W . A . P O N S , JR., A N D J O H N D. G U T H R IE Southern Regional Research Laboratory, N ew O rleans, La.
A method is described for the determination of inorganic phos
phorus in a variety of plant materials, Including those high in pro
tein. A trichloroacetic acid extraction and colorimetric evalua
tion by a modification of the Berenblum and Chain procedure are utilized. The molybdenum blue complex developed in isobutyl alcohol has absorption maxima at 6 2 5 -6 3 0 and 730 m/j, respectively, and is stable for 19 hours.
A
M E T H O D for determ ining inorganic phosphorus in p lan t m aterials was required in investigations on th e influence of th e phosphorus com pounds p resent on th e preparation of oilseed p roteins for in d u strial utilization. I t w as essential th a t th e m ethod be unaffected b y large am o u n ts of protein or by colored an d tu rb id extracts. A fter consideration of available m ethods, a trichloroacetic acid ex tractio n of inorganic phosphorus and colorim etric analysis by a modified B erenblum a n d C hain (8)Sulfuric acid, approxim ately N . D ilu te 114 ml. of concentrated sulfuric acid to 4 liters.
S tannous chloride stock solution, 10 gram s of stan n o u s chloride hexahydrate dissolved in 25 m l. of concentrated hydrochloric acid. Store in a sm all glass-stoppered brow n bottle.
Stannous chloride, dilute solution. D ilu te 1 ml. of stock solu
tion to 200 ml. w ith approxim ately N sulfuric acid ju s t before use.
Iso b u ty l alcohol. Com m ercial grade, w ith a boiling range 106°
to 110° C ., is satisfactory.
E th y l alcohol, 95% .
S ta n d a rd phosphate solution. R ecrystallize A.C.S. grade mono
basic potassium ph o sp h ate th ree tim es from w ater, d ry a t stable. D ilutions of th e stock solution are m ade as needed.
A N A L Y T IC A L PRO CED U RE
W eigh 1-gram sam ples of th e ground m aterial, 60-mesh or finer in to 125-ml. glass-stoppered E rlenm eyer flasks; -add exactly 50 ml. of 0.75 N trichloroacetic acid; and shake m echanically for 1 hour. F ilte r th ro u g h ashless filter p aper of m edium re te n tiv ity , discarding th e first portion of th e filtrate.
P ip e t aliquots containing from 0.005 to 0.045 m g. of inorganic
of inorganic phosphorus in th e range 0 to 0.045 mg. of phosphorus, obtained b y diluting stock p hosphate solution, in to 125-ml. sep
ara to ry funnels and developing th e color exactly as outlined in the procedure. T he logarithm s of th e per cent transm ission values of the sta n d a rd s m ay be fitted to a stra ig h t line by th e m ethod of
W hen using th e Beckm an spectrophotom eter calculations m ay be m ade from th e extinction coefficient. T h e extinction coefficient
Table I. Calibration Data for Evelyn Colorimeter
M g. of P in 50- a d ilu te sulfuric or hydrochloric acid e x tractio n of inorganic phos
phorus (S, 6, 11). Alcohol acidified w ith hydrochloric acid has also been used (5). F o r m aterial high in protein, however, ex
traction w ith d ilu te sulfuric or hydrochloric acid is unsatisfactory, since enough p ro tein is ex tracted to cause a p recip itate to form hydrochloric acid, protein being p recip itated from th e filtrates b y m aking them 0.75 N w ith respect to trichloroacetic acid before analysis. T h e sam e a m o u n t of inorganic phosphorus, 0.71 mg., extracted in each case, w as equal to t h a t ex tracted b y trichloro
acetic acid in th e concentration range 0.37 to 1.0 N . Experience w ith a v a rie ty of p la n t m aterials has shown 0.75 N trichloroacetic acid to be satisfacto ry for th e extraction.
M arch, 1946 A N A L Y T I C A L E D I T I O N 183 E x trac tio n tim e was investigated by extracting 1-gram sam ples
of a different solvent-extracted p e a n u t m eal for periods ranging
S atisfactory recovery of inorganic phosphorus added to peanut a n d cottonseed m eals in th e form of m onobasic potassium phos
p h a te , K H 2PQi, an d p rim ary calcium phosphate, C a(H 2PO();.- I i 20 , w as obtained as show n in T able II . S atisfactory recovery of inorganic phosphorus added to highly colored trichloroacetic acid ex tracts of goldenrod, sw eet potato, and tobacco leaves was also obtained. Tricalcium phosphate, Ca3(PO()i, is also soluble in 0.75 N trichloroacetic acid.
N o interference due to colored extracts has been observed.
C hlorophyll is n o t extracted by 0.75 N trichloroacetic acid. T he yellow color of m ost p la n t extracts did n o t pass into th e isobutyl alcohol a n d consequently w as discarded w ith th e aqueous phase.
In cases w here color rvas extracted by th e isobutyl alcohol, ex
am ples being cottonseed m eal, w heat straw , goldenrod, and sw eet p o tato leaves, th e extracted color had no absorption a t 730 mp. investigated contained reducing agents which caused a p artial reduction to m olybdenum blue prior to th e addition of stannous hours, and were th en again analyzed for inorganic phosphorus.
F rom th e resu lts shown in T able I I I i t is evident th a t glucose-1- phosphate is rap id ly hydrolyzed by th e 0.75 N trichloroacetic acid used in th e ex tractio n step. F ru cto se diphosphate is hydrolyzed very slowly, while sodium /3-glycerophosphate, adenylic acid, and p h y tin show no hydrolysis a fte r 24 hours in c o n ta c t w ith th e acid. were analyzed after stan d in g for various periods determ ined from th e tim e th e m olybdate was added to th e tim e a t which th e sh ak ing w ith stannous chloride w as com pleted. T h e sh o rtest interval, 4 m inutes, showed 0.07% hydrolysis of glucose-l-phosphate, 5
veloped colored solutions were allowed to sta n d overnight, show
ing th a t com plete separation of inorganic phosphorus from glucose-l-phosphate w as o btained in th e sh ak eo u t m ethod. T he shakeout m ethod should prove valuable w hen it is used to follow enzym atic synthesis by m easuring increase or decrease in in
organic phosphorus. A n alm o st insignificant a m o u n t of hy
drolysis of glucose-l-phosphate takes place in th e approxim ately 5 m inutes needed for analysis; and, once developed, th e colori
186 Vol. 18, No. 3 A lthough from the results in T able IV it is evident th a t easily
hydrolyzable organic phosphorus com pounds do n o t ap p ear to be present in an y significant a m o u n t in th e p la n t m aterials investi
gated, w ith o th er m aterials 1-hour extraction should bo com pared w ith overnight extraction to establish th e absence of these com
B, Standard phosphorus solution, 0.030 mg. of phosphorus C , Trichloroacetic acid extract of cottonseed meal, 0.031 mg. spectrophotom etric curves for each of the complexes extracted w ith iso b u ty l alcohol w as sim ilar to th a t of th e curves for th e u n reduced m olybdate m ethod of G erritz (9) are also given.
A C K N O W L E D G M E N T
T h e ■authors are indebted to R . T . O ’C onnor for th e sp ectro photom etric curves, to E . B. S chum an for preparation of th e figures, an d to T. D . F o n tain e for th e p rep aratio n of th e p ean u t an d cottonseed meals an d for a num ber of suggestions. T he sam ples of glucose-1-phosphate and sodium /3-glycerophosphate were o btained th ro u g h th e generosity of D avid M . G reenberg of