a long se r ie s o f work that had i t s be
ginning in 1922, A chem ical means o f d etection and
more e s p e c ia l ly , a chem ical means o f estim ation o f
vitam in A was u rgently needed to supplant or at le a s t
supplement the slow and c o s t ly feed in g experiments
which fo r years served as the on ly method o f estim ation .
In 1922 iruramond and Watson (1) demonstrated the
c lo s e re la tion sh ip between the d is tr ib u tio n o f vitamin
A and the development o f a v io le t co lo r when l iv e r o i l s
were treated w ith sulphuric a c id , a rea ction which came
to be known as the Drummond t e s t . I t was from th is
s ta rt that Fearon (2 ) began h is stu d ies which were to
1 , Jack C e c il Drummond, and Arthur Frederick Watson, "The b u lfu r ic Acid Reaction fo r Liver O i l s . ” Analyst, XLVil (1922 ), 341, through Chemical A bstracts, W f , Mo. 20 (October 20, 1922), 3550,
2, W illiam Robert Fearon, "Colour Reactions Associated w ith Vitamin A ," Biochem ical Journal. XIX. Ho. 6 (1925), 888.
2
r e s u lt in the development o f the phosphorus pentoxido
r e a c t io n and o f the te s t w ith t r ic h lo r a c e t ic acid in
com bination with p y ro g u llo l and other phenols* Fearon’ s
r e a c t io n using t r ic h lo r a c e t ic acid w ith p y ro p a llo l and
r e s o r c in o l was shown by W illim att, Moore, and wokee (3)
to be n o n -s p e c if ic fo r vitam in A* Tho c h a ra c te r is t ic
red c o lo r was found to appear with o i l s in which the
vitam in A had been destroyed , as w ell as w ith substances
that were r ic h in vitam in A. In tho ease report these
workers showed the phosphorus pontoxidc rea otion to bo
le s s se n s it iv e w ith ood l iv e r o i l than woe the arsen ic
t r ic h lo r id e r e a c t io n , applied to cod l iv e r o i l by
IkTOsmond, Rosenheim, and Coward (4 ) in 1925* A b r i l l ia n t
ultram arine blue w ith arsen ic t r ic h lo r id e was f i r s t
recorded by Kohlenberg (5 ) who in 1922 had reported come
very c h a ra c te r is t ic c o lo r rea ction s when ch o le s te ro l was
5* Stanley Gordon W illim ott, Thomas m o r e , end Frank Wokes, "S ffe o ta o f Various Agents on Colour Tests fo r Vitamin A*«* ^ io ch o o lca l Journal. IK . Bo* 6 (1916 ). 1292*
4* Jack C e c il Inuaaond, Otto P.osonheia, end Katherine H* Coward, 'The d e la tion o f S te ro ls to Vitamin A* ” Journal o f the S ociety o f Chemical Industrioa KUV (1925). I S . tforough $hs©ical Attracts. XiK. Ho* 157 (Ju ly 10 , 1925}, 2067.
5* Louis Kohlenberg, "On Some How C olor Rea ction s o f C h o lestero l* ” Journal o f B io lo g ic a l Chemistry. L II , No. 1 (May, 1922), ZiJT.
3.
treated with th is chemical* Tarurnmond, Rosenheim, and
Coward in th e ir work upon irra d ia ted s te r o ls used the
a rsen ic t r ich lo r id e reagent upon a ood l iv e r o i l , thus
s to r t in g the work open the rea ction s between vitam in A
end the ch lorid es o f certa in m etals. Subsequent stu d ies
on t3ie arsen ic ch lorid e re a c tio n by Rosenheim and rumraond
(6 ) proved that & tran sien t blue c o lo r was developed
when the c h o le s te r o l - fr e e , n on -aopon ifiab lo p ortion from
cod l iv e r o i l was treated w ith the substance, 'hen th is
fra c t io n fro© cod l iv e r o i l was d ilu ted 1 : £ ,000 ,000 ,
a blue c o lo r s t i l l appeared when treated with arsen ic
t r ic h lo r id e ( 7 ) . rtvamond and Coward (8) had p rev iou s ly
shown that the unoapon lfiab le fra c t io n o f cod l iv e r o i l
contained the vitam in A. At the same t i r e , Rosenheim
and Drummond (9) reported the development o f a permanent
blue c o lo r when the re* pent acted upon carotene.
6 . Otto Rosenheim, and J&nk C e c il iwtuaetond. "A In d ica te Colour He notion fo r the Presence o f Vitamin A .” Biochem ical Journal. XIX, Ifo. 6 f l9 £ 6 ) , 753.
7. I b id . , 754.
8 . J ok C ec il ru eon d , and Katherine H. Coward, "N u trition and Growth on le t s Devoid o f True r a t s . " Lancet. CCI, Pert I I , STo. 5118 (1921 ), 698.
9* Otto Rosenheim, and Jack C e c il Drummond,2£* £ & * » 6 7 8*>&.
4 ,
In 1926 Carr and P rice (10) reported th e ir
work upon ce rta in c o lo r reaction s sa id to be charac
t e r i s t i c o f , or a t le a s t in d ica t iv e o f , the presence
o f vitam in “*• Their ea rly work was done on the t r i
ch lo ra ce t ic acid re a c tio n , but in the ooureo o f th e ir
experiments they learned that the v io le t c o lo r was
not duo to an in te ra ct io n o f the t r ic h lo r a c e t ic acid
i t s e l f w ith the substance contain ing the vitam in.
Instead , the c o lo r was found to bo duo to the reaoticn
o f decom position products o f tho acid and a substance
associa ted w ith tho v itam in . Having found the c o lo r
developed w ith the impuro t r ic h lo r a c e t ic acid to be
o f a very f l e e t i n f nature, they turned to other coro-
poundo, some o f whloh had already been reported upon
by other w orkers, The arsen ic ch lo r id e was not in
cluded in th e ir stu d ies because i t did not lend i t s e l f
to d ilu t io n w ith a fa t so lv e n t , and fu rth er because o f
i t s noxious ch a ra cter. The compounds considered in
th e ir study included antisiony t r ic h lo r id e in chloroform
s o lu t io n , stann ic ch lorid e in chloroform s o lu t io n , an
hydrous f e r r i c c h lo r id e , anhydrous aluminum ch lo r id e .
10# Francis Howard Carr, nnd nr neat Arthur P r ice , "Colour K eactions Attributed to Vitamin A, 31ochemical Journal, XX, Ho* 3 (1926 ), 497.
5.
s i l i c o n te tra ch lo r id e , and phosphorus oxy ch lorid e .
They reports ""The co lou rs produced in a l l those re
lictions con sist in the main o f blue with varying pro
p ortion s o f r e d ,” (1 1 ),
The C arr-Prioe reagent i s a saturated so lu tion
o f antimony t r ic h lo r id e in ch loroform , 30 grams being
used in enough chloroform to make 100 c c . The rea ction
co n s is ts in the development o f a blue c o lo r when
substances contain ing vitam in A ore treated w ith th is
reagent* The blue developed in th is rea ction with vitam in
A soon begins to fa d e , o fo o t which has caused many
e f fo r t s to bo made to s t a b i l iz e the o o lo r , Carr and
P rice report that a trace o f saoisture, or the usual
q u a n tities o f a lco h o l presen t in the chloroform do not
in te r fe ro w ith the re a c tio n , U orris and Church (12)
rep ort c im ila rly*
The C arr-P rioe rea ction has been adopted to
the qu a n tita tive estim ation o f vitam in A through analy
s i s , by means o f a tin tom eter, o f the c o lo r p ro n e e d
fo r the p rop ortion o f blue and ye llow components. In
r u I b id . , 499.
12. liorl R, U orrle , and Anna B. Church, "A Study o f the Antimony T r ich lo r id e Color Reaction for Vitamin A .* Journal o f B io lo g ica l Chemistry. XXXXV. Ho. 2 ( JamviryT TSV-H ------------------------
6.
the caB© o f the co lo r with the vitam in , N orris and
Church (13) rep ort:
Vitamin A, or tho substance producing the "b lu e ” co lo r w ith antimony t r ic h lo r id e reagent, produces a green ish -b lue which fades to a c o lo r le s s so lu t io n ; the r a t io o f blue to yellow i s approxim ately 1*0 to 0*4 Lovibond u n it .
In an e f f o r t to elim inate the error due to
the fad ing o f tho co lo r in the qu an tita tive measure-
. ment o f vitam in A by means o f the C arr-P riee reagent,
N orris and Church (14) suggested that the rea ction take
p la ce at a terape^ture o f about 2° C* atta ined through
the use o f a mixture o f ic e and w ater. They were a lso
able to show that the concentration o f the antimony
t r ic h lo r id e reagent should be at f u l l sa tu tra tion fo r
the tenperature used, and that the reading o f the co lo r
should be aocoaplished w ith in 30 seconds a fte r beginning
the ad d ition o f the antimony t r ic h lo r id e to obta in con
s is te n t r e s u lt s . Brode and M agill (15) used anhydrous
13. Earl R* N orris , and Anna B. Church, op. c l t . , 489.
14. I b id . , 482,
15. Wallace R. Brode, and Mary A. M a g ill, "A C r it ic a l Study o f the Antimony T rich lorid e C olor Tost fo r Vitamin A ." Journal o f B io lo g ic a l Chemistrv. XCII. No* 1 (19«;1), 87.
7*
chloroform end r o d ls t i l l c d antimony tr ic h lo r id e in the
preparation o f the reagent* In the rea ction mixture
they Included a trace o f a c e t ic anhydride to react
w ith any h ydroch loric acid or water present* they a lso
allowed the rea ction to take p la ce at a temperature near
0° C* ?he3e workers, however, wore more in terested in
the absorption sp ectra than they were In the actual
c o lo r produced, although they did record fad in g o f the
blue c o lo r to red w ithin e igh t minutes at room temper
ature (1 6 ), Bro&e and 1 'a g ill (17) rep ort absorption
bands at 472 ou and a t 632 r,m in the faded or red so
lu t io n obtained by treatment o vitam in A w ith antimony
tr ic h lo r id e reagent* The ueo o f tho a ce t ic anhydride
i s recommended as a dehydrating agent which, I t appears,
tends to render tho co lored so lu t io n more b r i l l ia n t*
More r e c e n t ly , Levine and P.ichmcn (18) have
suggested that tho use o f aootio anhydride in a reaotion
mixture in vo lv in g antimony t r ic h lo r id e as a "chromogonic
16* Wallace E* Erode, and Mary A* M a g ill, op* c l t * , 94*
17* !&&&•* 95*18* V icto r E* Levine, and Radios ;ichmen, "The
Antimony T r ich lo r id e Eeaction with Compounds Containing five-merabored M onohetorocyolic B ings#” Journal o f B lo lo r lc a l C honlotry. C l, Jlo, 2 (1933), 7E7T7
8
ettsRiliktor*. These workers report a more c le a r ana a
more d is t in c t c o lo r whan the n oe tic anhydride i s In
cluded in the rea ction n a t u r e . Their work a lso in d i
ca tes that the presence o f an hydroxyl group in a co o -
pound reactin g with antimony t r ic h lo r id e slows up the
re a ctio n and lessen s the in ten s ity o f the c o lo r produced*
Morton (19) in 195£ suggested the in c lu s ion o f 7 -n eth y l-
in d o lo in the entitaony t r ic h lo r id e t e s t fo r vitam in A*
However, the use o f th is substance was indieatod on ly
when speotroaoop ic ©amain&tion o f tho blue liq u id mm
intended* *his substance in h ib its the form ation o f one
o f the absorption bonds given by the blue so lu tion * and
thus stakes i t p o s s ib le to in crease the accuracy o f th is
type o f determ ination*
J.'oeenthal and i2rdelyi (£0) hare mad© tho noet
recen t m od ifica tion o f the w e ll known antimony t r i
ch lo r id e reaction * They describe a te s t in which antimony
19* Bichard Alan Horton, MThe Use o f 7-M ethyl- indole in the Antimony T r ich lo r id e Colour Tost fo r Vitamin **#* Biochem ical Journal. XXVI. Ho* 4 (1932 ). H 9 7 . ----------- ---------
CO* Hugene Boson thal imd John ’ r d e ly l , "A lew Colour Teat fo r the * otorralnation o f vitamin A ,” B io - eher.ioal Journal* XX7IXX, Ho. 1 (1934), 41, and "Klne neue Ifeakiion sura Haohweis und sur k o lorin etr isch on Bee|^ptt|£ V|$|mlns A*" Bloohomliiche Z e lt a e h r l f t *
9.
t r ic h lo r id e in the presence o f pyroc& techia tsorvcf3 not
on ly to detect vitam in A but a lee to d istin g u ish i t
from curotano* ?h©y also suggest that th is re a c t io n
. may be adapted to a c o lo r in e tr ie method o f estim ation
tlirough comparison with an aqueous so lu t io n o f p ota ssiu m
permanganate*
In th eir t e a t , a blue co lo r in formed a t room
tof^ era tu ro in a chloroform talxture made up to a to ta l
volume o f f iv e o c* , containing a so lu t io n o f an o i l
r ic h in vitam in A, two oc# oi the antimony t r ic h lo r id e
reagent and on© oc* o f a re ce n tly prepared 0*5$ so lu tio n
o f pyrooateohin in chloroform * Immediately a fte r mixing,
the mixture i s tran sferred to a water bath and maintained
at a temperature o f 60° C* fo r one to two minutes* "uring
the treatment with heat, the o r ig in a l bluo c o lo r changes
to a v io le t - r e d , a c o lo r uhioh may be conven iently matched
w ith a 0*017* e o la tio n o f potassium permanganate* Those
in v e s t ig a to rs report the secondary v io lo t - r e co lo r to
bo more s ta b le than the o r ig in a l blue o f tho C arr-P rice
reaction * I'hia secondary v io le t -r o d c o lo r a lso appears
when other polyphenols or th e ir d e r iv a t iv e s are used in
the roaotion mixture* In a la te r report Bosonthal and
B rdelyi mentioned hydroquinone, v o r a tr o lc , and guaiaool
10.
being usable in the te s t (w l) .
In a fur ther report (i& )» a 8% solution o f gm&luool in oh lorofor® la roooaaenuod as being superior
to the pyrocatecU ta orig in a lly ouggeated. ?h© guaiaool
s o lu t io n was found to b© more sta b le than the p yro -
ca tcab in and was found to g iv e co lored so lu t io n s sh lch
remained c le a r fo r longer periods o f tltat then d id the
mixture w ith the pyrocatech in .
11. r.agone hosonthal, and John l.rdelyl t ’’Ifeltere Un t orsuchungen uher die none F&rbenreaktlon dee Vitamins A." iilooheaisohe K aitachrlft. CCLXXI (1934),. 414.
LZm Bttgeae nooenth&l end Hergaret Meitner, "Spectroscopio investigation <xf the Hew T1 twin A Colour Seat*" aioaheiaicol Journal. XXIX, Ho. 8 (May 1935), lOS1?^
CHAPTER I I
TUB PROBLBK
2he purpose o f the present paper Is to rep ort
a study o f th is new rea ction described by Hoeenthal
and ijrdelyi* An attempt w il l be made fa) to determine
what o f f o o t , I f any# the presence o f tho polyphenol has
in the rea ction mixt’ure wherein a co lo r i s developed
when vitam in A or oarotone i s treated w ith antimony
t r ic h lo r id e j and fb) to determ ine, i f p ossib le# whether
th is reaction i s due to vitm ain a o r to certa in ims&tu-
r a te ! fa t t y acids# fa t ty acid esters# or other substances
tonn& in o i ls *
Ilorris and Chur oh (23) in 1930, were o f the
op in ion that the blue c o lo r produced by the vitam in
faded to a c o lo r ie s s o lu t io n , and that the subsequent
red c o lo r was oaueed by some other substance present
in o l e i c acid so lu t io n or in o i l s con ta in ing unsaturatod
fa t t y acid components*
23* Burl R* N orris , and Anna B* Church# op, c i t . , 402.
cB jv m t i n
W®mTm®TkL MOBIL
parents and S o lu tion s .
1 . ch loroform , U .3 .P ., which contains about
3$ o f ethyl a lc o h o l.
2 . Chloroform , U .S .F ., washed with w ater, dried
over anhydrous calcium c h lo r id e , decanted and d i s t i l l e d .
5 . A rooont sample o f reagent grade antimony
t r ic h lo r id e .
4 . The C arr-P rico reagent fo r th is work was
prepared by adding 30 grams o f antimony t r ic h lo r id e to
100 c o . o f chloroform * The antimony t r ic h lo r id e was
washed with chloroform ?<nd drained o f excess so lven t
lu s t b e fore u se . Shis reagent was prepared using both
the U .S.P . grad© and the anhydrous a lc o h o l- fr e e ch loro
form .
6 . 2h© 0.5% pyrocateeh ln so lu tion in chloroform
was prepared each morning fo r the day’ s work using r o -
subllmed pyrocatooh in supplied by Merck.
t>. The cod l i v e r o i l used in th is work wee a
sample from Mead, Johnson and Coi^ptmy assayed end ad lusted
to contain at le a s t 1800 U .S.P. u n its o f vitam in A per
gram.
13.
7* Tho o l e i c a c id , lin seed o i l , cottonseed o i l ,
and o liv e o i l wore a l l o f U .8.P . q u a lity .
8 . The h a lib u t l iv e r o i l was a commercial sample
supplied by Pork©, la v is and Company, assayed end
standardised to contain e t le a s t 50,000 U .S.P . u n its o f
v i t a i ln A per pram. According to the la b e l , i t contained
no hydroquinone.
9. The "Vitamin A Conoentrate - d .L .H ." was pre
pared in England by the B r it is h Drug Bouses Lim ited, o f
London, and guaranteed to have a C arr-P rico blue value
o f not lo s e than 60 ,000. The co n tro l number o f the sample
used was 556995. This concentrate was stored in sea led
ampulea in the r e fr ig e r a to r prev ious to use.
10. The carotene was obtained from tho 8.H.A.
Corporation o f C leveland, Ohio, and con sisted la rg e ly
o f beta -caroten e with small amounts o f a lpha-carotene.
This m ateria l was stored in sm all evacuated v ia le in the
r e fr ig e r a to r previous to use.
11. Tho alpha-carotene and tho beta -caroton e
were lik ew ise obtained from the 8.1UA. C orporation , and
stored in the r e fr ig e r a to r previous to use.
IS . Tho gu aiaool was o f U.3.P* <pJ£Llty, a 5% by volume s o lu t io n in ch loroform being used.
13. Two samples? o f ch o le s te ro l prepared by Slerck
14,
wore used,
14, 2he potassium permanganate was a rooent
sample o f reagent grade.
Procedure -s
To observe the color® produced under the con
d it io n s o f th© to s t with an unoaturctel fa t ty acid and
w ith o i l s oompoeed o f varying amounts o f e s te rs o f these
uneaturatod fa t ty a c id s , a s c r ie s o f so lu t io n s o f such
substances was used,
A chloroform so lu tio n o f cod l iv e r o i l was
prepared o f such e concentration that one c c , o f the
so lu tion contained at le a s t 100 U ,3 .p . unite o f v itan in
A, For the p a r ticu la r o i l used , 1 ,4 grams was d isso lved
in enough o f the enhy&roas and a lo o h o l-fr o o chloroform
to make £5 c c . In order to have the other so lu tion s
con ta in about the seme amount o f fa t ty m a teria l, 1 ,4 grams
o f each substance, o l e i c a c id , o l iv e o i l , cottonseed o i l ,
and lin seed o i l was weighed and each was lik ew ise d isso lved
in enough o f the a lc o h o l- fr e e anhydrous ch loroform to make
26 o c .
To one c c , o f each so lu tion was added one c c , o f
the 0,©$> pyrocatoch in s o lu t io n , one c c , o f the anhydrous
a lc o h o l- fr e e ch loroform , and two c c , o f the antimony t r i
ch lo r id e reagen t. The co lo r which appeared on mixing the
15.
so lu t io n s was noted and the rea ction mixture was then
tra n sferred to a water hath at 50° c . , warmed fo r two
minutes and the secondary c o lo r n oted . This procedure
was a lso follow ed in a se r ie s in which no pyrocatech in
was used. The re su lts were recorded in Table I . When
U.S.P* chloroform was used as the so lven t and d ilu en t
in a sim ilar s e r ie s , the c o lo r s developed were the same.
As the r e s u lts which can be expected from an
experiment such os th is are on ly rouphly q u a n tita tiv e ,
no a t t e s t was made to ad ju st the concentration o f the
o i l and fa t ty acid so lu tion s to exact m olecular equiva
le n cy .
The o i l s were se le cte d as ty p ica l examples o f
ipixtures o f g ly ce ry l e s te rs o f fa t t y acids o f varying
degrees o f unsaturation . The approximate iod in e ab
sorp tion values o f the o i l s are: cottonseed o i l , 110;
o l iv e o i l , 85 ; lin seed o i l , 185; and cod l iv e r o i l , 160.
Suoh m ateria l was se le cte d as there i s some evidence (£4)
to in d loa te that the degree o f unsaturation o f the con
s t itu e n t fa t t y acids o f the o i l s has an e f f e c t upon the
c o lo r developed upon treatment with antimony t r ic h lo r id e .
In Table I I there i s recorded the r e s u lt o f a
24. Earl R. H orris , and Anna R. Church, op. c i t . .489.
16
TABLE I . COLORS PRODUCED TTfROUGH THE REACTIONof tmaft-TtmAriR) fatty acids and glyceryl estersOF FATTY ACIDS, OR RELATED SUBSTANCES /'ITR
ANTIMONY TRICHLORIDE.
00. 00. 00.1 . ™ — — ■----- 2 1 2 o d o r le s s c o lo r le s s
2 . — 3 0 2 o d o r le s s c o lo r le s s
3 . O le ic a cid 1 1 2 brownish*red
brownish**red
4 . O le ic a c id 2 0 2 b r o m i shred
brownish-red
5 . Linseed o i l 1 1 2 m ilky-gray m ilky-gray
6 . Linseed o i l 2 0 2 m ilky-gray m ilky-gray
7 . O live o i l 1 1 2 oloudy with yellow t in t
c le a r - fa in t amber
8* O live o i l 2 0 2 oloudy with ye llow t in t
c le a r - amber t in t
9 . Cottonseed o i l 1 1 2 amber with a suggestion o f pink
amber
10. Cottonseed o i l 2 0 2 amber with a suggestion o f pink
amber
11 . Cod l i v e r o i l 1 1 2 blue pink with a t in t o f v io le t
12 . Cod l i v e r o i l 2 0 2 blue red with a t in t o f v i d e t
17.
s im ila r experiment, awing t Pollution o f h a lib u t l iv e r
o i l in chloroform d ilu te s to represent at le a s t 100
U . f . 2 . units o f vitam in A per c c . ?o nake thin so lu t io n ,
0 .05 prom o f the h. 11 but l iv e r o i l ran d isso lved in
enoufh o f the a lcoh oS -free chloroform to make Zb c c .
A ©i i la r so lu tion o f o l e i c acid was prepared using?
0 .06 gram o f tho fa t ty acid*
5b learn the e a r l ie s t quantity o f the o le ic
acid which w i l l cause a rod c o lo r to ho developed upon
treatment with antimony t r i c h lo r id e , varying- q u a n tities
o f o l e i c acid were treated w ith the reap®»t in the seme
manner as p rev iou s ly d escrib ed . A so lu tion o f o l e i c
a c id waa prepared in the a lc o h o l- fr e e chloroform so that
on© o o . contained 0*08 nr an o f tho fa t t y a c id . The data
are recorded in Table III*
Using? a so lu tion o f ood l iv e r o i l and a so lu t io n
o f cottonseed o i l to which a quantity o f the vitam in A
concentrate hod been added, i t was o a e ib le to compare
the c o lo r s developed under aim llar con d ition s by these
two sources o f the v itam in , a record o f which ia found
in Table IV* Two prams o f the ood l iv e r o i l wee d ilu ted
w ith enouph o f the a lc o h o l- fr e e chloroform to make 100 cc*
Two proms o f cottonseed o i l with an added 0.0016 pram o f
the vitam in A concentrate was d ilu ted w ith a lc o h o l- fr e e
18.
TABLE I I . COMPARISON OF THE COLORS PRODUCED BY OLEIC ACID AND BY VITAMIN A WITH ANTIMONYTRICHLORIDE IN THE PRESENCE OF AND IN THE
ABSENCE OF PYROCATECHIN.
<d
s 3■pviVi o •<3 ©<Ue g oo S S SO P P o 3 w -»O 00 CD <rt
©a xr • o © o
BoVisO
1
|!
2s
& hoa a«4 O P <H
jj*H ©S 3
©P-1Ik' O P H © O ©045
00. 00. 00.1 . — 2 1 2 c o lo r le s s c o lo r le s s
2 . — ---------- — 3 0 2 c o lo r le s s c o lo r le s s
3 . H alibut l i v e r o i l
1 1 2 blue permanganate pink#
4 . H alibut l i v e r 2 0 2 blue permanganate pink
5. O le le a cid 1 1 2 oloudyc o lo r le s s
oloudy no c o lo r
6 . O le ic acid 2 0 2 oloudyc o lo r le s s
fa in tpink
* A permanganate pink Is a pink that i s t in te d with
v i o l e t . The c o lo r resembles that o f a very d ilu te
s o lu t io n o f potassium permanganate - 0.001 N o r
weaker.
19.
TABLE I I I . RESULTS OF AH EFFORT TO DETERMINE TICK SMALLEST QUANTITY OF OLEIC ACID THAT WILL
PRODUCE A PERCEPTIBLE RED COLOR HTH ANTIMONY TRICHLORIDE*
to© a «$
H O * 3 U O H C O **» o *d
U 3 O 0 * 4o h I p © ©l*s»
5
O tQo oO Hp o 0 • Ho o
esso
© 4*CjO
22mgO
m4* *45 U
4*
J0 49H ©S I
it0 49
H do ©0 /5
1* — -•*— —c c .2
c c .1
c c .2 c o lo r le s s c o lo r le s s
s • — — 3 0 2 c o lo r le s s c o lo r le s s
3 . 1*0 00. 1 1 2 pink pink v ith
4 * 1 .0 00* 2 0 2 pink.bro.m t in t pink v lth
5 .
iO•o
C O . 1 .5 1 2 fa in t pinkbrovm t in t fa in t pink
6 * 0 .5 C O . 2 .3 0 2 fa in t pink fa in t pink
7 . 0 .3 C O . 1 .7 1 2 c o lo r le s s c o lo r le s s
8 * 0 .3 o c . 2 .7 0 2 c o lo r le s s c o lo r le s s
The c o lo r o f the l iq u id in tubes f iv e
and s ix i s b a re ly p ercep tib le* Upon
warming i t becoiaes sonewhat in te n s if ie d .
JO.
TABU; IV. COMPARISON OF THE COLOR DEVELOPED BY A NATURAL SOURCE OF VITAMIN A WITH Tf!AT DEVELOPED BY AN OIL DILOTION OF THE CONCENTRATE WHEN SITRJ CTED TO fitE CONDITIONS OF TOT TEST.
CO. 00. 00.1 . 1 2 c o lo r le s s c o lo r le s s
2 . — ——— 0 2 c o lo r le s s c o lo r le s s
3 . 0 .5 co . o f the perannga-cod l i v e r o i l 1 .5 so lu t io n
1 2 blue nate pink*
4 . 0 .5 c e . o f the perraanga-ood l i v e r o i l 2 .8so lu tion
0 2 blue nate pink
0 . 0 .5 oo . o f the cotton seed o i l and 1 .5 vitam in A
2 blue1 permanganate pink
concentrateso lu t io n
6 . 0 .5 c c . o fthe c o tto n - 'seed o i l and 2 .5 0 2 blue pernanga-Vitamin Aconcentrates o lu t io n
nate pink
• A permanganate pink i s a pink that le t in ted with
v i o l e t , The c o lo r resembles that o f a very d ilu te
s o lu t io n o f potassium permanganate • 0.001 N o r
weaker.
£1
chloroform to stake 100 cc*
In order to study the c o lo r s developed with
antimony tr ich lo r id e no-r the H a l t o f s e n s it iv i t y , a
se r ie s o f so lu tion s in chloroform ««ere prepared so that
one c c . o f the so lu tion contained £0 mp# o f the substance*
o l e i c a c id , cod l iv e r o i l , cottonseed o i l , and cottonsood
o i l to which a smell quantity o f the vitam in A concen
tra te had been added, were used. One h a lf cc# o f each
so lu tion was measured from a a e ro lo p io a l p ip e tte and
w&e treated with antimony t r ic h lo r id e in the presence
o f and in the absence o f the polyphenol* The work is
recorded in Table V* Bach cc* o f the so lu t io n o f co tton
seed o i l w ith the vitam in contained 0.05 tag* o f the vitam in
A concentrate*
To determine the r e la t iv e s t a b i l i t y o f the co lo rs
developed w ith vitam in A end antimony t r ic h lo r id e , re
action mixtures with and w ithout a polyphenol wero comr
pared on a K lo tt co lorim eter tutting a 0*001 $ so lu tion
o f potassium pormanpaneto as the standard* The two
mixtures were oo.'spared at in te rv a ls as nearly simultane
ously as p o s s ib le , and were compared with the seme so
lu t io n o f potassium permanganate* Reaction mixtures
fo r th is purpose were prepared uainp the vitam in A concen
tra te as the source o f the chromogenio substance* Bach
22.
TABLE V. GO, PARIAN OF THE COLORS DEVELOPED EY SOLUTIONS OF OLEIC ACID, OLEIN, AND "IT/JHN A NEAR THE LIMIT OF SENSITIVITY OF T?TE TEST USING ANTIMONY TRICHLORIDE WITH AND WITHOUT
PYHOCATECHIN.
©Sip
©
g
©I B <m io •
© «d o c o © • u o ri pO O H cs (U o 6 p p o O 3 ©*4• 4 0 ^
O o m ffl *4
op
rap # d HCIS U © K O * r-4 P«O as 60 O o > u
H W . O© H.O d8 cr • X;o © o 8
5£o©pctio1A«
tj<riC O
P *4
5 5
Ou© *p g ,O Pr-J fl5 O ©o x :
©psO Pr--i cdS jg
1 . mmrnm mmmmimc c .2
c c .1
c c .2 c o lo r le s s c o lo r le s s
2 . 3 0 2 c o lo r le s s c o lo r le s s
3 . O le ic acid 1 .5 1 2 pink t in t pink t in t
4 . O le ic acid 2 .5 0 2 pink t in t pink
5. Cottonseed o i l 1 .5 1 2 c o lo r le s s c o lo r le s s
6 . Cottonseed o i l 2 .5 0 2 c o lo r le s s fa in t yellow
7. Cod l i v e r o i l 1 .5 1 2 blue fa in t pink
8 . Cod l i v e r o i l 2 .5 0 2 blue pink
9. Cottonseed o i l w ith the 1 .5 1 2 blue penaanga-
10.
vitam in Aconcentrate Cottonseed o i l w ith the 2 .5 0 2 blue
nate pink*
permanga-vitam in Aconcentrate
nate pink
♦ A permanganate pink Is a pink that Is t in te d 1th
v i o l e t . The c o lo r resembles that o f a very d ilu te
so lu t io n o f potassium permanganate - 0.001 N or
weaker.
mixture contained enough o f a chloroform so lu tion to
represent 0*16 rag* o f th is concentrate* The so lu tion
o f the concontrato nae measured from a £ c c , Ostwnld-
fo lir i p ipette* ry-ch • ix tu re cent' '• • the ir r:c M -ant'ty
o f tho antimony t r ic h lo r id e reagent neocurei from a
bu rotto ; ono contained the ch lo ro io rm so lu tion o f tho
polyphenol and tho other on equal volume o f chloroform *
Both so lu tion s wore.made up to a volume o f EG c c . A fter
heating the mixtures in the water bath fo r two minutes
at 60° C. the tubes were tran sferred to a water bath at
£0° C .f cooled fo r two m inutes, then a small p ortion o f
the rea ction mixture was tran sferred to the co lorim eter
cup and compared to the standard so lu tion se t at 20 ram*
Comparisons were made a t in terv a le o f f iv e minutes us
ing fre sh p ortion s o f the c o o l re a ctio n mixture obtained
from a stoppored veseel* The data are rocordod in Table
VI end the curves shown in Figure I were p lo t te d from
th ese data* The value used a» tho ordinate rets the
reading in m illim eters o f the co lored so lu tion contain ing
the vltavain A* The t i e reading in minutee wat? used as
the abscissa*
The d iffe re n ce in the readings recorded in Table
VI fo r tubes two, fo u r , and s ix can be accounted fo r by
the fa c t that some ox idation o f tho vitam in occurred
£4
TABLE V I. COMPARISON, USING A KLETT COLORIMETER. OF THE RATE OF PADIM OP THE COLORS PRODUCED BY TREATMENT OP VITAMIN A VYTH ANTI HOMY TRICHLORIDE IN THE PRESENCE OF AND
IN THE ABSENCE OF A POLYPHENOL.
S olu tion s number 1 and S each contain vitam in A, pyrocatech in , and antimony t r ic h lo r id e in a ch loroform medium. P - pyrooatech in .
S o lu tion number 5 contains vitam in A, gu a ia eo l, and antimony t r ic h lo r id e in a chloroform medium.G - g u a la e o l.
S olu tion s number 2a 4 , and 6 each con ta in vitam in A, and antimony t r ic h lo r id e in chloroform medium.
A l l so lu tion s were warmed at 6CP C. f o r two minutes b e fo re being coo led to 20 C. and compared a 0.001 N re fe ren ce so lu tio n o f potassium permanganate se t at 20 an . on the co lor im eter s c a le .
25
FIGURE I. GRAPHIC RECORD OF THE RATE OF FADING OF THE COLORS PRODUCED BY TREATMENT OF VITAMIN A WITH ANTIMONY TRICHLORIDE IN THE PRESENCE OF AND IN THE ABSENCE OF A
POLYPHENOL.
8 8 2 2 6
2*.
between the preparation o f the su ccessive seta o f re
action m ixtures. Further, a new and d if fe r e n t so lu tion
o f the vitam in concentrate was used in the preparation
o f the se t o f so lu tion s numbered fiv e and s ix ,
A so lu tion o f h a libu t l i r o r o i l was prepared
in U .3.P . chloroform oo that 1 o c . was equivalent to
100 O .3 .P . u n its o f vitam in A, This so lu t io n was used
in an e f f o r t to determine in a sea l-q u a n tita tiv e manner
the r e la t iv e s e n s it iv ity o f the rea ction w ith vitam in A
o f antimony t r ic h lo r id e in the presence o f and the absence
o f a polyph enol. Typical data are recorded in Table V II.
S olu tion s o f carotene using both the anhydrous
and the O . f . grade o f chloroform were prepared and
treated with antimony t r ic h lo r id e in the presence o f
and in the absence o f p yrocatach ln , the co lo rs being
observed upon n ix ing and a fte r heating. A rea ction
mixture con tain ing 0 .1 mg. o f carotene in a to ta l volume
o f f iv e co . gave a green ish -b lue t in t changing immedi
a te ly to blue and remaining unaltered on the a p p lica tion
o f h eat. Larger q u a n tities o f carotene gave deeper
shades o f blue. Both so lu tion s o f carotene behaved sim i
la r ly , there being no epparont d iffe re n ce in the in ten s ity
o f c o lo r developed. I t was noted that tho f u l l blue co lo r
was developed more ra p id ly in the absence of the pyro-
table; v i z - colors produced ti th t it amir a and antimony ra t chlorine i s r r r PRESENCE op and inTHE ABSENCE OF PYROCATFCHIN IN AN EFFORT TO
M N P THE SENSITIVITY Of T ? .
£ o ® 8 <m K Q H H O
©43I*»£
O©4JhO*-»Oo
*3
35
0.00 oo. 2 oc. X oo. 2 00. colorless colorless0.00 3 0 2 colorless colorless0.03 1.97 1 2 fainttint blue colorless0.03 2.97 0 2 fainttint blue faint pink tint0.1 1.9 1 2 blue - pink0.1 2.9 0 2 blue rose1.0 1.0 1 2 blue red1.0 2.0 0 2 blue violet-red
28
catochln.
A chloroform solution containing 0.025 raps. of alpha-carotene por ee* wm subjected to the action of the Carr-Price rearent in the pr sconce of and In the abeonco of the polyphenol. In every ease studied, a blue color developed within one minute, and this color oeeraed to bo intensified slightly by heating, then quantities of alpha-oarotone between 0.05 mg. end 0.5
0
rap. were used in the reaction, the oolor developed hod a faint lavender tint. The snail© st cjuontity of alpha* carotene which rives a definitely recopnizoble blue eo3or, or blue tint with lavender, was found to uc 0.05 rap.
Beta-carotene behaves m does the alpha-carotene used in the preceding experiment, this substance rives the typical ultramarine blue color which persists for hours at room temperature, n th the smaller quantities of betu-o&rotene, the lavender tint likewise appeared.
Cholesterol in chloroform solution had boon ob
served to react in a peculiar manner with the antimony trichloride reagent* ?o further study this reaction, solutions of antimony trichloride, purl cool, raid chole
sterol were prepared using the 0 . 8.P* grade of chloro
form an the solvent, ?h» antimony trichloride reagent
29*
was prepared as b e fo r e , the guelaool was a 6% by volume
s o lu t io n , and the ch o le s te ro l was o f such a concentration
that one oc* represented 0*01 grera o f the s te ro l*
One rea ction mixture con sisted o f f iv e c c . o f
the ch o le s te ro l s o lu t io n , one eo* o f the guaiaool eo
la t io n , four oc* o f ch loroform , ana f iv e cc* o f the
antimony t r ic h lo r id e rea g en t. I t s oorapanion mixture
was the euiae except that instead o f one oc* o f the
gu a iooo l s o lu t io n , an extra o c . o f ch loroform was added.
The t o ta l volume in each mixture was lb cc*
The o o lo r was noted ju s t a fte r addin*? the a n ti
mony t r ic h lo r id e , and again in two minutee ju s t be fore
transferrin*?: to the water bath* Each mixture was warned
at 60° C* fo r two m inutes, immediately tran sferred to
another bath at 20° C, where i t woo kept during the
observations* Keoord of the c o lo r was made a t f iv e
minute in te rv a ls w hile in the c o o l bath* The o o lo r
changes are recorded in Table VIII*
30.
TABLE VIII. COLORS DEVELOPED BI CHLORQFOJK SOLUTIONS OF ClfOLHS? ROL UPON TREATMENT * ITH AHTXHOKT TRICHLORIDE IN THE PRESENCE OF AND IN T'T ABfNNON 05 GUAXACOL.
Tide R eaction mixture w ith gu& iaool.
Roaotion mixture without g u a ia co l.
Upon a d d ition o f antloonytr io li lo r ld a
pink pink
Tso minutes a f t e r ad d ition o f antloony 1 t r ic h lo r id e
blue blue
Four minutes a f t e r a d d ition o f antimony t r ic h lo r id e(ram sd f o r two minutes)
v io le t -p in k green ish -blue
Fine minutes a f t e r ad d ition o f antLoony t r ic h lo r id e
v io le t -p in k cre e n irh -blue
Fourteen minutes a ft e r a d d ition o f antimony t r i ch lo r id e
v io le t -p in k green ish -blue
Tr-o hours a fte r a d d it io n o f antimony t r i ch lo r id e
v io le t -r e d v io le t
31
CHAPTER IV
DI3CU33I0H
1* O le ic a c id , and to a le s o extent co tto n -
seed o i l , and o l iv e o i l fsv e a roddish co lo r upon tre a t
ment w ith chloroform so lu t io n s o f antimony t r ic h lo r id e ,
both in tho presence o f and in the absence o f the p o ly
phenol# This red c o lo r i s somewhat in te n s if ie d upon♦
boatin f* I t thus appear© that unsaturated fa t t y a c id s ,
e s te rs c f those a c id s , or substances associated with
such m ateria ls arc ohroraopanic substances* The red
c o lo r which i s produced under the con d ition s o f tho to s t
with such m ateria ls i s a brownish-red in r e la t iv e ly high
con cen tra tion s , but near the l im it o f s e n s it iv i t y , the
co lo r i s a pink* The pink c o lo r which appears with small
amounts o f o l e i c a c id , resem bles tho co lo r which appears
w ith small amounts o f vitam in A* Cod l iv o r o i l in com
p a ra tiv e ly hlrfc d i lu t io n , upon treatment w ith antimony
t r lc h lo r id o reagent f iv e s a primary blue o o lo r which
Oh an pcs to a p a le rod or x ink upon warminf at 60° C.
f o r two minutes* -h is pa le red or pink co lo r may be
due to the uneaturetod character o f i t s con stitu en t
fa t t y a c id s , or noy bo due in p a rt to the presence o f
the v itam in . O le ic acid present to the extent o f 10 rof*
in f i r e ee* o f a re a c tio n m ixture, produoeo a deopor
rod c o lo r than does m equal weight o f a su perior grade
o f cod l i v e r o i l d ilu ted to the same volume*
2* V ita tin A with antimony tr ic h lo r id e f iv e s
a primary ultram arine blue stolch changes to a secondary
v io le t -r e d o r v io le t -p in k c o lo r upon heating at GO0 C,
fo r two miautos* *his sequence o f c o lo r s appears in
re a c tio n m ixtures contain ing polyphenols or d er iv a tiv es
such as pyrocatech in or guaiaool* These ch a ra c te r is t ic
c o lo r s a lso appear in rea ction m ixtures In which the
polyphenol has been om itted . The c o lo r s ere the same
whether anhydrous, a lc o h o l- fr o o chloroform or the U .S .p.
grade i s used as the so lv en t and d iluent*
S* She secondary c o lo r produced in the presence
o f a polyphenol i s apparently no more s ta b le than i s &
s im ila r c o lo r produced in the absence o f such a phenol*
the curves in f ig u re I that have been drown from the data
appearing in Table 7X are s u f f ic ie n t ly a lik e fo r oach
s e t to show that any s t a b i l is in g e f f e c t i s r e la t iv e ly
n o f l i f ib le * The c o lo r s which are produced in each case
appear to match the s o lu t io n o f potassium permanganate
p e r fe c t ly , although the so lu tion contain ing the p o ly
phenol changes in c o lo r (becomes a brow nich-red) to a
grea ter decree as time passes than does the so lu tion
33*
con ta in in g no polyphenol. In on© experim ent, i t was
p o s s ib le to match the co lo r produced in the absence o f
the pyrooatechin with the potassium permanganate so lu tion
a fte r as much as an hour had ©leased#
4# ’ /hen pyrooatechin or puaiaool i s used in con
ju n ction w ith antimony tr io h lo r id o so a te s t fo r vitam in
A, the in te n s ity o f the c o lo r produced is always s l ig h t ly
lo s . than i s the in te n s ity o f the c o lo r produced with
the same quantity o f vitam in A but w ith the phenol om itted,
fh is a c t io n as an in h ib it in g agent is In agreement with
the fin d in g s o f Levine and Richmsn (£5) who reported that
the presence o f an hydroxy group in a compound re noting
w ith antimony t r io h lo r id o acted to in h ib it the c o lo r pro
d u ction . Rosenthal and E rdoly i (£6) have a lso recorded
re s u lts which in d ica te that phenol® exert an in h ib it in g
of. e c t on the c o lo r p rod u ction . *?hey reported a s ca rce ly
d is ce rn ib le blue co lo r when antimony t r ic h lo r id e reacted
w ith a so lu t io n con ta in ing 0*001 fag. o f vitam in A per c c . ,
but upon the ad d ition o f gu a iaco l and tho e x p lica tio n o f
heat the c o lo r disappeared.
£6* V ictor 2* Levine, end Bu&loe Hiohnon, op. o l t . , 373.
£0. Eugene E osoathal, and John E rd o ly i, o p . o l t . , Bioche iiocho “ C lta o h r lft . CCLXXI (1934 ), 414.
C* ?he fa c t that the B a m v io le t -r e d co lo r i s
produced whether a polyphenol ia included in the rea ction
mixture or n ot, and the fa c t that the polyphenol does not
Doom to o re rt any d e f in ite s t a b i l is in g e f f e c t upon the
c o lo r that ia produced, c ota some doubt a- on the b e l i e f
that a polyphenol ia an e s s e n t ia l component o f the re
action m ixture. An in te re s t in g b i t o f date was fleaned
from the l it e r a tu r e regarding th ia p a r ticu la r aspect o f
the problem . In 1931, Brodo and Ms.pill (27) examined
ap eotroB oep ioa lly the red or faded so lu tio n obtained by
a llow ing a rea ction mixture in vo lv in g antiraony t r io h lo r id e
and vitam in A in chloroform so lu tio n to stand fo r a time
at room temperaturo, 'Theao workers reported the presence
o f absorption bands at 472 mi and at 532 mu ho son th s i
and -e ltn o r (28) examined by mo ana o f the spectrophotome
t e r , the v io le t - r e d co lored s o lu t io n they obtain®! by
warming at GO0 C, fo r two minutes a mixture o f vitam in A,
antimony t r i c h lo r id e , and guaiaool o i l in ch loroform so
lu tion # ’ hen the abaorption spectrum o f th ia secondary
v io le t -r o d c o lo r waa examined w ith in 10 minutes, bunds
27# Wallace B. Brode, end Mery A. if&gill, op. o i t * # 95.
28. Eugene R osenthal. and Margaret W eltner, op. P i t , . 1037.
were noted at 475 ctu ant at 645 ©a, Twenty minutes la te r
those bands had reso iree in to bonds at 470 nu, 485 nu,
535 m , and 55” tau*
*Jme# both grottos o f worker© rep ort bend© in the
asm r e la t iv e p o s it io n s . As the bm&m th a t sro shown by
the absorption spectrum o f tho bine s o lu t io n obtained by
treatment o f r i ton in A with antimony t r ic h lo r id e are known
to appear a t 572 m and a t 605 net (2 9 ), i t I s quit© e v i
dent that m m change has t; ken p la ce in tho ohromoren*
A© the change no eras to hare boon about the ansae in the
substances examined b the two groups o f w orkers, It ia
p o s s ib le that the rod so lu tion studied by iJrode and ifn g ill
m y here been tho sans ar tho v io le t -r o d so lu t io n studied
by Rosenthal and « a ltruer• In tho course o f tho present
•ork, i t has boon observed that In so lu t io n s o f vitam in
A concentrate where m m ox idation has taken p la c e , the
v io le t - r o d c o lo r obtained by su b jectin g tho so lu tion to
the condition© o f the re a c t io n , io more red and lo s s
v io l e t than ia the co lo r obtained with a frosh so lu t io n
o f v i twain A concentra te . This fa c t may account fo r the
29* le ia o r 'fo rr ia Ilo ilb ron , A lbert Tklward Gillora, end Bichard A im M orion. " S p e c i f i c i t y In Testa fo r T lten ln a. A Hew Conception o f the Chronogenie Constituents o f Fresh and Aged Liver O il* ” Biochemical Journal. 1ST,So. 4 (1931 ), 1252.
36
d iffe r e n c e In the terns need by the two Bote o f workers
to d escr ib e the co lored liquidfi#
6* fhe e f f o r t s to determine tho s e n s it iv i t y to
the presence o f vitam in L o f the C a rr-..r ice rea ction when
modified, by worming, in d ica te that about 6G TT.S.?* un its
o f vitam in A mast be present in order to obtain a d e f in it e
ly recog n isa b le v io le t -p in k oecondary c o lo r . \ hon & p o ly
phenol i s Included in a re a c tio n m ixture, the te s t i s
s l ig h t ly le a s ce n e it iv o as judged by the in te n s ity o f tho
c o lo r that ie produced. 1th q u a n tities o f Ice s than SO
u n ite o f vitam in Af a red c o lo r appears^ but i t is very
d i f f i c u l t to recogn ize i t a® being d e f in it e ly in d ica tiv e
o f vitam in A# The red c o lo r may be due to eone other
substance found associated w ith v i t m in A,
7* Carotene, a lpha-caroteno, and bota-coroterse
have been found to g ive r is e to a blue co lo r upon treatment
w ith antimony t r i c h lo r id e , the c o lo r p e rs is t in g unchanged
upon warming at 60° C. fo r tiro m inutes. This c o lo r appears
when a polyphenol i s included in tho re a ctio n mixture as
troll as when the polyphenol i s om itted . The use o f an
hydrous, a lc o h o l- fr e e or tho U .S.P. grade o f chloroform
i s op tion a l ss no apparent e f f e c t upon the co lor produced
has been observed# The report by Rosenthal end S zilard
that the blue c o lo r given by carotene w ith anti ony t r i
ch lo r id e disappears on warming w ith guu lacol (3 0 ),
has not beon confirmed In tho present work# In f e e t ,
tho bluo c o lo r has been observed to pore 1st fo r more
than two hours during numerous ox; crimenta*
0* C h o le s te ro l, or an associated m betn n cc,
because o f i t s p e cu lia r re a c tio n with antimony t r i
c h lo r id e , t r i l l undoubtedly In to r fo rc w ith the R osenthal-
X rdely i m od ifica tion o f tho ^ a r r - ir i c c re a ctio n fo r
vitam in A arid erroten e . Whon a ch o le s te ro l so lu tion
in ch loroform ie treated w ith antimony t r ic h lo r id e In
the presence o f g u a ia co l, a pink c o lo r appears that
Changes to bluo w ithin one minute# Upon heating V in
mixture fo r two minutes at 60° C# the blue changes to
a v io le t -p in k that gradually grows darker* '"hen a sim i
la r quantity o f the c h o le s te ro l so lu tion is treated with
antimony t r ic h lo r id e a lone, a pink co lo r appears that
changes to blue w ithin on© minute# However, upon
heating thin mixture at 60° C. fb r two minutes, a
groon leh -b lno c o lo r i s produced which p e r s is t s f o r at
le a s t 15 minutes# A fter standing fo r one hour at room
tem perature, a l l the s o lu t io n s , w ith and without the
30# Uogonc R osenthcl, and Catherine S s ilo r d ,"A Hen Method o f Determining tho Vitamin a Content o f 3 lo o ■," itocheu ica l Journal, XXIX, He# 5 (May, 1935) , 1039#
38*
puaiacol develop an Intenee v io le t co lo r* At th is time
the re a c tio n mixtures In which the fu a la co l has boon
used arc nor© red and len s v io le t then are th* n ix tore a
in which tho fu& laool has been omitted* The ad d ition
o f 0*1 or* o f a c o t ic anhydride to the rea ction mixtures
r e s u lts In so lu tion s tho s in e c o lo r , but more b r i l l ia n t
than thoce ^ust described*
cnApnm v
su w m r
1* l?hen vitam in A te treated w ith antimony
t r ic h lo r id e using ch loroform as the s o lv e n t , the
primary b lue c o lo r which develop© changes to a ©econ-
dary v io le t - r e d or v io le t -p in k xapon worming to 60° C,
f o r two minutes* The same sequence o f the same c o lo r s
appears whether pyrocatech in or gu aiaool ie included
In the re a c t io n mixture or not*
E. mien vitam in A or carotene i s troatod w ith
antimony t r i c h lo r id e , the presence o f pyroooteoh ln or
gu aiaool In h ib its to a s l ig h t degree the production o f
the c h a r a c te r is t ic co lo rs*
8 . O lo ic a c id , ce rta in low m olting f a t s , or
associa ted substances produce a rod c o lo r tin ted with
brown upon treatment w ith the antimony t r ic h lo r id e r e
agent* form ing to SO0 C« tends to in te n s ify the c o lo r
s l ig h t ly *d . !7hen the antimony t r ic h lo r id e reagent i s
used w ith gu a laco l fo r the d e te c tio n or estim ation o f
vitam in A through the produ ction o f a secondary v i o l e t -
red or v io le t -p in k c o lo r , ch o le s te r o l i s apparently on
in te r fe r in g substance.
40,
5. A p e rs is te n t b lue c o lo r 1® produced when
alpha or beta otirotene, or a mixture o f the tw o, i®
treated w ith antimony t r ic h lo r id e in the presence o f
or in tho absence o f pyrocatech in or fm a laco l.
6* The some c o lo r s are produced and the same
o o lo r oh napes take p la ce when anhydrous a lc o h o l- fr e e
chloroform and when the U*3*P* prade ie used a® the
so lv en t and d ilu en t in the reaction® described in th is0
paper*
B I B L I O G R A P H Y
BXSUOOaidBT
Brodo, Callao© R ., und I la g il l , Eery A ., "A C ritio& l Study o f the Antimony T rich lorid e Color Toet fo r Vitamin A** Journal o f B io lo g ic a l Chemistry* XCII, ITo, 1 (1951} , 15V~
Carr, Francis Howard, and P r ic e , Arneat Arthur, "Colour R eactions A ttributed to Vitrjrain A ," Bioohomioal jHOjurnriJL,, Aa , Ho* <& (19—*j) , 497*501#
ruraaoad, Jack C e c il , and Coward, Katherine H«,"n u tr it io n and Growth on D iets Devoid o f True F a ts ," Lancet, CCI, part I I , Ho. £110 (1921), 698-700.
Drurroond, J<*ok C e c il ; Hosonhclia, O tto ; and Coward, Katherine K ,, "The R ela tion o f a te ro ls to Vitamin
Lruiarrond, Jack C e c il , and v/atcon. Arthur F rederick , "The S u lfu r ic Acid Reaction fo r l iv e r G i ls ." A nalyst. X1VII (1922 ), #541, through Cholic-:-!» e 4l«« <U | *>#**+« l . VUAUU? H *■Vetracts. XVI, So, 2& (October 2 0 ,lU fi? j, §8b0.
Fearon, W illiam R obert, "Colour Reaction® Associated w ith Vitamin a* " J ioc3 ;oalcn l JonruTi. XXX, So, 6 (1 9 2 5 ), 888-895.
H eilbron , Is id or M orris; Oillara, A lbert Edward; andMorton, Richard Alan, " S p e c i f i c i t y in. Testa i'or Vitamin A, A Set? conception o f tho Chromogonic Conatiuontr. o f Fresh and Aged L iver O ils ,"Blocho: .lea l Journal. XXV, No. 4 (1951 ), 1562-1566,
"On Hone Sew Color Reactions o f C h o le s te r o l." Journal o f B io lo g ic a l Chemistry.L II , Ho. 1 (MayT T O sT ^ f e g g g * ------------
Kahlenborg, L ou is,
Levine, V ictor E ., end " ich n n , Tudioo, "The Antimonytr ic h lo r id e Hoaction with Coc^oundc Containing Five-rwmborod Vonohotor© cyclic Binge* " Jour m l of, B io lo g ica l C he:.ietry . C l, DO. 2 (ISSST, ' &7S-389.
43.
Morton, Richard Alan, ” 131© Us© o f 7-M othylinclole In the Antimony T r ich lorid e Colour Tost fo r Vitamin A ." B iochem ical Journal, XXVI, Ho, 4 (1932), 1197- 1201*
M orris. Earl IU, and Church, Anna £ * , "A Study o f the Antimony T r ich lo r id e C olor R eaction fo r Vitamin
LXXXY* m ' 2
Rosenheim, O tto , and Srucaaond, Jock C e c i l , "A •Plicate Colour R eaction fo r tho Presence o f Vitamin A*” B iochem ical Journal. XIX, Ho* 6 (1 9 2 5 ), 753-756*
Rooenthal, Eugene, and E rd e ly i, John, ” A Hew Colour Test f o r tho determ ination o f Vitamin A*” Biochem ical Journal. XXVIII, Ho. 1 (1934 ), 41-357
R osenthal, Eugene, and R rclelyi, John, "Bine neuo R eaction sum Haohwols und ssur h o lorin etr isch en Bestiranunp des Vitamins A ,” Blochenleohe S e lt -B clu .iit « CCLT/II (19 33 ), 119-1757
R osenthal, Eugene, and B rd e ly i, John, ” .o it c r e Unter- suchungen fther d ie neue Farbenroaktion des Vitamins A .” Bloohculohe R c it s c h r l f t . CCLEXX (19 34 ), 414- 419.
R osenthal, Eugene, and W oltner, M arrerot, "S pectros co p ic In v estig a tion o f tho How Vitamin A Colour T e s t .” B iochem ical Journa l . XXIX, Ho* 5 (1935 ), 1037-1032;
R osenthal, Eugene, and S s ila rd , C atherine, "A How Method o f Determining tho Vitamin A Content o f B lood*” B ioehouioal Journal. XXIX. Bo* 5 (May. 1925), 1025=1232;------------------
f f i l l im o t t , ntanley Gordon; itooro, Thomas; and Hokes,Prank, "E ffe c ts o f Various Agents on Colour Toeto fo r Vitamin A** Biochem ical Journal. XX. Ho. 6 (1926 ), 1292-12SG7------------------------