INDUSTRIAL a n d ENGINEERING CHEM ISTRY
A N A L Y T I C A L E D I T I O N
HARRISON E. HOWE, E D IT O R < ISSUED A B RIL 15, 1941 » VOL. 13, NO. 4 » C O N S E C U T IV E NO. 8
V IT A M IN S Y M P O S I U M
P h y s ic a l a n d C h e m i c a l D e t e r m i n a t i o n o£ V i t a m i n A ...J. B. Wilkie 209 R e c e n t D e v e l o p m e n t s i n M e t h o d s fo r D e t e r m i n
i n g C a r o t e n e ...Walter J. Peterson 212 C h e m ic a l M e t h o d s f o r D e t e r m i n a t i o n o f V i t a m i n
B , ... Douglas J. Hennessy 216 D e t e r m i n a t i o n o f V i t a m i n B 2 (R ib o fla v in ) . . . .
A. D. Emmett, O. D. Bird, R. A. Brown, Gail Peacock, and J. M. Vandenbelt 219 C h e m ic a l E s t i m a t i o n o f N i c o t i n ic A c id a n d V i t a
m i n B« . . Harry A. Waisman and C. A. Elvehjem 221 C h e m ic a l M e t h o d s fo r D e t e r m i n a t i o n o f V i t a m i n
C ... C. G. King 225 S p e c t r o s c o p ic M e t h o d f o r Q u a n t i t a t i v e E s t i m a
t i o n o f V i t a m i n D ...Nicholas A. Milas, Robert Heggie, and J. Albert Raynolds 227 O p tic a l A c t i v i t y o f Q u i n in e a n d S o m e o f I t s S a l t s in
M ix t u r e s o f W a t e r a n d E t h y l A l c o h o l ...
James C. Andrews and Bailey D. Webb 232 E ffe c t o f T e m p e r a t u r e o f A lc o h o l i n D e t e r m i n a t i o n
o f P o t a s h i n F e r t i l i z e r s ...
C. W. Hughes and O. W. Ford 233 R e d u c in g P r o p e r t ie s o f / - S o r b o s e ...
F. K. Broome and W. M. Sandstrom 234 D e t e r m i n in g C o p p e r a n d N ic k e l i n A l u m i n u m
A l l o y s ...Henry A. Sloviter 235 S i m p l i f i c a t i o n o f P e t e r in g - W o lm a n - H ib b a r d M e t h o d
fo r D e t e r m i n a t i o n o f C h lo r o p h y ll a n d C a r o t e n e . H. G. Petering, E. I. Benne, and P. W. Morgal 236 R e m o v a l o f T h i o c y a n a t e i n D e t e c t i o n o f H a lid e s
David Hart and Robert Meyrowitz 237 D e t e r m i n a t i o n o f F o r m a ld e h y d e w i t h 5 ,5 - D i m e t h y l -
c y c l o h e x a n e d i o n e - 1 , 3 ...
John H. Yce and Lewis C. Reid 238 P e r m a n g a n a t e T i t r a t i o n o f T h a l l o u s S a l t s . . . .
Robert S. Beale, A. Witt Hutchison, and G. C. Chandlee 240
M e a s u r in g O x id a t io n o f V e g e t a b le O i l ...
George L. Clark and Frank M. Rugg 243 O r g a n ic A c id s i n P l a n t T i s s u e s . George W. Pucher,
Allred J. Wakeman, and Hubert Bradford Vickery 244 R a p id D e t e r m i n a t i o n o f S t a r c h (R o o t) w i t h S o d i u m
H y p o c h l o r i t e ...R. T. Balch 246 R a p id D e t e r m i n a t i o n o f R e d u c i n g S u g a r s ...
S. A. Morell 249 D e c o m p o s i t i o n T e m p e r a t u r e s o f S o m e A n a l y t i c a l
P r e c i p i t a t e s . . . . M. L. Nichols and B. E. White 251 P r e c is io n C r y o s t a t f o r R a n g e — 3 5 ° t o + 2 5 ° C . . . .
Edwin E. Roper 257 I m p r o v e d E l e c t r o d i a l y z e r ...F. E. Brauns 259 P h o t o e l e c t r i c P h o t o m e t e r f o r V i t a m i n A E s t i m a t i o n
Allan E. Parker and Bernard L. Oser 260 S i m p l e V a c u u m T u b e R e la y . . . . Earl J. Serfass 262 D e v ic e f o r C o n t in u o u s L i q u id - L iq u id E x t r a c t io n
John R. Matchett and Joseph Levine 264 P e r fo r a te d P la t e C o l u m n s f o r A n a l y t i c a l B a t c h
D i s t i l l a t i o n s ...C. F. Oldershaw 265
M I C R O C H E M I S T R Y
M ic r o a n a ly s is o f G a s e o u s H y d r o c a r b o n s ...
Léo Marion and Archie E. Ledingham 269 I n t e r f e r e n c e s O c c u r r in g w i t h S e l e c t e d D r o p
R e a c t i o n s . . Lothrop Smith and Philip W. West 271 S t u d i e s o f M e t h o x y l D e t e r m i n a t i o n ...
Bert E. Christensen, Leo Friedman, and Yoshio Sato 276 M ic r o - a n d D r o p - S c a le T i t r a t i o n o f O x a la te . . .
Paul L. Kirk and Paul C. Tompkins 277 D e t e r m i n a t i o n o f D ie t a r y F l u o r i n e ...
J. F. McClendon and Wm. C. Foster 280
M O D E R N L A B O R A T O R IE S
L a b o r a t o r ie s o f B e s t F o o d s , I n c . . H. W. Vahlteich 284
T h e A m e ric a n C h e m ic a l S o c ie ty ass u m e s n o re s p o n s ib ility fo r th e s ta te m e n ts a n d o p in io n s a d v a n c e d b y c o n t r ib u to r , to i t s p u b lic a tio n s .
25 ,3 0 0 co p ies of th is issu e p r in te d . C o p y r ig h t 1941 b y A m e ric a n C h e m ic a l S o c ie ty .
P u b l i c a t i o n O ff ic e : E d i t o r i a l O f f ic e : R o o m 7 0 6 , M i l l s B u i l d i n g , W a s h i n g t o n , D . C .
T e l e p h o n e x N a t i o n a l 0 8 4 8 . C a b le : J i e c h e m ( W a s h i n g t o n )
P u b lis h e d b y th e A m e ric a n C h e m ic a l S o c ie ty , P u b lic a tio n Office, 2 0 th «fc N o r th a m p to n S ts ., E a s to n , P e n n a . E n te r e d as seco n d -class m a tte r a t th e P o s t O ffice a t E a s to n , P e n n a ., u n d e r th e A c t o f M a rc h 3 , 1879, as 24 tim e s a y e a r. I n d u s tr ia l E d itio n m o n th ly o n th e 1 s t; A n a ly tic a l E d itio n m o n th ly on th e 1 5 th . A c c e p ta n c e fo r m a ilin g a t s p ecial r a t e of p o s ta g e p ro v id e d fo r m S e c tio n 1103, A c t of O c to b e r 3, 1917, a u th o riz e d J u ly 13, 1918. .
A n n u a l s u b s c r ip tio n r a t e , I n d u s tr ia l E d itio n a n d A n a ly tic a l E d itio n
*old o n ly a s a u n it, m e m b e rs $ 3.00, o th e rs $4.00. F o re ig n p o s ta g e to c o u n tr ie s n o t in th e P a n A m e ric a n U n io n , S2.25; C a n a d ia n p o s ta g e , $0.75.
E a s t o n , P e n n a .
A d v e r t i s i n g D e p a r t m e n t : 3 3 2 W e s t 4 2 n d S t r e e t , N e w Y o r k , N . Y . T e l e p h o n e : B r y a n t 9 - 4 4 3 0
S in g le co p ies: I n d u s tr ia l E d itio n , $ 0 .7 5 ; A n a ly tic a l E d itio n , $ 0 .5 0 . S p ecial r a te s to m e m b e rs .
N o claim s c a n b e allo w ed fo r co p ies of jo u r n a ls lo s t in th e m a ils u n less s u c h claim s a r e re c e iv e d w ith in 60 d a y s o f th e d a t e of issu e, a n d n o claim s w ill b e allo w ed fo r issu es lo s t a s a r e s u lt o f in su ffic ie n t n o tic e o f c h a n g e of a d d re s s . (T e n d a y s ’ a d v a n c e n o tic e re q u ire d .) “ M issin g fro m # files'’
c a n n o t b e a c c e p te d a s th e re a s o n fo r h o n o rin g a c laim . A d d re s s claim s to C h a rle s L . P a r s o n s , B u sin ess M a n a g e r , 1155 1 6 th S t., N . W ., W a sh in g to n , D . C ., U . S. A.
4
H O S K I N S PRODUCTS
• The h e a tin g unit of this FH-303-A C o m bustion Fu rn ace is h e a v y — it’s 7 g a u g e C h ro m e l-A . Hence, it wil! sta n d a lot of h a rd g o in g . This ex tre m e d u r a bility c o nstitutes o n e fac to r in e c o n o m y of m a in te n a n c e . A n o th e r e c o n o m y f e a tu re lies in th e fact t h a t th e heatin g unit co n s ists m e re ly of th e w ire itself.
There is no refracto ry m o u n tin g . The c o m b u s t i o n t u b e p a s s e s d i r e c t l y th ro u g h th e helical coil w h ich is sur
ro u n d e d by h ig h - te m p e r a t u re in s u l a tion. . . . The f u rn a c e s h o w n here is a rela tively n e w m ode l. Due to its i n c re a s e d in su latio n , it h e a ts u p in o n e - third less tim e, u s e s 18% less p o w e r, a n d h a s a c a s e t e m p e r a t u re 120° F.
cooler (at 2 0 0 0 ° F.) th a n before. The fu rn a c e o p e r a te s on A.C. th ro u g h a sm all tran s fo rm er, w ith te m p e r a t u re control th ro u g h a rh eo s ta t. For m o re in form ation on this FH -303-A , of m in im u m m a i n t e n a n c e , w rite to y o u r d e a l e r or to us. . . . H oskin s M a n u fa c turing C o m p a n y , Detroit, M ichig an.
E L E C T R I C H E A T T R E A T I N G F U R N A C E S • • H E A T I N G E L E M E N T A L L O Y S • • T H E R M O C O U P L E A N D
L E A D W I R E • • P Y R O M E T E R S • • W E L D I N G W I R E • • H E A T R E S I S T A N T C A S T I N G S • • E N A M E L I N G
F I X T U R E S • • S P A R K P L U G E L E C T R O D E W I R E • • S P E C I A L A L L O Y S O F N I C K E L • • P R O T E C T I O N T U B E S
A pril 15, 1941 A N A L Y T I C A L E D I T I O N 5
THE SENSITIVITY OF QUALITATIVE REACTIONS
This chart will lie found valuable as a companion picce to the Merck Qualitative Analysis Chart. Containing a vast amount of useful information in concise form, it should he conveniently available in industrial, research, and college laboratories.
We will be glad to send you copies on request. The coupon is for your convenience.
M E R C K & CO. Inc. \d la n u fa c tu tu n a (j/ie m iit'i RAIIW AY, N. J.
New York « Philadelphia • Si. Louis ■ In Canada: Merek & Co. Ltd., Montreal anil Toronlo o
t A i& icA ;
L A B O R A T O R Y C H E M I C A L S
F IN E T O O L S F O R P R E C I S I O N M E A S U R E M E N T S
M
E R C K & CO. Inc. offers a com preh en sive line o f R e agent, C .P ., and other chem icals suitable for all industrial research, educational, and routine plant and laboratory uses. M erck C .P . and R eagent M ineral A c id s and Am m onia W ater are o f h igh est p u rity and are in dicated w herever these a cid s are used.
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Name...
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M y p o s i t i o n i s ...
Large free air displacement . . . 34 liters per minute • Tested to attain a vacuum of 0.1 mm or less. . . test data show all pumps produced so far attain much lower pressures • When compressed air is required . . . this pump will satisfy the need . . . 10 lbs per square inch. May be used to circulate or col
lect gases. . . fumes from distillations may be conducted to vents • These features are of value to the chem ist. . . and at a price lower than ever before • Specify No. 90510A for 115 volts 60 cycle current.
S C I E N T I F I C
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• Toronto • San Francisco
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April 15, 1941 A N A L Y T I C A L E D I T I O N 7
E q u a lly as u n v a ry in g is th e conform ity of M allinckrodt A. R. C hem icals to their pre
determ ined standards of purity. W h ere absolute accuracy in a n a ly tica l w o rk is essential, M allinckrodt reagen ts insure the purity th a t facilitates precision in the laboratory.
Send for new catalogue of M allinckrodt A nalytical R e ag e n ts and other chem icals for laboratory use. C on
tains detailed descriptions of chem icals for every type of analytical w ork . . . gravim etric, gasom etric, colori
m etric or titrim etric.
M A L L I N C K R O D T C H E M I C A L W O R K S
ST. LOUIS • PHILADELPHIA • M O N T R E A L
CH ICAG O • NEW YORK • TORONTO
Unvarying Standards
Congress, under a Joint Resolution o f July 27, 1866, provided that each o f the states be furnished w ith a com plete set o f weights, running fro m 10 kilogram s to 1 m illigram . . . u n varyin g standards o f w eig h t throughout the states.
8 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
“P Y R E X " is a registered tra d e-m a rk a n d indicates m an u fa ctu re by R em em b er that“ pet” funnel you liked so w ell?
Y o u probably tried many before you found it.
M aybe you still have it, o r perhaps you’ve lon g been searching for another just as good.
Search no longer. N o w you can have any number o f “ pet” funnels without searching for them . . . use P yrex brand accurate
6
o° fluted funnels.T h e y are precision-shaped, pressed to an accurate 6 o ° angle w h ich permits close fitting o f filter paper, and the inside flutes double the effective filtering area. A ll these characteristics make for rapid filtering.
M ade o f P yrex brand Chem ical Glass — the Balanced G lass— every funnel is m echanically strong, chem ically stable and, o f course, thermally resistant. A ll are stronger, too, because o f beaded edges and heavy wall stems. Furthermore, automatic m oulding per
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T r y the accurate 6 0 ” P yrex brand fluted funnel and you’ll agree that it is the “ pet” o f the funnel family. Consult your regular labora
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C o r n i n g
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Ji Research in Glass
A pril IS, 1941 A N A L Y T I C A L E D I T I O N 9
H E V I D U T Y E L E C T R I C C O M P A N Y
IH EAT T R E A TIN G FURNACES E L E C T R IC E X C L U S IV E L Y
M I L W A U K E E , W I S C O N S I N
One hundred and forty-five years ago the first chemical laboratory in the world for undergraduates w a s established at N assau Hall b y Dr. John M aclean. N assau Hall still stands as a monument to this cradle of chemical education though the fine Frick Chem ical Labo
ratory now houses new modern laboratory equipment. It is gratify
ing to us to note that Princeton, like m any other leading universities, uses Hevi Duty Laboratory Furnaces for chem ical study and analysis.
HEVI DUTY
F U R N A C E S
10 I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
F .R .I. M IC R O M O D E L
W I L E Y L A B O R A T O R Y M I L L
A c o m b in e d c u t t in g a n d screen in g a p p a ra tu s f o r use in q u a n t it a t iv e a n a ly s is
1276-1).
H o p p e r fo r h a n d lin g la r g e r q u a n titie s of m a te r ia l t h a n c a n b e a c c o m m o d a te d in th e r e g u la r feed tu b e .
C o lle c tin g J a r f o r la rg e r s a m p le s th a n o b ta in a b le w ith m e ta l re c e iv e rs B , C a n d D .
LABORATORY MILL, Wiley-F.R.I. Micro Model.
A co m b in e d c u t tin g a n d sc re e n in g a p p a r a tu s fo r u se in q u a n t ita tiv e a n a ly sis. A s d esig n e d a n d u se d in th e F o o d R e s e a rc h I n s t i t u t e of S ta n fo rd U n iv e rs ity , fo r th e m illin g of v e r y sm a ll q u a n titie s of o rg a n ic m a te r ia ls fo r m ic ro -a n a ly sis. A fte r m illin g , th e r h e o s ta t c a n b e sp e e d e d u p to blo w o u t th e la s t p o r tio n of th e sa m p le a n d th e r e b y m a k e p o ssib le re c o v e ry of p r a c tic a lly th e e n tire a m o u n t — a n im p o r ta n t c o n s id e r a tio n w h e re sm a ll q u a n titie s a re a v a ila b le . T h is m ill h a s b e e n su c ce ssfu lly u se d in p r e p a rin g h o m o g e n e o u s sa m p le s of ce reals, le av e s, b a rk s , ro o ts , c r a b shells, fish scales, b o n es, d e s ic c a te d a n im a l tissu e s, e tc . D rie d m a te r ia ls a r e g e n e ra lly e s se n tia l.Consisting of a polished iron chamber, within which a rotor with two steel cutting edges is so mounted as to revolve at high speed past a stationary steel blade and the cutting end of a steel feed tube, producing a shearing action. This rapid rotation, i.e. up to 5000 r.p.m., keeps the sample agitated and causes it, when cut to sufficient fineness, to fall through the sieve top of the removable delivery tube mounted below the grinding chamber.
The capacity of the feeding tube is 5 ml. The chamber is 31.5 mm diameter X 16 mm deep, its capacity for one charge being about ten kernels of wheat or several leaves. The delivery tubes are offered with sieve tops of 20, 40 and 60 mesh and with removable receivers which afford convenient collection of samples and ease in cleaning. No. 4276-F collecting jar, 2 oz. capacity, with adapter for attachment to the delivery tube, can be substituted for the metal receiver when larger samples are required. Contamination of samples after milling is avoided by the use of this jar as the threaded top with adapter can be removed and replaced by the plastic cap regularly supplied with the jar.
A polished glass plate, held in place on the face of the mill by a swing-aside screw clamp, closes the cutting chamber and at the same time permits observation of the sample during grinding and, when removed, exposes the entire interior for easy cleaning. The mill is coupled by direct drive to a high speed motor, with speed control rheostat, all conveniently mounted on the same base.
See W. H. Cook, C. P. Griffing and C. L. Alsberg, “ A Mill for Small Samples,” Industrial and Engineering Chemistry, Analytical Ed., Vol. 3, No. 1 {Jan. IS, 1931), p. 102.
4276. Laboratory Mill, Wiley-F.R.I. Micro Model, as above described. Complete with wooden plunger A, three de
livery tubes with sieve tops of 20, 40 and 60 mesh, respectively, and removable receivers (B, C and D of illus
tration), extra stationary blade E, extra front plate of glass F, and lapping cylinder G for sharpening the cutting end of the feed tube, tommy pin and wrench. With universally wound motor for 110 volts a.c. or d.c., and with detailed directions for use... 115.00 4276-1. Ditto, but with universally wound motor for 220 volts a.c. or d.c... 120.00
4 2 7 6 -D . H o p p e r , o n ly , of n ic k el p la te d b ra s s , w ith a lu m in u m c o v e r, c a p a c ity 5 0 m l. F o r h a n d lin g la r g e r q u a n titie s of m a te r ia l th a n c a n be a c c o m m o d a te d in th e feed tu b e re g u la r ly s u p p lie d ... 5.25 4 2 7 6 -F . C o llectin g J a r , co n s is tin g of g la ss ja r , 2 o u n ce s c a p a c ity , w ith m e ta l a d a p te r w ith o p e n in g to fit lo w er e n d of th e d e liv e r y tu b e . F o r
c o lle c tio n of la r g e r s a m p le s t h a n o b ta in a b le w ith th e m e ta l rece iv ers B , C a n d D , w ith o u t th e n e c e ss ity of tr a n s f e r r in g a f t e r m illin g a s th e th r e a d e d to p c a n b e re m o v e d a n d r e p la c e d b y th e p la s tic c a p r e g u la rly s u p p lie d w ith th e g la ss j a r ... 3 .1 0
Copy o f P a m p h let EE-107, “ W iley Laboratory M ills,” describing tw o larger m odels, s e n t up o n request.
A R TH U R H. T H O M A S C O M P A N Y
R E T A I L — W H O L E S A L E — E X P O R T
LA B O R A TO R Y A PPA R A TU S A N D REAG ENTS
W E S T W A S H I N G T O N SQ UARE, P H I L A D E L P H I A , U . S . A . C a b le A ddress, “ B a la n c e ,” P h ila d e lp h ia
INDUSTRIAL a n d ENGINEERING CHEMISTRY
A N A L Y T I C A L E D I T I O N
P U B L I S H E D B Y T H E A M E R I C A N C H E M I C A L S O C I E T Y • H A R R I S O N E. H O W E , E D I T O R
P a p e r s f r o m t h e S y m p o s iu m o n V it a m in s p r e s e n te d b e fo re t h e j o i n t m e e t in g o f t h e D iv is io n s o f B io lo g ic a l a n d A g r ic u lt u r a l a n d F o o d C h e m is t r y a t t h e 1 0 0 th M e e tin g o f t h e A m e r ic a n C h e m ic a l S o c ie t y , D e t r o it , M ic h ,, p a g e s 2 0 9 -3 1 .
Physical and Chemical D eterm ination o f Vitamin A
J . B. W ILKIE
U. S. Food an d Drug A d m in istra tio n , W ashington, D. C.
T
H E existence of vitam in A was biologically established about 25 years ago, and since th a t tim e its importance for health and nutrition has become generally recognized. Coincidentally three general methods for its assay have received much attention. One of these, the biological method, is well established and is now recognized by th e U. S. Pharmacopoeia as official. However, because of th e time involved and the expense associated w ith the biological method, much effort has been expended upon possible physical and chemical pro
cedures for this vitam in. T he literature upon the subject is relatively voluminous and contains m any contradictions, but enough work has now been completed to indicate either the causes or th e remedies for many of the earlier difficul
ties. I t is a principal purpose of this review to indicate the current sta tu s of the physical and chemical determinations of vitam in A.
The accum ulated knowledge of the vitam in A determina
tion is largely empirical and not until recently has its struc
ture been determ ined. A study of this structure m ay help to determine causes of error in th e physical and chemical esti
m ation of this substance. W e see by reference to the struc
tural formula th a t it consists of a beta-ionone ring attached to an unsaturated chain with methyl groups and ending with an OH group.
H,C CHS
C CH, CHS
H s < J l > —C H = C H —C = C H —C H = C H —k = C H —CH2OH liaC G—CH,
Y
H,Antim ony Trichloride Reaction
The earlier efforts tow ard th e estim ation of vitam in A dealt largely w ith chemical reactions. The observation was m ade th a t cod liver oils would give color with various types of reagents Buch as arsenic trichloride (89), sulfuric acid (46)>
2 0 9
and antim ony trichloride (49). As early as 1880 a chemical test for the identity of cod liver oil in the U. S. Pharmacopoeia was based upon the action of th e oil w ith sulfuric acid. Thus it appears th a t a te st for vitam in A was available before the vitam in was discovered biologically.
Carr and Price (4) in 1926 studied the analytical possibilities of such a reaction more thoroughly than had been done pre
viously. They established the use of antimony trichloride in chloroform as a quantitative reagent but found th a t the color resulting from its reaction with vitamin A faded rapidly. When this color was examined spectrophotometrically the 620 and 583 in^ bands were found to predominate in concentrates while 606 and 572 bands were found in unconcentrated oils. The antimony trichloride test has been published as a recommended method for concentrates in the British Pharmacopoeia.
A 20 per cent solution of the oil is prepared a t a specified tem
perature (20°); 0.2 cc. of this solution is dropped into a 1-cm.
cell and the oil is placed in a colorimeter or a tintometer. Then 0.2 cc. of the reagent (antimony trichloride) in dry chloroform is added in such a manner as to mix rapidly.
M any other investigators (%, 3, 7 ,1 7 ,1 8 , 21, 80, 82, 33, 34, 86, 37, 43, 45) have shown th a t th e antim ony trichloride re
action is normally susceptible to various factors affecting the developed spectral bands. Changes in these bands m ay be due to interfering materials, such as inhibitors, or to lack of proper precautions in technique, particularly control of tem perature, improper preparation of reagent, or lack of speed in making readings.
For certain classes of m aterials the numerous investiga
tions indicate th a t the antim ony trichloride reaction w ith the proper precautions will give results of quantitative signifi
cance having about th e same accuracy as th e ultraviolet spectrophotometric m ethod considered below.
Several attem pts to modify the Carr-Price method have been made and significant changes have included the use of catechol (40) and guaiacol (41). The test performed with guaiacol was claimcd to provide greater specificity relative to carotene which might be present and to improve persistence of the color formed.
The effect of 20 different modifications of the antimony trichloride reaction has recently been reported by Pacini and Taras (88).
Dann (1938) applied the Evelyn (9) photoelectric colorimeter to the antimony trichloride reaction. His contribution to the
technique, especially in the use of the photoelectric colorimeter, undoubtedly provides needed objectivity by eliminating errors due to personal judgment. While the dependability of such a method may be established for a certain type of material, the possibility of interfering substances in other products may still be significant. Also, speed and special precautions are still needed, though undoubtedly not so urgently as they were origi
nally.
Ultraviolet Absorption
Essentially the only physical characteristic of vitam in A utilizable for its determ ination is its absorption in th e u ltra
violet a t 32S m/x. I t appears th a t the 328 m ¡x ultraviolet ab
sorption characteristic was first discovered by Takahashi in 1925 in fish liver oil concentrates (46). Subsequent British investigators, startin g with M orton and Heilbron in 1928, showed a correlation between absorption and biological ac tiv ity (7, 12, 32, 35).
The conclusion from early evidence largely accumulated by the British workers indicated the ultraviolet absorption to be a more reliable index of vitam in A content than an ti
mony trichloride color reaction with its atten d a n t complica
tions. In 1934 the International unit of vitam in A was established by the H ealth Organization of the League of N a
tions as 0.6 microgram of beta-carotene. Since th a t tim e a large am ount of effort both in England and in this country has been directed toward establishing a possible q uantita
tive relationship between biological units and the unit of ab
sorption a t 328 m/i which has come to be known as the value. T his problem has been the subject of individual stud
ies as well as collaborative effort both in this country and in England (1, 23,26, 27). I t is now ordinarily assumed th a t the vitam in A ultraviolet absorption follows Beer’s law in concen
trations suitable for spectrophotometric examination. Ex
perience in this laboratory, as well as reports and collaborative d a ta from other laboratories, has indicated the validity of this assum ption whenever certain precautions are taken, such as th e m aintenance of purity of th e measured radiation. Gener
ally, then, either th e use of value or a factor for con
verting value into biological units is an accepted prac
tice for comparing results. The factor m ay be established by direct comparison w ith biological assay or more generally by th e use of a secondary standard whose biological value has been established. Such a secondary standard has been avail
able in th e U. S. P. reference cod liver oil biologically stand
ardized against the International standard beta-carotene.
A curious lack of agreement between English workers and American workers as to what the conversion factor should be has persisted until recently. The British workers have found 1600 to be an acceptable factor, while in this country the value most generally used approximates 2000. Differences in biological pro
cedures may possibly explain the discrepancies, although the question of a possible deterioration of improperly stored samples of the U. S. P. reference oil must be considered (81). The experi
enced British workers, Underhill and Coward (8), in 1939 deduced from biological studies involving crystalline vitamin A esters and the International standard beta-carotene th a t the conversion factor should probably be approximately 2000. The crystalline vitamin A prepared by Holmes and Corbet (24) was found by one of their collaborators to have a maximum value of 2100.
With a factor of 2000, this would mean th at the pure crystalline vitamin A has a value approximating 4,000,000 units per gram.
Biological evidence obtained with solutions of this pure vitamin A indicates th at it does in fact have a potency of between 3,500,000 and 4,000,000 International units per gram.
T he collaborative investigations have in general not yielded correlations as close as could be desired. The study completed in 1937 by the American Pharm aceutical Association yielded
¿¡¡itsn, values from 1.4 to 1.79 for the U. S. P. reference oil.
T he study completed last year by th e American D rug M anu
facturers Association yielded essentially th e same values and th e same am ount of variation when both true spectrophotom eters and modified ultraviolet absorption apparatus were jointly considered. However, when only the true spectro
photom eters were used, the spread was reduced to =*=8 per cent. A t the present tim e another collaborative study by the U. S. Pharmacopoeia com mittee on th e spectrophotom etric vitam in A determ ination is nearly completed. This should afford a more acceptable conversion factor and some addi
tional insight regarding previous difficulties, since this is the first study in which only true spectrophotom eters standard
ized with potassium chromate have been exclusively used.
No doubt the greatest cause for discrepancies in either the ultraviolet absorption m ethod or a colorimetric m ethod lies in the labile nature of the molecule of vitam in A itself. In terrelationships w ith vitam in A . and other m aterials ab
sorbing in the ultraviolet or interfering w ith a color te st furnish additional complications.
Morton, Webster, and Heilbron treated a vitamin A concen
trate in alcohol with hydrochloric acid and were able to obtain from the resulting solution the derived product which they iden
tified as 1,6-dimethylnaphthalene. From this it was concluded th a t vitamin A had undergone cyclization (13, 15, 22). This change was associated with the development of numerous bands in the ultraviolet region. I t appears th a t this degraded material may be formed in an oil under various normal conditions, espe
cially if fatty acids are present, or it may occur naturally (29).
The blue values for vitamin A are not changed by cyclization.
The cyclized material has considerable ultraviolet absorption with the principal peak shifted to the region of 368 mu. Some workers in this country have referred to this material as spurious vitamin A, since it has no biological activity.
A compound very similar to vitamin Ai has recently been de
scribed and is known as vitamin A2. Lederer and Rosanova (1937) found substances in certain Russian fresh-water fishes in which antimony trichloride bands a t 690 and 645 van predomi
nate (29). This finding was confirmed and elaborated in 1938 by Edisbury, Morton, Simpkins, and Lovem (14), Gillam, Heilbron, Jones, and Lederer (19), and Lederer and Rathman (2S). Vitamin A or Ai has a peak around 620 or 605 mjj in oils low in vitamin A and a direct absorption maximum at 328 m/t, while vitamin As is responsible for a 690 to 697 m^ antimony trichloride band as well as one at 640 mu- I t was also found to possess direct ab
sorption at the correspondingly longer ultraviolet wave length of 345 to 350 m/i together with another band in the region of 290 m^.
Gillam el al. judged th at vitamin A2 had the same formula as vitamin Ai except that it had two or more carbon atoms or one more ethylene group. Contradictory evidence has been re
cently obtained (1939) by Gray to indicate th at the differences may be explained by the presence of merely one additional double bond and th at vitamins Ai and A2 therefore have the same num
ber of carbon atoms (20).
Although vitamins A, and Aj differ considerably in their ultra
violet absorptions and antimony trichloride values (19, 20), the absorptions of the cyclized compounds have been found by in
dependent investigators to be identical. However, it would appear th at these cyclized compounds are not identical, since Embree and Shantz (lfi) have proposed a quantitative determina
tion of each of the constituent vitamins when mixed, based on the differences of their respective adsorptions on alumina. Vitamin Ai is more strongly held by adsorbing agents. Wald has proposed another method for the estimation of these two vitamins, using simultaneous equations based upon the differences found in the antimony trichloride absorption maxima (47).
F ortunately vitam in A2 preponderates in fresh-water fish while vitam in Ai preponderates in salt-w ater fish, although there appear to be some cases where both vitam ins m ay be present in significant am ounts. T hus a more satisfactory technique for the estim ation of these two compounds in the presence of each other would be helpful.
Stability o f V itam in À
T he stability of vitam in A is influenced principally b y oxi
dation and by light. Vitamin A is destroyed rath er easily by oxidation and certain substances, which appear to occur in
N E E R I N G C H E M I S T R Y Vol. 13, N o. 4
April 15, 1941 A N A L Y T I C A L E D I T I O N 211
natural oils, can inhibit this oxidation. Also, the naturally occurring esters appear to be resistant to attack. I t is com
mon observation th a t an air space in the top of a bottle of fish liver oil can cause a very significant drop in strength in a few weeks, as evidenced by a decrease in the 328 m/i absorp
tion and a corresponding decrease in biological potency even though th e oil is stored in the dark in a refrigerator.
The oxidative deterioration is distinguished by a decrease in absorption a t 328 mju w ithout the development of new bands as in the case of cyclization.
With reference to the effects of light, some investigations (6, 10, 11) have shown th at irradiation can partially destroy vitamin A, while others have shown that it can totally destroy it.
Consequently, attem pts have been made to determine vitamin A by measuring absorption before and after irradiation. The method seems to lack confirmation, although the possibilities in this direction do not appear to be exhausted.
Smith (42, 44) and associates have reported some interesting studies concerning the stability of vitamin A in solution. They found th at decreases in 328 m^ absorption caused by irradiation were partially reversible, indicating isomeric equilibrium af
fected by light. They also reported similar reversible changes by specific solvent action. For example, in one experiment a vitamin A-containing material was first made to volume in chloroform, the absorption was measured, the chloroform was re
moved with a vacuum, and the material was again made to vol
ume with diethyl ether. In this experiment the latter ether solu
tion was found to have an appreciably higher absorption value than the original chloroform solution. Such results have been attributed to isomerism. I t would appear expedient to have in mind the possibility of such phenomena occurring inadvertently before or during a spectroscopic examination.
In the preparation of vitam in A solutions sufficiently pure for light-absorption measurements, it is ordinarily necessary to use extraction and saponification methods (43, 48)- (A fish liver oil, for example, m ight be saponified for 20 minutes with alcoholic potash, extracted four times with diethyl ether, then washed to remove soaps. The ether would then be evaporated alm ost to dryness but never to dryness, and made to volume w ith a solvent more suitable for spectrophoto- metric use.)
These m ethods have received considerable study and al
though n o t completely satisfactory a t the present time, they are usually dependable for m ost materials in the hands of experienced workers. The fact th a t maximum correlation in collaborative work has not been attained with the nonsaponi- fiable and extracted portions of oils suggested th a t further improvement in these methods is desirable. In the separa
tion of carotene from vitam in A in solutions to be used for light-absorption measurements, both the selective action of solvents and th e adsorption of carotene on charcoal have been reported to be satisfactory. A ttem pts have also been made to separate vitam in A by means of selective adsorption. In general, however, this m ay be a dubious practice since there is some evidence th a t chromotographic separation results in uncertain molecular changes in the vitam in component (5, 25).
The evidence thus far accumulated indicates th a t quantita
tive m easurem ents of absorption a t specific wave lengths either before or after chemical reaction can be fairly satis
factory for a given product such as cod liver oil. On the other hand, it is ju st as evident th a t more complicated procedures are both possible and necessary for more accurate analytical work with miscellaneous vitam in A-containing materials.
For such a program a more rapid and accurate definition of entire spectral curves throughout the ultraviolet and visible regions is regarded as fundam ental. The author is therefore a t present actively concerned w ith apparatus, and while his work in this direction is probably far from complete, he feels th a t progress is being m ade along this line. I t is expected th a t such apparatus will be the subject of subsequent publication.
Literature Cited
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Recent D evelopm ents in Methods for D eterm ining Carotene
W A L T E R J . P E T E R S O N
K a n s a s A g r ic u lt u r a l E x p e r im e n t S t a t i o n , M a n h a t t a n , K a n .
M
OST procedures th a t have been devised for the determ ination of carotene and other carotenoid pigments have been based upon th e discovery of Borodin (3) in 1883 th a t the carotenoid pigments could be separated into alcohol- soluble and petroleum ether-soluble fractions. E arly methods for th e quantitative determ ination were also reported by A rnaud (1) and by M onteverde and Lumbimenko (30), b u t the m ethod which has been used most wridely as a starting point in the development of new techniques is th a t of Will- sta tte r and Stoll (36).
Lest it be assumed th a t the latter method, which was pub
lished in 1913, is only of historic significance scientifically it should be pointed out th a t in m ost essentials la ter extraction m ethods have offered no strikingly new contributions. The extent to which this is true is shown by the following brief sum m ary of the W illstiitter and Stoll method.
The fresh plant material is ground finely in a mortar with quartz sand and 40 per cent acetone. The ground material is then filtered and washed with 30 per cent acetone until the fil
trate comes through clear. The extracted material is finally washed with pure acetone, removed from the filter, macerated once again under pure acetone, and filtered a second time. The combined acetone extract is then treated with ether and the ace
tone saponified with methyl alcoholic potash, which is removed from the ether solution by washing with water. The ether ex
tract is evaporated to dryness with vacuum, the residue taken up in petroleum ether, and the extract poured into a separator)' fun
nel. Xanthophyll is then removed from the carotene by washing first with 85 per cent methanol, then 90 per cent, and finally 92 per cent methanol until the washings are colorless. The xantho
phyll which is present in the alcohol phase is then brought into ether solution. Both the carotene and xanthophyll solutions are washed free of methanol with water, dried and brought to volume and the concentrations are determined colorimetrically, using a 0.2 per cent solution of potassium dichromate as the colorimetric standard.
Numerous modifications of th e W illstâtter and Stoll m ethod have appeared (6, 11, 15, 28, 29, SO, 32). Of these, th e G uilbert m ethod (11), which was first presented in 1934, has been m ost widely used as a starting point for recent modi
fications. In this m ethod, ethyl ether is used as an extraction solvent from w'hich the pigments are transferred to petroleum ether. Rapid and effective saponification and extraction are accomplished sim ultaneously by boiling th e sample for 0.5 hour w ith saturated alcoholic potash.
M ore recently Peterson, Hughes, and Freem an (26) in
troduced a modification of th e Guilbert m ethod which is con
siderably shorter, eliminates several possibilities of carotene loss in manipulation, and gives results which are readily re
producible. The original ether extraction of th e Guilbert m ethod has been eliminated entirely in their modification.
Instead, petroleum ether (boiling point 40° to 60°) is used.
This obviates th e necessity of carrying on a single solvent evaporation during the course of the determ ination, and ex
cludes the possibility of carotene decomposition which might occur during the ether evaporation required in the original method. The m ethod is considerably shortened, inasmuch as the chlorophyllins, flavones, alkali, and xanthophyll can be removed directly from the petroleum ether exactly as G uilbert describes their removal from ether and petroleum ether, re
spectively.
Since th e la tte r modification was published a num ber of
changes have been made in the m ethod. Some of these changes were a development resulting from experience gained in th e carotene analyses of several hundred samples yearly. O ther changes were suggested by published modifi
cations.
Early in the author’s investigations it was found, for example, th at the original method, which was highly effective in the ex
traction of carotene in dry feeds which had been run through the Wiley mill, extracted only 50 to 80 per cent of the total carotene from fresh tissues such as pasture plants, grasses, silages, etc.
With these materials it was found advisable to grind the residue from the original digest under alcoholic potash with sea sand in a mortar and to reflux again for an additional 15 minutes to remove all the carotene. If the residue is only ground under alcohol but not redigested, 95 per cent of the carotene is removed. If, in
stead of grinding after the original digestion, the sample is ground before digestion the extraction is not so complete, usually 90 to 95 per cent of the carotene being extracted. I t is always a safe practice in the carotene analysis of strange materials to re
peat the digestion of the thoroughly macerated residue to de
termine whether all the carotene has been removed by one ex
traction. This technique has also proved valuable in testing the efficacy of other extraction techniques which do not involve the use of alcoholic potash as an extraction and saponification me
dium.
In the original method of Peterson, Hughes, and Freeman, it was customary, after digestion of the sample was complete, to decant the liquid extract from the sample residue into a separa
tor}' funnel. The residue was re-extracted several times with petroleum ether and the solvent similarly removed. This opera
tion, from a routine standpoint, was slow, and with finely ground meals stopcocks of the separatory funnels were frequently clogged. Sintered-glass filter funnels were found to be excellently suited to this step in the procedure, since the residue can be re
peatedly and conveniently washed and stirred on the funnel plate until the solvent comes through clear. Alcohol and pe
troleum ether, used alternately in small portions, are particularly effective. The use of Buchner funnels fitted with the appropriate paper is also an improvement over the older decantation tech
nique.
At the suggestion of Buxton and Dombrow (4) it was found that chlorophyllins, flavones, alkali, and xanthophylls could be re
moved from the original petroleum extract effectively by direct extraction with 90 per cent methanol. I t is not necessary to re
move first the water-soluble constituents by preliminary washings with water. This also reduced appreciably the number of wash
ings with 90 per cent methanol, since it appears th at xantho
phylls are more soluble in the presence of small amounts of alkali. In the extraction of xanthophylls, no advantage can be found in extracting the petroleum layer with 85 per cent meth
anol preliminary to washing with 90 per cent methanol.
Since most petroleum ethers ordinarily used in the extraction of carotene are partially soluble in 90 per cent methanol, consider
able difficulty is frequently encountered in the serious reduction of volume in the petroleum phase during the removal of the xantho
phylls. Skellysolve B, a widely used commercial grade of pe
troleum ether, has a solubility of 12 cc. per 100 cc. of 90 per cent methanol. Where this mutual solubility of the two solvents is troublesome it is advisable to saturate the methanol with pe
troleum before use.
Revised procedures m ay now be presented for the extrac
tion of carotene from dry and fresh plant tissues.
Extraction of Carotene from Dry Plant Tissue
Weigh out the samples (1 to 5 grams, more or less, depending on the relative potency), transfer to a 250-cc. Erlenmeyer flask, and add 100 cc. of freshly prepared, 10 per cent ethanolic potas
sium hydroxide. F it the flasks with reflux condensers, and boil the contents on a steam bath or hot plate for 30 minutes. If portions of the sample collect on the sides of the flask, wash down
with alcohol from a wash bottle. Cool the contents of the flask, then pour them into a sintered-glass filter funnel, applying a vacuum only until most of the solvent has come through. Then wash the residue alternately with 25-cc. portions of Skellysolve B and absolute alcohol until the filtrate comes through clear.
The suction should a t no time be applied unless the sediment is partially covered by solvent. After the addition of each wash portion of solvent, more complete extraction may sometimes be obtained by stirring the sediment on the funnel plate with a stir
ring rod before applying suction. Transfer the filtrate to a 500-cc.
separatory funnel.
Pour gently about 100 cc. of distilled water through the alcohol- Skellysolve solution in the separator}' funnel. Draw off the al
kaline alcohol-water solution from the bottom of the funnel and re-extract three times by shaking gently with 30-cc. portions of Skellysolve B, using two other separatory funnels. Combine the Skellysolve extracts and wash free from chlorophyllins, flavones, alkali, and xanthophylls by shaking thoroughly with 30-cc. por
tions of 90 per cent methanol (five washes are generally suf
ficient) and re-extract the first methanol portion with 50 cc. of Skellysolve B. Wash the Skellysolve B solution once with 50 cc.
of distilled water to remove the alcohol and filter into a volu
metric flask through filter paper upon which is placed a small amount of anhydrous sodium sulfate. After making the carotene solution up to a definite volume, determine the concentration by the spectrophotometer, photoelectric colorimeter, or colorimeter by comparison with 0.1 per cent or 0.036 per cent potassium di- chromate.
Extraction of Carotene from Fresh Plant Tissue
Cut fresh plant tissue finely with shears and mix as thoroughly as possible. Weigh samples of 4 to 10 grams into a 250-cc. Erlen- meyer flask and proceed with digestion and washing as for dry materials. When the residue on the filter plate has been thor
oughly washed, transfer it into a deep mortar with a well-defined lip, add 10 cc. of a saturated solution of potassium hydroxide in ethanol, and macerate with the pestle. Add quartz sand and grind. From time to time wash down the sides of the mortar with a stream of alcohol from a wash bottle and grind until the tissue is fine. Transfer the contents of the mortar to the Erlen- meyer flask used in the original digestion, using more alcoholic potassium hydroxide to effect the transfer. Add alcoholic po
tassium hydroxide until the total volume is 100 cc., digest for 15 minutes, and proceed as indicated for dry tissue. Combine fil
trates from both digestions and washings before extraction of xanthophylls.
Determ ination of Carotene Concentration
Of the three methods available for the determination of carotene concentration, colorimetric, spectrophotometric, and photoelectric photometric, the colorimetric method has probably been used m ost extensively (1, 11, 21, 26, 27, 28).
Accurate results have been obtained by this method. The spectrophotometer, however, has an additional advantage in th a t no standard solution is needed for comparison. One needs only determine the absorption coefficient for pure carotene in the solvent to be used a t some convenient wave length, usually in the region of one or both of the absorption maxima.
In laboratories of this station, as in many others, the photo
electric colorimeter (31) has almost completely replaced the colorimeter and spectrophotom eter in the determination of pigm ent concentration.
T he spectrophotometer, however, has not lost its value in th e study of m any aspects of the carotene problem. I t is use
ful in th e determ ination of related chromogens which fre
quently are found in carotene extracts, and w'hich may or m ay not possess vitam in A potency, and may also be used to advantage in the calibration of the photoelectric pho
tom eter. I t is in th e la tte r use of th e instrum ent th a t some significant observations regarding carotene solutions have
been made.
The photoelectric photom eter is usually calibrated for the determ ination of carotene by the preparation of a curve of transmission rs. carotene concentration in which the carotene
April 15, 1941 A N A L Y T I C
concentration of a number of solutions has been determined or checked by means of the spectrophotometer. Crystalline /3-carotene, which gives a normal absorption spectrum, is usually the standard. Rarely, however, does one find in practice a carotene extract which has absorption maxima and a minimum identical with th a t of true /3-carotene, nor are the ratios of the optical densities a t various wave lengths correct.
The value of log ~ a t 4500 Â. is frequently too large with respect to th a t a t 4700 A. (27) while log j a t 4800 A. is usually less than th a t a t 4700 A. In Table
I
are shown the relative spectral absorptions of a num ber of carotene extracts from different sources expressed in percentages of absorption a t wave length 4700 A.Ta b l e I. Re l a t i v e Sp e c t r a l Ab s o r p t i o n’ o p Ca r o t e n e Ex t r a c t s
(E x p ressed in p e rc e n ta g e s o f a b s o r p tio n a t w a v e le n g th 4700 A .)
N o . o f ,
S am p les S a m p le *1500 A. 4700 À . 4S 00 A.
I, E D I T I O N 213
5 /3-C arotene 118 100 105
20 S p rin g g rasses 117 100 103
21 S u m m e r grosses 111) 100 103
11 F a ll g rasses 129 100 106
6 B u tt e r 114 100 104
42 46
E g g y o lk D e h y d r a te d a lfa lfa
117 119
100 100
92 105
16 P r a ir ie h a y 138 100 103
3 S o rg h u m silag e
Y ello w co rn 123 100 100
6 116 100 87
27 M iscellan eo u s co m m e rc ia l feed s 120 100 96
I t is apparent from an inspection of Table I th a t a carotene concentration obtained by means of a photoelectric pho
tometer, calibrated w ith true /8-carotene, m ay agree w ith th a t obtained by the spectrophotom eter a t one wave length, and n ot at all a t some other wave length.
Reasons for these peculiar changes in the absorption spec
trum of /3-carotene are n o t readily explained. On all adsorb
ents the main pigment fraction adsorbs in the same position as /3-carotene and cannot be distinguished from it when the two are adsorbed simultaneously. W ithin the usual lim its of experimental error in th e ra t assay for vitam in A potency it appears th a t the main pigment fraction has a vitam in A po
tency equivalent to th a t of true /3-carotene. The main pig
m ent fraction on removal from an adsorption column possesses an abnormal absorption spectrum approaching more nearly th a t of a-carotene rather than /8-carotene. Zechmeister and Tuzson (38) have m ade the observation th a t carotenoids readily undergo certain isomerization processes in certain solvents, particularly when heated. These changes are said to be accompanied by a m ovement of th e extinction maxima toward the shorter wave lengths. I t is claimed th a t this phenomenon is not induced by th e adsorption process as de
scribed by Gillam and co-workers (10). All attem p ts to separate petroleum fractions from a num ber of the materials listed in Table I under conditions known to prevent the solvent, heat, and adsorption effects described resulted in no improvement in the absorption spectra. I t seems clear th a t the abnormal absorption spectra are an intrinsic property of th e pigments as found in the original m aterial previous to extraction.
No discussion of methods for th e determ ination of carotene would be complete w ithout some consideration of techniques suggested for the separation of “ true’' /3-carotene from the noncarotene chromogens found in m ost petroleum extracts of feeds and silages.
Hegsted, Porter, and Peterson (12) have described a method involving the extraction of the petroleum extract w ith aque
ous diacetone.
