S e e Special N o t i c e s o n p. 2 5 8 V o l u m e 6 9
N o . 8 2 1 , P a g e s 2 2 9 — 2 5 8
p- 11¡h h
A u g u s t
T H E A N A L Y S T
T h e J o u r n a l o f
The Society of Public Analysts
a n d o t h e r A n a l y t i c a l C h e m i s t s p u b l i c a t i o n C o m m i t t e e
C h a i r m a n : J . R . n i c h o l l s, d. Sc., f.r.i.c. N . L . A L L P O R T , F . R . I . C .
F . W . F . A R N A U D , F . R . I . C . A . L . B A C H A R A C H , M . A . , F . R . I . C . R . C . C H I R N S I D E , F . R . I . C .
H . E . C O X , D . S c . , P h . D . , F . R . I . C . B E R N A R D D Y E R , D . S c . , F . R . I . C . F . W . E D W A R D S , F . R . I . C .
B . S . E V A N S , M . B . E . , M . C . , D . S c . , F . R . I . C . E . H I N K S , M . B . E . , B . S c . , F . R . I . C .
■© on . S e c r e t a r y : L . E Y N O N , B . S c . , F . R . I . C .
7-8, Id o l Lane, E.C.3
E . B . H U G H E S , D . S c . , F . R . I . C .
D . W . K E N T - J O N E S , B . S c . , P h . D . , F . R . I . C . S . E R N E S T M E L L I N G , F . R . I . C .
G . W . M O N I E R - W I L L I A M S , O . B . E . , M . G . , P h . D . , F . R . I . C . A . M O R E , I . S . O . , A . R . C . S . ,
A . R . T . C . , F . R . I . C . W . H . S I M M O N S , B . S c . , F . R . I . C .
E R I C V O E L C K E R , A . R . C . S . , F . R . I . C . K . A . W I L L I A M S , B . S c . , F . R . I . C .
© o n . t r e a s u r e r : G . T A Y L O R , F . R . I . C . P eck House, 20, Eastcheap, E.C.3 E d i t o r : C . A I N S W O R T H M I T C H E L L , D . S c . , M . A . , F . R . I . C .
T h e Close, Weedon, A y le sb u ry , B u ck s.
S e c r e t a r y a n d a s s i s t a n t E d i t o r : J . H . l a n e , B . S c . , f.r .i.c. 7-8, Id o l Lane, E.C.3
H b s t r a c t o r s
S . G . C L A R K E , D . S c . , P h . D . , A . R . I . C . B . S . C O O P E R , B . S c . , F . I n s t . P . E . B . D A W , B . S c . , A . R . I . C . J . G R A N T , P h . D . , M . S c . , F . R . I . C . A . O . J O N E S , M . A . , F . R . I . C .
J . W . M A T T H E W S , P h . D . , D . I . C . , F . R . I . C . E . M . P O P E , B . A . , B . S c .
F . A . R O B I N S O N , M . S c . T e c h . , F . R . I . C . W . R . S C H O E L L E R , P h . D . , F . R . I . C . D . R . W O O D , F . R . I . C .
Published for the Society by
W . H E F F E R & S O N S L T D . , C a m b r i d g e , E n g l a n d
I M P O R T A N T N O T I C E T O S U B S C R I B E R S (Other than Members of the Society).
All Subscriptions a n d renewals to the J o u rnal should be sent t h r o u g h a Bookseller or direct to
W . H E F F E R & S O N S L T D . , C A M B R I D G E , E N G L A N D . Price 3/6; or to Subscribers in advance, post free, 3 5 / - per a n n u m .
N.B.— M e m b e r s send their subscriptions to the Hon. Treasurer.
E n t e r e d a s S e c o n d C l a s s a t N e w Y o r k ,
U.SA.,
P o s t O f f i c e P r i n t e d i n E n g l a n dii T H E A N A L Y S T
R E I G A T E B R A N D
Registered Trade Hark i N A T U R A L I S O L A T E S :
E S S E N T I A L O I L S : S Y N T H E T I C S :
F L O W E R O I L S : Citral, E u g e n o l , G e r a n i o l s ,
l o n o n e s , M e t h y l l o n o n e s , H y d r o xy c i t r o n e l l a l S O L E M A N U F A C T U R E R S : —
W H I T E , T O M K I N S & C O U R A G E , L T D R E I G A T E , S U R R E Y
E sta b lish ed 1841 'P h o n e
R e ig a te 224 2
'G r a m s E ssw h ite , R eigate
S A V O R Y & M O O R E L T D . M A Y FAIR A B R A N D
A L U M I N I U M O X I D E o f British M a n u f a c t u r e
S t a n d a r d i s e d for C H R O M A T O G R A P H I C
A D S O R P T I O N A N A L Y S I S
a n d S E P A R A T I O N Further information from :
S A V O R Y & M O O R E L T D . L A W R E N C E RD., T O T T E N H A M , N.15
D y e s t u f f s
for p a r t i c u l a r p u r p o s e s
G U A R A N T E E D H A R M L E S S
E d i b l e C o lo u r s
I n c l u d i n g t h e U S A G E R T r a n g e c e r t i f i e d b y t h e U . S . A . G o v e r n m e n t
D y e s o f B . P . C . S t a n d a r d
T D
W i l l i a m s ^ ^ L -
E s t a b l i s h e d 1 8 7 7
H O U N S L O W - M I D D X . , E N G L A N D
T H E A N A L Y S T 111
T H E
G - L - C C O N S T A N T V O L U M E G A S A N A L Y S I S A P P A R A T U S
A new apparatus, simple to operate, for use in the accurate analysis of practically any gaseous mixture
• A m o d ific a tion o f t h e " B o n e and W h e e l e r ” ap paratus, d e v e lo p e d in th e Fulham L a b o r a t o r y o f th e G a s L ig h t & C o k e C o . as a r e s u lt o f m any y e a r s ' e xp e rie n ce .
• U s in g m e r c u r y as th e c o n fin in g liquid, it is a p r e c is io n apparatus, essential in g a s w o r k s and w h e r e p r o d u c e r and o t h e r ga s p la n ts are in op e ra tio n .
P R I N C I P L E : A w a ter-ja ck e te d b urette, an a b s o r p t io n p ip e tte and t w o gas-heated rea ction t u b e s c o n ta in in g sp e c ia lly p re p a re d c o p p e r o x id e and a c o p p e r o x id e - ir o n o x id e catalyst.
A D V A N T A G E S : A c c u ra c y , rap id ity, sim p lic ity , fre e d o m f r o m s o lu bility, a d s o r p t io n and c a lib ra tio n e rr o rs , c o m p le te a b s o r p t io n a ssu re d by th e use fo r each an a lysis o f fre sh reagents, etc.
Ref. J.S.C.I., 1938, 5 7 , 388
Please apply for Booklet GTI302/0I
G R I F F I N a n d T \ T L O C K
L O N D O N MANCHESTER G L A S G O W EDINBURGH Kemble St., W.C.2 19 Cheetham Hill Rd., -4 45 Renfrew St., C.2 7 Tevlot Piece, I
E s t a b l i s h e d as S c i e n t i f i c I n s t r u m e n t M a k e r s i n 1 8 2 6
T H E A N A L Y S T
CAMBRIDGE p H
INSTRUMENTS
P O R T A B L E INDICATING
A N D CO N TRO LLIN G
BEN CH PA TT ER N S RECO RD IN G
A N D ALARM
y H E f u l l R A N G E of Cambridge instruments for the measurement and recording of p H in all branches of research and industry, is described in our L |S T |j.,08
This book also contains valuable notes on the principles of p H measure
ments and its applications in m a n y industries, with a useful bibliography.
M A Y W E S E N D Y O U A C O P Y ?
For solutions of H I G H ALKAL I N I T Y , use the n e w A L K I electrode.
C A M B R I D G E I N S T R U M E N T C O M P A N Y L I M I T E D 13 GROSVENOR PLACE - LONDON - S VV I. :
T I T R A T I O N B E N C H Incorporating Nivoc “ W.J.” type burette holders, with cupboard for standard solutions
W . & J . G E O R G E L T D PROPRIETORS OF F. E. B E C K E R & C O
17-29 Hatton Wall, 157 Great Charles St.,
London, E.C.l Birmingham 3
Trade Mark
L A B O R A T O R Y
I N G and Manufacture Laboratory Furniture For Ail General & Special Purposes
AUGUST, 1944. Vol. 69, No. 821
T H E A N A L Y S T
P R O C E E D I N G S O F T H E S O C I E T Y O F P U B L I C A N A L Y S T S A N D O T H E R A N A L Y T I C A L C H E M I S T S
T h e D e t e c t i o n a n d D e t e r m i n a t i o n o f A u x i n s i n O r g a n i c M a n u r e s
P a r t II.— E x t r a c t i o n o f A u x i n s f r o m M a n u r e s , a n d A p p l i c a t i o n s of t h e P e r c h l o r i c A c i d T e s t f o r £ - I n d o l y l A c e t i c A c i d a n d of t h e
W e n t P e a T e s t
B y J. H U B E R T H A M E N C E , Ph.D., M.Sc., F.R.I.C.
(Read at the Meeting, M a y 3, 1944)
In Part I of this series1 a n e w test based on the use of a perchloric acid reagent was described for the determination of /3-indolyl acetic acid and its homologues, and also a n e w technique employing this reagent in conjunction with chloroform for the determination of /3-indolyl acetic acid in presence of the propionic and butyric homologues. This communication is devoted to a description of methods devised in our laboratory for the extraction of auxins from organic manures an d the methods of examining the extracts thus obtained.
Although very little work on this subject appears to have been carried out, sufficient knowledge concerning auxins in general wa s available to predict that the total a m o u n t of auxins in fertilisers wa s not likely to exceed a few m g per 100 g of organic manure. It followed from this and from the general nature of organic manures, farmyard manure, dried blood, etc., that tests could not be applied directly to the manures and that the auxins must be extracted and concentrated before they could be detected and estimated. Moreover, it also became necessary to employ somewhat larger quantities of material than are usual in analytical processes. A m o u n t s ranging from 50 to 500 g have been used in our expts.
At this stage it is appropriate to discuss certain small modifications that it wa s found necessary to introduce into the original perchloric acid technique in order to m a k e it applicable to all types of organic manures. In some of the early expts. the failure to obtain complete recovery of added /3-indolyl acetic acid with some fertilisers was found to be due to an in
hibiting effect brought about b y impurities extracted together with the auxins.
This fact w a s established as follows. T h e final extract from a specimen of hop manure to which a k n o w n a m o u n t of indolyl acetic acid had been added was divided into halves.
In one half the /3-indolyl acetic acid was determined b y the perchloric acid test; to the second half an additional k n o w n quantity of /3-indolyl acetic acid was added, and the total /3-indolyl acetic was then similarly determined in this portion. Th e following amounts of /3-indolyl acetic acid were found: first half, 0-010 m g ; second half plus 0-05 m g of added /3-indolyl acetic acid, 0-026 mg.
This proves that some very strong inhibiting action takes place, since in this expt. the auxin added to the final extract could not have been lost in the extraction process.
A very large n u m b e r of expts. were m a d e to find a me t h o d of separating the inhibiting impurities from the auxins. In view of the method of extraction employed it was rightly assumed that the impurities were acidic substances, and accordingly methods of separation as lead and calcium salts were investigated. T h e separations were carried out in neutral and acid solutions, in water and in alcohol, but without achieving the desired result. It is worth noting at this stage that the lead separation in dil. acetic soln. was very effective in removing colouring matters; this point will be referred to later.
Chromatographic resolution was then tried, employing columns of alumina and m a g nesium carbonate as the adsorbants; various different solvents were used such as ether, acetone, etc., but still without success. Separation b y distribution between immiscible solvents, such as water a n d petrol, and the use of adsorbent clays and charcoal were also
229
230 hame n c e : t h e detection a n d determination of auxins in organic m a n u r e s investigated. It soon became apparent that the interfering impurities had properties very similar to those of /3-indolyl acetic acid and that they could not be readily separated.
T h e difficulty was finally overcome in a comparatively simple m a n n e r by modifying the perchloric acid test. It was observed that the interfering bodies tended to form a ppt. when the hot perchloric acid was cooled and that the ppt. adsorbed part of the red colour produced by the action of the perchloric acid on /3-indolyl acetic acid. It was found that this pptn.
could be prevented if the test was carried out in a solution containing 2 0 % of alcohol.
T h e use of alcohol gave very m u c h improved recoveries, but the real solution of the problem was found to lie in addition of a trace of ferric chloride to the alcohol soln. The function of the trace of ferric chloride in the perchloric acid reaction is b y no m e a n s clear;
possibly it m a y act as a catalyst in the perchloric oxidation just as the impurities act as inhibitors.
Mo d i f i e d Pe r c h l o r i c A c i d Te s t— Place 1 - 5 m l of the soln. to be tested in a test-tube, add 1 - 0 m l of alcohol, 1 drop of 0 - 2 % ferric chloride soln. and 2 - 5 m l of 2 0 % perchloric acid and boil gently for 1 5 sec. Cool immediately and extract with 5 m l of chloroform. Then m at c h the red colour of the chloroform extract in a 1-cm cell in a Lovibond Tintometer.
Apart from ameliorating the inhibiting action of the impurities the use of ferric chloride together with alcohol has the effect of increasing the sensitivity of the reaction.
T h e propionic and butyric compounds yield yellow colours in the modified test and, as with the acetic compound, the sensitivity is increased. W h e n the yellow solutions are extracted with chloroform only a very slight pink colour is imparted to the chloroform by the propionic compound, whilst the butyric c o m p o u n d yields a slightly green chloroform extract. T h e production of this green colour provides a me t h o d of detecting the butyric c o m p o u n d in presence of the propionic body, which was not possible by the original process.
Unfortunately the modified procedure suffers from the disadvantage that indole, if present in sufficient quantity, m a y lead to erroneous results, since it will impart a distinct pink colour to the chloroform extract. Indole m a y be separated from the indolyl acids by ex
tracting an alkaline soln. of the acids with ether. This stage has been included in the final purification process of the auxin extracts to which reference will be m a d e later. Indole m a y also be separated b y the distillation process of Holt and Callow.2
Me t h o d s o f E x t r a c t i o n— A s w a s pointed out in Part I (loc. cit.) apparently no attempts have hitherto been m a d e to extract auxins from solid manures. T h e classical methods of Kogl, Haagen-Smit and Erxleben3 for the isolation of auxins from urine are too long to be employed as routine analytical processes, but nevertheless give most valuable indications of the type of extraction process most likely to be successful. Holt and Callow2 described a m e t h o d of separating indolyl acetic acid from urine by direct continuous extraction with ether. Whilst this process is eminently suitable for the extraction of auxins from urine, preliminary expts. indicated that it would not be suitable for some types of organic fertilisers.
T h e first process that w e tried w a s alcoholic extraction on the lines of the classical Stas-Otto toxicological technique. Farm y a r d m a n u r e and hop man u r e were used for the tests. Portions (100 g) were acidified with citric acid and extracted with alcohol, and the process was continued as in the usual determination of aspirin, or veronal in viscera. This process was abandoned w h e n it w a s found that the two manures employed for testing gave unworkable emulsions on treating the residue from the alcoholic extraction with water.
Apart from the obstinate emulsions it wa s considered that, in view of the unstable nature of auxins, a process not involving the evaporation of large volumes of alcohol, even under reduced pressure, would be preferable.
Cold Lime Extraction— A s auxins are acidic bodies which form water-soluble salts, it was considered that extraction with dilute alkali should remove the auxins from the bulk of the fertiliser. Lime water removed less fatty material and less pigments from the manure than dil. caustic soda soln. and was accordingly adopted as the solvent for the preliminary extraction of the auxins from the fertiliser. Exhaustive extraction was found to be un
necessary. B y adding a k n o w n volume of water to a mixture of the fertiliser and lime, filtering, and taking an aliquot portion of the soln., added auxin could be satisfactorily recovered. After a solution of the auxins in lime water had been obtained the auxins could be concentrated satisfactorily by acidifying the soln., extracting with ether and evaporating the ethereal extracts. After the acidification it was found desirable to employ a clearing agent to remove traces of fatty material, etc., prior to extraction with ether. T h e choice proved very difficult, since most of the usual clearing agents carried d o w n with them part
of the auxin, presumably b y adsorption. This difficulty was finally overcome b y the use of previously pptd. zinc ferrocyanide. T h e zinc acetate and potassium ferrocyanide reagents were prepared b y the me t h o d of Moir and Hinks4 and were mixed together in a small volume of water before being added to the auxin solution. There was no adsorption of auxin with this reagent, w h e n used in this manner. After acidification of a portion of the filtrate from the lime water extraction with sulphuric acid a suitable quantity of the clearing agent was added, and the solution was m a d e up to k n o w n vol., well shaken and filtered. A definite volume of the filtrate was then extracted with peroxide-free ether, and the ethereal extract was washed with water and finally evaporated.
Modified Alcohol Process— W h e n at an early stage in this investigation unsatisfactory recoveries of added /3-indolyl acetic acid were obtained with fresh farmyard man u r e it seemed possible that micro-organisms might be responsible for the' small losses that occurred and that therefore an additional process, in which the activity of micro-organisms was suppressed, would be useful. Accordingly the Stas-Otto process was modified in the following manner.
The man u r e w a s acidified with saturated citric acid soln. and then extracted with alcohol.
The alcoholic extracts were united and m a d e slightly alkaline with strong caustic soda soln., and the alcohol was distilled off under reduced pressure. T h e residue wa s dissolved in water, the soln. was acidified with sulphuric acid, and zinc ferrocyanide clearing agent was added.
Reference was m a d e above to obstinate emulsions frequently obtained at this stage; it was found that these could be avoided b y adding sodium chloride to the soln. before m a k i n g it up to a k n o w n volume; clear bright filtrates were thus obtained. T h e filtrate was then extracted with ether as described in the cold alkali process.
Ex a m i n a t i o n o f Et h e r e a l E x t r a c t s— After the auxins have been obtained from a large bulk of organic m a n u r e in the form of an extract weighing between 1 and 50 mg, the final stage is to determine their proportion. T h e /3-indolyl acetic acid is determined b y the per
chloric acid m e t h o d as follows. T h e ethereal extract is dissolved in a small k n o w n vol. of dil. caustic soda soln. and the modified perchloric acid test is applied direotly to an aliquot portion, say, 1 ml. T h e remainder of the extract is retained for the determination of total auxins and for other tests.
T h e problem of the detection of the homologues of /3-indolyl acetic acid can n o w be considered. In Part I of this series1 the proportion of indolyl propionic and butyric acids was calculated from the n u m b e r of yellow units required to match the colour in the perchloric acid test before extraction with chloroform. T h e same technique m a y be applied to the extracts obtained from organic fertilisers, but extreme care must be exercised in the interpretation of the results. T h e reason for this is that almost all the extracts obtained by either the lime or the alcohol met h o d from organic manures are slightly yellow, some more so than others, and allowance must be m a d e for this initial, colour by matching the colour of the soln. obtained on mixing the reagents for the perchloric acid test prior to boiling.
Experimental work is fiow in hand to produce a satisfactory me t h o d of determining phenyl acetic acid. T h e odours of w a r m extracts from a n u m b e r of organic manures indicate that they contain phenyl acetic acid in small proportions.
Total Auxins— T h e usual method for the determination of auxins in plant physiology is by the Avena or oat test, and the m e t h o d is ideal for the determination and detection of the comparatively small quantities of auxins involved in that work. W h e n this investigation was undertaken it w a s decided that the Avena test was too sensitive for the problem in hand and that the W e n t pea test, which is capable of dealing with comparatively gross quantities of auxin, would be more suitable.
The W e n t Pea Test— This test, described b y W e n t 5 in 1934, is based upon the fact that when elongating organs, particularly stems, are split longitudinally in the growing zone, the two halves curve outwards in water and inwards in auxin solution (see Fig. 1), the split stems being completely immersed in the solutions. T h e outward curvature is due to tissue tension, the epidermal cells being normally under tension and the pith cells under pressure. T h e inward curvature is a differential growth phenomenon of a complex nature.- It is not proposed to describe here the m e t h o d of growing the peas and the preparation of the stems in detail, but only to give our m e t h o d of applying the test. T h e peas are grown on moist sand in the dark in an incubator at 23° C. T h e y are ready for the test when the plants have developed two nodes each bearing a scale, and one at the top bearing a leaf. In the first place it must be borne in m i n d that m a n y substances interfere with the
h amence: th e detection a n d determination of auxins in organic m a n u r e s 231
232 h a m e n c e:t h e d e t e c t i o n a n d d e t e r m i n a t i o n o f a u x i n s i n o r g a n i c m a n u r e s
C— No auxin D — Auxin present
pea test and that therefore only the final ethereal extract from the lime or the alcoholic process must be used for the test. T o give a few examples of interfering bodies, W e n t and T h i m a n n 6 find that traces of copper, nickel, manganese and zinc salts practically inhibit the curvature. Alcohol also interferes.
Little attention appears to have been paid to the reaction of the solution in which the test is carried out. O u r expts., however, have sho w n that the test is most sensitive in slightly alkaline soln. T h e extract from the organic manure is accordingly treated with water and neutralised with 0-1 N sodium hydroxide until the solution is just alkaline to litmus.
In our experience it is very difficult to assess the quantity of auxin in a solution by comparing the curvature given by a pea stem with standard curvatures given by stems in solns. of k n o w n auxin content. W e have found that the most reliable me t h o d is to prepare a n u m b e r of dilutions, similar to those used in the M c C o n k e y test for B. coli in waters, and then determine the dilution which just shows a slight positive inward curvature. T h e auxin content of this limiting dilution is determined by a control expt. with different dilutions containing k n o w n quantities of pure /3-indolyl acetic acid.
In our laboratory the dilutions are all m a d e up to a final volume of 10 ml and should be alkaline to the extent of 0-05 ml of 0-1 N sodium hydroxide per 10 ml; this volume is sufficient for the complete immersion of the stems. Three split stems are employed in each test dilution, and are observed after standing immersed for 12-15 hr. in the incubator at 23° C. If a total of 15 m l of neutralised extract are available for the pea test, w e generally use exploratory dilutions containing 5-0, TO, 0-2, 0-04 m l of extract and then m a k e further intermediate dilutions w h e n the limiting range has been found. It is not our practice to standardise each batch of peas grown, but each n e w batch of seed is checked and also re-checked from time to time. W e find that the am o u n t of /3-indolyl acetic acid in 10 m l necessary to give a positive curvature varies between 0-001 and 0-002 mg. The distilled water used in the test is twice distilled from glass, a crystal of thiosulphate (“hypo”) being added in the first distillation if the tap water used contains m u c h free chlorine.
D e t a i l s o f Pr o c e d u r e
Co l d Li m e Pr o c e s s— Place 100-300 g according to the nature of the fertiliser in a 1-litre W a g n e r shaking flask, add 5 g of calcium hydroxide and 500 m l of water. Shake at intervals for 2 hr. and leave overnight. Then shake well and filter through a W h a t m a n No. 12 fluted paper. Place 200 ml of the clear filtrate in a 250-ml flask, acidify with dil. sulphuric acid (a piece of litmus paper m a y be used as indicator), add 20 m l of pptd. zinc ferrocyanide mixture, prepared by adding 0-5 m l of zinc acetate soln. and 0-5 m l of potassium ferrocyanide soln.4 to 19 m l of water, and m a k e u p to 250 ml. Shake well, leave for a few min. and filter through a W h a t m a n No. 12 fluted filter. Extract 100 m l of the filtrate with 120 m l of ether in a separator; draw off the bottom aqueous layer into a second separator, shake it with a second 120 m l of ether and reject the bottom layer. Place a second 100 m l of the clear filtrate in the first separator and shake well; run off the aqueous -bottom layer into the second separator, shake well and reject the bottom aqueous layer. W a s h the ether in the first separator with 20 m l of water, draw off the washing liquor into the second separator and shake again. W a s h the ethereal layers with a second wash of 20 m l of water. Allow the ethereal layers to drain, evaporate the ether off in a 100-ml flask and add 0-2 N sodium hydroxide until the residue has completely dissolved and the resulting soln. is slightly alkaline to litmus. M a k e the soln. up to definite vol., say, 4 ml., e.g., b y adding water until the soln- in the flask weighs approx. 4g. Use 1-0 ml for the modified perchloric acid test and, if
h a m e n c e: t h e d e t e c t i o n a n d d e t e r m i n a t i o n o f a u x i n s i n o r g a n i c m a n u r e s 233 necessary, repeat the test on 0-5 ml. Dilute 2-5 m l of the soln. to 15 m l with water and use this diluted soln. for the pea test in the dilutions described above under The Went Pea Test, adding 0-05 m l of 0 T N sodium hydroxide to all the dilutions.
If the solution of the ethereal extract in 0-2 N sodium hydroxide is highly coloured, it must be purified as described below under “Highly Coloured Extracts” before the perchloric acid test is applied. W h e n calculating the auxin content of a manure, allowance must be m a d e for its moisture content.
Al c o h o l Pr o c e s s— Place 50-100 g of the organic m a n u r e in a 400-ml beaker and stir in 10 m l of saturated citric acid sohi. A d d 200-300 m l of 9 0 % alcohol and leave in an in
cubator at 37° C. for several hours. Filter off on a Buchner funnel and suck dry with the pump. Return the residue to the beaker, re-digest with 200 m l of 9 0 % alcohol and filter.
Unite the alcoholic filtrates and add 3 0 % sodium hydroxide soln. until the mixture is just alkaline to litmus. Evaporate the alcohol, preferably under reduced pressure. Transfer the aqueous residue in the flask to a 200-ml flask and rinse in with water. Acidify with dilute sulphuric acid, add 20 g of sodium chloride and 20 m l of clearing agent prepared as .described under “Cold L i m e Process” and m a k e up to the mark. Shake well and filter through a W h a t m a n No. 12 fluted filter. Extract 180 m l of the filtrate in two portions of 90 m l with ether as described for the cold lime process. T h e n examine the extract as described under the cold lime process.
H i g h l y Co l o u r e d Ex t r a c t s— O n e of the advantages of the cold lime process over the alcohol process is that the final extract (containing the auxins) from the former usually con
tains considerably less pigments than the extracts from the latter. Highly coloured extracts cause trouble in the perchloric acid test; not only is the colour of the test soln. yellow, but occasionally the colour goes into the final chloroform layer. If the final ethereal extract contains appreciable quantities of these pigments it m u s t be purified before the perchloric acid test, can be applied.
O n e m e t h o d of purification is as follows. Dissolve the ethereal extract in 0-2 N sodium hydroxide, m a k e u p to 10 ml, and acidify with dil. sulphuric acid. Stir until the ppt. has coagulated (a little sodium chloride assists coagulation), filter and wash the filter with 5 m l of water. M a k e the filtrate distinctly alkaline with 1 0 % sodium hydroxide soln. and extract twice with 20 m l of ether to remove any indole. W a s h the ethereal extracts separately with 3 m l of 0-2 N sodium hydroxide. A d d the small a m o u n t of alkaline washings to the bulk of the soln., acidify with dil. sulphuric acid, extract twice with ether, wash the ethereal extracts with water and evaporate.
T h e following lead precipitation process is useful with highly pigmented extracts, which are not readily purified b y the process described above. Dissolve the ethereal extract in 0-2 N sodium hydroxide and m a k e up to a k n o w n volume. Withd r a w a portion for the pea test and to the residue add 3 drops of 1 0 % aqueous lead acetate soln., and 3 3 % acetic acid drop b y drop, until the soln. is acid to litmus. Filter through a small filter. In absence of indole this soln. m a y be tested directly b y the perchloric acid test. This process suffers from the disadvantage that a small proportion of the auxin is co-precipitated or adsorbed in the lead ppt. Experiments on the chromatographic separation of impurities showed that the pigments could be easily removed b y this means, but the technique for the recovery of auxins has not yet been worked out.
Re s u l t s— T h e figures given below show the types of results obtained and also the results
of expts. in which k n o w n quantities of /3-indolyl acetic acid had been added.
Co l d Li m e Pr o c e s s
Dried blood
*Hop manure Peruvian guano
T o t a l a u x i n s b y t h e p e a t e s t e x p r e s se d
a s j3 -in d o ly l a c e tic / 3 -In d olyl
/ 3 -In d olyl a c e tic a c id
A d d e d /J-indolyl a c e tic a c id
a c id a c e tic a c id a d d e d re c o v e r e d
m g / 1 0 0 g m g / 1 0 0 g m g / 1 0 0 g m g / 1 0 0 g
0 -25 0-17 0 -25 0 -2 0
0 0 6 le ss t h a n 0-03 1-00 1-12
0 -1 4
(if a n y )
0 -1 4 0-5 0 0-47
* A w e ll d e c o m p o s e d s a m p le .
Th e results given under the heading of /3-indolyl acetic acid recovered in the last column were obtained b y deducting the /3-indolyl acetic acid naturally present in the organic manure
234 h a m e n c e: t h e d e t e c t i o n a n d d e t e r m i n a t i o n o f a u x i n s i n o r g a n i c m a n u r e s
from the total found in the test mixture. Definite evidence of the presence of the propionic or butyric c o m p o u n d in the sample of dried blood was obtained.
T h e following results show the type of recoveries which m a y be expected from the W e n t pea test.
W e n t Pe a Te s t
T o t a l a u x i n e x p r e s se d T o t a l a u x i n e x p r e s se d a s /J-indolyl a c e tic / 3 -In d olyl a s /3-indolyl a c e tic
a c id f o u n d in th e a c e tic a c id a c id f o u n d in
m a n u r e a d d e d m ix t u r e
m g / 1 0 0 g m g / 1 0 0 g m g / 1 0 0 g
D r ie d b lo o d . . .. 0 -2 5 TO O I -00
H o p m a n u r e . . . . 0 -06 0 -5 0 0-62
Co n c l u s i o n s— It will be seen from the results given above that satisfactory recoveries
of added /3-indolyl acetic acid have been obtained by the cold lime process. Satisfactory recoveries of /3-indolyl acetic acid have also been obtained from mixtures to which, in addition to /3-indolyl acetic acid, /3-indolyl propionic or /3-indolyl butyric had been added.
Th e quantitative determination of the proportions of the propionic and butyric homo- logues from the n u m b e r of yellow units given in the perchloric acid test is, however, not entirely satisfactory, as nearly all the results are high. A s stated before, extreme caution must be exercised in the interpretation of the results for these two derivatives.
T h e results given b y the W e n t pea test, while not of the same degree of accuracy as the results given b y the perchloric acid test, m a y nevertheless be regarded as reasonable, having regard to the nature of the met h o d and the very large multiplying factor involved.
T h e alcohol process has only recently been developed and it is therefore too early yet to draw an y useful comparisons between the two extraction processes. It is quite certain, however, from the comparative experiments that have been made, that the extracts obtained b y the cold lime process are m u c h cleaner and freer from impurities than those obtained by the alcohol process.
It seems quite likely that with some manures the alcohol process must be used; in the same w a y it m a y be found that the alcohol process m a y prove successful for removing auxin which is in a combined form and not completely extractabl'e b y the lime process. In testing c o m p o u n d fertilisers containing a m m o n i u m sulphate and superphosphate the alcohol process is more suitable than the lime process.
T h e two processes described determine the free auxin present in the fertiliser, but of course do not take into account the auxin that m a y be produced b y decomposition of the organic matter in the soil.
Soils— T h e cold lime water process has been found quite suitable for the determination of auxins in soils, but, owing to the very small proportions in which these substances are present, it is necessary to work on at least 500 g of the soil.
Peroxide-free Ether— T h e peroxide-free ether used throughout this investigation was prepared from methylated ether (0-?2) b y the m e t h o d of Garbarini,7 which consists in shaking the ether with an aqueous suspension of ferrous hydroxide and then distilling.
I wish to thank Mr. George Taylor, F.R.I.C., for his interest an d helpful criticism through
out this investigation.
R e f e r e n c e s 1. H a m e n c e , J. H . , An a l y s t, 1943, 68, 356.
2. H o lt , P . F „ a n d C a llo w , H . J., I d . , 1943, 6 8 , 351.
3. K o g l, F ., H a a g e n - S m it , A . J., a n d E r x le b e n , H ., Z . p h y s i o l . C h e t n . , 1 93 4, 2 1 4 , 241.
4. M o ir , D . D ., a n d H i n k s , E ., An a l y s t, 1935, 6 0 , 439.
5. W e n t , F . W ., P r o c . K o n . A k a d . W e t e n s c h . , A m s t e r d a m , 1934, 3 7 , 547.
6. a n d T h im a n n , K . V ., " P h y t o h o r m o n e s . " T h e M a c m ill a n Co., N e w Y o r k . 7. G a r b a r in i, G ., C h e m . Z e n t r a l b l . , 1909, 80, 1126.
A P P E N D I X — Si g n i f i c a n c e o f A u x i n s i n Re l a t i o n t o Pl a n t Gr o w t h
A l t h o u g h t h e f o r e g o in g p a p e r is c o n c e r n e d w it h t h e d e t e c t io n a n d d e t e r m in a t io n o f a u x i n s in o rg a n ic m a n u r e s , a b r ie f r e v ie w o f o u r p r e s e n t k n o w le d g e o f th e s ig n ific a n c e o f a u x i n s in r e la t io n t o p l a n t g ro w t h i s c o g n a t e t o t h e s u b je c t. T h e e x is te n c e o f s u b s t a n c e s a ffe c tin g p l a n t g r o w t h , o r g r o w t h s u b s t a n c e s other t h a n n it ro g e n , p h o s p h o r ic a c id a n d p o t a s h a n d t h e m in o r e le m e n ts, w a s p r e d ic t e d a s e a r ly a s 1 8 8 5 b y S a c h s . T h e w o r k o f B o t t o m l e y in E n g l a n d f r o m 1 9 1 4 o n w a r d s , w h ic h u n f o r t u n a t e ly w a s la r g e l y ignored, c o n fir m e d t h e s e p r e d ic t io n s , b u t o n l y in t h e la s t 2 5 y e a r s h a s t h e e x is te n c e o f th e se s u b s t a n c e s b e e n fir m ly e s t a b lis h e d a n d t h e ir c o m p o s it io n e lu c id a te d . T h i s b ig a d v a n c e w a s m a i n l y d u e t o t h e w o r k o f B o y s e n - J e n s e n , W e n t , K o g l a n d T h im a n n . A s a r e s u lt o f t h is w o r k a u x i n s m a y n o w b e r e g a r d e d a s g r o w t h
h a m e n c e: t h e d e t e c t i o n a n d d e t e r m i n a t i o n o f a u x i n s i n o r g a n i c m a n u r e s 235 s u b s t a n c e s w h ic h fu n c t io n in a m a n n e r s im ila r to t h a t o f h o r m o n e s in t h e h u m a n b o d y a n d t h e y are, in fact, s o m e t im e s re fe rre d t o a s P h y t o h o r m o n e s . I t is b e s t t o r e g a r d t h e m a s o n e o f t h e m a n y f a c t o r s n e c e s s a r y fo r t h e o r d in a r y g r o w t h p r o c e s s o f p l a n t s a n d t o p o s t u la t e t h a t w it h o u t t h e m th e re is n o g r o w t h . T h e p r in c ip a l a u x i n s a re a u x i n a (a u x e n t r io lic a c id ), a u x i n b ( a u x e n o lo n ic a c id ), a n d h e t e r o - a u x in o r /8-indolyl a c e tic a c id . T h e s e th re e a u x i n s h a v e a ll b e e n is o la t e d f r o m u rin e , a n d u r in e h a s f o r s o m e y e a r s b e e n r e g a r d e d a s t h e ir ric h e s t n a t u r a l sou rce . I t w ill b e se en t h a t t h e y a re a ll a c id s u b s ta n c e s, a n d i t is in t e r e s t in g to n o te t h a t t h e ir e ste rs a r e f r e q u e n t ly in a c tiv e .
A s .w ith o t h e r n a t u r a l p r o d u c t s , a n u m b e r o f s y n t h e t ic s u b s t a n c e s h a v e n o w b e e n m a d e w h ic h p o s s e s s so m e o f t h e p l a n t g r o w t h p r o p e r t ie s s h o w n b y t h e n a t u r a l a u x in s . T o g iv e b u t o n e e x a m p le o f t h e s e s y n t h e t ic s u b s ta n c e s , I m a y m e n t io n a - n a p h t h y l a c e tic acid , w h ic h h a s th e p r o p e r t y , in c o m m o n w it h t h e a u x in s , o f a c c e le r a t in g t h e f o r m a t io n o f r o o t s o n c u t t in g s . P h e n y l a c e tic a c id is a n e x a m p le o f a s u b s t a n c e w h ic h f a lls in t o a n in t e r m e d ia t e g r o u p b e tw e e n t h e n a t u r a l a u x i n s a n d t h e s y n t h e t ic s u b s ta n c e s. I t o c c u r s n a t u r a lly in u rin e , a n d o u r w o r k s h o w s t h a t i t p r o b a b ly o c c u r s in a n u m b e r o f o r g a n ic m a n u r e s.
A n u m b e r o f w e ll- k n o w n effects i n p l a n t p h y s i o l o g y a re r e a d ily e x p la in e d b y a u x i n a c tio n . A u x i n s p l a y a v i t a l p a r t i n ce ll e lo n g a t io n a n d m u lt ip lic a t io n , b u t a s y e t t h e m e a n s b y w h ic h t h e y d o t h is is b y n o m e a n s clear. T h e b e n d in g o f a p l a n t t o w a r d s t h e l ig h t is d u e t o u n e q u a l d is t r ib u t io n o f t h e a u x in , w h ic h r e s u lt s i n u n e v e n g r o w t h . A u x i n s a ls o f u r n is h a n e x p la n a t io n o f w h y r o o t s g r o w d o w n w a r d s in t h e soil.
T h e y a ls o p r o v id e t h e b e s t e x p l a n a t io n o f b u d in h ib it io n . L a r g e q u a n t it ie s o f a u x i n s p r o d u c e a n u n u s u a l b e n d in g o f t h e le a ve s. F r o m t h e h o r t ic u l t u r a l p o in t o f v ie w a t t h e p r e s e n t t im e a u x i n s a re b e s t k n o w n b y t h e ir p r o p e r t y o f a c c e le r a t in g t h e f o r m a t io n o f r o o t s o n c u t t in g s .
I t h a s b e e n s h o w n b y t h e A m e r ic a n w o r k e r s t h a t n o t o n l y a re a u x i n s p r o d u c e d n a t u r a l l y b y t h e p la n t s , b u t t h a t p l a n t s a ls o h a v e t h e p o w e r o f t a k i n g u p a u x i n s f r o m t h e soil. A v a s t a m o u n t o f w o r k h a s b e e n d o n e o n the, r a t e o f p r o d u c t io n o f a u x in s , t h e ir d is t r ib u t io n a n d t h e ir effect o n d iffe re n t p a r t s o f t h e p la n t , b u t t h e p r o b le m o f a u x i n a b s o r p t io n f r o m t h e s o il h a s b e e n v e r y la r g e ly ig n o r e d . W e k n o w t h a t m in u t e a m o u n t s o f a u x i n a c c e le ra te r o o t g r o w t h a n d t h a t la rg e a m o u n t s c a u se a c o n s id e r a b le r e t a r d a tio n . W e k n o w t h a t a u x i n m a y h a v e a p r o f o u n d in flu e n c e o h t h e p la n t , a n d t h a t t o x i c effects a r e p r o d u c e d b y a n excess, b u t a t t h e m o m e n t w e h a v e p r a c t ic a ll y n o k n o w le d g e o f t h e p a r t p la y e d b y a u x i n s i n t h e s o il o r in t h e fe rt ilise r s w h ic h a re a p p lie d to t h e soil. T h e u se o f s e e d -d r e s s in g c o m p o u n d s c o n t a in in g a u x i n s is c la im e d t o p r o d u c e a n in c re a s e in t h e y ie ld o f t h e c r o p s t o w h ic h t h e y a re a p p lie d .
I t is q u it e c le a r f r o m t h e w o r k t h a t h a s a l r e a d y b e e n d o n e t h a t n o d r a m a t ic effect, s u c h a s a n e n o r m o u s in c re a se i n p l a n t g r o w t h , is li k e l y t o r e s u lt f r o m t h e a p p lic a t io n o f a u x i n s t o t h e soil. B u t i t is q u it e p o s s ib le t h a t t h e fa ilu r e o f s o m e c r o p s a n d p o o r y ie ld s o f o t h e r s o n c e r ta in s o ils m a y fin d a n e x p la n a t io n i n t h e a u x i n b a la n c e w h ic h e x is t s i n t h e soil.
H e t e r o - a u x in is p r o d u c e d b y , a n d a ffe c ts t h e g r o w t h of, a n u m b e r o f m ic r o o r g a n is m s w h ic h o c c u r in t h e soil. H a s t h e a u x i n t h u s p r o d u c e d a n y effect o n s o il f e r t ili t y ? B e f o r e s u c h q u e s t io n s c a n b e a n s w e re d i t is n e c e s s a r y t o k n o w t h e a u x i n c o n t e n t o f t h e s o il a n d a lso o f t h e fe rt ilis e r s w h ic h a re a p p lie d t o t h e s o il;
h e n ce t h is in v e s t ig a t io n in t o m e t h o d s o f d e t e r m in in g a u x i n s in th e se m a t e ria ls . . I t s e e m s p r o b a b le t h a t in t h e p a s t t h is w o r k m a y h a v e b e e n la r g e l y ig n o r e d o w in g t o t h e v e r y s m a ll p r o p o r t io n s in w h ic h th e se a c tiv e s u b s t a n c e s o c c u r in t h e fe rtilise rs.
I n v ie w o f t h e s o m e w h a t u n s t a b le n a t u r e o f a u x i n s a a n d b , i t s e e m s u n l ik e l y t h a t t h e s e s u b s t a n c e s w ill o c c u r t o a n y g r e a t e x t e n t i n o r g a n ic fe rtilise rs. A u x i n a , f o r in s ta n c e , is c o n v e r t e d in t o a n in a c t iv e is o m e r ic c o m p o u n d in a m a t t e r o f w e e k s. H e t e r o - a u x in , o n t h e o t h e r h a n d , is f a r m o r e sta b le , p a r t ic u l a r l y in t h e a lk a lin e c o n d it io n , a n d , a p a r t f r o m t h e n a t u r a l l y o c c u r r in g h e t e r o -a u x in , f f- in d o ly l a c e tic a c id a n d it s h o m o lo g u e s a re p r o d u c e d b y t h e d e c o m p o s it io n o f o r g a n ic m a tte r, p a r t ic u l a r l y o r g a n ic m a t t e r c o n t a in in g t r y p t o p h a n a n d o t h e r a m in o a c id s. I t w a s fo r t h is r e a s o n t h a t o u r a t t e n t io n w a s m a i n l y d ire c t e d t o w a r d s th e e s t im a t io n o f h e te r o -a u x in .
U n f o r t u n a t e ly , a t t h e p r e s e n t t im e n o c h e m ic a l t e s t s e x is t f o r t h e e s t im a t io n o r d e t e c t io n o f a u x i n s
a a n d b , a n d t h e b e s t w e c a n d o is t o o b t a in a r o u g h a p p r o x im a t io n b y d e t e r m in in g t h e t o t a l a u x i n s p r e s e n t b y a b io lo g ic a l m e t h o d a n d t o d e d u c t f r o m i t t h e h e t e r o - a u x in f o u n d b y t h e p e r c h lo r ic a c id m e th o d .
L a b o r a t o r y o k D r . B e r n a r d D y e r a n d P a r t n e r s J u n e , 1 94 4
20, Ea s t c h e a p, Lo n d o n, E . C . 3
T h e P h o t o m e t r i c D e t e r m i n a t i o n o f C o b a l t , T i t a n i u m a n d I r o n i n U n s i n t e r e d M e t a l C a r b i d e s
By H. C O X , A.M.C.T., A.R.I.C.
Th e determination of cobalt, titanium and iron in hard metal carbide powders, as carried out by the usual analytical processes, is an involved and lengthy procedure, and a simpler and speedier m e t h o d of analysis is desirable.
Photometric determination appeared to offer the most promising me a n s of effecting substantial saving in time, and a composite me t h o d has been successfully developed b y which these three constituents m a y be determined on one initial sample with an accuracy c o m parable to that realised b y the methods employed hitherto. In addition, the procedure for the preliminary treatment of the sample has been simplified and effects further saving of time.
Advantage has been taken of the pioneer work of V a u g h a n 1 in connection with the general application and operation of the Spekker Photo-electric Absorptiometer and of the work of H a y w o o d and W o o d 2 on the determination of cobalt in steel.
R e a g e n t s — (1) Sodium carbonate, anhydrous, AnaiaR. (2) Potassium bisulphate, AnalaR. (3) Nitroso-R-Salt, B.D.H.— 2-0 g dissolved in water and made' up to 1 litre.
(4) Sodium acetate trihydrate, A n a l a R — 500 g dissolved in water and m a d e u p to 1 litre.
(5) Hydrogen peroxide— 30 volumes. (6) Potassium thiocyanate, A n a l a R — 200 g dissolved in water and m a d e up to 1 litre. (7) Nitric acid— 1-4 sp.gr. (8) Sulphuric acid— 1 0 % b y vol.
Pr o c e d u r e— Thoroughly oxidise in the muffle furnace 0-5 g of carbide weighed into a
platinum crucible. Fuse the oxides with 5 g of sodium carbonate, extract with boiling water, filter and'wash with hot water. Discard the filtrate, which contains the tungsten. Removal of tungsten is necessary because, if kept in solution with phosphoric acid this acid affects the titanium colour produced with hydrogen peroxide and also because pptn. of tungsten is not avoided w h e n buffering with sodium acetate for the development of the cobalt colour with nitroso-R-salt. Ignite the filter-paper and contents in the original crucible and fuse with 5 g of potassium bisulphate. Transfer to a 400-ml beaker and dissolve the melt in 100 m l of water and 50 m l of conc. sulphuric acid. Cool, transfer to a 500-ml graduated flask an d m a k e u p to vol. with water. This is referred to subsequently as solution A.
Cobalt— Pipette 25 m l of soln. A into a 200-ml graduated flask and m a k e u p to 200 ml with water. F r o m this, pipette 20 m l into each of two 250-ml beakers. T o one beaker add 10 m l of nitroso-R-salt solution and 10 m l of. sodium acetate soln. T h e addition of the nitroso-R-salt must be m a d e accurately from a burette. Heat to boiling, add 5 m l of nitric acid and boil for 1 to 2 min. Cool and dilute with water to 100 m l in a 100-ml graduated flask. Treat the contents of the second beaker, the blank, in the same m a n n e r but omit the nitroso-R-salt. Measure the extinction on the absorptiometer, using 1-cm cells and Ilford Spectrum Blue filters No. 602. Obtain tjie cobalt % by reference to the cobalt graph.
Titanium— Pipette 50 m l of soln. A into a 100-ml graduated flask. A d d 5 to 10 m l of hydrogen peroxide and m a k e u p to volume with sulphuric acid (10%). For the blank, pipette a second 50 m l of soln. A, omit the hydrogen peroxide and m a k e u p to 100 m l in a 100-ml graduated flask with 1 0 % sulphuric acid. Measure the extinction on the absorptio
meter, using 1-cm cells and Ilford Spectrum Violet filters No. 601. Obtain the titanium % b y reference to the titanium graph.
Iron— Pipette 100 m l from soln. A into a 250-ml beaker. A d d accurately from a burette 5 m l of potassium thiocyanate soln. For the blank, pipette a second 100 m l of soln. A, omit the thiocyanate and add 5 m l of water to preserve equality of volume. Measure the extinction on the absorptiometef, using 1-cm cells and Ilford Spectrum Blue-Green filters No. 603. Obtain the iron % b y reference to the iron graph.
It is r e c ommended that the absorptiometer be operated with the d r u m aperture fully open {i.e., d r u m reading = 0) for the coloured soln., so that the d r u m reading for the blank gives the extinction value directly.
Ca l i b r a t i o n Gr a p h s— In order to establish the graphs, two sources of the respective metal were used in each instance. For any given % of metal over the ranges covered, the max. observed difference in extinction value wa s 0-01 d r u m division.
Cobalt— Solutions were prepared from (1) A n a l a R cobalt sulphate a n d (2) a cobalt metal powder of pre-determined cobalt content, such that 1 m l contained 0-000025 g of Co (i.e., 1 m l = 1-0% of Co on 0-5 g of the original sample). A m o u n t s (from a burette) equiv.
to from 4 to 1 4 % in steps of 1-0% were m a d e up to 100 m l after being buffered with sodium acetate and the colour was developed with nitroso-R-salt. A second series of solns. in which the nitroso-R-salt wa s omitted, was prepared for the respective blanks. T h e extinction values were obtained and a graph w a s prepared.
Titanium— Solutions in 1 0 % sulphuric acid were prepared from (1) a British Chemical Standards Ferro-titanium (22-8% Ti) and (2) a sample of titanium oxide (98-75% T i O z), such that 1 m l contained 0-00025 g of Ti (i.e., 1 m l equiv. to0 - 5 % of Tion0-5goftheoriginalsample).
F r o m 8 to 30 m l in steps of 2 m l were run from a burette, coloured with hydrogen peroxide a nd m a d e u p to a final vol. of 100 m l with 1 0 % sulphuric acid. A second series, in which the peroxide was omitted provided the respective blanks. T h e extinction values were obtained a n d a graph was prepared giving titanium % from 4 to 15.
Iron— Solutions in 1 0 % sulphuric acid weye prepared from (1) A n a l a R ferrous a m m o n i u m sulphate and (2) A n a l a R ferric alum, such that 1 m l contained 0-0001 g of F e (ferric), (i.e., 1 m l s= 0 - 1 % on 0-5 g of the original sample). F r o m 1 to 10 m l in steps of 1 m l were re
spectively m a d e u p to 100 m l with 1 0 % sulphuric acid and' then coloured with 6 m l of 2 0 % potassium thiocyanate soln. measured accurately from a burette. A second series, in which 236 cox: t h e p h o t o m e t r i c d e t e r m i n a t i o n o f c o b a l t, t i t a n i u m a n d
IRON IN U N S I N T E R E D M E T A L CARBIDES 237 5 m l of distilled water replaced the thiocyanate soln., provided the respective blanks. T h e extinction values were obtained, and a graph prepared giving iron % u p to TO.
C o m p a r i s o n o f R e s u l t s— Table I gives a comparison of the results of determinations m a d e photometrically with those obtained by the methods employed hitherto (gravimetric iron and titanium and electrolytic cobalt).
Ta b l e I
C o b a l t % ' T i t a n i u m % I r o n %
A _________ A_____________ _ _____ -A_________
t \ r t t
S a m p le P h o t o m e t r i c E le c t r o ly t ic P h o t o m e t r i c G r a v im e t r ic P h o t o m e t r i c G r a v im e t r ic
A 5-70 5-56
— —
0 -3 0 0-32B 10-95 10-96
— —
0-17 0-16C 9-02 8-98 5 -25 5-27 0 -2 2 0 -2 4
D 11-65 11-75 13-00 13-2 4 0 -2 9 0 -27
E 6 -58 6-52 14-35 14-31 0 -2 0 0 -2 0
F - 10-23 ' 10-18 9 -40 9-43 0-32 0-35
G 7 -45 7 -43 9 -80 9-86
— —
H 9-37 9 -40 5 -80 5-86
% — —
I 8 -94 9-00 5-35 5-61
— —
J 6-76 6-75 14-35 14-12 — —
N o t e s— T h e use of nitric acid as an oxidant for ferrous iron is to be avoided. This acid produces a colour with potassium thiocyanate (in absence of iron) due to the formation of nitrous fumes. A t a concentration of 5 m l of nitric acid (sp.gr. 1-4) in 100 m l this colour is rapidly produced. For this reason potassium permanganate was used to oxidise the ferrous a m m o n i u m sulphate in the preparation of the graph for iron. In the composite method described the initial oxidation of the sample avoids the need for subsequent oxidation of the iron to the ferric condition.
Th e addition of thiocyanate must be m a d e accurately. T h e intensity of colour increases with increasing concn. of thiocyanate. For example, 0-0002 g of iron m a d e u p to a final volume of 130 m l gave d r u m readings ranging from 0-13 to 0-335 with thiocyanate additions ranging from 5 to 30 ml.
T h e colour of the iron thiocyanate is not permanent a n d fading is detectable photo
metrically after about 3 hr. Extinction measurements should be m a d e therefore within 2 hr.
of developing the colour.
I a m indebted to Dr. A. P. M. Fleming, C.B.E., Director, and Manager, Research and Education Departments, a n d to the C o m p a n y for permission to publish this work.
Re f e r e n c e s
1. V a u g h a n , E . J., " T h e U s e o f t h e S p e k k e r P h o t o -e le c t r ic A b s o r p t io m e t e r in M e t a ll u r g i c a l A n a l y s i s . "
M o n o g r a p h p u b lis h e d b y t h e R o y a l I n s t i t u t e o f C h e m is t r y , 1941.
2. H a y w o o d , F . W . , a n d W o o d , A . A . R ., " T h e R a p i d P h o t o m e t r ic D e t e r m in a t i o n o f C o b a l t in S te e ls, u s i n g N i t r o s o - R - S a l t , ” J . S o c . C h e i n . I n d . , 194 3, 6 2 , 37.
Ch e m i c a l La b o r a t o r y, Re s e a r c h De p a r t m e n t,
Me t r o p o l i t a n- Vi c k e r s El e c t r i c a l Co., Lt d., Tr a f f o r d Pa r k, Ma n c h e s t e r M a y ,>1944
A S i m p l i f i e d D e t e r m i n a t i o n o f t h e A l k a l i s i n S i l i c a t e s By J. J. S. C O R N E S , B.A., B.Sc.
I n t r o d u c t i o n— In the determination of alkalis in silicates, if the silicate is decomposed by the method of Berzelius (1824), using hydrofluoric and sulphuric acids, sulphate and m a g nesium must be removed b y special treatment with barium chloride and barium hydroxide.
For that reason the m e t h o d was largely replaced (in 1871) b y that of J. Lawrence Smith, in which from the outset m a gnesium is m a d e insoluble b y excess of calcium hydroxide. That method still required, however, removal of calcium, with the consequent disadvantage of having to remove a m m o n i u m salts later.
Modern methods are n o w available for the precipitation of both sodium and potassium as complex triple salts (sodium magnesium uranyl acetate and potassium sodium cobalti- nitrite), in presence of calcium. Hence it is possible to dispense with removal of calcium in the Lawrence Smith procedure. Modifications in this direction have been devised b y Miller and Traves1 a n d b y H a s l a m and Beeley.2, In both modifications the aqueous extracts from separate ignitions are acidified and evaporated, and sodium and potassium are pptd. as the
