A n a l y t i c a l E d i t i o n V o l .
7, No. 2
M a r c h
15, 1935
I n d u s t r i a l
AND ENG INEERING
C h e m i s t r y
VOL. 27, CONSECUTIVE NO. 10
Pu b l i s h e d b y t h e Am e r i c a n Ch e m i c a l So c i e t y Ha r r i s o n E . Ho w e, Ed i t o r
P u b l i c a t i o n O i t i c b : Eatton, P a .
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C O N T E N T S
16,100 Copies of This Issue Printed Calcium Hypochlorite as a Volumetric Oxidizing Agent . .
. . ...I . M . Kollhoff arid V. A. Sterujer 79 A New Method lor Determining Invertase Activity . . .
. . . W. R. Johnston, Sutton Redfern, and G. E. Miller 82 Determination of Mercaptans in Hydrocarbon Solvents. .
. . . . William M. Malisojf and Claude E. Anding, Jr. 86 Determination of Gas, Coke, and By-Products of Coal . .
... W. A. Selvig and W. II. Ode 88 A Rapid Test for Chlorate I o n ...II. It. Ojjord 93 Iodine Value of Tung Oil . K. Ho, C. S. Wan, and S . II. Wen 96 Direct Determination of Total Oxygen in O i l s ...
... Maurice E. Marks 102 Determination of Free Sulfur in R u b b e r ...
...A. F. Hardman and II. E. Barbehenn 103 Metallic Salts of Ursolic Acid . II. M. Sell and R. E. Kremers 105 Determination of Lactose in Mixed F e e d ...
...D. A. Magraw and C. W. Sievert 106 Determination of Copper in Organic M a t t e r ...
. . . Olive Sheets, Robert W. Pearson, and Marvin Gieger 109 Adsorption of Organic Liquids by Cellulose Products . .
...I. Wiertelak and I. Garbaczowna 110 Determining Correct Weight of Sample in Coal Sampling
...Louis S. Kassel and T. W. Guy 112 Viscosity D ata for Commercial Rosin and Abietic Acid . .
. George S. Parks, Monroe E. Spaght, and Lois E. Barton 115 A Rapid Method of Preparing Biological Materials for
Phosphorus D eterm inations...II. W. Gerritz 116
Recovery of Silver and Iodine from Silver Iodide . . . . ... Joseph R. Spies 118 Improved Gas Burner Top for Ignition in Determination of
Potash in Fertilizers . . . L .E . Horat and 0. W. Ford 119 Employment of Potassium Ferrocyanide in Standardization
of Dilute Potassium P e r m a n g a n a te ...
...Edwin J.deBeer and Axel M. Hjort 120 Separation and Detection of Cyanide...
...L. J . Curtman and S. M. Edmonds 121 A Simple Photoelectric T herm oregulator...
...William L. Walsh and Nicholas A. Milas 122 Determination of Uric Acid in the Mixed Excrement of
Birds...lames C. Fritz 123 Benzoyl Aura mine G . John T. Scanlan and J. David Reid 125 Identification of Certain Solvents by the X anthate Reaction
...Willet F. Whilmore and Eugene Lieber 127 Syringe Pipets... August Krogh 130 Precise Determination of Oxygen in W ater by Syringe
P i p e t s ...•... August Krogh 131 Fluorescence of Gaseous Acetone as a Test for Traces of
Oxygen... Glenn II. Damon 133 A Six-Atmosphere Mercury M a n o m e te r ...
... Walter Scholl and R. O. E ■ Davis 135 Preparation of Sintered Glass Filters . . . . Paul L. Kirk 135 Use of Sintered Glass Disks in Distillations . M .M attikow 136 A Simple Inexpensive Galvanometer Suspension...
. . . • ...II. Roswell Jones 136
S u b s c r ip tio n to n o n m e m b e rs . In d u s t r i a la n d En g i n e e r i n g Ch e m is t r y, J 7 .5 0 p e r y e a r. F o re ig n p o s ta g e $ 2 .1 0 , e x c e p t to c o u n tr ie s a c c e p tin g m a il a t A m e ric a n d o m e s tic r a te s . T o C a n a d a , 7 0 c e n ts . An a l t t ic a l Ed it i o no n ly , J 2 .0 0 p e r y e a r, s in g le co p ies 7S c e n ts , to m e m b e rs 80 c e n ts . F o r e ig n p o s ta g e , 3 0 c e n ts ; C a n a d a , 10 c e n ts . Ne w s Ed it i o no n ly , S I .50 p e r y e a r. F o re ig n p o s ta g e , 6 0 c e n ts ; C a n a d a , 2 0 c e n ts . S u b s c r ip tio n s , ch a n g e s o f a d d r e s s , a n d c la im s fo r lo s t cor>ies s h o u ld b e re f e r r e d to C h a rle s L . P a rs o n s , S e c r e ta r y , M ills B u ild in g , W a s h in g to n , D . C . T h e C o u n c il h a s v o te d t h a t n o c la im s will b e allo w e d f o r co p ies of jo u r n a ls lo s t in th e m a ils, u n le ss su ch claim s a re re c e iv e d w ith in 6 0 d a y s of th e d a t e o f is s u e , a n d n o c la im s w ill b e allo w e d f o r issu es lo s t a s a r e s u lt of_ in s u ffic ie n t n o tic e of 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 r e q u ir e 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 f o r h o n o rin g a c laim . I f c h a n g e o f a d d r e s s im p lie s a c h a n g e of p o s itio n , p le a s e in d ic a te it s n a t u r e .
The A m e r i c a n C h e m i c a l S o c i e t y a ls o publishes the Journal of the American Chemical Society a n d Chemical Abstracts.
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6 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. 7, No. 2
SPECIAL A P P A R A T U S
EX T R A CT IO N A P P A R A T U S
S T O P C O C K S
IMPORTANT 1 FACTS FOR THE CHEMIST ON ^
F U N N E L S
I H E COST o f all lam p-blown chem ical apparatus is relatively high. I t involves more handwork th an on th e more sim ple-shaped beakers and flasks. Your in vestm en t in co stly apparatus is best protected b y the “ P Y R E X ” trade-mark.
In the Corning Lamp Shops skilled workers are aided b y th e la te st developm ents in glass technology. “ P Y R E X ” brand glass allows the fullest application o f their skill; th ey h ave th e benefit o f equip
m ent specially designed for working “ P Y R E X ” glass; and construct apparatus m ostly from m ould blow n blanks o f uniform wall th ick ness instead o f less uniform and less rugged blanks fabricated from tubing.
Their finished product goes through tim e-and-tem perature co n trolled lehrs, insuring correct annealing.
T h ey have th e aid o f technologists w ho understand b oth the chem ist’s requirem ents and th e art o f glass blow ing.
T he unusual chem ical and physical properties o f “ P Y R E X ” brand glass, com bined w ith skilled workm anship guided b y scientific supervision, insure th e production o f laboratory apparatus o f o u t
standing quality.
B e sure th a t th e “ P Y R E X ” trade-m ark below is reproduced exactly on every piece o f laboratory apparatus you buy.
PYREX
LABORATORY GLASSWARE
“ P y r e x ” i s a t r a d e - m a r k a n d i n d í c a l e s m a n u f a c t u r e b y C o r n i n g G l a s s W o r k s • C o r n i n g ,
N. Y.
March 15, 1935 A N A L Y T I C A L E D I T I O N 7
HOSKINS
Electric
FURNACES
Eltçtrlcàl Htat PossibleThe WirtFrom the Mine, to Us- and B ack Again
F ro m th is “ In c o ” m in e com es th e o re l h a t su p p lies th e 80% n ic k e l c o n te n t o f C h ro m e l h e a tin g - e le - m e n ts . I n o u r p l a n t i t is alloyed w ith 20% c h r o m iu m a n d d ra w n to w ire t h a t is u se d in all H osk in s F u rn a c e s. T h e n b a c k to o n e o f th e I n te r n a tio n a l N ickel Co. la b o ra to r
ies w e n t th e fu rn a c e sh o w n below .
T h is f u r n a c e , FD-203, h a s coiled u n i t s o f C h ro m e l, o p e ra te s o n lin e voltag e, a n d is good fo r u se u p to 1800° F . T h e u n i t is a o n e-p ie ce h e lic a l coil t h a t is w ra p p e d a r o u n d a grooved m u ffle . T h u s i t is very sim p le to re n e w . S en d y o u r i n q u iry to y o u r d e a le r. H o sk in s M fg. C o., D e tro it.
Wc have a h an d y little g ad g et called a H e a tin g -U n it C alcula
to r. T ells how to design coiled u n its u p to 1000-W, u sin g C h ro m el w ire. Ask for F orm -K Y .
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 Vol. 7, No. 2
COMPLETE ASSORTMENTS OF “PYREX” AND
“EXAX” LABORATORY GLASSWARE
IN OUR STO CK FOR IM M E D IA T E S H IP M E N T
“PYREX”
B R A N D
LABORATORY GLASSWARE
Of the 1,157 items of
“ p y r e x ”Brand Laboratory Glassware listed in the manu
facturer’s catalogue, 610 are already listed in the current 1044-pp edition of our cat
alogue LABORATORY APPARATUS AND REAGENTS—published before the Corning catalogue.
W e now m a in ta in , i n a d d itio n , c o m p le te sto c k s o f th e re m a in in g 547 ite m s lis te d in th e “ P yrex” c a ta lo g u e , fo r im m e d ia te s h ip m e n t, in o rig in a l p ac k ag es, a n d a t m a k e r ’s c u r r e n t p rice s.
T he
“ p y r e x ”items n ot already listed in our catalogue m ay be conveniently ordered of us under the m ak er’s num bers as printed in the ccPYREx”'Catalogue which has already been distrib u ted directly to consumers by the Corning Glass W orks.
BLUE LINE “EXAX”
GRADUATED GLASSWARE
k *
0 Î <EXAX> Ü B L U E L I N E
O f th e 353 ite m s o f B lue L in e “ E x a x ” G ra d u a te d G lassw are lis te d in th e m a n u fa c tu r e r ’s new c a ta lo g u e now in d is trib u tio n , 107 a re a lre a d y lis te d in th e c u r r e n t e d itio n o f o u r 1044-pp c a ta lo g u e L A B O R A T O R Y A P P A R A T U S A N D R E A G E N T S w ith o u t, h o w ev e r, th e d e s ig n a tio n ,
“ blu e lin e ,” w h ich w as n o t a d o p te d u n til a f te r th e p u b lic a tio n o f o u r c a ta lo g u e in O c to b e r, 1931.
T h e se ite m s are now fu rn is h e d w ith b lu e line g ra d u a tio n s.
W e n o w m a i n t a i n , in a d d itio n , c o m p le te s to c k s o f th e r e m a in in g 246 ite m s o f B lu e L in e “ Exax” w a re fo r im m e d ia te s h ip m e n t, in o rig in a l p a c k a g e s, a n d a t m a k e r ’s c u r r e n t p ric e s.
T h e “ E x a x ” ite m s n o t a lre a d y lis te d in o u r c a ta lo g u e m a y be c o n v e n ie n tly o rd e re d o f us u n d e r th e m a k e r’s n u m b e rs as show n in th e new c a ta lo g u e o f B lu e L in e “ E x a x ” G r a d u a te d G lass
w are re c e n tly p u b lis h e d a n d d is tr ib u te d d ire c tly to co n su m ers b y K im b le G lass C o m p a n y .
C o m p le te a s s o r tm e n ts o f b o th th e 1,157 ite m s o f “ P yre x” B r a n d L a b o ra to ry G lassivare a n d o f th e 353 ite m s o f B lu e L in e “ Exax” G ra d u a te d G lassicare are n o w
o n p e r m a n e n t e x h ib itio n in o u r sa lesro o m .
ARTHUR H. THOMAS COMPANY
R E T A IL — W H O L E S A L E — E X P O R T
LABORATORY APPARATUS REAGENTS
W EST W A SH IN G TO N SQ UARE P H IL A D E LP H IA , U.S.A.
C a b le A d d ress, “ B a la n c e ,” P h ila d e lp h ia
•»Tji " "... ...
A N A L Y T I C A L E D I T I O N
---*
I n d u s t r i a l
( p O U T E C h ; wAND ENG INEER ING
X 4 £ K A $ >Vo l u m e 7
( j h e m i s t r y
Pu b l i s h e d b y t h e Am e r i c a n Ch e m i c a l So c i e t y Ha r r i s o n E . Ho w e, Ed it o r
M a r c h
15,
N u m b e r 2
--- =
1935
Ü++-
Calcium Hypochlorite as a Volumetric Oxidizing Agent
Stability and Standardization of the Solution. Determination of Ammonia
I . M . K o l t h o f f a n d V. A . S t e n g e r , S chool o f C h e m is try , U n iv e r s ity o f M in n e s o ta , M in n e a p o lis, M in n .
I
T HAS come to th e authors’attention th a t “H . T. H .,” a c a lc iu m hypochlorite of high available oxidizing strength m anufactured by th e M athie- son Alkali Works, New York, yields stable solutions. This being th e case, they have a t
tem pted to m ake use of the prod
u ct in th e quantitative oxida
tion of various reducing sub
stances. In general, hypobro- m it e a c ts m o re r a p i d l y in oxidations th a n does hypochlo
rite, b u t is m uch less stable.
Since hypochlorite reacts quickly w ith bromide to form hypobro-
Solutions o f hypochlorite have fo u n d little application in volumetric analysis since in general they have been fo u n d unstable. I t was fo u n d that “II. T. I I ” calcium hypochlorite yielded solutions which were quite stable. B y adding an excess o f bromide to the sample to be titrated, the added hypochlorite behaves as hypobromite. A systematic study is being made o f the application o f standard hypochlorite to the determination of various reducing substances. I n this paper the standardization o f the solution either in acid or weakly alkaline medium, with Bordeaux as indicator, is described and its use in the determi
nation o f am m onia investigated.
m ite, th e advantages of both sta
bility and rapid oxidation m ay be obtained by keeping a stock solution of calcium hypochlorite and adding an excess of alkali bromide to each sample before titration. B y this means calcium hypochlorite m ay be expected to be
come useful in any reaction a t present involving th e use of sodium hypobrom ite or bromine-bromide solutions. (It m ay be m entioned th a t K . Jellinek and W. Krestoff, 4, used so
dium hypochlorite as a standard reagent. T h ey k ep t th e stock solution in clear glass bottles and found after 17 days of standing a decrease in strength of about 2 per cent. Possibly the reagent would have been found m ore stable if protected from light.) The authors are now engaged in a study of the application of calcium hypochlorite to th e determ ination of various inorganic compounds; and it should be useful as well in q u antitative organic analysis. In this paper are discussed th e stability and other general properties of calcium hypo
chlorite solutions, indicators for use in weakly alkaline and acid m edia, and an application to th e determ ination of ammonia. Subsequent papers will deal w ith th e determ ina
tion of several other inorganic substances.
Re a g e n t s
Ca l c i u m Hy p o c h l o r i t e. The solid “ H . T. H . ” was obtained from the Mathieson Alkali Works in cans, but as these after
opening soon became corroded, the material was transferred to glass- stoppered bottles. The solid is not stable, losing strength gradually whether or not it is exposed to light. J. J. Lingane, of this labo
ratory, has analyzed a sample of uncertain age, probably over 4 years, with the following results ex
pressed in percentages: moisture, 2.10; available chlorine (as OCl~), 31.5 (active chlorine, 43.4); total chlorine, 45.5; total calcium as Ca, 22.36; Na, 10.59; Fe10,,0.54; free CaO, 3.17; CaCO,, 6.08. The fresh product contains about 60 per cent of available chlorine.
To prepare approximately 0.1 N solutions, about 16 grams of the solid are taken for 2 liters. I t was found convenient to stir the solid into about 500 cc. of water, allow to settle, and filter through paper in a Biichner funnel, then dilute the filtrate to about 2 liters and refilter if necessary.
Calcium carbonate which may appear as a turbidity is not harm
ful, but soils burets rather quickly. The solution is preferably kept in a bottle painted black on the outside, or a t least pro
tected from light as much as possible. A reagent prepared in the foregoing manner upon analysis was found to be equivalent to a solution 0.0267 M in calcium hypochlorite (0.1068 N oxidiz
ing strength), 0.0019 M in calcium hydroxide, 0.0086 M in calcium chloride, and 0.0306 M in sodium chloride. The ratio of sodium to calcium is variable in different solutions, as varying amounts of calcium are removed in filtration.
So d i u m Ar s e n i t e. Pure arsenious oxide from the Bureau of Standards was dissolved to make 0.1000 N sodium arsenite solu
tions according to the directions of Kolthoff (5), and 0.0100 N solutions were prepared by appropriate dilution.
In d i c a t o r s. G. F. Smith of the University of Illinois kindly furnished samples of the indicators studied by himself and Bliss (10) for use in bromate titrations. These and various other indicators were used without further purification, in 0.2 per cent aqueous solutions to which equivalent amounts of sodium hydroxide were added if necessary.
Am m o n i u m Co m p o u n d s. Determinations of ammonia were made using stock solutions of ammonium hydroxide or ammo
nium chloride. The former were prepared from redistilled am
monia and were kept free from carbon dioxide, being standardized acidimetrically. The latter were prepared from carefully dried c. p. ammonium chloride.
The other salts used, potassium bromide and the buffering 79
80 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. 7, No. 2 agents, were c. p. quality, recrystallized if necessary. The
potassium bromide should not contain over 0.01 per cent of iodide, as this is oxidized to iodate by hypobromite, thus causing an error if the solution is not subsequently acidified. Conduc
tivity water was used in all experiments.
St a n d a r d i z a t i o n o f Ca l c i u m Hy p o c h l o r i t e So l u t i o n s
The standardization may be made either of several ways, corresponding to various methods which may be followed in actual determinations. W ith use of hypochlorite for direct titrations in acid solution, any of the nonreversible indicators used in bromate titrations of sodium arsenite are satisfactory, provided an excess of bromide is present. F or use in indirect determinations, the hypochlorite is best standardized by pipetting a known volume into excess potassium iodide, acidifying with 10 cc. of 4 N sulfuric acid, and titratin g with standard sodium thiosulfate to the disappearance of the blue color with starch.
It is a particular advantage of hypochlorite th a t determ ina
tions may be made entirely in basic solutions, thus eliminating interference of m any oxidizing agents such as cliromate, iodate, and bromate. The procedure for standardization is simple, and the same whether direct or indirect titrations are involved in the determinations.
To 25 cc. of standard sodium arsenite in a 200-cc. Erlenmeyer flask add 10 cc. of a solution containing 10 per cent potassium bromide and 5 per cent sodium bicarbonate. Titrate a t a moderate speed with hypochlorite until near the expected end point, add 0.05 cc. of a 0.2 per cent solution of Bordeaux, and titrate dropwise with thorough stirring until the color fades.
Then add another 0.05 cc. of indicator and, if it does not fade, add more hypochlorite cautiously until a split drop causes the solution almost to “flash” from pink to colorless or light yellow-green.
Since these indicator reactions are not reversible, one must make certain that the end point has actually been reached when the color has faded; this should be tested by the addition of more indicator. I t is advantageous to use as little indicator as pos
sible and, when the approximate end point is known, one can per
ceive the end point sharply with only one or two drops. The total amount used should be known and the indicator correction made on the basis of the results below.
The standardization may also be made potentiometrically against arsenite in bicarbonate medium, using a bright platinum electrode, agar-potassium chloride salt bridge, and calomel half
cell. A sharp rise in potential appears in close agreement with the end point obtained with Bordeaux indicator.
In d i c a t o r sa n d Co r r e c t i o n s
As mentioned above, any of the common bromination indi
cators m ay be used w ith hypochlorite in acid bromide solu
tion. Bordeaux serves very well; 0.10 cc. of 0.2 per cent Bordeaux solution requires 0.012 cc. of 0.1 N hypochlorite for decoloration, good proportionality holding for other am ounts regardless of moderate change in dilution.
For use in weakly alkaline solutions containing bromide the indicators given in Table I have been chosen, largely from the list studied by Sm ith and Bliss (10).
Ta b l e I . In d i c a t o r s f o r We a k l y Al k a l i n e Me d i a In d ic a t o r
B o rd e a u x
B r illia n t P o n c e a u 5 R N a p h th o l b lu e b la c k C o n g o C o rin th I n d ig o c a rm in e
Br it i s h Co l o u r I n d e x N o .
88 185 246 375 1180
Co l o r Ch a n g e
v e ry f a i n t y ello w -g reen colorless
v io le t-p in k --- >*color!ess
> - lig h t yellow
■^►green——f l i g h t y ellow
been rejected as bromination indicators since they react either too rapidly or too slowly w ith hypobrom ite in alkaline solu
tion. None of the common reversible oxidation indicators have been found useful under th e conditions employed. In any case the indicator m ust be used in aqueous rath er than alcoholic solution.
St a b i l i t y o f Ca l c i u m Hy p o c h l o r i t e So l u t i o n s
Experim ents have shown th a t the stability of calcium hypo
chlorite solutions decreases w ith decreasing concentration of th e solutions, with exposure to light, and w ith frequent open
ing of th e containers. Chapin (3) found th a t potassium hypochlorite has its maximum stability a t a pH of 13.1.
The 0.1 N solutions employed b y th e authors probably have a pH between 11 and 12. In general, calcium hypochlorite solutions are more stable th a n sodium hypochlorite and far more stable th a n hypobrom ite. T he results in T able II
Ta b l e I I . St a b i l i t y o f Ca l c i u m Hy p o c h l o r i t e So l u t i o n s Tim eAFTER Ap p r o x im a t e l y 0 .1 N Ap p r o x im a t e l y
Pr e p a r a t i o n So l u t i o n s 0 .0 1 N So l u t i o n s
M o n th s D a ys
1 0 . 1 2 6 2 0 . 0 8 9 5 0 .0 0 9 1 9 0 . 0 0 9 1 8 °
5 0 . 1 0 4 7 ____
15 0 . 1 0 4 4 ____
1 0 . 1 0 4 2
1 15 0 . 1 0 4 1 0 . 0 0 9 0 5 0 .0 0 9 0 5
2 0 . 1 0 4 0 ---
3 0 . 1 2 5 8 0 . 0 8 8 4
4 15 0 . 0 0 8 8 0
6 0 . 1 0 3 5 0 . 0 8 8 1 0 .0 0 8 9 3
8 19 0 . 0 0 8 2 2b
8 2 0 0 .0 0 8 2 0 &
8 2 6 0 .0 0 8 1 4 &
9 3 0 . 0 0 8 0 8 &
15 0 . 1 2 3 0
10 0 . 1 2 3 0
24 0 . 0 0 5 3 5
° T h is s o lu tio n c o n ta in e d a la r g e r excess of c a lc iu m h y d r o x id e th a n th e o th e r a n d a p p e a r e d to b e m o re s ta b le .
& D e te rm in a tio n m a d e b y E . B . S a n d e ll.
Ta b l e I I I . Io d o m e t r i c De t e r m i n a t i o n o f Am m o n i a
Nit r o g e n Tim eof Nit r o g e n
Ta k e n Bu f f e r St a n d i n g Fo u n d Erro r
M g. G ram M in . M g . %
1 4 .0 1 0 . 5 N aH C O a 1 1 3 .9 6 - 0 . 4
1 4 .0 1 0 . 5 N aH C O a 15 1 4 .0 2 + 0 . 1
1 4 .0 1 0 . 5 N aH C O a 5 1 4 .0 1 0
1 4 .0 1 0 . 5 N aH C O a 5 1 4 .0 4 + 0 . 2 “
1 4 .0 1 1 .0 N a jH P O < 5 1 4 .0 2 + 0 . 1
14 .0 1 1 .0 N a jB iC h .lO H jO 5 1 4 .0 5 + 0 . 3 6
1 4 .0 1 1 .0 N aO A c .3 H iO 5 1 4 .1 4 + 0 . 9
0 .7 0 0 0 . 5 N aH C O a 5 0 .7 1 0 + 1 .4
Bordeaux is recommended most highly. F or titrations in solutions containing 0.5 to 1.0 gram of potassium bromide and 0.5 gram of either sodium bicarbonate, disodium phosphate, or sodium carbonate, the indicator correction for Bordeaux is approxim ately 0.02 cc. of 0.1 N hypochlorite for each 0.10 cc.
of 0.2 per cent indicator, plus a constant am ount of 0.01 cc.
if the total volume is 50 cc. or 0.02 cc. if th e volum e is 100 cc.
Nearly all of the common pH indicators as well as several dyes have been investigated, b u t all except the above have
tt I n p re s e n c e of 30 m g . K N O a.
6 R e tu r n of s ta r c h co lo r a f t e r e n d p o in t in d ic a te d n it r ite f o r m a tio n .
show' th e stability th a t m ay be expected when approxim ately 0.1 N and 0.01 ~N solutions of “ H .T . H .” are kept in glass- stoppered bottles painted black on th e outside, a t an average tem perature of 25° C. w ith occasional variations of =*= 5 ° C . A 0.01 N solution exposed to average room light lost 9 per cent of its strength in 2 days.
Ap p l i c a t i o n t o De t e r m i n a t i o n o f Am m o n i a
The iodometric determ ination of am monia has been in vestigated thoroughly by several groups of workers (1, 3, 6, 7, 8, 9, 11), th e general conclusion being th a t the oxidation of ammonia to nitrogen and w ater
2NH, + 30Br~ — >- N , + 3H ,0 + 3Br"
takes place quantitatively in excess hypobrom ite a t a pH close to 8. The authors have obtained good results using hypochlorite instead of hypobrom ite as standard solution, by first adding an excess of potassium bromide to the weakly alkaline ammonia solution. T he m ixture is alknved to stand 3 to 5 m inutes w ith a slight excess of hypochlorite, then treated w ith potassium iodide and acid, and back-titrated
w ith standard thiosulfate. Table I I I shows the effect of varying conditions, 1.0 gram of potassium bromide having been used throughout.
Teorell (11) has introduced a sensitive micromethod for ammonia, back-titrating the excess hypobrom ite with naph- th y l red. I t seems probable th a t calcium hypochlorite could profitably be substituted in this m ethod as well, being more stable than th e sodium hypobrom ite originally specified.
March 15, 1935 A N A L Y T I C .
Ta b l eIV. De t e r m i n a t i o n o p Am m o n i ai n Al k a l i n e Me d i u m
Nit r o g e n Tim eo f Nit r o g e n Sa lts
Ta k e n BOFFER St a n d in g Fo u n d Error Pr e s e n t
M g . G ram s M in . M g . % M g .
1 0 .5 1 5 N aH C O a 3 1 0 .6 1 + 1 .0
1 0 .5 1 3 N a liC O a 3 1 0 .5 5 + 0 .4
1 0 .5 1 1 N a l l CO* 3 1 0 .5 1 0 . 0
1 0 .5 1 1 N a H C ( ) 3 12 1 0 .5 2 + 0 . 1
1 4 .0 1 0 . 5 N aH C O a 15 1 4 .0 6 + 0 .4
1 4 .0 1 0 . 5 N aH C O a 5 1 4 .0 4 + 0 . 2
1 4 .0 1 0 . 5 N a H C O j 5 1 4 .0 5 + 0 .3 30 K N O a
1 4 .0 1 1 N ajB iO r.lO H iO 5 1 4 .1 2 + 0 . 8
1 4 .0 1 1 N a 0 A c .3 H 20 5 1 4 .2 2 + 1 .6
1 4 .0 1 1 N ajH P O * 5 1 4 .0 3 + 0 . 2
1 4 .0 1 1 N aaH PO « 3 1 4 .0 0 - 0 . 1 27 C u C h
1 4 .0 1 1 N ajH P O « 3 1 4 .0 2 + 0 . 1 32 FeC la
1 4 .0 1 1 N a 2H PO« 3 1 4 .0 1 0 . 0 28 K B rO a
1 4 .0 1 1 N ftjH PO « 3 1 4 .0 1 0 . 0 10 K 2C r20?
Oxidizing substances interfere w ith the iodometric method.
Therefore, a procedure in which the solution could be kept alkaline throughout W'ould be of advantage, since the inter
fering action of various oxidizing agents could then be avoided. Accordingly, the authors attem pted the direct titratio n of am monia w ith hypochlorite using excess bromide and Bordeaux as indicator, b u t the reaction was found too slow and th e indicator faded prem aturely. B y first adding excess hypochlorite, and then a known excess of arsenite, the titratio n could be completed w ith Bordeaux as indicator ju st as in th e standardization of hypochlorite against arsenite.
The authors have adopted th e following procedure, in which 0.1 N hypochlorite is used for samples containing 5 to 25 mg.
of nitrogen and 0.01 N hypochlorite for sm aller am ounts:
To about 25 cc. of the nearly neutral ammonia solution in an Erlenmeyer flask add 10 cc. of a solution 10 per cent in potassium bromide and 5 per cent in sodium bicarbonate, and titrate with hypochlorite until a permanent light yellow color appears, indi
cating a slight excess. Allow to stand 3 to 5 minutes, then add from a pipet 10 cc. of 0.01 N sodium arsenite. Shake well and add 0.05 cc. of 0.2 per cent Bordeaux, which should impart a pink color to the solution. If the color fades, more arsenite and indicator should be added. Continue the titration with hypo
chlorite to the end point as in the standardization against arsen
ite. Calculate the amount of hypochlorite corresponding to the arsenite and indicator added, and deduct this from the total volume used. The difference represents the amount of ammonia oxidized on the basis of three equivalents of hypochlorite per mole of ammonia. (1.0 cc. of 0.10 N hypochlorite corresponds to 0.467 mg. of ammonia nitrogen.)
The results in T able IV show the accuracy of th e m ethod under various conditions. E vidently th e oxidizing agents added did not interfere. Disodium phosphate instead of sodium bicarbonate was used in th e presence of iron and copper, since the color became less troublesome. M ore th a n 10 mg. of potassium dichrom ate rendered th e indicator change indistinct. The potentiom etric m ethod (back-
Op a c i t y St a n d a r d s f o r Pa p e r Te s t i n g. The development of glass standards for the calibration of instruments used to test the opacity of paper by the contrast ratio method, as specified in the standard method of the Technical Association of the Pulp and Paper Industry, is described by Deane B. Judd in the Sep
tember issue of the Bureau of Standards Journal of Research.
According to this method the reflectance of the material backed by a perfectly absorbing surface divided by its reflectance when backed by a highly reflecting material such as magnesium oxide is taken as an index of opacity. Magnesium oxide, itself, is,
titration of the excess arsenite with standard hypochlorite solution) yielded excellent results and is especially advan
tageous in th e presence of colored compounds or in the de
term ination of extremely small am ounts of am monia w ith reagents 0.01 N or even more dilute.
T he d ata in Table V show th a t quantities of am monia as small as 0.5 mg. in a volume of 25 cc. can be determ ined w ith a . maximum error of about 2 per cent, using the general pro
cedure described above. T he iodometric procedure yields errors of th e same magnitude b u t is preferable in th e absence of other oxidizing substances, since the color change of th e iodine starch is sharper than th a t of Bordeaux in these very dilute solutions. Blanks should be run along w ith th e de
term inations, as traces of reducing substances are likely to be present in the w ater used as a solvent and some bromine or oxygen m ay be lost by decomposition of the hypobrom ite on longer standing.
l L E D I T I O N 81
Ta b l e V. De t e r m i n a t i o n o p Sm a l l . Am o u n t s o f Am m o n i a Nit r o g e n
Ta k e n K B r N aH C O a
Tim eof St a n d i n g
Nit r o o e n
Fo d n d Er r o r
M g . Grama G ram s M in . M g. %
1 .0 5 0 1 1 5 1 .0 7 1 + 2 . 0
1 .0 5 0 1 1 3 1 .0 6 8 + 1 .7
1 .0 5 0 1 1 15 1 .0 7 7 + 2 . 6
1 .0 5 0 1 3 3 1 .0 6 8 + 1 .7
1 .0 5 0 1 3 15 1 .0 7 4 + 2 .3
1 .0 5 0 3 .1 3 1 .0 5 7 + 0 . 7
1 .0 5 0 3 1 8 1 .0 5 9 + 0 . 9
0 .7 0 0 1 0 . 5 5 0 .7 1 1 + 1 .6
0 .4 2 0 1 1 3 0 .4 1 9 - 0 . 2
Co n c l u s i o n s
Solutions of “H .T . H .” calcium hypochlorite are quite stable if kep t in the dark and are recommended as standard solutions in volumetric analysis.
H ypobrom ite oxidizes more rapidly th a n hypochlorite;
the form er is obtained from th e la tte r by adding an excess of alkali bromide.
Hypochlorite m ay be standardized against arsenic trioxide using Bordeaux as indicator, in acid or weakly alkaline solu
tion.
Procedures have been given for th e determ ination of quan
tities of am monia from 0.5 to 20 mg.
Li t e r a t u r e Ci t e d
(1) A rtm a n n , P ., a n d S k ra b al, A ., Z . anal. Chem., 46, 5 (1907).
(2) C h a p in , R . M ., J. A m . Chem. Soc., 56, 2211 (1934).
(3) D o n ald , M . B ., A n a ly st, 49, 275 (1924).
(4) Je llin ek , K ., a n d K restoff, \V., Z . anorg. allgem. Chem., 137, 333 (1924).
(5) KolthofT, I . M ., a n d F u rm a n , N . H ., "V o lu m e tric A n aly sis,”
Vol. I I , p . 363, N ew Y o rk , Jo h n W iley & Sons, 1929.
(6) K o lthoff, I . M „ a n d L a u r, A ., Z . anal. Chem., 73, 177 (1928).
(7) M eulen, J . H . v a n dcr, Chem. W eekblad, 27, 551 (1930).
(8) N a n ji, D . R ., a n d S haw , W . S., A n a ly st, 48, 473 (1923).
(9) R u p p , E ., a n d R ossler, E ., A rch - P h a rm ., 243, 104 (1905).
(10) S m ith , G . F „ a n d Bliss, H . I I ., J. A m . Chem. Soc., 53, 2091 (1931).
(11) T eorell, T „ Biochem . Z„ 248, 246 (1932).
(12) W illard , H . H ., a n d C ake, W . E ., J. A m . Chem. Soc., 42, 2646 (1920).
Re c e i v e d J a n u a r y 24, 1935.
however, commonly not used as the white backing because of its fragility; nor is the white backing placed in actual contact with the sample. Because of these and other sources of error, opacime- ters frequently give erroneous results. Standards of opacity made of permanent material serve to check and to calibrate such instruments. Such standards made of opal glass are described, the theory of their application is given, and results of tests by their use reported. It is found th at TAPPI opacity corresponds to a reflectance of white backing in contact with the sample of about 0.89.
A New Method for Determining Invertase Activity
W. R . J o h n s t o n , S u t t o n R e d f e r n , a n d G. E. M i l l e r , The Fleischmann Laboratories, New York, N . Y.
T
H E R E are several m ethods now in use for estim at
ing invertase activity (4, 7,9 ,1 1 , IS, 13). These methods have as a basis the determina
tion of either the unimolecular constant k or time values required for a given percentage hydrolysis by a definite am ount of invertase preparation. In addition, Nel
son and Hitchcock (7) have pro
p o se d a n empirical c o n s t a n t which is proportional to enzyme concentration over a wide range of concentration.
The methods utilizing the unimolecular k are n o t valid, as has been dem onstrated repeatedly by several investigators (1 ,5 ,8 ). The methods employing tim e values are sufficiently accurate, b u t in general are time-consuming with respect to experimental measurements and calculations. The empirical constant of Nelson and Hitchcock (7) is not easily evaluated, and while it is proportional to enzyme concentration it is not easily expressed in terms of actual enzyme performance.
In the authors’ study of invertase they have developed a method of activity measurement which is characterized by extreme simplicity of measurement and calculation. The method accurately evaluates enzyme activity in term s of the rate a t which the invertase hydrolyzes the sucrose, thus giv
ing an activity measure which is directly related to enzyme performance.
I t has been established by several workers (6, 8) th a t the initial rate of hydrolysis of sucrose by yeast invertase is di
rectly proportional to the concentration of the invertase.
The authors have elaborated on this work by studying the rate of hydrolysis very near the sta rt of the reaction and have shown conclusively th a t the rate of hydrolysis a t zero time—
th a t is, a t th e very beginning of the inversion— is directly pro
portional to enzyme concentration over a wide range. Using this as a basis they have defined a rational enzyme unit termed the “inverton.” The “inverton” is th a t am ount of yeast in
vertase which will hydrolyze sucrose a t the rate of 5 mg. per minute a t zero time, under the specified experimental condi
tions. Since initial rates are proportional to invertase con
centrations, we m ay use the inverton u n it as a measure of the concentration of enzyme in a given preparation. The num
ber of invertons per gram of preparation give a rational and logical measure of the strength of the preparation. This method of treatm ent has been previously applied to the study of alpha-amylase by Johnston and Jozsa, whose results will soon be published.
Ex p e r i m e n t a l
In order to establish the validity of the inverton, it is necessary to carry out careful m easurem ents of initial rates of inversion.
The measurements were made on a 5 per cent sucrose solution which was buffered to a pH of 4.6 by a Walpole acetate buffer (2). The sucrose solution was prepared by dissolving 100 grams of sucrose in distilled water, adding 50 ml. of Walpole acetate buffer of pH 4.6, and making up to 1 liter a t 25° C. with distilled
A new method fo r the determination o f yeast invertase has as a basis the determination o f the number o f enzyme units, termed “invertons,”
per gram of preparation. The enzyme units are based upon the initial rate o f inversion o f the sucrose substrate. They are quickly and accu
rately evaluated, and are o f practical significance with respect to the actual performance o f the enzyme.
The method has been applied to three different commercial preparations with excellent results.
water. A commercial e n z y m e preparation preserved in glycerol was used. This was diluted for use by making up weighed samples to a definite volume with distilled water. The dilute enzyme solu
tions generally contained, from 1 to
10 mg. of original enzyme concen
trate per ml. of solution. In car
rying out a rate measurement 25 ml. of diluted sample were pipetted a t 20° C. intoa200-ml. flask. The sample was placed in a thermo
sta t a t 25° C. and after coming to temperature, 25 ml. of a 10 per cent sucrose solution were added a t 25°
C., noting the time. The sucrose solution was added from a fast pipet a n d th e m ix tu r e thor
oughly shaken. The mixture was incubated a t 25° C. for inter
vals of 1, 2, 3, 4, 6, 8, and 10 minutes. The reaction was stopped and m utarotation accelerated by the addition of 0.5 ml. of 15 N ammonia. After standing for several minutes (longer than 5 minutes, less than 2 hours) the solutions were polarized in a 4-dm.
tube, using a Fric saccharimcter with a Ventzke scale. The initial or blank polarization was determined in each case by polarizing a mixture of 25 ml. of sample, 0.5 ml. of ammonia, and 25 ml. of sucrose solution, mixed in the order named.
The initial reaction m ixture corresponds to a 5 per cent sucrose solution, which is the optim um concentration for invertase activity. T he d a ta in Table I were used in plot
ting the rate curves for th e preparation used as a standard.
Ta b l e I. Ra t e o f In v e r s i o n b y St a n d a r d In v e r t a s e - Su c r o s e In v e r t e d-
I n v e r ta s e C o n c n . I n v e r t a s e C o n c n . I n v e r ta s e C o n c n .
Tim e 2 .5 m g ./c c . 5 m g ./c c . 10 m g ./c c .
M in . M g. M g . M g . M g. M g .
1 31 36 67 67 141
2 66 71 139 138 281
3 101 101 206 210
4 137 137 278 282 505
6 208 208 417 4 10 744
8 279 279 540 517 952
10 350 350 665 624 1197
P lotted on a large scale, th e ra te curves indicate th a t over the range tested th e initial ra te of action of the invertase is directly proportional to its concentration. This is illustrated in Figure 1. The results of Table I I were obtained from the large-scale curves.
Ta b l e I I . In i t i a l In v e r s i o n Ra t e s o f St a n d a r d In v e r t a s e Co n c e n t r a t i o n o fi n
v e r t a s e Pr e p a r a t i o n
M g./rrd.
2 . 5 5 . 0 10.0
Ha t e o r In v e r s i o no p Su c r o s ea t Ze r o Ti m e
M g ./m in . 35 70 143
These results enable us to choose a rational enzyme unit which m ay be applied over th e experimental range. In order to obtain a value approxim ating 100 enzyme units or invertons per gram for th e standard commercial sample, the authors chose th e inverton as equal to a rate of inversion of sucrose of 5 mg. per m inute a t zero tim e a t 25° C., under the conditions specified. This gives 112 enzyme units per gram of th e standard invertase.
To determine the inverton concentration of a given sample w ithout resorting to ra te measurements, it is necessary to 82
March 15, 1935 A N A L Y T I C A L E D I T I O N 83 establish a relation be
tween some easily obtain
able quantity and the num
ber of invertons present.
The am ount of sucrose in
verted after 0.5 hour a t 25°
was chosen as the easily obtainable quan tity . Di
lutions of th e standard invertase were m ade and solutions of known inver- to n concentration w ere u s e d a s o u t l i n e d pre
viously, except th a t the re
action was stopped after 30 m inutes in each case instead of after variable tim e intervals.
D ilutions were m ade so th a t th e c o n c e n t r a t i o n varied from 1.4 to 33.6 in
vertons per 25 ml. of solution, corresponding to a variation of 0.5 to 12 mg. of standard invertase per 25 ml.
C alibrated glassware (Bureau of Standards) and distilled w ater were used in all these
dilutions in order to in
sure accuracy. Vosburgh (10) found th a t errors were introduced upon dilution w ith w ater c o n t a i n i n g small am ounts of electro
lytes. If an accuracy of 1 per cent is to be realized, distilled w ater m ust be used. In carrying out the determ inations a standard 25-ml. pipet having an out
flow tim e of 35 seconds a t 25° C. was used. Time was counted from the mo
m ent of introduction of th e sucrose. The mixture was shaken vigorously dur
ing the addition.
T he values of Table I II were used in plotting the
inverton-sucrose curve shown in Figure 2.
Ta b l e I I I . Su c r o s e In v e r s i o n' a s Fu n c t i o n o p In v e r t o n Co n c e n t r a t i o n
Su c r o s e In v e r t e da f t e r
In v e r t o n s Mi n u t e s
PER 25 Ml. A B
M g . M g .
1 . 4 2 0 9
2 . 1 3 Î 5 3 2 1
2 . 8 4 2 2 4 2 3
4 . 2 6 0 4 6 0 6
5 . 6 7 8 2 7 8 4
8 . 4 1 1 0 7 1 1 1 5
1 1 . 2 1 3 9 2 1 3 9 5
1 6 . 8 1 8 1 3 1 8 1 8
2 2 . 4 2 0 7 2
3 3 . 6 2 3 3 1
I t was found th a t the curve could be best fitted by two equations. A least-squares solution for th e portion of the curve up to 1025 mg. gave th e following equation:
log I = 1.0667 log S - 2.3368 I = number of invertons per 25 ml. of sample
S = mg. of sucrose inverted after 0.5 hour reaction'at 25° C.
F or the range from 1025 to 2000 mg. a logarithmic equa
tion was derived:
log I = 0.0004289 S + 0.4490 S and I have their previous significance.
In using these equations, one first calculates th e num ber of milligrams of sucrose inverted and then chooses the proper equation to fit th e sucrose value. T he inverton value calcu
lated represents the num ber of invertons per 25 ml. of diluted sample. I t m ust be calculated to a basis of 1 gram of original sample; so th e dilution factor m ust be applied to the inverton value obtained from th e equation. For example, if 10 inver
tons are calculated per 25 ml. of diluted sample and the di
luted sample contains 125 mg. of original preparation per 25 ml., there are 80 invertons per gram of original sample.
Ta b l e I V . Ra t e o r In v e r s i o n b y In v e r t a s e No. 1
(2 .5 m g . of i n v e r t a s e p r e D a r a tio n p e r m l. u s e d in e x p e r im e n ts A , B , a n d C ; 6 m g . p e r m l. in D a n d E )
A B
--- S u c i C
t o s s In v i
A v.
SRTED---
D E A v.
M in . M g . M g . M g. M g. M g. M g . M g .
1 4 1 . 1 4 1 . 1 3 6 . 0 3 9 . 4 7 2 . 0 8 2 . 1 7 7 . 1
2 7 1 . 8 7 1 . 8 7 2 . 0 7 1 . 9 144 144 144
3 108 113 10 8 1 1 0 211 2 2 1 2 1 6
4 139 144 1 4 4 142 2 7 8 2 7 7 2 7 8
6 2 0 0 2 1 6 2 0 6 2 0 7 4 1 2 4 1 1 4 1 2
8 2 7 7 2 8 2 2 7 3 2 7 7 5 3 5 5 3 9 5 3 7
10 3 4 4 3 4 9 3 4 3 3 4 5 6 5 9 6 5 7 6 5 8
In order to establish th e method of m easurem ent it was necessary to study the rate curves of other commercial invertases and investigate the constancy of th e calculated inverton value on dilution of the e n z y m e p r e p a r a t i o n . Three commercial invert
ases were studied. Itw ras found th a t the activity of all three invertases could be accurately determined by applying the inverton method. The results ob
tained w ith commercial in
vertase No. 1 are given in Table IV.
T he d ata for invertase No. 1 were plotted in Figure 3. The initial slopes of these rate curves showed th e same property as those of th e standard curves. The in v ert
ase concentration was di
rectly proportional to the rate of inversion a t zero time. In order to check the behavior of the prepa
ration on dilution the d ata given inTable V (invertase No. 1) were obtained.
W hen plotted, the re
sults in Table V gave the straight line of Figure 4 ,
showing clearly th e linear relation between concen
tration and num ber of in
vertons.
Another commercial in
vertase, No. 2, was pur
chased in the open m arket and checked exactly as in
vertase No. 1 (Table V I
Fi g u r e 1. Ra t e Cu h v e sf o r St a n d a r d In v e r t a s e
A , 10.0 m g . p e r m l.
D , 5 .0 m g . p e r m l.
C, 2 .5 m g . p e r m l.
Fi g u r e 2 . In v e r t o n- Su c b o s e Cu b v e
F i g u r e 3 . R a t e C u r v e s f o r I n v e r t a s e No. 1
A , 5 .0 m g. p e r m l.
B , 2 .5 m g . p e r m l.
