F. H . S M IT H , State C ollege o f A g ricu ltu re and Engineering, R aleigh, N . C.
Spectral absorption curves are presented for the aniline derivative, o f pure gossypol and gossypol extracted from cottonseed meal and cottonseed meats with 6 0 % alcohol containing ether in the W aring Blendor. These curves represent the difference between the ab
sorption spectra o f the an iline derivative o f gossypol and of gossypol or gossypol extracts at the same concentration. The absorption curve for the cottonseed meats extract is practically identical with that o f pure gossypol ex cep t in the region of 3 8 0 to 4 1 0 mp. The curve for the cottonseed meal extract is shifted slightly to the right but is very similar to that of pure gossypol.
Cottonseed meal samples were extracted by allowing them to stand 1 0 minutes in 3 0 % alcohol, then adding sufficient 7 2 % alcohol to give a 6 0 % alcohol to which ether was added. A fte r blending fo r 5 minutes in the W aring Blendor, the extracts were filtered and m ade to 1 0 0 m l. A liq u o ts were taken for the blanks and deter
minations. The dianilino-gossypol color was developed in the latter b y heating after adding aniline, then the per cent transmittance was read on the Colem an spectrophotometer, from which the gossy
p o l content was determ ined . D uplicate determinations of gossypol in cottonseed meal or cottonseed meats may be com pleted in 2 hours with re a d ily reprod ucible results.
A
N U M B E R of m ethods, b o th gravimetric (2, 6, 7) and spec- tro p h o to m etric (7, 5), for the estim ation of gossypol in cottonseed m eal a n d cottonseed m eats h ave been published. The gravim etric m ethods depend on the reaction of gossypol w ith ani
line to form a relativ ely insoluble dianilino derivative. Lym an, H olland, a n d H ale (6) reported a colorimetric m ethod based on th is reaction. B oatner, C aravella, and K yam e (I) proposed a sp ectro p h o to m etric m ethod for estim ating the gossypol content o f cottonseed m eal a n d cottonseed m eats based on th e reaction of gossypol w ith an tim o n y trichloride. All these methods require a considerable period of tim e varying from 48 to 120 hours for
■completing th e analysis. T h e am ount of gossypol found depends upon b o th th e e x tra c ta n t an d th e length of tim e the extraction is continued.
T h ree processes of ex tractio n have been used: simple equili
bration, in te rm itte n t flushing in th e Soxhlet-type extractor, and continual w ashing in th e B u tt extractor. T he gossypol content
■of cottonseed m eal determ ined by th e la tte r tw o m ethods depends so m ew h at on th e m oisture content of the m eal and th e am ount of alcohol an d w ater in th e eth er used for th e extraction. In order to o b tain satisfactory reproducible results, the m ethods cited m ust be followed w ith care. M oreover, the gossypol found in a given sa m p le of cottonseed m eal varies considerably when determ ined
by th e different m ethods.
A rapid m eth o d is proposed for determ ining the gossypol con
te n t of cottonseed m eal or cottonseed m eats in which the extrac
tio n is carried o u t in a W aring Blendor w ith 75 ml. of 60% ethyl alcohol containing 15 ml. of e th y l ether. T h e filtrate is made to volum e and aliquots are ta k e n for th e spectrophotom etric deter
m ination b y a m odification of th e dianilino-gossypol m ethod of Lym an, H olland, an d H ale (5). T his modification gives a more com plete developm ent of th e dianilino-gossypol color which is .sta b le for 24 hours or longer. D uplicate or triplicate determ ina
tions of gossypol in a sam ple of cottonseed meal m ay be com pleted in 2 hours; cottonseed m eats require slightly less tim e. T h e re
sults are readily reproducible. T h e composition of th e e x tra c t
a n t need n o t be held to extrem ely narrow limits. V alues alm o st as high as those given in T able I were obtained w ith a n 85% a l
cohol m ixture. T he 60% alcohol m ixture has m uch b e tte r foam ing characteristics, which gives a good suspension of the sam ple in th e solvent an d results in less splashing.
E X P E R IM E N T A L
In th e w ork reported here, 25 mg. of pu re gossypol were dis
solved in 5 to 10 ml. of peroxide-free eth er; m ost of th e e th e r was rem oved u n d er reduced pressure, an d th e solution d ilu ted to 250 ml. w ith 95% alcohol. T his, o r a sim ilar solution, w as used for th e sta n d a rd transm ittance-concentration an d spectral ab
sorption curves. A liquots of 2 m l. of th is solution were p ip etted to tw o 25-ml. volum etric flasks. T he color was developed in one of th e flasks by heatin g th e aliq u o t w ith 0.5 m l. of aniline for 40 m inutes a t 60° to 65° C., as outlined under Procedure, a n d the oth er w as used as a blank for running a spectral absorption curve on a B eckm an spectrophotom eter, using th e hydrogen discharge tube. T h is is represented by curve 1, F igure 1.
T hree preparations of pu re gossypol containing 0.2 mg. in 25 ml. gave an average value of 0.569 for log j a t 445 m p when th e color was developed w ith aniline an d read on th e B eckm an spec
trophotom eter using square cuvettes having a d ep th of 0.999 cm.
T h e i J i value a t 445 m p is 712. T he concentration of gossypol
in gram s per 100 ml.
log j a t 445 m p
712 X d ep th in cm. T he am o u n t of
gossy-o o
o
o
W AVELENGTH IN M ILLIM ICRONS Figure 1 . A b s o rp tio n Curves o f A n ilin e
1. Pure gossypol In alcohol-ether mixture. 2 . Gossypol extracted from cotton
seed meal with an alcohol-ether mixture. 3. Gossypol extracted from cottonseed meats with an alcohol-ether mixture. In each case absorption read against respective
untreated solution
44 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 Vol. 18, No. 1 to the rig h t occurs when hydraulic-expressed crude cottonseed oil is th e source of the gossypol (8). T h is sh ift is so slight a n d th e spectrophotom etric estim ation of gossypol in cottonseed meal.
C urve 3, Figure 1, for cottonseed m eats follows th a t for pure
photom eter a t 445 mg, using th e blanks containing th e correspond
ing a m o u n t of gossypol. T h e values were expressed as m illi
A N A L Y T I C A L E D I T I O N 45 per cen t of gossypol in cottonseed meal. In the case of cottonseed m eats, th e m illigram s of gossypol found per 25 ml. tim es 8 gives
therefore, i t is recom m ended th a t two separate charges of cotton
seed m eal be carried th ro u g h th e entire process w ith the color being developed in only one aliquot from each extract. For control work involving large num bers of samples, however, single determ inations should be satisfactory. m ethods also differ in th a t th e proposed m ethod requires only one fourth as m uch aniline an d th a t h e a t is used to prom ote the com
plete color developm ent w hich is stable for a considerable tim e.
In th e Lym an, H olland, an d H ale m ethod, th e color appears to develop more readily for pure gossypol th a n for t h a t extracted from cottonseed m eal. A bout 20 to 24 hours are required to reach the m axim um color in ten sity b y th e la tte r. E th y l alcohol solu more effective th a n 50 for th is purpose.
T h e filtered extracts will become slightly tu rb id if too m uch e th er is lost during th e filtration. A liquots used for th e blanks will also become tu rb id when diluted to volum e w ith a m ixture of 1000 ml. of 60% alcohol and 70 ml. of ether. On th e o th er hand, if th e dilutions are m ade w ith 95% alcohol, tu rb id ity frequently occurs. These difficulties are n o t encountered when all dilutions and washings are m ade w ith th e 72% alcohol-ether m ixture (1000
Chemical Determination of Vitamin A in M ixed Feeds
reaction and the use of a prelim inary chromatographic purification.T
H E analysis of m ixed feeds for th eir v itam in A co n ten t is com plicated by th e relatively low v itam in A level often prese n t, th e large num ber of different ingredients p resen t in th e m ix
tu re, an d th e wide v ariatio n in th e physical an d chemical charac
teristics of these ingredients. These com plications have pre
ven ted the use of spcctrophotom etric m ethods for v itam in A m easurem ent. T he blue color reaction produced by v itam in A w ith antim ony trichloride offers the b est approach to th e problem a t th e present tim e.
chloride under conditions of th e usual v itam in A test.
T he reaction has been utilized for th e q u a n tita tiv e m easure on v itam in A solutions should be carried o u t in subdued artificial light, preferably in am ber glassware) for 4 hours .in a B u tt-ty p e wash the filter p ap er w ith additional solvent.
Pu r i f i c a t i o n. If little or no color is presen t in th e final ex 2.5-cm. (1-inch) lay er of calcium carbonate. T h e dibasic calcium phosphate should be tested prior to.,use a n d show n to retain v ita