heels of (Yiraell .»Yerar& Mousey) Approved
74
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Transcript of heels of (Yiraell .»Yerar& Mousey) Approved
h e e ls of (Y ir a e l l .»Yerar& Mousey) Approved
e t ^ _________ l?r« Lt B» BroughtonIFroIe^Sor "in "e lia rge "o f ' t h e s i s
AH IHT Si’ll 9a£lOM & « » APPMOACTGH W MSs i t m u i . w9 S i m & to a M m m m m M M W 'mmme 0 ? OAHQI3B0IS n m m m ib fh o u s abb a m m a n m o m a i B , ~
M . .
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Timokm s u t a i t t e d .*4 , th e ? M 8 &.ty *£ %u®' tjrsd&mt# s c h o o l o f th e £ f * a i f a o f Ma,i*^layad.dh n a^ M al folflttmmM of the ref0lr«03£td £0# "t&#
' d e g r e e o f B o a t e r « f P h i lo s o p h e r
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t i l
$k© author mtmhmm to Ms *#pr©©l*ti©ii to
Dr* «* B. ttfelt© ant Jtr* #* Im» Bttvoll* food nut Drag
Admlnt strati on ©f the Qfeltot dtai©© Dofartaottt ©f
Agrlsaltur©* and or who©© smponrisioa this work was tons*
©at to Dr. L* B* Broughton ©at Dr* D* L* Drak© for
©at ©©&©%rMtlro oritisisas ©a th© prepar
ation. fhsak© &r© also da© t© Mr# I5* A* Clifford for
aasist&ne© in ©onstruotioa ©at ©trio© on th© memtrml
wo&g© photoiaotsr ©n& to Dr* B* A* Brio© far ©trio© ©a
*©ri©u© pkfsioal proBX©©©*
fM i tla a o rta t& o ja i s p r sso m ts t with th© p o r m iss io a
o f th e CM #£ o f t&« F o o t ©at Drag M w ia i ©tr& tioxu
iv
s e c t io n -.'ageT 'j k i - ; :k ?c% * t ' 1 • "|«L ft X. *,,* ,&«. '; V. LJ *’V t. ■*+ • .:•* **'*©*»*#*-**»»$*#*■* <J„
X X * rxl ,.'; X' '- ' i i i 0 i t i ' * ® * # * * * # * * # » * * » » * * * * * » * # * • * • • • • * * • * * 41
xli® aieasoram ent o f ttia oarotanoicl. pi uient'3in f lo u r• . • * .................................. • . * « . * • . . * • • • 4
C h a ra cter ! s a t io n o f th-a c a r o te n e id p i gment® o f f lo ur* I * * * * . * . * * * * * * . * . . . * * * * * . . . * * * .......... 7
if. 1 * \i rej.i ..Hk.'ili jj i. J W LiCf :-ii Oil • .»**-*#*#***** a «»!•♦»■* s * * .- * • * O
0 u t l i am o f i n v e s t i gat i on « * * * * » • . • * * • • « • , • • • . 8
f henry o f O o lo r im etry , Photom etry anda 'p O C t r O XiO t c :ll€ fb ry * * * * * * * * » * # * * • • • * * * # * • * ♦ • * * 8
d e s c r ip t io n o f liea tr& l ue&ge Photom eter* •* • » 11
D e s o r ip t i on o f 1:h.o to e l e c t r i c dp m tr o pho to - mater* IS
P r o p e r t ie s o f c a r o te n e id p ig m en ts • * * • • 13
IT . .hr -HI P P ff AL. ..................... IB
Oh&rsct#ri*a t i on o f the carotexioid pigments o f f 1 our by apr 1 i c a t i o n of tks v i l l h t i t t e r and a t o l l petroleum ethc?r~alcohol p a r t it io n p rin cip le* . . a* * . * * * * * * * * * . * * * . * . . . . * * * * . * * * .
Ch&raeterl nation of the ©arotenoid pigmeni© o f f lo u r by appl i ca t i on o f Qhromatographic A nalyst s * . « * * * . « * • • « • * « • . . . . . . « • * * • • * * . , « • • •
O h araaterisation of the earoten oid pigments o f f lo u r by absorption spectrum of the f lo u r e x tr a c t* ................
B e lec tio n o f f i l t e r f o r Neutral ./edge Photo- meter* ............................................................................ST
a tand&rdi 2a t i on o f Ifeatral a edge .Photometer fo r measuring carotene con cen tra tion * 33
Method developed in th is study fo r the determ ination of added carotene to f lo u r and macaroni products* ............... * • * • *• *• • «• • 36
18
S3
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of the '.wmtMk for ofQ m r o t B B B *. * • • » • * * .* « « • • • •* « * « * • .•* * • • ** * • » S f
'Pml®1 on of til#. *••**»*+**•»• ♦ * * *♦ »• 4S
fii# aoooraoy of t&# @atfaD&»«*•*•••*«•*•*#«•***» 46
Eosmlto of 4et«dn*&ft&*lQja of @©-0mlX#t oarotoo*1b ® m hk® m m h® & t l o m w * . f a r ia m * m r n m l l x m *m m m m m m lt # € f # s a t a o o & l# # * * « * * * * * * * * #* * * * * * * * * 4 6
I m t m T f o T m m ® * * • • * # * * * * «*• * * # * * ** * •» • * * *% *** * * * * 66
'Wmrtl&m, apflloatiom! of ■ %h® foutral s#tg#* ,* * » * * * «: * . . * « ' $%
? * m m s m * * * * * * * * * * # * ♦ * > * m
JUX *j'l* or XudA J: age
I S o l u b i l i t y Jn mg per l i t e r a t 25 d eg r e e s 0 o fcareten © and x a n t h o p h y l l * 15
I I a b s o r p t i o n o f o a ro te rn s and h a n t h o p h y l l * * 26
X XI A b sorp tion c o e f f i c i e n t in 30,a a b s o lu te a lc o h o land 20,Y anhydro us e t h y l e th e r o f a lp h a e arc ta n a ,A e ta ca re t© a s and x a n ih o p h y ll* • • « • » .* • • * • * • • • * • 23
IY h eaau reu en t o f O p tic a l C en tro id fo r ho* 511C orning g la s s 5 m m th ick * * • • * * • • • « • • • • • • • • • * 30
Y Ih o to m eter s e a l a r e a d in g s f o r ©anal co n cen t r a t io n s o f a lp h a and b e ta c a r o te n e and x a n th o p h y ll u s in g ]Jo* 445 f i l t e r * * * * * * • • • • • • • • • 33
YI D ata used in c o n s t r u c t io n o f r e fe r e n c e cu rve inA igura 4 • *••«.* a*************a****.*****.******* 34
Y1I h©suits on *iaearonl with added C aroteneex p re sse d in p . p . a * * • * • « • * * • • » * • • • • * • * • • ............ 44
f i l l R e s u lt s o f d eterm in ation . o f bo- c a l l e d c a r o te n ei ii mi»-)i©ached i.lour«•••••»»•***•*•«»**•*•••*•*• 4 f
u l t s f o r t o t a l c o lo r ( c a lc u la t e d as c a r o te n e ) and c a r o te n e in p*p*m* on a u th e n t ic eggyo lhs ir o a various ports o f the c o u n t r y * • # * « • » • • • * • • • * * • • • 49
XL fh e r e s u l t s o f th e a n a ly s is o f a u th e n t icm acaroni p rod u cts* ex p ressed in p #p«a«• • * « • • • • * 50
XI R e su lts of a n a ly s is o f com m ercial sam p les o faaaro ih 51i**-
A ll R e s u l t s o f a n a l y s i s o f egg n o o d le s * • * • * « « • * * • • • 58
AX11 C om parison o f r e s u l t s on f l o u r o f >«lntonv & g a s o l i n e c o l o r and t h o s e o b t a in e d on th e p iio to n e c e r • * • • • * • • • • * » • • • » • • • • • • • 5 3
v i i
OF W im nMpuge
i* absorption apootra of th-B aomcm airoliinaiis* B9m
Bm ^ m m t i tM t l i r ® absorption ourvos for oarotanoid pigsieafca« 30a©* ^raomlaaioj* at&rve for Oomiag glaaa f i l te r 1 0 * ©1 1 *
§ mm tMvkimmw* Si£m
4* Conversion almrt for n®atrml tto&g* ^lotoaotor# Oar# tana90i»*B$TBtion against male rcsa&ing. 34a
WXmmr im generally u&orstood a® the product o fetal nod
from tii© endosperm of wheat after certain milling operation
hare been performed* fh© term *fl©ar* when as#& la tills
artiel# will* therefor©* refer to whest flour# In mmmmu
with many other food® flour ten beei* defined by %he Urnitedm
Stmts® itopertaent of Agrlesltnre ssfallow ss "Flour, wheat
f lour, white fleer , i s the fine ground ywdeet obtained la
the eoem©ratal e l l Hag of wheat, and ©onaiata ©ssentially of
the stmroh mad gluten of the ©adoep©raw I t coat alas not more
than lifS of moistmr©, m t loss than l>§ of nitrogen, not isor©
than 156 of ash, sad not »f@ than 0#5p? of f iber* w s®m#
cereal eheaiet ha® ©Imrso t ori »©d floor a® ”» complsx biologi
cally aetiwe eeHel&el. material-** fii.# mmplmM matarc of
floor 1® at least partly represented by the chart published I B )
by Briggs* Urn list® ©Tor SO lagradla&t* la floor including
f ir s Tltaa&tto, pi gmcat® and about twenty*©®© ensyees*
fhere arc sereral grades ©f floor prodmoed la the com-
mere1*1 milling, ©©samonXy classified mm short patent, patent,
straight, f ir s t e l ear and mmmmi clear* the latter two
grades are of l i t t l e import ait®© in this study sines the
f ir s t clear la largely sold to Xmkmwm and the seeemd clear
generally need for wardens mmmmmXmX perjmemkss* fixes®
warloaa grade® of flour are pro a used from the some lo t of
wheat by at&nipuiotxon of the Tari ©as stream® daring the
milling process, rionr of the ammo grade any possess
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w ith th e la r g e c a p a c ity o f our f l e e r mi 11a today i t JLa
ap p aren t why a r t i f i c i a l b le a c h in g hua become so p op u lar a n d
w id esp read among th e f l o a r m i l l e r s s in c e a similar e f f e c t ,
a t l e a s t on c o l o r , mm be produced in a few hours w ith &rhl~
f iC a l b le a c h in g a g e n ts *
F lo u r b lea ch ed w ith n itr o g e n p e r o x id e { a ls c -p p r o c e s s )
was made the aubjjact o f e a r ly in v e s t ig a t io n s i n c o n n e c t io n
w i t h th e en fo rcem en t o f th e F ed er a l fo o d and Drugs Act o f
1908* F o llo w in g t h e s e in v e s t ig a t io n s th e c o o r o ta r y o f
a g r ic u l t u r e is s u e d food In sp ect* on D e c is io n Bo* lv-:-o in 1908,
w hich in b r i e f d e c la r e d f lo u r a d u lte r a te d when b lea ch ed wi t h
n itr o g e n p e r o x id e b ecau se o f th e a d d it io n o f a p o iso n o u s
su b sta n ce* in an a ttem p t to e n fo r c e t h i s d e c i s io n a e a s e
is&s l i t i g a t e n i l th e way to th e 8 aprons C ou rt, r e s u l t i n g in
a d e c i s io n th a t in th e aaoajit a s M , n itr o g e n p sro x id o was
n o t d em on strab ly harm ful to h e a lth * As & r e s u l t o f th e
duprome O ourt d a e is io i i Item 3 8 0 , s e r v ic e and He d i la t o r y
denouncem ent Ghem* 2 8 , was is s u e d in 1920* in th o s e e a s e s
mb or® b le a c h in g ca n n o t be shown to add a harm ful cu b atiu ioe ,
to red u ce f l o u r q u a l i ty o r to c o n c e a l ' in f p r io r i t y , the
announcement s t a t e s t h a t b lea ch ed f lo u r in i n t e r s t a t e coat—
moreo ca n n o t he h e ld in v i o l a t i o n o f the F ed era l Food ana.
Drugs -,o tf i f p la in ly and c o n sp ic u o u s ly la b c lo d to chow
th a t I t i s b leach ed * amm s t a t e s mmn r e q u ir e th e dec 1 stra
t i on o f th e ty p e of b le a c h in g a g e n t used and some f o r e ig n
c o u n tr ie s p r o h ib i t o n t ir o ly %he u se o f a r t i f i c e ! b le a c h in g
agents.
fhas® req u irem en ts for a r i l £is a l l y b lea e h sd flour bring
up th e problem of m ethods for the det©rmination of art!fleal
bleaching*
K I^ fO R IC IA L
52x® measurement of the ear©ten®!4 pigslent la flour*
Ih® f i r s t a ttem p t to d i f f e r e n t i a t e ble&ohsd ana un-
b l e a c h e d f l o u r w a s b y t h e m e a s u r e m e n t o f t h e c o l o r o f f l o u r *14 1
bin ton C19111 e x tr a c te d the y e llo w color from flour with
o o lo r l^ s s gasoline and compared i t w ith an O.OOIga potassium
a hr© mute solution in a c o lo r im e te r * Ho © a llo d th e v a lu e
o b ta in e d the ’’g a s o l in e o o lo r v a lu e 1* and o a lo e la te d i t on. the
basis of an a r b i t r a r i ly a s s ig n e d r a i s e of 1*0 for the a hr© stats
s o lu t io n * £h« o r i g in a l H inton method, s l i g h t l y m o d ified in
the; book of Methods, K#0*B*0*t 1935, is s t i l l ascid and h&s
been o f c o n s id e r a b le v a lu e* However* duo to th© d i f £ arenae
in hua betw een th e ohrom&te so 1 s t io n and th s g a s o l in e ©xtr&ot
of flour, mtJaor d iv e r g e n t result* arc f r e q u e n t ly obtained
within a group of analysts* Jinny of forts have been imde to
im prove the u ia to n m ethod, with special emphasis on s e e k in g
a r e f ©reuse a t ra a rd w ith th e earn# hue a s th e f lo u r e x tr a c t* ( 5 )
Jorgenson m o d ified >*latan*ii stan d ard by b u ff ©ring th e( 6 ;
ehroa&te so lu t io n t o & d e f in i t e pH* Kent-Jon os and Herd
u sed a aixtur-ci o f p o ta ss iu m ©hr© mat© and e o b a lt ni ir a te *
V ariou s o th e r skM aM i have been used in © arot^noid studies,
©uon aa a l i z a r i n in o h lo r o f o r m t nnpthoX y e l l om and orange
Q in water* and /*3 0b#n2ene in a lc o h o l* koac of th o s e
r e fe r e n c e s ta n d a rd s a re s a t i s f a c t o r y a s l a ovidonoed by
c o n t i n u e d work on t h i s problem*i f }
F e r r a r i and B a i l ay 11989} p u b l i s h e d a q u a n t i t a t i v e
method f o r i l ia &©b#Ffaia&tf on o f c & r o ta n o ld s i n f l o u r * c a l c u
l a t e d a s aarobeii#* a f t e r e x t r a c t ! , cm o f t h e p ig m e n t with, a
m ix tu re o f 9 3 a n a p h t h a and 7,9 a b s o l u t e a lc o h o l , t h e i r p r o
c e d u r e t o r t h e oi'JHcnuvraej&t o f e & ro to n o id n igmtmta web b a s e dI B }
on th e v.-orlc. o f be h a r t s * who measured th e amount o f o& rotons
l a s o lu t io n by use o f th # apa©trophotom© t e r « T his n e c e s s i
ta te d * a t t h a t tim e* th e i s o l a t i o n o f pure c r y s t a l l i n e
o s r o tc n e w hich was p repared from c a r r o ts* ihown d e f i n i t e
am ounts o f c r y s t a l l i n e c a r o fe a e wore d is s o lv e d in v a r io u s
s o lv e n t s and th e tr a n sm iss io n o f th e u i f f a r e n t oonoon-
t r a t io n a wore -a m sored i n th e ©pec tro pho torn# t e r a t ao
d e f i n i t e %im a le n g th * 435*1*.* from t h i s d ata a r e f e r ana e
cu rv e f o r th e r e la t io n s h ip betw een c o r e to n e c o n c e n tr a t io n
siid tra n sm iss io n , t a m e s ta b lis h e d * i a r r a r i and B a llo y * c
work shows t h a t ex e s H e a t r e s u l t s a re p o s s ib le by t h e i r pro
cedure* 2h© r e s u l t s arc ex p re sse d in part® p er sal 111 on o f
c o r o te n o id p igm en ts a s ©&rot<mo« fh s p r in c ip a l d isa d v a n ta g e
s s m s to bo th e us# o f & sp ec tr o pho to motor w hich i s a v ery
e x p e n s iv e in stru m en t aim ©a# not gatifiraXly a v a ila b le *(9 )
Q-e&des a t &1 119214} compared th® s p e c t r a l d i s t r i b u t i o n
c u r v e o f f l o u r e x t r a c t w ith t h e Win t o n p o t a s s i u m o h r a mate*
6
th e Yim%~jo in ts 0 h r o m te * 0 o M l t n itr a t© and. th e dprague
n a p h th o l y o l Xow~arch 1 , standard®# fh© ca n ra s o b ta in edo
c r o s s &t th e m ercury &re .lliu :, 4ah8.at arid th e a u th o rs con
c lu d e th a t a l l th r eo standur& s ar@ s a t i s f a c t o r y from th e
s ta n d p o in t o f i d e n t i t y o f s p e c tr a l c h a r a c te r is t ic ® a t fhe
m ercury arc l in e # However, when d a y l ig h t i s used e o n s id e r
a i l a d i f x o r ©no® e x i s t s , w hich a cco u n ts f o r th e d i f f e r e n c e
in hue betw een f lo u r e x tr a c t and th e r e fe r e n c e stan d ard s*
Ca the basic of reproducibility and permanence, pct& ssiuia
ehromate ta a oho sen as their sta n d a rd , fheir procedure 0 0 1 1 -
sis ted o f extracting w ith a mi art ore of 92i> naphtha and 7p=
a b s o lu te a lc o h o l and matching, in a colorimeter or M e ss ie r
tu b e s , th e f lo u r e x t r a c t w ith chromato s o lu t io n , u s in g a
mercury are aa a source o f light# fhe direaate s o lu t io n
was previously standardised a g a in s t a rotens and t h e i r
r e s u l t s on flo u r ' e x t r a c t s w ere , therefore, ejspressed in
part© oar m il l i o n of e&rotemoid pigments as carotene#C10)
F e r r a r i and Ores® (X t34) r e p o r t that s. f l o u r extract#
prepared by using a so lv en t composed o f a mixture of 9Sh
cleaner*© naphtha and 7> ab so lu te a lco h o l and stored £91
days in a brown, g la s s ©toppered bottle# showed no ®ig~
n if ia a a t change aa determined by the spectrophotom eter#
2hey proposed th a t a standard f l o u r ex trm ct be us ad. as a
r e fe r e n c e in making com parison© in th e c o lo r im e te r or
l e a d e r tubus* b oas la b o r a to r y equipped w ith a ep eo tro ~
photom eter w ould o f c o u r se be n e c e s sa r y to d i s t r i b u t e th e
V
sta n d a rd f lo u r e x t r a c t a t d e f i n i t e in te r n a ls *
Ohsr s©t©r i i a t i ©a o f the caroteneId pigm snts o f flo u r*
fh e q u a n t i t a t iv e mens or omenta o f th e c a r o te n e id p igm ents
in. f l o u r have fo r m e r ly been s t r e s s e d in p a r ts par m i l l i o n( 11 )
o a lo u l& to d a© ca ro te n e * iioni or-'*; i H i am© (19 1 £ ) r e p o r te d
th a t ©arctan© was th e p igm ent o f wheat f lo u r * Ferrari and (7}
B a l l s y (1989) compared th e a b so r p tio n s p e c tr a o f a p rep a-
r a t i on o f pure c a r o te n e w i t h f l o u r e x tr a c ts * l*hey c o a e lu d e
t h a t ?,th e s p e c t r a l d i s t r ib u t io n cu rv e f o r th e f l o u r ©attract
p o s s e s s e d c a r o t in c h a r a c t e r i s t i c s , b u t e v id e n c e was o b ta in ed
o f th e p res once o f a n o th er p igm ent i n a d d it io n to c a r o t in * '*
U k eir e v id e n c e ten d s to e s t a b l i s h th e p igm ent o f f lo u r ex
t r a c t a© c a r o te n e «d th l i t t l e i f any x a a th o p h y ll* U ark icy ( 12)
ni'id B a i le y (1955) mad® a p p l ic a t io n o f th e b l l l s t & t t e r andf 13)
w t o l l method o f s e p a r a t in g c a r o te n e from xan b h op h yll baaed
on th e p r e f e r e n t ia l s o l u b i l i t y in a naphtha-meth&no1 m ixture*
f h e methano 1 used in th e m ixtu re was 90^ m ethanol and 10)4
w ater* f h e i r a n a ly s i s o f fo u r sam ples o f w heat showed 12*8
to 34*8;* o f th e t o t a l p igm ent to be ca ro ten e* a b so r p tio n
carve© w ere do term ! nod on th e naphtha ( ©arc to n e ) and
aieth&nol (x a a th o p h y ll) f r a c t i o n s t bu t th ey d id n o t draw any
d e f i n i t e c o n c lu s io n a s to th® n a tu re o f th® pigm ents*
B oth F e r r a r i and ;-iarlloy a ttem p ted to e h a r a © to r ise th e
c a r o te n e id iiigstan t In f lo u r by chroma to grap h ic a n a ly s is *
X a a th o p h y ll i s adsorb ed by F sw ett Ca 0%. tow er from a h ydro-
8
carbon solvent* Ferrari and. Bailey failed to get adsorption( 18)
o f a- f lo u r e x tr a c t on a Qa tow er* h a r te l^ y s a d Bai l e y
found p a r t o f th e f lo u r p igm ent was adsorbed in p a s s in g
th rou gh a 42** 0& 0 0_ tower*o
(* .14 . It An ~>I. •> J J o . > i ' B|
Out 1 in© of' in v e s t ig a t io n *
I’ho n e u tr a l wedge p h o to m e te r a s d esig n ed and d o v o lo p o d
by C li f f o r d and B rio© , h a lte d s t a t e s Food and d ru g /Ldaitsi»~
tr& tlon * bepartaaent o f A g r i a a l i t i r © , i s a r a th e r in e x p e n s iv e
ap p a ra tu s and h as m m m f p r a c t i c a l a p p lie a tI o n s * ??he m e a su re
ment o f th e c o lo r o f f l o u r t o a © too t b lea ch in g * and o f th e
e o l o r o f macaroni p r o d u c t s t o d e t e c t a d d i t i o n o f c a r o t e n e *
i s e s s e n t i a l * A c c o r d in g ly , th e a p p l ic a t io n o f t h i s i n s t r u
ment to t h i s work h as been attem pted* t h e a ccu rst© isu a a u r t*
m $ n t o f th e e x tr a c te d c o l o r 1© b u t one p h a s e o f th e problem*
Anoth e r pirnse e q u a l l y im p o rta n t, i s the d evelopm en t o f m
m ethod f o r t h e e x t r a c t i o n o f c a r o t e n e from m acaro n i
p rod ucts*
fheory o f c o lo r im e tr y , photom etry find n p e e tr o photom etry .
tthmi a be&ra of raonochrom&tie l i g h t fa lls on a homogene
ous layer of substance, part of the light In ref 1 retail, part
is absorbed* and part is tr a n sm itte d * lit most cases the r e -
flooted light can be neglected* hence* for practical purposes*
th e i n i t i a l light e q u a ls th e absorbed l i g h t plus the
9
traasmltte& light* Fresnol*a formula shown the relation
betw een I n i tia l l i g h t an t reflected l ig h t * Th& sm a ll
amount of scattered l i g h t any also b© n e g le c te d *(1 4 ) 115)
The law s o f Lambert and o f Boor p la y an Im portan t r o le
in c o lo r im e tr y , p hotom etry and epeebr© photommtry* Lambert* s
law : The asiount o f monooliromat 1 o l i g h t absorbed by a
substance i s p r o p o r t io n a l to th e Intensity of th e incident
light* represents transmitted light and I Incident
light*
I t QC10 or 1.1 « i I o
The f a c t o r A g i r e a th e fra© tion o f I n c i d e n t l i g h t tran s
m itted by a l a y e r 1 cm* i n th ickness* This fa© to r i s s a i le d
th e transmission coefficient* The in te n s i ty o f the tran s
mitted l i g h t decreases in geometric progression whoa the
thickness of t h e l a y e r through which the l i g h t t r a v e l s
increases i n arithiaetrio p r o g r e s s io n * T h e r e f o r e , t h e f o l lo w
in g r e l a t i o n e x is t s between I , and It o
I . • I ALt oin w hich 1 d e n o te s th e le n g th o r th ic k n e s s o f th e la y e r *
I f light o f i n t e n s i t y 1 fa lls ©a an in fin ite ly th in la y or
dl of the absorbing madias, the decrease in intensity d 1
o f th e incident light is proportional to I and d lt
di % ~r I ©1 oin which 1 i s a p ro p o rtio n -a ll,ty fa c to r *
I n te g r a t in g th e letter equation.
Th.B o 0 m l i R t k i s o f t e n c a l l e d th e a b so r p tio n c o e f
f i c i e n t # Changing from n a tu r a l to B r ig g sia n , lo g a r ith m s
i m i 0 no <g XQ T 1.0 o r I • | lo g- L 1o
Bunsen and Rose®® c a l l e d th e c o n s ta n t is th e e x t in c t io n c o e f
f i c i e n t # I t i s seem th a t J I s th e r e c ip r o c a l rain® o f th e
t h ic k n e s s o f th e la y e r in cm* a t w hich i s eq u a l to i / i a
Beer1a law: bo far the l i g h t absorption and trans
mission hare been a fu n c t io n of the th ic k n e s s of the layer#
However* we are m ain ly interested In. solutions# Beer found
th e same r e l a t i o n betw een tr a n s m is s io n and c o n c e n tr a t io n
that Lambert had found betw een tr a n s m is s io n and th ic k n e s s
of th# layer# b’e# th e r e fo r e # have th e came relation as in
e a s e o f Lam bert*« law w ith (eJ c o n c e n tr a t io n , s u b s t i t u t e d— cfo r I D th ic k n e s s - 10 #10 * C om bination o f th e two
— id •» ie q u a t io n s , I t • I 0 #10 *’ and 1. ^- I #10
g iv e s th e fun dam en tal eq u a tio n o f c c l o r l a e t r y and s p e c t r e -
piic te a # t r y
- k 1 ,,e i fo
fit# v a lu e o f .J depends upon th e manner o f e x p r e s s in g tit®
c o n c e n tr a t io n # I f o i s e x p re sse d a® m olar eon® ea tr& ti on *
11
B dsn©bos m olar e x t in c t io n c o e f f i c i e n t aM i s equal to th o
r e c ip r o c a l v a lu e o f th e th ic k n e s s i n ca* o f a 1 m olar
s o lu t io n o f w hich I* i s 0*1 o f I . 1m ! M s pros on t stu d y fa )b» O
i s expressed i n m g/l and -S d e n o te s e x t in c t io n c o e f f ic ie n t*
C olorim etric measurements may fe© a t i t i n & c o l o r i m e t e r
which i s an instrum ent i n i to io h th e th ick n ess o f t h e l a y e r s
c a n h a changed and measured e a s i ly , and sM © h a l l o t s com-
pari son o f t h e am ount of transm it tad l ig h t * Two s o l u t i o n s
Of m c o l o r e d su bstance having ooxm en b ra t i on s and e g c a n
b® o p t i c a l l y b a l a n c e d o r s e t a t t h e asm# c o l o r i n t e n s i t y *
^ t n * * t * ~ 1 ° 1 s 1 1 0 ° 8
fhom 2 ® C onsequently, w ith the as© ©f a
solution of known oon©©ntr&tion, the measurements of the
depth, o f the known and unknown s o lu t io n a t equal [email protected]
a llo w s the c a l c u la t i o n o f t h m con eeatr a t i on o f the unknown
so lu t io n by the l a t t e r equation*
Uesoription of Heutral dodge Jfiofecaseber
T M m photonieter i s s i m i l a r t o th e on# d e s ig n e d fey
C l i f f o r d and B r ic e * It c o n s i s t s of a wooden box IB in c h e s
l o n g , 14 inch#© w ide and 10 in c h e s h igh* ‘1Thm t e c h i s
fitted w i t h a solid ground o p a l glass for the r e f l e c t i o n
o f t h e l i g h t , furnished by f o u r 100 w a t t projection bulbs* f l i i s l i g h t shinee th r o u g h two h o l e s i n t h e f r o n t o f t h e
box fo rm in g two beams, i n the m $m ® vertical piano, w hich
ere brought together by an eyepiece* fhe eyepiece is the
1 z
comparator heed o f a Dsnaeh and Bomb Ho* $600 hemoglobi*-
noma tap* A s u ita b le f i l t e r i s used in th e #yepi«io® to
l i m i t the ab sorp tion to a s narrow a spectral baud a s p o ssib le*
fh e f i l t e r chosen f or t h i s work i s a d o m i n g g l a s s # Ho* $ 1 1 ,
v i o l e t , § am th ickness* On t h e f r o n t o f th e I n s t r u m e n t j u s t
be low tli® ey ep iece i s a co m p artm en t c o n t a i n i n g a ca rr i age to
which i s a t t a c h e d a n e u t r a l wedge and a so a le* The wedge i s
a Wratten g e l a t i n e wedge c o n s i s t i n g o f a g e la t in e f i l m
cemented b e tw e en two s t r i p s o f g lass* I t i s shaded f ro m a
l i g h t t o a dmrk end w i t h an absorption c o e f f i c i e n t o r d e n s ity
r a n g e o f 0 *1 # f h # s e w edges a r e n o t permanent due t o gradual
f a d i n g o f t h e dye oomponent by h e a t and l i g h t * However,
t h i s iiedge h a s b e e n used a y e a r and f o u r m onths w i th m s i g
n if ic a n t change* I t i s now p o s s i b l e t o o b t a i n permanent
w edges m ate o f n e u t r a l g l a s s * f h e c e l l s u sed f o r t h e
s o l u t i o n s are made o f pyreac g l a s s w i t h fu sed on corex
windows# The s o l u t i o n I n t h e c e l l i s placed i n a h o l d e r in
on© beam o f l i g h t w h ile t h e o t h e r beam o f l i g h t p asses
through t h e n e u t r a l wedge* $h© two f i e l d s a r c b a la n c e d by
r a c k i n g t h e wedge and t h e s c a l e r e a d i n g i s t a k e n .
D escr ip tio n o f .P h otoelectric 3'pectrophotomet@r
fh c p h o to © la c tr io spectrophotom eter meed in th e sp ectre*
p h o to m etr ic measuremsatB mum d es ig n ed and assem b led by B* A*
B r ic e , U* b . ie'sod and Drug Adati ui. s trm t i on* L ig h t from a
© m il tu n g s te n bulb i s fo c u se d by s Ia n s on to th e b i l a t e r a l
m
outran## milt ®t m §-is#pt*i*r oomt&st 4#*l*tl#s m&mmhr® rat# r *
0 # le rs & g l m m t u t o r s # r a so#& oh## aorXtiig: i s %h® h X m m *n&
r#4 @fiii of tli# spootrom to rotsso© srrara 4m# to stray light#
tfoxtMhraratls l i g h t morn## ftt« tfe# tiXato ra l exit s l i t of
tli# lastrusioRt# the width of both s ilt# 1# s&Josted at sash
wseo Xo>ls. settlag to git# * hasA #f tk# epostrasi aitotrfc 40/i°
v ita for «#e2i MMmreitiecit* flat# voiitahresKtl# bean passes
throttgh m 11 ght«t i gh% metal eovpartaioat ehleh eost&i as two
similar g la s s ©all® with plan# parallel oa&s# on# fo r t h m
solrattt and #m for the solution.# 4 @1141 m I#
provided to 'bring either ##11 in to the 1ighb beam# th e ##11#
or# E#§ ea* Is length* After pm®ml mg tftreiigh os# of the
s e l l s th e l i g h t toon f o i l # ou a gaoH filled petssfiimisi phste~
eleetrie tmbe* I hot#eleetrie ewreats or# measured by moos®
of m ea se l tiw # a tr r a r ealvaxieasier w ith timsopmromt e s s le *
fh e trssom ittM oy i* determined by flooloMI a w ire ser o sa o f
knows traaam lssio ii, whisk i s appraxiaateljr s t e a l to th e ea**
knows# under th e ##11 soataisl&g th e salvest# fh# m la tl ob
o o l l 1# #114 under the bmm o f l i g h t and the d e f le e b io s read«
lag t akaa* I s I lk # vanner m defleetistt reading i s taken fo r
tit# eoiveat ##11 pirns oorooii# fh# r a t io o f thm ssaple a#-*
f lo e t ie s * tim es the knows transaissioxi o f th e a i t a g l v m
%ha traneml tt&sey o f th e
( 1 6 )Properties of Oaretomid Fipwsbs*
A kaowl edge o f th e prepmtlee o f the earatefield
pigMBUt® 1# e s s e n t ia l# espeeially for the eharastorlsatios
14
o f th o pi g /ie n ts i n f l o u r # th e developm ent o f it laatfcod
f o r th ^ ir «txtr& etlon aM nminnrm^mtm Thm t e e r r in e ip e l
oar 0 tar1 0 .lt p i a a w t s o o n o o m o t in the s t t t | ar* o a re tfm e
end mn.tv > h y il# a lth o u g h rrypt^scanthltte oouor© in, itoo& lee
t«© to th e ogg preaant* X'h&ee pigment© « .l l harm m&Xmnlm®
w ith 40 carbon atosw** Si hydrog w ntom# mmA 11 d o u b le ben&a*
.^jwRtfeoohyll lia s two a lo o h o l group& aM th in e on®
a lc o h o l group* £h© o& rotene aaafitlioph^ll form © r y e ta le
o f m e t a l l i c lu e t r e tsi th a c o lo r o f v i o l e t f o r mmrotmm to a
f o l lo w f o r m ath apA flX # I n m%r th#y a e t o x ld i s e w| th
I f ic r e a c e In w i g h t end Xomm o f c o lo r arid or* *. t ^ l l i e a b i II ty*
For mob a high degree of tiasat oration, thee© pi* are
quite ©table* !i*he fomala f o r carotene in s3_ IL,* and4u Pisxantho p h y ll d^.. 0^ e a t Icryptaxuijtliia© a ,„ 0* jii#
Cl*?)e t r u c t u r a l 4 o r •■sola f o r b a te aero tea© dov«.tlo^ed by h& rr«r#
# t a l , t la a lran*
crtr c«j o*>,c#J'C r« , c # , <^J c *4, e ,A rt H / H h H • H H H H ‘ J? S 'S r 7 i / Cl* c - c = C- " C - C . - O - C , - c l —c - c r C .“ C s C - 6 « C - C 5 C - C ; c « C C. v
H*.c °-'c % b e ta c&ro t one ^C cM,. Wv
faro th r o e a a re ben* iso a a r ii* .^Ijilm aaro fc^ne t i f f ore
from b e ta in th a t th e r e 1® one aoe^ saaetrie $ atom a t
p o s i t io n 6 on one o f th e rJUigsj cl.tin to a ohiffc o f th e
d o u b le bond fr o si ;pii:t. than 6 to p o s i t io n 4 t and one M atom
f-M It from € to 6* h&ntho \ 1 1 o r I n ta in i s Blm llm t to
a lp h a c a r o te n e b u t w ith one o }| .group in p lan a of a H atom
IB
i n p o s i t i o n 0 o n s & a l i r i n g * Z tm s t r u c tu r a l Q h n r m s t o r i s t i e s
o f t h e © B T o t o n o S t i e a re I I ) t h e l a r g e m im b m * of d oub le b o r i d e ,
( B 1 the? r o ■: p e t i t i o n o f the? ! s o p r u n e r e a l do.© w i t h t h e l a t e r a l
210t h y l g r o u p , ( S ) a p u r e l y a l i p h & t l e u o n f 1 g u r & t l o n , o r t h e
in©lust on of at most two h y d r o a r o x a & t l © rings (gsn-arally
&1 nim or beta. ion -on e), and (4) the preferential s i t u a t io n
o f oxygen , ©lion prea#at, at p o s i t io n th r e e in the ring o r
r i n g s , eo a n tin g frora th e % a s te r n a r y e&rbon atom, c a r r y in g
two m ethyl groups* Phe oxygon in th e tr u e o a r - o t o n o i dm l a
e i t h e r hydro x y l i e or k e to n ie o r both* fh& o n r o te n o la s a re
s o l a b i a in th e o r d in a ry f a t s o l v e n t s and I n s o l u b l e i n w a t e r ,
w h l o h f o r t h e . I r a e o w a u l a t i o n I n l i p o i d - * r i c h
m a te r ia l* Carbon d i s u l f i d e and © h lo r o fo m t r e th e b e s t! 1 8 }
s o lv e n t s * c o h o r ts gira© th e fo l lo w in g t a b le o f s o l u b i l i t y ;
f a b l e I
c o l a b i l i t y in mg per l i t e r at 3 6 d e g r e e s 0
b o l T e n t C a r o t e n e , -* & n th © j> h y l l
Patrolearn e t h e r 62d 9*S>I h s o l a t e e?fhyl a lc o h o l lb# a BOX* ha b s o lu te ra e th y l a lo o h o l n e a r ly ia s© 1a b le 134*9..thyl e th e r 1006 9£>3*0
2 h # f l g u r n s o n s o l u b i l i t y e b o w t h e b a s i s fo r t h e e o p e r a t i o n
o f ear©ten© and x n n t h o r h y l l by th e .v i l l a t a t t e r and c t o l l
p a r t i t i o n t a e t h o d * wh S. a h a o u n i a t s o f s h a p i n g m p e t r o l e u m
e t h e r s o l u t i on o f th e s e two p i ( $ a e n t 8 w ith aqueous m ethanol*
By p r e f e r e n t ia l s o l u b i l i t y t h e ear©ten© g o es in to th e h y d r o
c a r b o n la y e r find t h e a o a r t t h o p h y l l in t h e aqueous m ethanol
1 6
la y e r * 0110 hundred p e r c e n t q uan ti ta fc iv 0 s e p a r a t io n can
not toe mad a by th is proo ©dure bat th e sep ara tion 1® suf~
f l c l e n t l y co m p lete f o r m ost purpose®* 6 m h p i g&ent® a®
lyoopcnc, ca r o ten es , arid xnnthophyll e s te r s go in to the
hydrocarbon layer*, ant th® aqueous methanol la y e r s con ta in
th e fre® xanthophyll* such as lu t o ia , se&x&nthine* sM
b ix in and cro c a tin*
Ia c id e n ta lly * referase© i s mad® to a recen t e& rtitlon(19)
method toy Clausen and iio Coord (1936) ap plied to caroten e
and acantho p h y ll separation* the procedure in v o lv e s on ly a
s in g le shake-out between two im m iscib le liq u id s* hexane and
aqueous d ia ceto n e alcohol* f M i s in g le op eration I s made
p o ss ib le because of the favorab le d is tr ib u t io n c o e f f ic ie n t
fo r th ese two liq u id s*
fh e in* p* o f caroten e and xaathophyll i s q u ite h ighI 20)
and th e v a lu es in th e l i t e r a t u r e are variab le* Im ith g iv e s
iiim ■»* f o r alpha and beta carotene as IS? d egrees Ccorr* )C 21)
and 104 degrees (corr*) respectively* Miller gives a* p*
fo r xsn th op h yll mm 190 degree® 0* fh@ beta caroten e and
xaathophyll a re o p t i c a l l y in a c t iv e w h ile aloha c a r o te n e i s(23)
o p t ic a l ly a c tiv e* B alth g iv e s i t s s p e c i f ic o p t ic a l r o ta t io no
bb +311 In benzene a t wave len g th 7066&* l*h@s® pigment®
show d i f f e r e n t absorption sp ec tra , which are more or lo s s( 2 2 } '
c h a r a c te r is t ic o f the pigjraent* bmith g iv e s , fo r alpha
caroten e in d(};,> a lco h o l and e th y l e th er , maximum absorp
t io n &t wave len g th 44BG and 476Gd® and fo r beta caroten e
17
at 4530 sod 4610ii* I n the same aolve.ot the valae® forC M }xanthophyll. read fro® mil®*" 8 © w a s , are 4470 and 476GA
(.rigiire XI*
Carotene and xanthophyll solution® are fairly stablein patrolman ether and alcohol solution* Caroton® is.{IB |hoitavar§ more stable than nnthophy 1 1• Sohart* found no appreoi able change, by cpootrophotorn®trio measurement©, for carotene in petrol earn ether or alcohol stored in ice boxover a period of ISO days.
fhe alpha and beta carotenes arid fcryptoxayrithlno are biologically act!re a© a source of vitamin A, probably duo to ©nsyismii© hydrolysis. Xafithophyfl sat the other omrotonold pigments produce no vitamin A effect* Caroteneis considered the mother substance {pro-vitamin) of vit&min
{ B5)A* Culer ©t* ml*, showed the vitamin A eff ect from batscarotene was twice as much am from alpha carotene* This-
woold be exuoctod from the formula* Beta carotene shouldgive two vitamin A emit© and alpha carotene and, Irypto-xanthine should give only one vitamin A unit* thm role ofo&rotenola® appear to bo important la plant met&bali ssi.talthough as yet abacare* 1'he occurence of the oarotonoi dssare principally in the plant kingdom* Usama carotene israrely encountered, and the majority of leave a contain, beta
< 86)carotene* MaeKImnay found, in §9 different varioties off 27}
plants, that the alpha carotene varied from 0 - Shu* daith report® the pifpaaat© of tea leave® and hors© chestnut loavesas nearly mil alpha carotene*
18
Oharactsrisstioa of the earct©noid pigment© of flour by application of the *<111© tat ter sad Atoll petroleum eth©r-slack© 1 partition principle*
In connect ion with t h e study of dlf f erentiatlon ofbleached and unbleached flour the raenlts of the &m>li cation
(13)of t h e Willst-atter and 8to 11 p a r t i t i o n method 'fey Mmyltley and (IB)Bailey on the flour pigment© load to the idea that a now
possibility might exist for th© tiffercnti&tion of bleached a M unbleached flour* Heretofore the reduction la total pigment color has been used as an aid in the detection of bleaching# Am cited shove «&ar&ley find Bailey reported that the carotene on four sample© of wheat ranged from 12*8 to 34#8,B of the total pigment* She question naturally arises what the relation 1® for flour* It Is conceivable that the so-called carotene fraction in flour might be more or less constant, or at least more so than the total color* Also it may be that bleaching agent© have a greater effect on the so-called carotene fraction than on the so-callcd xanthophyll fraction or via# versa* *>ueh information would bo of value- In flour bleaching studios.
Bartley and Bailay*e procedure for the separation of the flour pigments in 93 - naphtha mud absolute alcohol, was applied to several samples of flour# Bhe emulsions formed m e re so disturbing and time consuming that finally a 95A petroleum ether and f p alcohol mixture wa© substituted for the naphtha and alcohol solvent, which resulted
19
In i i j diats separation with no emulsion# ilany samplm
{approximately SO) ware analysed "by this procedure, namely*
extraction of the pa * sts from the flour with the patrolsum ether-aleohol solvent and a separation of the pigments similar to 5arkl@y*s procedure# The so-called carotene ranged from 5*0;- of the total color in dor am flour to 54g of the total oolor in the bread flours# The bleached flours showed by this procedure a reduction in both the so-called
carotene and xanthophyll fractions* ^hile the so-called
carotene could be duplicated i f the check analysis were
made at the same time* i t frequently happened that incon
sistent results wore obtained when the ©ample® were re- analysed at a later date* It «e® believed that possibly the saponi f I cation during the procedure Might be a factor*
Therefore, the method used for determination of carotene in
macaroni products was applied to flour# fills method requires
the saponification and extraction of the pigments fey heating
on the steam bath with a potassium hydroxide ©atorated
solution of ethyl alcohol# The results obtained by this procedure gave only a trace of the so-called carotene
fraction* The results of analysis of 68 ®ample© of flour
and semolina gave a maximum value for the ©o-called carotene am OmBO p*p*a«* the great majority of ©ample© being around
0*10 p#p#m# The©© aaousts are rejpoaiad as a trace* Three
sample© analysed by a procedure similar to ii&rklcy and
Bailey but using petroleum «thor-alooho1 solvent gave a
so-called carotene figure of 0*88* 0*90 and 0*90 p#p*m#
30
l * h s s s saa# © e l u t i o n s w a r # in a vacuum %o aboutID e e , sad two i t r e t r e a te d wi th bO 0 0 o f & sa tu ra ted .
a lc o h o lic potassium hydroxide s o lu t io n 0 s the steam hath
& a l t l i # s e p a r a t i o n c a r r i e d o u t a s i n t h « s & p o n i f i o a t i o n
m e t h o d f o r d 0 t e r a l m a t 1 on o f o&rot&ae* t * > r e s u l t s f o r iso--*
a i i l i e S o & r o t a n e w e r e 0*X i? a n d 0 * 1 9 p * p * » * j?h© t h i r d
solution was treated with bo 00 a lc o h o l i c potassium h yd rox id e
and a l l o w e d to ataod o v e r aiaht at rooai t e m p e r a t u r e and the
separation carried out by t h e nmmm method for the d e t e r a i l -
n a t i o n of c a r o t e n e * B10 r e s u l t f a r t h e so - c a l l #4 e a r n t o m e
f r & o t l c a w e # 0 * 1 0 p « p * e u t w o s s a p l e a o f t h e i « a e f l o u r
w e r e e x t r a c t e d d i r e c t l y b y t h e s a p o n i f i c a t i o n s i e t h o d f o r
d e t e r m i n a t i o n o f c a r o t e n e * S h e ra sa ltn on. t h e s o - o s l la t
e s r o t e n e f r a c t i o n w a r # 0 * 1 3 a r id 0 * 1 4 p * p * & * S h i # s a s t p i ® o f
f l o u r w a s c h o s e n s i n e # t h e eo<~e a i l e d c a r o t e n e f r a c t i o n r e p -
r e s e n t s S40 of % he t o t a l c o lo r* on# o f th e M g lis is t
©neoon'tcred* o th e r f lo u r s gar# n ija i ls x rceuX ta*(2 8 i
K e r r o r and iililkwa (1 9 3 0 ) hers shown that x a n t h o p h y l l
i e v e r y aoiasionly f o u n d a s a n o e t e r o f th e f a t t y a s i d e * F t i h n C 39}
and Broeloaaon (11)33) have shows that the xcmthophyll esters,
h o lon ien and phys& lien would appear mm car#tone in the
petroleum eth er la y e r , and th a t the fr e e xeiith&phyrll would
m pt\pr la the alcohol layer* I t is thus ecseltxdod that the
high and Inconsistent results ohtainod by the petroleum
#ther*HDleO'hcl p a r t it io n method* s im ila r to the &m& \m ^4 toy
*Xar5cley and B a ile y , arc due to incom plete e a p o n if ic a tio n o f
S I
x a n th o p h y ll e s t e r s # One argum ent a g a in s t t h i s c o n c lu s io n -
may be th a t p ro b a b ly th e sm a ll amount o f s o - c a l l e d c a r o te n e
was d e s tr o y e d a t th e tem p eratu re o f b o i l in g a l c o h o l i c
p o ta ss iu m hydroxide# t h e r e f o r e , a s o lu t io n c o n ta in in g l#5t>
p#p*m* o f car© tone prep ared from th e c r y s t a l s woe added to'
th e r e s id u e from t h i s earn© f lo u r {w hich had a s o - c a l l e d
c a r o te n e c o n te n t o f 0*90 p*p»m*)• £he c a r o te n e o b ta in e d by
th e 8 apen i . f i c a t io n method f o r th e d e t e m in a t io n o f c a r o te n e
was 1*S0 p#p#m*, o r a reco v ery # T h is d a ta to g a th e r
w ith o th e r experim ent® on la r g e r amounts o f added careten ©
show th a t c a r o te n e in n o t d e s tr o y e d by h e a t in g to th e
b o i l in g p o in t o f a l c o h o l i c p o ta ss iu m h y d ro x id e f o r o n e -h a lf
hour*
On th e b a s i s o f t h e ir b eh a v io r l a th e p etro leu m o th e r -
m ethanol s e p a r a t io n , i t m&y ho s t a t e d th a t p r a c t i c a l l y a l l
th e ea r© to n e id p igm ent la f l o o r i s x a n tticp h y ll# C e r ta in ly
f o r th e p urpose o f th e d e t e c t io n ©f added c a r o te n e to f lo u r
and m acaroni p ro d u c ts i t may be assumed th a t th e o a r o te n o id
pigm ent o f f l o u r i s x a n th o p h y ll* I n c id e n t a l ly , th e f o l lo w
in g f in d in g s on th e r e s u l t s o f b lo & sssy t e s t s on f l o u r a re{30}
o f I n te r e s t* B eck er and von Bangal - ds&bo (1936) co n c lu d e
th a t r a t s n o u r ish ed o n ly w ith f l o u r , showed more o r l e s s
c l e a r in d ic a t io n ® o f a la c k o f v ita m in s A and ix* In
a d d it io n to th e sto p p a g e o f grow th, xero p h th a lm ia 5;r e t ic e n t ly
appeared in d ic a t in g fe e d poor in v ita m in A* d in e a th e
c a r e to n e id p igm ent o f f lo u r a cc o rd in g to th e method o f
scvpmratloa aa©& I s ao&rly a l l :^mth©phyll# %hm &ppli®&ti©a
©£ siiali a method f o r tiff© ra& ti& tlon of bleaeh©* and mi**
bl®a©h©4 f lo u r in ©XI
Qlmrno t a r ls a t i ©a o f fch® ©arotoaoid pigment* s i f l o w toy s r r i ioa tlox i o f OtirosmtograpM© Analysis*
Doflait© proof tkmt m n th ap h yll i s bha Mrotftfnold
pl&^ent la f lo u r i s looking# tto rrfo ra ., th© fo llo w in g
©toomato&raphi © pro© ©dor© to® b#«a £® i low©6« The so 1 s t I: one
o f %tm i lou r p lg m n ts ©btninod toy th o n n p o n i t i e&tion a©thod
on th® ot&mm b a th , am &lv©& In t h i s jmi sj% m y ©arry l e s s
IctffTf©ring a& tarlal tk&n th ose sand by /© rrari ant uarkl«y#
A eoordiagly , in th is study a 300 g-i* sasapl© M e troatod by
t t o a e p o a if io a t lo a aat&od and th© potroieina ©thar so lu t io n
of th© | i i©nt wt# poor-sadl o n to m Os 00^ aaaorption tow or*
Dto adLaorptlon was a li$ U t and fa r from tfc© © h& rseiw isti©
ad sorp tion o f a © olu tloa o f ©rygt&llin® x&nthophyll in
p&trolonte srtbor* h j ©on® u itm tlo n o f t'h# ui i*s it in th©
flour oxtru&t was n t x f f lo to a i to list# shown adsorption*
l.o Da J >, adsorption towor® wor« prepared by pouring a©aspens! on o f tfcts dried Ca 00g in naphtha Into the tab#
f 311and or & wu&k s tr a in (1934) h&© ©tuidi ©& the as# of
d if f e r e n t «d®orbing age*t© fo r fefc® »®p&ration o f o&roten&ld
pigpa©ntfcs* Dmoii «d»orp«nt» as rln Og* ub 0g.t §r^ 0^t o to r w a l*
fo liar*© ea r th , hydr&lo to l^ 0^} and lig 0 ware invoatig©t®&»Ho f in d s & mixtur® o f »u&l a r ts o f o (a p e o ia l pr&ps-
r a t i o n ) and h y f X©~imper©sll t o th© i m o t o d e s i r a b l e f o r i i i#
/ooporotloa o f ©aroton® mat or for ©oj»a?©ti©<k
• f to© imstoro*fM@ H H m we© oMortlagXy ©sot la eo&iiiifiotlosi o f
tMi oto&r to pm m k W m adsorption tower* aat floor ojctroot* $r spar at otatlorljr to too-oos ad oa to© 0 a 0 0 tooor*#&© ©oisrot onto to© towor M U ©ll^kt omtiOA o$$ll«ft* 4
i^olXow to a t m o ©fetoimt a t to# top wMok we© ©omowh&t
■ wprmM, ©of ®mA m t ©atiroiy Im ipviitti ml to to© tout ©In* jtolnot fr&m a solution of por# somtootojrll, PtiM^ %hm
, mmtmm town «& to potro lom other ©aooot ora© ©©para t i on* tot «o ooporotloa m f f lo lm t lj f t o f l alt© to p retf too o f
oarotoci#* Hy&ralo was ©Iso tr io t w ith m feottsr rooolto*
flus. OMlf* prototl© latsrforioo #©tot#noo© la floor oaetroot molt soon to to tomfMHrtfttoto-. mMo**- ©at ■■tot© l o -proomt la #m!i © m il M i l l to to oom
Gtitarmtwlsatioft of to# oamtoiioit ptgnmto of floor% absorption sptot^si of too flour mtrmt*
■ flu im jfom to to four o r ito r iii for € i f t ©roabioMoii of too ©arotM olt plgmmtm mlmm. in too on© t e l l in# fora*
•n m o lf i «u p*f option! rotation* btoarlor o f to o ir oolotloft
t t « f i o to o to lto ©«*»i© to# o tom otorlo ttM o f too :Optotrol absorption oorro* An oxift©mm«ftil attempt m m mAo
’;to orrotoll&i* ■©©«# of too 'p&gMttt fr©» floor ' oxtroot* ©#m** too © m il amount o f plgmxtt prmmrnt mmh to© nature
■ \;,:.# floor it ooowo alomt m m m m m rm k lo to mgwot to otto la f^otolo# . IMi ©Halmt#© too a* p* mat optloul rotation
anA loorm for ©tuAp too, oWrorpbtoa spsotro of too floor
m
fh# m bm rptttm mpmtvmm wm dotomlMft on tho
xaathophjrll fm a t ie m mul th* mmxmtmmm frostlorn mm mmmrmX
f lo u r m&trm&t® preparsO thm m&pomlf 1 o o tiom m®%h®4> so #4
in th is staty* ''£w®m®% I mg ths sp#s trophoi&obri o tebm oW
t* ia # t w il l a n t # no tassfsl f iB f i i s mtmm th s agrssmsat mi%h th s va lu ss fo r absorption obtained by 410 0 0 1 Ting the
orystiax iii# pifpMmt ia so lu tion I t not r@ry o lo so . B»««*ar,
th s region of n tadtm absorption fo r th t oo~saXX#€ wntfco*
phyll f m otion sorrasponds Tory a lo so ly with that for
months thjrll a t 440OA* m l fa ir ly e loso a t 4TO®a* fIntro la a lso mlmmm 0 0 rrsspomisnsm in th sss regions with ths months**
phy 11 fraotion. obtains* fro© ths trsatMnt o f froth aplnash
by th# §w»# trtto&iuteft- f'fe* tt«#ttlloA samtsm fvmtlsn gar#
absorption m ® M $m im tb s t mmm p osition as mmthaphyll*'This mmmm s ib h s r th a t t h s %m ass o f p is n o u t l o f t i n th # so**
s a i l # * a a sfe tsn * f r o o t l o a la tm sm traotsi, ipsathojhyXX ..or
a lp h a s a r o to a s * B oth f r a s t i s n s from th # f l o o r s m tm o f show
a b so r p tio n mmlmm ®igxjJLfim®ntl$ d l f f s r a a t from th a t f o r
b e ta a a r s ta it s s r y a t a la l a s o lu t io n * a n t from th s s a r o ta a s
f r a c t io n o b ta in s * from th o tr im tm m t o f s a r r o t* and sp in a ch
by th e sums p r o s s ta r s * I t l a p erh ap s to© m s h to sm pest
th a t an e x t r a s t prepared from f l o o r sh o a l* a&rsa p s r f s s t l y
w ith th # a b s o r p t io n s w r * o b ta in s * from m n t h s p h y l l s r y s t s l a
l a s s ln t lo n * »« bus© o f th # i m m * o f jn n th sp h jr ll in
ap iaash * an* a l s o o f t h s p r sa o a sa o f s s r o ta a # l a a p ln a sh
and sa ro b sita In s a s r o t s * i s knew a l s o th a t th s r * im th#
ammo la a k o f 0 X0 0 0 n g m sm ii* b stw san th # a b s o r p t io n 0 1 tr r o s
25
o f h i g h p u r i t y c r y s t a l l i n e m a t e r i a l , and " c a r o t e n e ” m e re ly
e x t r a c t e d f ro m s p i n a c h o r c a r r o t s , o r " x a n t h o p h y l l ” ex
t r a c t e d f ro m s p i n a c h . I t would seem t h e r e must be j u s t
enough i n t e r f e r e n c e t o c a u s e t h e v a r i a t i o n s o b t a i n e d . As
p r o o f t h a t t h e method o f t r e a t m e n t i t s e l f d i d n o t a c c o u n t
f o r t h e v a r i a t i o n , t h i s e x p e r i m e n t was p e r f o rm e d :
d e l a t i o n s o f c r y s t a l l i n e c a r o t e n e and c r y s t a l l i n e
x a i i t h o p h y l l w ere mixed and t r e a t e d by th e method u se d f o r
e x t r a c t i o n o f f l o u r , and t h e a b s o r p t i o n c o e f f i c i e n t s were
d e t e r m i n e d , The same c a r o t e n e and x a n t h o p h y l l s o l u t i o n s
w ere a l s o mixed and t h e c a r o t e n e and x a n t h o p h y l l w ere
s e p a r a t e d by s h a k i n g w i t h aqueous m e th y l a l c o h o l , e x a c t l y
a s i n t h e m e thod . A g a in t h e a b s o r p t i o n was d e t e r m i n e d i n
t h e p h o t o e l e c t r i c s p e c t r o p h o t o m e t e r . I n b o t h c a s e s t h e
a b s o r p t i o n s g iv e n below a r e t h e r e s u l t o f d u p l i c a t e m e a s u re
m en ts o f t r a n s m i t t a n c y . (The a b s o r p t i o n i s t h e n e g a t i v e
l o g a r i t h m of t h e t r a n s m i t t a n c y )* The column head ed
" c a r o t e n e 1 " r e p r e s e n t s m a t e r i a l s e p a r a t e d f rom t h e column
h ea d ed " x a n t h o p h y l l I " , by s h a k i n g w i t h aqueous m e th a n o l .
The m a t o r I a l h a n d l e d i n t h e columns head ed " c a r o t e n e I I "
and " x a n t h o p h y l l I I " was p u t t h r o u g h t h e r e g u l a r e x t r a c t i o n
method and t h e n s e p a r a t e d . o s s i b l y t h e b e s t way to show
t h e a g r e e m e n t i n co lum n I and I I u n d e r b o t h c a r o t e n e and
x a n t h o p h y l l i s to c a l l t h e maximum a b s o r p t i o n 100 and
c a l c u l a t e - th e p e r c e n t a g e a b s o r p t i o n of t h e maximum f o r oach
wave l e n g t h . These v a l u e s a r e g i v e n i n t h e column marked
p e r c e n t a g e . J x a m i n a t i o n o f t h i s column shows good a g re e m e n t
2 6
and i t mmj them fea eonalfcui&& u t t e matted o f tract t Ion
te a no s ig n i f ic a n t o f f s e t on th e oarotono and x&nthopftyll
pigm ent.
Hafcle II
j.r *-*r • '■■ **.&. a, X l iipwiwrjiigMO i > mwwwwi’i n mm*—**!***.
■■■te length. Ca» tana I > Carotana 11 >.s?
4 100.4 • 25 8 4 0 . 3 ♦ 272 46* 64150 *SS3§ §4*9 ♦ 313 §3.74 200 *377 63 .6 .266 63* 2*&'» *411 69 .4 • 404 69*343 * • 429 72*5 * 4 20 72*14anu *460 T U I .456 76*14400 *614 86*8 .606 06* 644 5 0 *§64 9§.3 .556 96*44500 .592 100.0 • 563 i'%- '*04550 .674 97 .0 .668 96*44 6 C 0 .530 59*9 .619 i:h *■ * 14650 .501 84 .6 *491 94. 24 7 0 0 .604 85. 2 *496 65*14750 * 6 25 05 .4 *511 87* f4 0 0 0 .§16 §7.8 *607 87*04550 .463 78.2 * 44 9 77.04900 .072 s t . a .362 60*0
-s v s len g th *kMB fcte p h i'll 1 ’,§ Ijuejth o p h y ll I I ^
4loO& .£66 §1*2 ♦ 870 47 .141§0 • 314 60*0 .368 63*94200 *348 67 .2 ♦ 397 09 .54B60 *364 70*8 ♦ 411 71.74300 *565 70*4 .4 1 2 7 1 .94360 .4 1 0 8 0 .3 *464 81 .04400 *461 92 .9 *636 93.94.450 . 618 1 j.O • 573 100*04500 *498 96*2 .§§1 9 6 .24660 .440 85*0 *482 B4* 14600 *391 76*6 ♦ 431 76 .24060 ., 39ci 76.8 .437 78*54700 *441 85*8 *486 84.84760 *447 8 6 .3 *§20 87 .04800 *404 78*0 *433 76.64850 *299 § 7 .a *321 66*64900 .197 6 8 .0 * {.n I O 36* 7
Tim Bmmm porsuaaiwa that Is
th© saaential anrottfnoid pigment in f lstir. Certainly tor
pwtp&mmt of do boo ting added e&rotearn in flour and si&oaroai
prodsota i t mey bn bo QOftaidorod*
J&lootton of F ilter far ffeuferal fredgo i\ho toaster
fh© previous etpeotrophotofjiobrla mBaBnr&mmnto node by
ftiTiFi and also G#44#» for tho dot&rsiination of o&roboiioia
plg&cmta w#r© and© at 43M #° It I® impomihlo to » l t a
f i lte r whiflfe w ill .giro str ictly leonoohrombio light of a
narrow wars length baud and at the 8 » time hare a aatis-
factory degree of tranaaia-Blon* Heweror* f ilte r s
sufficiently none chromatin a an bo obtained for most par-osee•
i t Is not asecmbi&l that the absorption band bo at 4368a®
fMs wave length has bean us«d prerioualy fe&e&nee It happens
to be In the region of high absorption and in also an im
portant oomponont of th© nera&ry are I whioh of eaorae i« a
powerful source of light)* It is sieooesary or highly
desirable to as© some war# length in the region of imMaaia
absorption for two reason*? first* where th# absorption im
great oat one obtains moot aeonrut© mono ur ©meat © # particularly
when ths qujantity of the absorbing aubetaiui* is snail;
aooondlyv i t is this region whleh oontainw th« wave length
p e c u l ia r ly e h a r a o t e r i s t io o f th e n n le o a le*I 34)m il or 11934} published the quaa 11 babl to absorpti on
apootra of tha eomraoa oarotanoido* alpha oarotena* beta
e&rotene* lyeopaae and l e a f ' x m th o p h y ll# rilimm ®wrem mrm
ahown in Figure 1* A similar b®% of ® sn § s wer© obtained
In th# present ©ta&y umlmg a photo#leetrie spectrophotometer*
Ah© so lv e n t m&& %hm saiaa &b oa#d by i&iller* im o ljr: atl.
absolute aleehol and tCM eafeydreua ethyl ether* fhe alpha
asi beta ■isrolsiif erystala were purehaaod from the a* A# A#
Corporation and th# x&nthojshyll ©rystale from th® Ata rio&ti
Chlorophyl Company* Alia alpha and the beta ©aretem© wore
s s t i as pureh&aod. ffe© xanthophyll ©ryatala ©iaaolred
in a m all amount of &leohol ant reeryetallieod toy addition
o f pebroleam other* fho ©ryatals wore filtered onto m
hardened f i l te r ;papnr and triad In a manass deaieeator for
two hoare* Th& following 10' spi** 10*4 mg* a id
10*3 ȣ# of a lp h a onroteaa* to e ts oarotona and xanthophyll*
r< *• n ©fcSwaly* were weighed and transferred to a 100 ©o
glaae stoppered* volumeter!* f l a s h , disaolrad in a few o©
o f ethyl ©th<?r and mada to volume a t t o degreas £ w ith
p etro leu m e th er*
Ataorptiea eoeffioient i n SO.- a heelate aleohol and fOA anhydroae ethyl otn©r*
.:i ? t X aagth Alpha C aroten e B eta O&rotone X anth oph yll
T ab le i l l
6*400 m g /l 1*416 m g /l 0.413’ms/l
.1 6 4*16?*104*SX§*£30*24S
.161.IT S *109 . t i l . la • 6*o
29
f a b le XII (c o n tin u e d )
&ave length Alpha Oarotene Beta aarotone Ajsnthofhyll0.400 m g /l 0 .4 1 6 m g /l 0 » « l * ' m / l
48 .8 4 1 .2 4 3 .2 4 960 .2 3 8 .2 6 3 ,8 4 066 .2 0 8 .2 4 8 , 20860 .1 7 9 .2 3 3 ,1 8 266 .1 7 6 .2 1 3 .1 8 670 .1 9 9 .3 0 9 .2 0 876 .2 1 5 .2 1 8 .2 1 480 .1 9 4 .2 2 0 .1 9 186 .1 4 6 .2 0 2 .1 4 090 .0 9 2 .1 6 3 .0 8 9
0ne mm portion o f each o f then# s to c k a©Autlona wera
dilated to 8b0 oo wi th a mixture of 80$ absolute alcohol
and 20$ anhydrous ethyl ether* fhe resulting concentrations
©£ 0*40 ng of alpha carotene* 0*41i ag of beta oarotone and
0*4X2 ag of xanthopiqrll per lite r were as©#, for the determi
nation of the absorption spectra# fit© otmr«s obtained are
g iv s n i n rigar© 2* f h e data used for tk#©# ©urvaa a r e
given in fable III* A compar ison of those curves m th (241
Mi Her* s show general sloes agreement* Sit© agreement*
however* at the shorter wav® lengths below 4bQ0A°i» not
very sloe©* fhe exact data msed by Miller for Ms e u n n(m)
mm n o t a v a i la b le # However* the d a ta used by ©alth in
construction of III a curves for alpha and beta carotene was
available# fhe absorption caeffieiente of alpha caroteneo
at wave lengths 4480 and 4&00A obtained i n this study were
#24d$ and *18*^4* r e s p e c t iv e ly * as a g a in s t diaith*a results
of *2440 and *1992* © difference of 6*6 and 6*9$* A
abso
rptio
n co
frrcjE
Nf
29a
ao
ALPHA CAROTENE BETA CARO TE It LVtOPENELEAF XANTHOPHVLL
WAVE LENGTH IN A. U.
Fio. 1. Quantitative absorption spectra of the common carotenoids.
similar ©ompariaon for beta esrot jb© mt 4460 and 4600.rt
gmr® *2264 mxA #2277, raapeotlvoly» a© k «ioat anibtilsi
▼aloea of *g2?6 and #2342 ** a differ©®®# &f 0*62M and
2* 5 * fltene particular war© langtfta war a a ho am el now
tli@ absorption Qooffialoat* happened to Imre boon j&©B*t*r©&
at tho sam# netting for both ©eta of data# $'ho to#ba
oarotono ralnea agroo ¥«ry oloeely and nay therefore be
©onaidorod to indie®to pur© @oaro# of si&teri&l* kata on
absorption ©oeffioionto for xanthophyli i s f t not ar&ilabia,i £4 i
bat estimated riilms fro.ii Mi Her* a anrra.war* .mod foro
©omparlaejft* et ttifi longtha 4460 and 4600a the row-alt©
obtained «cr# *2i334 and *1663, respeatAtsly* aad Ms renalta
merm *11140 and *1610 or a differon*© of 8*3 and S*0 ,
MeeorMAag to these vein©* th© jpm thophyll ery&tala my not
haw© boon loo..* pur©* !ii# .a* p's** ©orrmtod, for the alpha
arid th® bate oar© ten® Mr© 176 &«d 177 degrees 0* roapsot-
ire ly , and f o r the xanthnphyll ISO 4®gr©*® 0* j-Mh further
ladia&tea the xanthophyll my nob b® IC/Om pur#* o «rrer*
ther© art @® many iaoaor© of leaf xanthophyll that one
©an net state with aortalnty tfeat this sample of xaivfchophyll( 2 1 1
was Impmm* Miller (1936) gl.ir#« absorption eurrea for
these three pigmmitm whiah 4s not exactly agree with Ms
former eurves* -'deamination of th© mmrvmm wh&mm the ab
sorption ©oeffiolenta to bis oqu&l for alpha ©arstene beta.o i ** )
©aroten® and xanthophyll at 446CM in tbm oa&® of Mill r#e
worl, but only approximately a® in the oarve© in Figure 2*
Absorption C oefficient
O pH ifibQ c+ S3 H*
H «*O p ef c+CJ> H*b <1o <s>H*
^5 «58 H» O& *P<D ct b K* ct O CO {3 * O
QK
H*
6csHi
ro
ct
O
30a
sx
( M lOn th s b& BlB o f roaalia I t ultornM smJc® m
wfcloh plgMUt Im sasaaurod* alpha o* h#tao
0 mrotsa# ox m ntb& vfayll at w«wo Xsn&th 44ti0A* fa«r©loro#
alnoa At 1 9 not pmmit lowly knows which pJUgjasttt ecmsstStats*
th s TOl&rlag * t to r o f f lo o r , 44SQO mmm ohoooo f&r till®
work* How#?0*% tho m m m tilm ting svl&onos on th© oharaotor
of tho o sro tsa o id p ignon ts Ata f lo u r icca o to hm m rm and
worn pmrmmmmi'wm th a t ac&n th op h yll rathor than o&rotsa* im
th& m-tijor earotonold im t l o ur# indosd ovidtmeo
ofeta2 nod in th io study Andlostoa that xantho.flay 11 o en a ti*
ta t es M iir ly a l l tfeo jr«il©w oarotoiteld fdga®«t A a flou r#
ffcroo flltaw w wmrm noaotxrod An an attow pt to g o t #110o
w ith, an aptio«*l oantrold at 44iOA# Dmtmmirmtiom of th e
optioal oontroid XirroXo** tho «*»«»ur i « t of tho
sil a n io n a t different wmm longth* with a photoaiootrio
©I *str#pli® to meter* A waXao o f t§00 dogroaa f has bomn
assumed for ths temperature of the light aonree* i^wii i B m ( 3 3 )
and. CMiifsoa g ir o th e T a la a a f o r en erg y C -I mad Jodd giro©
t h s v a lu e * f o r r l s i hi 1A ty i f I*
i tW#
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TP mo
Wigmr® I I I
t r a n s m is s io n ca rv e f o r C orningg l a s s f i l t e r Mo* 5 1 1 # 5 mm t h ic t e # s s »
of. reading oqoal oono antratlon® o f th« thro# pigments In m
mixturo of' B0 e ahooluto aXoctal and BBg> anhydrous ©thyl
othor*
mm.M ?
Xtatoastor soul# readings* for oqn&X oonoontrationa of
«-lpha end bata ear®ton# mad xn< tt o hyil using ta* 445 flitor*
Soul# rmilng on . iuttomotor*
■Cone* sag/l alpha Carotas# Bats Cairo tana xanthophyll
0*30 5*61 S. #4 0*430*40 4*t*9 4*9® 4*?€0*50 6*03 6*05 1>.?1
fii© r&laoa for alpha and tats oarotocia m$pF®m well, tat
tit# xanthophyll ralnae ar® lo«* Hfcwovor, in tit® aotua 1
d#t®rmln&tl®xi of murmtmemi t •plgmmtm in flour- or n&earoni
produo to this xanthophyll roadlag oorroapon&a to oiilj mtmmt
4;B orror in tit® o&leul&tod roealta# ?£hes« aoal® roadInga
rojtraswt approxlisat©ljr th« arorsge rang# on gelation®
aoaaurod in th® datoraiaatloa of oarotonold plgmonts in
flour and a&asroni prodnoto*
dtan&ar&isatl oxi of <* atral vtodge ?hotoa#ter far ao&auring dares tan# Gonoontratioa*
On© huadrod ssg* of mataml taixtar# of alpha and bets,
oaroton® fd*M*4* Corporation, CloirolanA, f'M§, 5 was die-
aolrod in b • 6 o@ ® ** ** aboolnto othyl oloohol
was ad dad, &®ol®4 In th* l®@ tax for about two hour® to
m
I m m M m x m and f i l t e r e d ©s m hard
f i l t e r paper# 'Sh&mm © sraten a o r ^ s t a ls wtsr© d is s o lv e d in
I - 5 cq 40 0 © p etro leu m e th e r was added* © ooled in
th # lo© h<m a s b e f o r e , and f i l t e r e d ©a imrd f i l t e r -pepor#
?h© o r y a t s l s w «f« d r ie d in VMnitai &©ai©<mt©r f o r 1 hour#
fw e lv a mg. w ere ? 1# hod so e u r t© ly sad washed w ith 10
eo ©th^l e th e r In to a 100 ©e grad no tad f l a e l # Th® w ash ing
w ith p etro leu m e th e r was eo n tin n ed and th e f la s h was mad©
t o t o I mm w ith a d d it io n o f petro leum e th e r mm soon mm a l l
© areten # was d i s s o lv e d * fhim s o l u t i o n mm uosiifin t d as
© te e ! s o lu t io n *
oix ©Oijo@«tratios# from this stool eolation war® mad#
% adding, tli# following amounts 'to WO ®m graduated flash©:
0 *Sf>* 0*6* Q#f§t 1*0* itSS and l* i0 so and made to volume
Kith petroleum ebhar* 'fh@s# reimesont ©omentr&tloits 0* lst
0*84* 0 *$&t 0*48» 0*d0 and 0*?3 »g* pnr liter* ?hose
solutions w s re r e a d in a M its* se ll u s i n g f i l te r Ko* 44b
In th# photoisieter 03re.plset* ?®s reading© on eaek solution
w»v« tah#n and the average reading plotted against son-
©sstration* I M s gave m straight I I 11# a s shown in. Figure 4*
fable VI
iplii ssud in e o n s t m e t lo n of r e f© r a se s ©nrve in f ig u r e 4*
Ooiio* mg* p er 11 t e r in p e tr o 1sum e th e r
Seal© reading in am* using f i l t e r llo* 44b
0* @41*44E.tO
■■■■■■■■■■■■■■■■■■■■■■■aIIII111II1II ■■■■■■■■■■■■ ■■■■■■■■■■■■ ■■■■■■■■■■HI ■■■■■■■■■■■■ ■■•■■■■■■■■■ ■■■■■■■■■■■I ■■■■■■■■■■■■ ■■■■■■■■■■■■ ■■■■■■■■■■■I ■■■■■■:■■■■■ ■■■■■■'if it ■■ ■■■■■■'iiria ■ ■ ■ ■ ■ r^ iir ■■ ■■■■■■jcjpii
■ *4 U A pi IB* (HP
: ‘. 4 i 1 :-U
W-i
$4 £ & 0 ©*HO $ 4» M
1k S3 *d © . <£ ©0 H* dla u o o © a*ri 4*IS
0 W© 0 S36 o d 6 4 fpQ Ps 83
'fab le VI {c o n t in u e d }
Come* t* par l i t e r i n p€%TQly\m ©th&r
P#a 1* re a d in g Im 0 ®* u s in g f i l t a r 0 * 445
4* 29 b*?B6*988*6?
the equation o f the ear v s o&loul&ted frota th is data, by
th© method o f sversge© i i I s **005 * *0-6® J£ (Y * Oomo#
la ssg* $»«r XItor and o * 0 0 aI® readings I s am*}
Th& oarvo obtained p lo ttin g , th e sons ontratl 032$
against the seal© readings {Uigurs 4} given a straight lino
whieh lndl.o«t©0 th a t Boor*s la® i s ©bey>sd over t h is rang#
o f eoaeen tratlon a* 8 0 hart© has a lso shown th a t B oer's lorn
holds up to 3*0 mg/l*
Method dor sloped in this study for the determination of addst Carotene to flour sad i&saronJ. Prodasta*
Reference throughout this paper has boom snad© to the
method used for extraetlem of the 002*0tmasld pljsjaisiite from
flo o r* "ihe method im given here fo llow ed by s t u d l o n l i e
d ev e lo p m en t, p r e c i s io n , a ssu r a e y and u ses*
j . r era r a t io n o f ^aianl.e
aaaroiU. aiad noodles were gr©«iisi to &p near flour
fim€H0©n as possible In the ordinary eoffee- t% 0 mill* K©
d lff lenity 000or©d with, proa us ts eontsinlng egg* but plain
m&s&roui prodsote required several grind lugs* Cars was
taken not t© set the mill too tight, 0© that the heat
( U}
36
gdam tdt amy not fee sufficient to d&smgo ths plgnents#
I;#brol#a» :thsr IB#p . * 30-60)
■ .IkyX Bbiuir
Saturated Alcoholic J'OE (10 fgm* kOH per 1 0 0 ®q BB^- aldehyde-
free alcohol}* The al&ehyd«N*free alcohol mmm prepared as
directed in' b# a* Phanaaoepoea Vol* .X*
9ZP saethy 1 alcohol (8 ce MO ♦ 9S ae abaci ate siethyl alcohol) •
Aatiydr© a» Kag i50 #
Appa rates
IBB oc separatory f onnsls
BOO cc separatory funnels
B§© oo distillation flask
dieohanloal slimier
Meatral *ied§p§ Photoraster and absorption cull
30 cm* cro odenssr
BfeO as ©action flask
Bttchner type fritted glass f i lte r ClldS)
Broaedurc
ifwenty gni* of flour t a one 11 na or &Boareni, o r 10 gm*
egg noodlest or B gn* egg yolk wore transferred into a i t i
cg -.•Jrlaweyer flask* §0 ee of csb£irat«d alcoholic KOH adiod*
ant heated on the &%®mm bath far ono-half hour with the
flask fitted to a condenser* The flask mm rotated
occasionally taring this tins* being mm careful as pesslhie
to keep the sample from collecting on the site® of ths flmssk*
37
ffeii fissile m&.B % cm# ooo^ed to room temajiaimtMr#*
fit# wolntioa was filtered throa^h o Bhah&or typo f r i t tad
&1&M f i l t e r C ll.5S | u.sl#i|i © notion# t r m m f e rr i* s$ a l l
aiatori&l m tin, & l i t t l e 9£> aleohol froia & wash bottle* Oh#
Motion mm-M uirn^d off* »ii th© flash riiMied with tS &m
mth&l other &ud iiottr@4 Itil *j glmse filter* ©birring th®
Material la tha f i l te r wi th a rod to allow the #th«r to eima
In ooatmt with a ll |#»rtloji** fhie woa filtered o f f and
tlilm proa©dura repeated twioo aoro* fh# f il tra te waa tr&aa**
ferrsd to a 350 oo glans stoppered a©para tory fuaael*
r l s s l i i f with, a b o u t MB m o t h y l eth& r* ro o ia o u o am*
tarictl t s i oisrtfg&rdad* 'fhoii 17b ## wold %mp wntew mmm
mldod* o e r e f a i l f i& ro r tw d to d r o t a t e a w e r a l t i» o o * whaa
th o a g u a e tm~ s la o h a 1 aod © th a r lR y # rs hmm ** r r a t e d # fcha
lower a oeo8*»&l*ofeftl lay or m raaorod# fliia mmmmmm oloo-hol leyer was aoetraoted again. with BB mm- ethyl other
and tho lower layer diaoardad# 1%0 r n t m r was added to the
original ether aoliitloa* fli© ether mum tsik# ©nee toy Merely pouring i>0 00 top w i#r through ii« After s#pr*
atieii of the Iayera the HO iay&* *&« withdraw* m4
di&a&raea# Fifty 0 0 of peiroleuM #%fcOT wna added ttt the
e t h y l eth«r ooiw & ien and t h e M ix tu re waahed f i r m timmm
with §0 mm of tap waher* aising gently by .Inverting and
r o t a t i n g t h e fu& iial* A l l E^O I n j u r e w are
flig h t #itmlsi©as way owaur hut aaa bo dlaragar&ed# fhw
wthar»p* trol ww abhor mixture mmm % w m m £® Tw m & to a dhO 00
d l s t i l ia t io n fla sk * '2 ho f la s h w a stoppered aM placed i s
m hmaker of water Bt around 4 0 - 0 0 degrees 0* a id s
«:m was 0 Ojut«O't»iA wi %h rammsm um the e o la t io n soi!M*e«trab#d
to about 10 - IS os* Till a remoYM th e e th y l ether# t h i s
s o lu t io n was f i l t e r e d through a filter paper g/s filled e i t h anhydrous iSfaio into a 100 on relum etrio f la sk * f h m
flash wan made to ?©!»§ w ith p& trolm mm other which tod boon passed port I. ©nwi ee through the f i l t e r and
the ab sorp tion ©oil mam f i l l #d w ith a pi potto, si no# non# of this s o lu t io n should hm lost during tit# f i l l i n g , and then
road in th e n eu tra l photometer* fh e so lu t io n in th e
©#11 wmrn returned to the f la s k , a f t e r reading fo r total so lo r , and th e so lu t io n tr a n s fe r rod to a Ilf* mm separatory
fu n n e l, r in s in g w ith & l i t t l s petroleum ether* f i f t e e n mm
o f J 4 methyl a lo o h o l wmm added to th e petroleum eth er
s o lu t io n and the mi acturs was shaken on a m echanical shaker
fo r 1 0 m inutes, removed and the separatory fu n n el allow ed
to stand i s upright posits on a minute or two u n t i l the
a lco h o l and eth er la y e r s separated* rha low er la y e r con
ta in in g san th op h y ll mm wi tMrawn and th e e x tr a c tio n
repeated f i r # more tim es, or u n t il the a t a## as methyl
a lc o h o l la y e r i s c o lo r le s s* The f in a l methyl a lco h o l
la y e r examined in a t e s t tubs or or a w hite background
to b# aura i t was c o lo r le s s * o im ex tr a c tio n s were u su a lly
enough ©n f lo u r and m acaroni, but noodles w ith h igh egg
con ten t » | r e p a ir s te n ex tra stio n e* Ike petroleum e t to r
39
so lu tion containing a l i t t l e methyl alcohol was washed
with tS oo 11 0 by inverting the separatory funnel several
tin®®* Thm H ^p luyar fa s discarded* Hat» procedure w a s
r^paated twice £aor©« Altar the la s t waahlfig to® petroleum
ether layer was i»oare& in to a S&o «o d is t i l la t io n flask*
She fla sk i t s pi mood, in a beaker of Ko0 &% about 4,> - bolla,
degrees 0 * s t o p p e r e d a r id t h e p a t r o l s u m e t h e r o o .n o o u t - r a t e d
with a p p l l e & t i o n o f visetimn mm b e f o r e * Mbor e e n e o n t r a t l o n
t o a b o u t 10 o r I S a s i t w e * f i l te r# ! t h r o u g h a d r y f i l t e r
p a p e r two** t h i r d s f i l l e d w i t h t m h y d r o u s b € ^ t i n t o a I S
o r a SO @o v o l u m a t r l s f l a s k , d e p e n d i n g o n c o n c e n t r a t i o n o f
e a r n t e n s # ® iie f i l t e r p a p a r w a s w a s h e d w i t h p e t r o l e u m o t h e r
i n s ta k in g ? t o v o l u m e * Th& c o n t e n t s w o r e m i x e d b y i n v e r t ! n
t h e f l a s k a n d t h e o o i m e n t r a t i o a s w e r e r « n d i n a n e u t r a l
w e d f f o p h o t o m e t e r , u s i n ® f i l t e r h o * 4 4 & * From t h e s t a n d a r d * *
i c a t i o n c u r v e p r e v i o u s l y c o n s t r u c t e d the e o n e e n t r a t l o n o f
o a r o t e n e w a s d e t e r m i n e d * IF M ft e o n e e n t r a t i o n - w a s d i v i d e d
by 4 or 3, cl spending on v o l u m e a d j u s t m e n t , t o obtain t h e
c o n c e n t r a t i o n o f o a r o t e n e i n 1 0 0 so* f h i s v a l u e w a s
©ml t l p l i e d by f i v e to c o n v e r t to. p#p*nu in t h e s a m p l e If
& 20 ®fm s a m p l e was u s a d , a n d by XO for a 10 gpraa s s s a p l s ,
e t c «
h e v o l o i v i e f t t of t h e l i s t h o d for ^ © t e r m i n a t i o nof v ,« m# J era t an © *
I f t h e o r d i n a r y f a t s o l v e n t s u s e d f o r © a t t r a c t i o n a r e
a l l o w e d t o s t a n d o v e r n l £fet i n c o n t a c t w i t h t h e m a t e r i a l
low results fo r a<!s**tsi w ill result# *iuah solvents
wtf8 tried ms acetone* 80# acetone - water mixture* alcohol
70>i a lco h o l — water mixture* petroleum eth er &n& variou s
mi act arcs o f petroleum eth er and a lcoh ol* mXJ wore unmatis»*
fa c to r y fo r one reason or another* but p a r t ic u la r ly because
o f low recovery o f added e&rotams fo r mmomr-Qml* I t
th a t most o f th® d i f f i c u l t y la recovery was due to i t m b i l l t
to mtIbA the h art hom y stacm-rcai to a b e f f i c i e n t l y f l e e
eon d ition * s t r a ig h t © xtrnotioii o f the pigments o f f lo u r
w ith petroleum ether and 7,d a le e hoi a t roe® tem perature
gaw© good r e su lts* A sample o f f lo u r was thus analysed
fo r t o t a l co lo r w ith a r e s u lt of £•&& p*p*m* The s&me
sample ground in a h a l l m ill two homris gave g*40 p*p*»*#
which shows b o s ig n i f ic a n t See true t l on o f pigment#
daoaroai ©an 'bo grofind to f lo u r f i smmmm i s i b a ll m ill in
two hoars, itnel m&oar©ni gat# much detfeor e o io r r c so v o r le s
by str&i&ht e x tra c tio n as used on flou r* However, th is
method of appro ash i s to© t i m e lo n s wmlng from & roi *ine
regulatory- standpoint#
Table I in d ic a te s th a t SO ©o o f e th y l a lc o h o l i s
s u f f i c i e n t to -d is so lv e the pigments pros out* 1!h® amount
present,. »*#ft In ease o f a noodle Kith double egg eontent*
is only shout 0*8 mg* in a 10 ga* Sams 1«# and 60 ao of
e th y l a lcoh o l w i l l d is s o lv e or on f o u r tim es t h i s amount o f
ear© tens C th e le a n t so lu b le o f th& two pigments}* However,
la s s than SO cc o f so lv e n t i s im p rest!cab le due to the
bulk of sample*
42
l?he p r a a i s i on o f tk e n e u t r a l wedg© pho f o a e i a r i n .read
in g a c a r o te n e s o l u t i o n o f 0*66 m g/2 m y hm I l l u s t r a t e d by
th e a v era g e of t e n actu a l. r e a d in g s ©£ n in e d i f f e r e n t c o l l
f i l l i n g s * R eadings: 6*60* 6*52* 6*63* 6*56* 6*6? , 6*55,
6*56 , 6*60 ant 6*66* fh i© spread from 6*62 to 6*60
c o r r e sp o n d s to & d S f f crstra© o f 0*02 m g / l o f c a r o te n e* On
th e b a s i s o f th e amount of sample used f o r macaroni and
m o l l e e i t i s a d i f f e r e n c e o f 0*05 p*p*m* and 0*10 p*p*m*,
r e s p e c t i v e l y * Otxl © i s r ^ p r e a c n ta t l v© o f r o a a iu g s up to
a o n o o n t r s t io n s o f 0*8 m g/l c a r o t in s * above which t h e e r r o r
becomes la r g e r *
in order to chad: the #ff@ct of various stags in t h e
development of the method, the following experiments war© carried out* On# hundred 0 0 of m stooh solution, of car©ten© (©one* 0*66 mg/l) with scale reading o f 6*89 wan transferred to a 250 0 0 distillation flash and concentrated at 40 * 50 degree© 0 under vacuum to about 10 ©e, filtered through anhydrous a;ri4 a d i to volume again* a£> TBreading of 6*80 in d i c a t e d p r a o t i o a l l y complete recovery*
Oonceotratioa under viicaum mud. filtration through Ba^n^
a re thus permissible* a 100 cc portion o f th e Bt.mm
o&roten© solution wa® nixed with 9B.d GHOH and ©halon o u t
w ith three 85 c© portion© o f H 0 to remove traces of this
alcohol* I t was than ©on©ont r a t ©&t dried by filtration
thro ugh Ka 10 and mad© to volume* A r e a d in g of 6*81n «kshows no interference dm© to washing w ith Xnol-
dentally, another 100 ©0 portion of th e same car©ton®
s o lu t io n washed w ith 92;- 'OHyDH, concentrated* filt& r ed
through dry f ilte r paper and tmd« to volume pt® % reading
of 6*8€* rhl© m%ild indicate th e small amount of 0Si.OH-*P
prm&mt in the petroleum ether, ant the tree© of stater not
absorbed by the f i lte r paper, had no effect on the results*
However, to be in complisne© w ith the so lv e n t used in
preparation of the referee©© ©hart for ©nrotoae ~ soul#
reading ©onversieaa, and to eliminate any doubt in a ll
cases, the Miblag and drying ov#r fen d-c were in©luded in
the pro© ed itre#
i’h© 11 term tore on the separation of xsnthophyll from
©areton© la hydroe&rhoa solvent., refer© to the use of 0b,
d f , § 0 and 92h aqueoos a©than© 1 * bom# worker© start with
8t> and inorsase toy ©tops to fC) sad than #8^* .^periw«mt»
with a ll those per© exit e gee showed that tt;o methanol gave
bettor separation© In the least number of extra©tions* A
9& methanol could not be tised dua to too large a oarotone(34)
loss {fl> recovery) • Miller found by apostrophe torn© tr io
measurement© that 9z wethan©1 leave© lee© xanthophyll in
the petroleum ether layer than methanol#
mm investigation of the amount of 98 i O.I^ok that
woa.lt most efficiently and ©oat ooonoasiieally ©operate the
xanthophyll and carotene, finally showed that mmilt
amount© of 1© ee ftf* &L-$ 1 were .moat satisfactory* Four
different period© of separation #11 a mechanical shaker
w#f© studied with shaking ©ontlnaed for lf>, 10, b and 2
43
mltmtco* in eoiae oases & m l a t i i m wore sufficient and u su a lly h minute© were s u f f i c i e n t , but e c n s ls te n t w ith the
least number of extrastione and to to on the safe ©id© in all o a s e s , the period for s h a l lng sae fixed as 10 minutes*
A period of 2 minute© hand ©haling gmva similar results to 5 minutes on the ©haling machine* fho fiOas lersuaw e)i&3:«r
was used* By arranging the holders properly and using
slumps to holt in the stoppers, the separatory funnel© esa he pi.seed horisontlally* Fire sample© can ha ©hair on Tory
conventsntly in th is way*
2 e» P * :ra ti tro variation© suoh as are likely to coo or in
reading th e s o lu t io n © w i l l ft©?© l i t t l e e f fe c t* same
s o l u t i o n r e a d a t t empcraturo o f 17 decree® and 30 degree©
C g&vo se a l© r e a d i n g s o f 6* 6a and 6*36 , r e s p o o t i Y e l y , - a
n e g l ig ib le differs©©©*
Irani a I on of the Method
"i?he r e s u l t s of r e p lle a t# a n a ly s is , by th is method, o f
sample© of macaroni and noodles co n ta in in g added ear©tan©
are given in fablm ¥1, these mxsples wars analysed at different In te r v a ls and may be considered as re pres eta ta t i v © o f th e precision of the method* rf o t a l color is c a lc u la te d
as carotene*
Imhlm VII
Reenlt© ©n il&earofii with addod Cerotene ^xpreaaedIn p*p»®m
Total 00 lor 3*4® t &*bb, £*$£, 3 .4 4 a a r o t e n e 1 .O 0 , 1 . 0 b . 0*u8# 0 .9 S
fetal eelor 9.10. 9.40. 9*£0. 8*60# 9.0 Osiroten© 4.4b. 4 . *50, 4. 4?, 4* l>0 , 4.4&?,. 4*40
K©salts on &ooai«© with added ©arctan© eacprausod in p*p.su
Xotnl oolor 10*8 - « ~Qmrotmm 4 .4 0 * 4 * 3 6 . 4.*bO, 4 * 6 0 .
1 6 . 5 , 10*4 4 . SO. 4.50
ffe average deviation of a single result is the m m of
the dovl& tiO f® d i v i d e d by t h e num ber o f r e s u l t s * t h e
deviation being the d if fe r©nee between the itcjF tit of mil result© and a ©ingle result* The average dev in ti on for
jean© nr v>ni w i th the i n t e r n e d let© aaot.mt of ©d&ed ea r© tan©
Cl.te) is 0* 0? p.p.n* 1?h© arlthraetle&l mmmM rived fro»
n measurement© of equal reliab ility is fli tiaaee as reliable
mm any ©1ngl© result. ffh© reliab ility of the saeen of two
result© la . therefore^ *0? or 0*05 p«p.isu Oka mmmm ©slum-
lation appli©d to the maearonl with larger amount of
added @&rot€ta# (4*43) gives m\ average deviation for a
©ingle result of 0*0? p*p»m. ffc© average deviation of the
dean of two result© 1® thu© 0*05 p.p.m. i*h® prool©1 on of the method for detemlnntlan of added enr©ten© siay, there
for#, b@ considered mol* In general. the rell&blli ty of
A,
u ©tngl® d e f e r a l n a t io n . m * $ h © c o n s id e r e d to be v e ry c l o s e
to p i os o r m inus 0*10 p*p*m* O.ho va lue® f o r t o t a l ©a lor
a r e n o t ao p r e o l o e , b u t w,j a r o n o t p r ia a a r l i n t - r e u t e d i n
them i n t h i s srto&y* ]low®v®y# th e y n r a s u f f i o i e n t iy p r e
c i s e and a c c u r a t e f o r th e p o r m e o f o r v..-hj c h th e y may fee-
used*
fh© ^©curacy o f the method*
k c a r o t e n e s o l u t i o n p ro t-a re d from c r y s t a l l i n e c a r o t e n e
c o n t a i n i n g » 7 2 m g/1 was added to 60 ae a l c o h o l i c r CMC and
e a r r t c& th r o u g h th.® m ethod m i t h a r e c o v e r y o f 0*695 m.g/l o r
9&*b:,-* f l ie s a n e c a r o t e n e s o l u t i o n pirns a s am ount o f
a r y a t n i l i f i e x a n t h o p h j l l c o r r e s p o n d in g to t h a t i n PiO gm* o f
f l o u r was t r e a t e d l i h o w i s a w i th a r e c o v e r y o f 0*?P§ m g /l
o r loor«6/v#. f.ha r e c o v e r i e s n r m t h u s v # r y good fro m a l a o h o l
and t h e r e w?i© t m I n t o r f e r e n e # # 2he a c c u ra c y o f t h e method
when a p p l i e d to f l o u r c o n t a i n i n g h t m m n a * a n t s o f u&aed
c a r o t e n e was n e x t t e s t e d * 0 sam p le o f f l o u r p r e v i o u s l y
a n a ly s o d fey t h l e m e th o d , and fou n d t o h&v© a s m a l l c a r o t e n e
h lao l: ia .s used* hiwo d i f f e r e n t c o n c h a t r - l i o n s o f a u r o i o n t
s o l u t i o n s v t K T o &dded to 00 grams o f t h i s f l o u r and r e
c o v e re d fey t h e method* t h e am®mats o f c a r o t e n e added w ere
0 . ? h m g /l and 0*50 m g / l and th e am ounts rh o o v e re d as
c a r o t e n e , a f t e r d@ duot.ios o f t h e s m e l l b la n k , m e r e 0*6S mg/X
and 0* 2 8 m g / l , r e a p o o t i v e l y # o r 94*few and 93*4,a* F u r t h e r
expcrimonte on the accuracy o f the method co n s is te d in ax-
t r e a t i n g ' t h i s same H o u r w ith 95.. p e t ro le u m e t h e r smi ?>••
44
a ls s t o l ovsr n ig l t t f i l t e r in g off th© flo o r malAas* wssb-
lug m i l with jistm lM * sthsr and drying* fsmtjr gram of
tMa flour rwida® i t s »&d#d to * ®&rot©m# solotI©a of 0 *M
ag/l und 0*S8f nf/t# fham t© ©ash aampl®. tt® a&&*4 an
jEtnthophjril m l s t l m fpropmTof tmm %h® s r y s t s ls ) ©orro* ©ponding to th® aaouat o f xmothojphyXl in th® flo o r
©H&lastljr* 'fh# 'mmPwmlm of .after h s lsg o&rrl@&through th® rogular methodt war© ®*00 and 0#B4 a g / l# or 9B»S
and '9g*#]t# tfom® rimdL%m that a r#*
of a t lo a s t 9Sj£ of - th® aM:#4 ta m ta m tan fc* shtai&sd
whoa this plgmimt 1® p m « s t up to th® ©stoat of apprsxi*
i i l t i l $*§0 p*p#m* on th® f lo o r h asls ( # f t s g / l ardi*fh# total oolor salsalattd a© tsrstms- umy h® ©sod in
® stilt in g th® m sast of ximt&ophyll* Ih# acmthophyll i s
tohea a® th« diffsromo httmm th® total oolor and
ssrstoao*.
tssslhs o f Petormiia&tioa of @s*sall«€ te r s to m in UablomlMNt Flours*
lm or dor to d ttw * th© addition of oar® ton® to
mmomml and oss&lss i t i s no®©star? to know th® natural
rang# of th® st~sall®& • w o ton® in floors# i*h« rm alts
halos ar® on 40 sm plss of van#®® floors roprmsatisg
sta m rsK il hi ©Ms going to th® m ssm & l isftsstr? and is*
dividual variiios ©xp» r im s ta l ly m illod fef Br* Oolmstt, 0*
a* D tpsrtm a* of tgrlssltsr** Iks s m a lt s igiv®i* la fafelt
t i l Im ilaatm thaVt& s traatlom as sa r s tm s bp th ismethod s i l l not © »ati. 0*t p+p»a»
4 f
tmblm m i l
A ll wmmI t s Imas mmmtmtmrn
S«*. 3tiba%g&ii©a ¥ * t * l -Q slsv .' S«-««11*K
1 F la w «•*«* 0 .1 0s 0 .0 83 *f HIM — «• 0 .0 74 « .*K».ll»; 0 .0 7i ft: 0 .0 84 « <*».mum 0 .1 0f w m-mm- 0 .1 6i .# m m mu 0*08t i» 8 * 8 0 .1 810 « 3 .4 . 0 .1 8u * 3# 43 0 .1 8IS ' a o n * 0 .1 0IS *t 0 .1 014 M «N«MIW 0 ,1 81# f* 0 .1 016 m ,i„tor,fe 0 .1 0I f «r 0 .1 61# * ■ 4NNW>«* 0 .1 8I f # 0 .1 8m a «WNWNW' 0 .0 821 a r«*?pww* 0 ,1 8m e* «nkM» . 0 .0 9m ft 0 .1 0m a 0 .1 0m m 0 .1 6s * m 6 .4 6 0 .8 0i f m ® .*o 0 .1 8m m 8 .« 0 0 .1 0m t» 8*66 0 ,1 6m '* 3*80 0 .1 8s i « 8 .9 0 0 .1 *s t » 8 .1 6 0 .1 833 it £ • •0 0 .1 8m m 8 .8 0 v-'-v ■ 0 .1 8m m 8 .1 8 : 0 * 1 8i i m 8 .4 6 0 * 1 8S f •** ■„ ■ 1*76 - 0 .0 6s s m ' : 8*06 : i 0 .9 0gas*3$p « 1 .7 6 0 .0®4 0 n S . 88 0 .1 841 a 8 .0 .. . '0 ,1 14 i a • v. v '■ , / 8 .7 , ■ 0 .1 2
4a
T a b l e V I I I { o o a t i j i u o d
S i © . •ubotajacto Total Oolor «o«Mt a i le d O&rotone
4a44 4 6464?4ti49hOd1h%.
§4
h*t&em6061626364 € 6 66 67 6 6
lOGT
>®m» I j. b,0-
S . 8
2 . 12*3 2 .4 6 2* lbE* 60
3.462 . TO2* 6Ci2 * 76 8*90S* I tS .6 0s»ao2*642*78 8 # 402 . HO 2*94 3* 80 ft#4E
oas 0*06 0.10 0* 00 Q.XS U « O l> O. 00 0* 10 0 .1 0 0*08 0 .1 0 0 . D:8 0 .10 0.14 L-# XO 0 .1 1 0.06 O.08 0*0 8 0 .1C 0 .1 4 0 .1 2 0*10 0* 10 0,06 0*06
oggs* m normal in groa l m.% &f noodles* oont&Xn &
e o r ta ln amormt o f p i g m m x % will e h ro s m ln s In thm pmtrol o i m
eth er l&^or* thvm boitm ©&X« d ie ted mm ocurotona. Ulllam(38)
and K elltron rep ort thim pigment mm lar^^ly fcryptox&nthlno
9*1 th n t t l © oar© tw o * r-n& of th&lr s i s s ly s is e l re s O'
-f;.f *§i* * to ta l pi 1*9 p.p.m . 1 ryptoxanthine « i l 0 .16
r .p .a . ca r o ten e . fhe 0 0 lo r o f in d iv id u a l Mg j?ol$r v a r i0 0
gra& tly from & p ale y e llo w to a deep rcddieh emnge*-
Oomri* jroial ^ollo ml#© * 0 1 0000 J dor able variation &0 sl'imm
fe;v t h e f i g u r e f o r t o t a l c o lo r * I n m b l o IX * o n 1 7
49
ant&oatls mmmmml&X fmmm® yoUes ant &rl*4 yelfcs#?lio mkmlmmmmmM ®M *g$ soil&s in tho
stw&*r& for noo&lo© im &•&;* on iho try tasls# ©r 0#$8 gm*
•sli4» im 10 i»# ©f aooais* ©s©& in IM» pvsootixro far
&®%®rmlmmtt®m ©f ©©lor* Oa tfea'bMi* of ths- Mroton*
figure fount ©» tli® mm ssnpX© of Mgh#®t oolor, in falsi #
IX, . mrntrn mlght W.«xp«*t®4 a so-oaXXot oar©ton© fig®*®
of 0*6$ p*p*ta* an mm mm- nootl©* shargoatolo to th# ogg*
flum %n#r# in sligh t mount oh&rgoafel®. to tfe®
floor i t s e l f , th is ©ar©t#ii© Monk s i l l rary dopoaOiag on
mtmun% of ogg solid®* tho eolor of tfe© ogg ant tfe© 00lor of
tit© flow #
Kssoltft'fer total oolor .(oal®«tlnt*&. mm ©or©tons)am o*Mt*n® In p*p*m. on aitthoati® ©mplm fro a rsrlom parts of th® 00 on try#
In# S u ta ta m ©
1f34 §§f§910 11 IS IS 14 1ft 14 I f
« #** «<t 1*© son am* «n **St *I# *■*# »** #* *ftf »* n*t *Sf *m ««
fatal 00 lor t*#*» Oar©ton© p*p*Umiminlar© fro® tmi©}- 1 ■*** 0 I'' * 8
Oil: ?8*0 4#Sa- 4f*ft S*T*t 41*0 4* 1m ftf*f 4*#
yoMc 81* S 4*0«r 1&4»0. 10*3tt ' §4*4 -8*0«t §#*§ 4*3** 6**3 0*0*f it* « 4*4* t i *0 6 .0*t 101*0 4*8m it*© ft# ft:vM 43.4 3*1m 44*0 3*4a 41*0 I*i- a ': ■#§*# ; 8*0
O'}
The l a s t f o u r m m p l ® ® o f fv&xen &pipm i n t h i s t a b l * a r e
.. mmitl® Coast -sob# OF. vuluoa fo r both t o t a l o o lo r tuid
eoTsto-B# f raati.&J% oonaldorabl* b&low th n© Iron otner
«*oti on®, of tho QonntTy* n&UiTmlXg on m.mh limited data no
$ m w r a l 1.§§53 t l o«@ «m.n fe0 m&d a •
fr tl# X
fh® roauIts of tiio aoaXyai* of aaths/itia ssao&roiii pro'4sots# eacprsMei. tm p+p#:o#
F lour uart*4 1 n produets f e t a l 0 © lo r O.or-0 ten©Cl - 1 1 in e lu e lv e ) 4 # 4 0*1 b
1 ^isomroiii 4 .0 3 O.SG8 ..aoaroni glum o ir i t s f i# # .4 4 .303 •» VS 1 0#4 4 . SO4 poouXm t*| tii 3 > froeea egg f o i l m lt& M l 7*7 O* Po5 • * • O.b * ‘ * ' * 10 .9 0.35e " iUO * rf ’* P 10* 8 0* oO? * ’gUUO xa»*i 0*60a * 5 * 6 whole eitg o o lid s 1 0 * f 0 .409 m . o 1 4 .b o *10 o«b egg yolk so 1 1 up p lus
earetene 14*4 4 « PA ■1 1 "* 5*§ d ried «o*| yo lk so 1 1 da 7 .7 0 .43
F l o u r used In p ro te staCIO - 14 Xnolaiif're) 2 .23 0 . 1 2
IS * a**?oni 1 .15 0 . 1 1IF ao ii*onl plus addad oareten s 2 .35 0 . 8 8*i > V* U‘ i# *# »*■ 3 . 20 1 * 50
fh # t o u t i n g artaljnios o f igoss© r sp r ^ a a n ta tl r« ooj»-
i ro ia l m aples of me*m>i*i giro un Idea o f th* uisu*l m g #
o f t o t a l ©olor oad o*srot?ma f iraotlait. oho t o t o l o o Io f
a a rot am imy bo l o w than th a t o f tli# o r ig im l row as*t-©rial
In.# la r g e ly to deatmxaticm of b&a p ig n u t utiring the a r | in s
prooaep*
XaJtl* si
S». S B b a U a t* T o ta l O«lor i>.j>.au Carotena fraation p. p.ra.
1 e .o s ' . 0.16I m ■ 18.46 0.161 m a . 10 0.1©€ ■» 0.60 m 00lo rI w 8.M 0.1*$ *t 1 .6 0.16? » 1 .6 0 .06a m 6 .0 0*1© ■t m 1.^. 0 .06i t w U f 0.0611 » l.S 0.06m . m 0 .S 0 .06m m 1 .0 0.0*M m 1*6 0.06I B m 8.0 0.08%$ m 1 .6
Tha rsau lt* o f ?«bl© E aay > • .of Talus froa tho stand-
fo lB t of setjibllolttng m maa&wm T»lm« fo r to ta l so lo r in
aaoaronl tmttfes a ia laaa ralao aajr tea m | low ia&se*. Sho
s&rotoao fra stio o * mvm o f tfe# saas ordsr o f wajsaltads ae
h©n® Qbt&lae&oii floor*
fi*0 fallo«iaig r««iiils 0*1 jfe$rM*a%afl70 mmmmmitskk
jMMNtlMr lilmtkmm/mkmw tall* wit# vwrlatieii la total aalar*
aiiti thm 1mm wrla&l#* fh«® 1# i*aaef %hm p i m m 1% la $v*$*aatt 0a #f mxmULm* &a
• l i o r a ,hw . a a f t . t h * la* f c H e a e f a a e *
IMwrialy. tm# %® la •»!•* #f mm- mm%rnsetml til f«ri»etion la woal #£ egg ®©ll&a*
x x i
6®. 5uh0tan®e Itot&l Color p.p.st* Caro too# p. p. si.
1 Hoodie® 18* 10 0.808 cf S*£ 0. 603 *■■*• 11*6 0. 704 *» 9 * B 0*606 <» 7*6 0.506 6.6 0*807 *f 7 * B O* 6H8 m 10.6 0* 009 n u* o 0.8010 n 8*0 0.4§11 n 8*8 O.B712 tt 11 *4 0*5013 11. f 0* 5614 rt 10* 7 0.48
fh* analysis o f 68 sample® of to w [email protected] a®@4 i n
mae&ronl mmnutmtare and sixteen samples of sa&earoni 9 show
m $M3cimw& rain© for the e&rotszie fm otion of 0*10 p.p*m#
Oener&lly i t was »ooh lower# Xu the preparation of
antheisti© noodle® a m otlt with 11X *&& solid® was i>r spared
from a highly eolered agg yolk* Thl® ®a£$>le oar* a ear®tens
froetioa of 0*60 p. p. a* and nay ho eonsidfsred ©lose to the
apper Tales expeeted on a eosuseraial noodle* Howewor* the
fable XU eon tains four Talne® somewhat higher than 0* 601
the highoot w&lae toeing 0*80 p*p*m« Th.*se four samples may
oont&in some addad e&r©b€?a©.
Intorf ersnee*
other oolors may of aounte bo addid to noodles, and
the lnfl&enso of %hose eolor® on the determination of
added oisrotoste had to bo determined* The n&o&l ©olora
addad ar© annate, tarmaria, saffron. t&rtarsiiie* naphtha1
yellow yallow a .a ?ni yellow ■:•* h* However, orange I
iM itms0l yellow a* B* w#r« also tasted* liaphbhol
y#Xl©w - t tarnerle, saffron, oraogo I* ©unset yellow h* 0*
i?* and tartars ins war# add «& 'to maaroni and o&rri«&
through tho method# k 11 oolor wan rmaoved in tfm aleohol -
water layer, and lid m>% ©hang# the total color* Annato
treated likewise was 1« r 1y raoovod in th© also hoi -water
layer, th© rmt being r«wv#d fey the moth&nol, serato thu©
oau&«& n® intsrfwonos in the osrotefi© determination*
Y ellow a * B* and ■ • B* ar© b o th o i l s o l a t i a dye© « M o h r # -
m ain i n t h e p e t r o le u m e t h e r l a y e r and ®ro t h e r e f o r # m easured
ms to tal c o l o r * They d o f however* s o t aislXarly to 38mnbh0~
p h y l l , feeing rem oved in t h e &©u© methyl a l e o h o l so It. i t ion#
f kair pros©no© does not, th®r»f©r## interfere with the
meaoureaont of th© oar© tens fraction*
P n r t l w r application of the. Beutrsl ^odgc . hotosiotor*
By a ffo rd !a n & eonveni ©at and aoaarats maasurennsnt of
th e t o t a l so lo r o f f lo u r t i t F i t l * the n au tra l w«dg*
photometer 1 ® an a id in th© d if f e r e n t ia t io n o f ties©Jhod and
untolea©hod f let tar. eg s ta te d in the e a r l ie r mention o f this
su b je c t , w intern* a gaao lin # c o lo r va lu e method has g iven
r&thcr IneonisX s t e n t r e sa lts* k ooiapari son o f th e r as a l t s by
th e .Vinton g n so lin * c o lo r v m lii* and those obtained on the
photo mo ta r i s given I n fa b le aXXX-- Tko earns fou r so 3- u tio n s
M
were OQttparod in th® ooloriantor % ttio Hiiioit prooe&mre
mud then read la %hm tiatitrsl wedge slmildr toy f©®r different A&elrwtw* there is mmeldermhly. hotter agreement ewetig eiielrete'with th® letter preeedwe*
IMs oho tome ter ®mm h® need t© itissrf the-eeretewe ©ae~
treetod frnm rmriom food f f s i i i s l s sM foede* la eonxioeilon
with eiiidi.ee ®m l i t re la tio n to vitmiaia A« Im l ik e manner I t «*m ho owed to moaamro xmrithophjrll aM ehlerogOiyll* I t
ss| heeOM wettfwl la Mewwrlmg ® h X ® im p h $ X % m m %hm% tk ro la seas « $ e r i ssonlel ©rid©nee that p a l a t a M l i o f dried
p t s s s s depend* sa the! r ehlorophyll ooatoat*
. . . 1 , \ ,
%&®®%km® deter fetmee*{Mvmhmr® obtain©! hf uomp^Timm t i H & eieitderd o f a rb ita rtly mmlm®d rwlme o f 1*01 :
1 * 3 4■dmeljet I #94 *4S-- 1*4# 1*91
a 1*0# *4# um i»*§3 1*31 tuieftle to mmmp.ere #*04 #*##4 1*M »M #«4# #*49
^otom eter mime® la #*#*«* eelew leted m **ret*&*»daaljmt 1 1*S# *49 s*97 2»dfi
£ 1*S£ *44 #*§4 3*390 1*04 *44 #*4# 3*3#4 1*1# *49 £*43 3*3#
Smamsa p
&rlden*e he* bmm i*r©s#itt©d fo r the Mod o f a rela* t ir o l^ ©heap instffmsmnt for the eeoisrwte measurement of
the add@4 osrotsao in f lo o r mad m ewrM i prod note* Thm
m
mppMmtlm o f t&a neutral wet go photometer to mmh mmmmm m@m% ham ham deaeribed* thim larelred m rntmw ©f the pigmsmt mmtmmXXf amorlmr la flo w * follow ed by the »@~1 action of a f i l t e r for the jdMrteaeter and the otoo4ortl«
notion of the photometer againet pare •arehoa* a# lotion# A
general method for the extraotle& of added oarotoao from
floor ant meareal. pre&aat* ha® tow dereiopedt ahewing high prewiaio* and good m m @ |» ft# ap p lication of the
method to the dateetiem of added carotene ro%airoe a ocmpre-
honeire etadjr of raw materials end fia ieh ed jMre&awte*Eooatto ly t t lo method fo r to ta l co lor ant fo r eareteae arc
proaanted for' floor# farina* eomolina* macaroni* egg jrelto
ant m ediae# f rom thaee roamlle a ralm# mm to ea leeted a to re wMoh i t can to concluded that oar© tone hae toon
added#
{X j o * • -si* * ii • v 2
(2 ) B r i g g s , G. K. F o r t h w e s te rn M ille r , Jan. 2 2 t P . 97 (1936)
(3 ) Monsoy, ?. A. J . A. 0 . A. 0 . 1 8 ,4 0 9 {1935)
(4 ) w inton, a * A. U. 8 . B a r . Ohem. Bui. 137 (1911)
(5 ) J o r g e n s e n , Holger Cereal Ohma. 4 ,4 6 8 -4 6 9 (1927)
( 8 ) K e n t , J o n e s , Dm P . and H erd , 0 . o . A n a ly s t 5 2 ,4 4 3 —4 5 2 (1927)
( 7 ) F e r r a r i , 0* Q» and B a i l e y , 0 . E . C e re a l Ghem.6 ,2 1 8 -2 4 0 (1929) 6 ,3 4 7 -3 7 1 (1929) 1 0 ,2 7 7 -2 8 6 (1933)
(8 ) B chertz, F* M. J* A g r i . He s e a r c h 2 6 ,5 8 3 ,4 4 0 (1925)
(9 ) Gadda s , e , F . Binmington, I>. 0 . and [email protected], A .B .C .C e r e a l Chea. 11, 1 -2 4 (1934)
(1 0 ) F e r r a r i , G. £ . aim Croze, A. B. C e re a l Ohen.1 1 , o i l - 8 1 4 (1 9 8 4 )
{11) monier - W illiam s, A. u m E e p t s . L ocal Govt. Bd.( G r e a t B r it ia n ) Pub. H e a l th and .Med, Bubjs. F. B a r .,n o . 73 p * p . 10
(1 2 ) Markley, M. 0 . and B a i l e y , 0 . H. C e r e a l Ohesa.1 2 ,3 8 -3 9 (1935)
(15) A i l l s t a t t e r , R. and 8 to 11, A. lint areuehungen ubar c h l o r o p h y l l J u lia s B pringer, B e r l i n (1 9 1 8 )
(1 4 ) L am b e rt Photom etria s i r e d© m easo ra @t gra&ibus lum inis c o lo r mu ot umbrae (1760)
(1 5 ) B e e r Ann. Phyaifc. 8 6 ,7 8 (1852)
(1 6 ) A e c h m e is te r , L . Carotinoi&e (1934)
(17) E a r n e r , e t a l H elv et. China. A c ta 15, 1084 (1930)
(1 8 ) co h o rts , P. M» J . A g r i . R e s e a rc h 50 , 4 6 9 -7 4 (1923)
(1 9 ) C la u s e n and MoCoord J . B. 0 . 1 1 3 ,8 9 -1 0 4 (1936)
(3 0 ) C m ith , J • H. 0 . J . A. 0 . 3 . 5 8 ,2 4 7 -2 5 5 (1936)
(2 1 ) M i l l a r , R« 8 . Botonlc&X G a z e t te 9 6 ,4 4 7 —467 (1935)
(88) 3mi til, 3 . K. 0. J. B. 8 . 104,48? (1934)
(S3) Smith, J, H. 0. tvlvat* Commanieatien
(84) Millar. A. a. Plaat Physiology 9,498 (1984)
(86) ialer, Farrar, 3abrys,Hairat. Ohim. iota 17,24 (1984)
(84) Maoirtimoy, 0. 3. B. 0. 111,78 (1938)
(87) Smith, 3m a. 8. 3 . 3. 0. 96,35 (1938)
( SB) Ksrrar, P. and Ishikawa, s . Holt* Ohira. iota,13.1099-1102 (1930)
(89) f'uhn, 8, and Brockmama, K. 3. Physiol, chaau ■'806,41-44 ;(!9f8) :i '
(30) Boolfcor, A. and 7on Haagai 3aaho A Unborn d Lebons 71,681-30 (1936)
(31) S t r a i n , M* H. * 3 . M a i . O h m . 106,623-34 (1934)
(32) Barla, a , sad Olhaon, K, 3, a lso . Pah. Bureau of standards ilo.114
(33) Jttdd, I), B. 3m Optloal 300. of Aaer. 23,368 (1933)
(34) Millar, Mm 3. J. A. 8. 3. 67,347-349 (1938)
(86) Si Ham sod Hielbron Bioehom. J, 89,1064 (1936)
jmmmt
Mi imMfiQ&rnou of tm abpmoa^iob of m$MmmAL PMotoMAfAR io ,,mji tmismMM*• OF OAROf MQ1D PXGM^m I I &L0VH .A1Q MaOAEOMI MQiKHm* . / ;■ : . _ ;; •, ;
Th® determination of th® ad&X t lon of o&roion© to flour,
floor mixtures, macaroni, egg noodles, eggs and other cereal
products depend on th© accurate Isolation and measurement, of
carotene* Thom® products contain a natural 00X0 ring fac
torial* classified as oarotenoid pigments, which was charac
ter! a od for the purpose of th© detection of added carotene
as practically a ll xanthophyll* Th® total caretenoid
pigments wore extracted, followed by the separation a d
measurement of the carotene* Th® method extra©ted the
esrotenoid pigaents by heating on the steam bath with
alcoholic potassium hydroxide, alcohol filtered o ff, the
residue extracted with ethyl ether, th® pigment transferred
to the ethyl ether by addition ©f water, the ethyl ether
layer mixed with petroleum ether, followed by remoral of
ethyl ether by evaporation to small value, dilution again
with petroleum ether and the xnntbephyll removed by ex
traction with aqueous methyl alcohol* fh® carotene
remaining in the petroleum ether was measured* Thi®
measurement was obtained by use of an instrument described
as a neutral wedge photometer* fills instrument was
calibrated against pare carotene solutions of Xcnowti strength
and the calibration ear?© given for th© conversion of the
photometer so a le readings into caroten e con centration s*
Th© neutral wedge photometer c o n s is t s of a box with a
ligh t source which sh in es through two holes* Attached to
the box i s a h older fo r the absorption c e l l , the compart-
ment co n ta in in g th e graduated scale and neutral wedge, and
the photometer eyepiece* i'n® of the beams of l i g h t pass
through the e o la t io n to be measured and the other through
the neutral wedge* Th© wedge can be moved such th a t the
two fie lds In th e ey ep iece are of equal in te n s ity * The
application of th is photometer depends on the use of proper
f i l t e r s in the eyepiece* The choice of the f i l t e r was
based on the study o f the absorption spectra o f the
o&rotenoid pigiaonta and th© g la s s f i l t e r chosen gives an
absorption in s region o f maximum absorption for both
carotene and x&nthophy1 1 #
The precision and accuracy of the method are good and
there is no se r io u s interference resulting from the use of
the commonly added yellow colors to foods*
The evidence for c h a r a c te r isa tio n of the © srotenold
pigment in flour was baaed on chromatograph!a analysis,
the absorption sp ectra curves obtained and th® behavior of
the pigments in the petroleum ©ther-m ethyl alcohol separ
ation* Tables on the natural carotene con ten t o f flour,
macaroni, noodles and eggs are given*
B i OORAPilY
Vir& ell Gerard lunaey mam bora Goto her 8 , 1899 a t
U&w G a stle , Main©* H@ graduated from L incoln Academy,
Few C a s t le . Main®, In 19X8 and rece ived th e degree o f
B achelor o f Bolenae In Chemical ih g in eer in g from th e
u n iv ersi ty of J a in e , Ororio, Aaine, In 1924 and received
the degree o f an,star o f Boience from George Washington
U n iv e r s ity , Washington, D* 0* in 1931. He has a lso
rece iv ed ten c r e d it hours in H ucation from the U n iv ers ity
o f C in c in n a ti, C in c in n a ti, Ohio.
P u b lica tio n s: -
s tu d ie s on F lour B leaching - J.A.O .A.C. 18 , 4o9-502,1935
Beteriai n a tio n o f iAilh 3 o lid s (Fat Method) J.A.O.A.C*18 , 873, 1938
lic d lf lc d • fta'alo Method fo r d eterm ination o f dtarch - Booh ' v o f Methods^ A* Ok A.G. , 1938 • edi t io n , -p. ' £13*
General R eferee Report on Feed 3tuffs, J#A*0»A*C. 14,141, 1931.
General Referee- Report on Feed 3to ffs, J.A .0.A.Q# 18,213, 1932*
General R eferee Report on Feed B tu ffs , J .iU O .A .0. 16,177, 1933.
General R eferee Report on Feed B tu ffs , J .A .O .ii.0* 17,172, 1934.
General R eferee on Feed 8t u f f s , J*4.0#A .a . 18, 333, 1934*
Report o f Associate Referee on Btarch, J.A.0.A.C* 17,400 , 1934.
Report o f A sso c ia te R eferee on 3ta r ch , J .A .O .A .0 , 18,570, 1935.
Report o f A sso c ia te Refers® on Btareh, J.A.O.A.G. 19,547, 1936.
Report of General Referee on Cereals, J. A. 0.A.C. 19*538* 1936*
Associate Referee Report on Fat in Macaroni* Breadand Baked Products, j.A.O.A.C. 19* 550* 1936#
Report of Associate Referee on Milk Bolide, J.a.O.a.0 .18, 573, 1935.
Associate Referee Report on Milk Bolide* J.A.O.A.C.19* 553* 1936.
He was employed as Acienoo teacher and Athletic
director of Brigham Academy, Bakersfield* Vermont, 1924-25,
and also of Waterbary High School* W&terbury* Verfaont* 1925*
26*27. Baring the summer of 1926 he was employed as a paper
mill chemist for the International Paper Company* Crlens
Falls* Hew Vork. He was employed by the Food and Brag
Admini stration* Uni ted 8 tates Department of Agricaltar® *
Cincinnati* Ohio* in 1927 and is s t i l l Employed at Washington*
B* 0** as an Associate Chemist.
