ACIDS A N D ALKALIES. PART II. The Use of Indicator Papers. A Note ()n l~othera.'s Test

for Acetone and Aceto-Acetic Acid.*

By WILLIASI Ft.:AROX, Ph.ldsiolo,dic~,l Lr Tr'i.it~ d College, Dub!lb,.

I N laborator 5 work, the re.action of liquid is gener~dly found by either the electrometric method or tile, co.levi-

metric m-ethod. The eleetrometrie method is an absolute method: it consists in measuring directly the potential ,lifferenee se~ up between a platinum pla.te coated with hydrogen, generally spoken of as the hydrogc~ electrode, immersed in the unknown solution, and a similar plate immersed in a standard solution of known hsdrogen-ion eon- ,entration. In actual practice, the standard solution i~ r~.placed by a standard calomel battery or cell. Bv these means the hydrogen-ion concentration corresponding to, a given po.tential difference can be ascertained.

Various refinements and alterations in the apparatus are being continually introduced. The method requires careful manipulation .and attention, which renders it unsuitable for ~eneral laboratories, but it is the final tribunal as regqrds ~he hsdrogen-ion concentration of solutions.

The e~Ao.rimetrie m'.,tbo~t, which is ea.pable cd much wider ~q}pl[eation, depends on the discovery of a number of dye- st~fffs, which can change co!our sharply at different degrees ,d hsdrogen-ion concentration. By means of standard solu- tions and a series of special indicators, it is now possible to d,.termine rapidly and with a very fair degree of aeeuraey ~he reactions of many important solutions.

The convenience of litmus papers when the rapid deter- ruination of the relative reactiort of a solution is required, ,~lggested that a serie,s of papers might beprepared inpreg- hated with various indicator dyes, so a~ to extend the range. Such a series was briefly described by the pr.esent writer in n paper published in this Journal (October, 1918) on the s~,bjeet of " I ' r inary Amylase. ~'

" P a r t I . o f t h i s p a p e r a p p e a r e d i n t h e '" D u b l i n J o u r n a l of M e d i c a l LN_,ience " f o r D e c e m b e r , 1920.

202 D U B L I N JOURNAL OF MEDICAL SCIENCE

Indicator Papers. Litmus paper is impregnated with a. dye which changes

colo.ur in the region of true neutrality (P• 7). For H-ion concentrations above 4 x 10-- 6 (or P~ 5.4), it is definitely red; for concentrations below 2 • 10 ---s (or pH 7.8), it is definitely bl.ue. In the region between pr~ 5.4 and pH 7.8, its shade varies from pm'ple to violet. These tints may be affected to some extent by the presence of concentrated electrolytes (" salt~ en'or "). The limitations e,f litmus paper will be obvious from the foregoing. ]?or any acid solution from 1 )~ 5.4 downwa,rds, the paper will turn red, and will not serve to distinguish by its shade the difference between such solutions as nephlqtic urine of P~ 5.2, infa.nt's ga,strie juice of pH 5.0, deeinornml acetic acid of pH 2.87, decinor- real hydroehtoaqe acid o~ P~ 1.08, and adult 's gastric juice e.[ Pg 0.9 to pH 1.6. The same holds for alkaline solutions above P:g 7.8. Li tmus paper will be bllte in the presence of all of then1. In short, outside its limited region of inter- mediate shade% litmus can give no direct quantitative infor- mation regarding the degree of the acidity (~r alkalinity which it records.

I t is owing to the fact~ that its turning point is in the region of true neutrality that, litmus paper is o,f general clinical use.

These objections hold for all indicators and indica~to.r papers; outside a narrow zone, they respond equally to vary- ing degrees of acidity or alkalinity.

This region of eolour change varies with different indica- tors, and by co~nstructing a series of indicator papers in the e,rder of their turning points it is possible to determine the reaction of a solutioaa with great rapidity .and a. very fair degree of accuracy.

For example, phenol phthalein is eolourless in solutions below PJ~ 8.3, between prt 8.8 and P a 10.0 it p~sse.s from palc piM~ to bright " cerise," above this it. does not change, unless the alkaline solution is ve~ T strong, when the colour of the indicator will be discharged, prot~ably owing to mass- action.

Phenol phthalein paper is now on the market. If a, solu- tion be tested with litmus and phenol phthalein papers and

ACIDS AND ALKALIES. 203

found to react, alkaline with the litmus and acid with the- phenol phthalein (i.e., a blue colour with one, and no change. with the other), its H-ion concentration must lie between P~ 7.8 and 8.3.

Theory. o] I,~dicators. The theory of indicators has already been discussed in

this Journal (W. G. Smith, September, 1915), and it is no.t proposed to consider it any further in the present, com- munication.

As employed in colorimetrie measurement, the indicator method is, practically, a comparative method, using the electrometrie method as a standard. I t is not necessary to know the chemical constitution, or the dissociation eon- sta.nts of the various ind;ieators employed, provided that the H-ion concentrations which bring about the eolour changes are known.

[For a more detailed discussmn, the reader is referred to a paper bv A. A. Noyes, 1910, J. Am. Chem. Soe., 32, 815.]

Theory of Indicator P~lpers. This subject does not appear to have received much atten-

tion. I t is a complicated subject, and may be resolved into two distinct problems : - -

(1) The nature of the process by which the indi'eator dye is taken up by the paper.

(2) The nature of the interaction between the ions of the solution under investigation and the indicator held by the. paper.

The first problem belongs to the physical chemistry of dyeing. In the ease of the pigments under consideration, the union between dye and paper is almost, certainly tha t o.f an adsorption or condensation of the dye-stuff on the surface of the cellulose fibres in the paper. Bayliss has investigated very thoroughly the adsorption of the dye, " eo.ng(~ red," by filter papers. (1906. " BiochemicaI Journal ," Vol. I. , p. 175.)

I t does not follow, however, that the interaction between the adsorbed, indicator and the ions of the solution under' examination will be the same as the interaction between free indicator and solution-ions. To begin with: selective

f104 D U B L I N JOURNAL OF MEI) ICXL SCIENCE

adsorption may take place in certain solutions. Filter p~per iminersed in aqueo.us so.lufions carries an electro-negative surface charge. If tile solution contained an acid, one might Expect that some of tile hydrogenions of tile acid, in virtue ,Jt their electro-positive charge, might be drawn to the sur- :face of the paper.

This phenomenon is termed " electro-capillary adsorp- tion," and is known to occur in many systems.

Again : suppose that the solution eo.ntains some substance eapuble, o.f lo,wering sm'faee tension---for ex~mlple, if w~ are trying to, determine the relative amount o.f lactic acid in a specimen of ga;~tric juice free from hydrochloric acid. The substance will be concentrated o,n the surface, aeec~vding *co a. well-known physical prineiifle of Willard Gibbs and .J. -J. Thomson. This is an .example of o.rdinary or meeb_'mieal :~.dsorption.

If either o.f these adsorptions ta,ke place, it will fcdlow that the indicator dye o,n the paper will be in the pr;.~enee ,)f a layer of solution o,f quite a different eoneentratio~ from ~hat in the main body of the solution.

Th.e relationship between the concentration of the ~c,~ute ,,n the surface o.f the paper a.nd tba.t in the main bc, dy of ~:h,_" solution may be represented as fol lows:--

A = Ce n Where A is the concentration of absorbed material on the

sm'faee of the paper. C is the concentration of the same m~iteria! in tb.,_ ~olu-

Zion. e fs "exponent ia I e , " t h e b a s e of nstural logarithm% amt

has tile value 2.7182 n is a. constanl; depending on the particular paper ~urfaee

~.a~J,] the nature of the solute. The method of obtaining tile above equation i~ rather

~.omplieated, and requires some mathem'/ t iea! know!edge. The equation ires been verified by ])onna,n for an air:'liquid ~u:faee, and by W. MeC. Lewis for a liquid/liquid surface. ]leeently the present author has devised a method for hi,ply- ing it to. liquid/solid surfaces.

This simplified equation will serve to iIlu.~trat.e some ~f -the difficulties to be overemne if the indicator paper meth~d i~ to be o.f general clinical u.ae. For example, urine ;.on-

A C I D S AND A L K A L I E S . 20~;

tab~;:'g bile salts, which are able to lo, wer smfaee tension con.-.i!crabls- , ma}- give a eompletet}- misleading reaetion when examined by l i tmus or other test papers. This mas' s~.,e~,~. ~,> be a small ma t t e r in view o.f the slight, import.anee often at tached to the result o,f determining the reaction of a san:ple of urine. It. may be ment ioned in passing that, this d~pl'eeiation o,f the results o,f qua.lita.tive urinm'y analysis is: ~n realit5:, a. depreeiatio,n o4 the method o,f anals-sis and n<~t ,A the result so.ught, for.

it'}2 hs"drogenion eoneentra.tio,n of the urine at ans- pa'c- ticu}:.~.,_" time is a funct ion of the equilibrium eonstant o* the acid~ and bases in the blood�9 This is strikingl5- shown by the >,~]]-recognised " acid tide " in the urine befo.re meals ~md t}~e " alkaline tide " after meals. I n each eondit.ion, the }.~!>o..t remains cons tant a.s regards hydrogenion eoncen- t r a t im, but o,wing t.o the withdrawal o.f hydrogenio.ns te fi~',~_ zhe hsdroehlo,rie acid of gastric juiee, some of the 'a:id ,~,tores of the body are depleted, and the superfluo, us atlk:fii i~ excreted in the urine, so, that the blood reaetio.n is tma'~v:ed.

r ( 7 �9 H~ .~r%emon nlea.surements ha.ve given aeem'ate infornm- tion .,S the variations in +he rea.etion of normal and a b n o r m a l

�9 1 7 m~..~- during long periods; it now rema.ins fo.r the ehemi~.~ t~ , ;-A~mine the changes in salt con ten t responsible for the altc~:c:ions in urinar 5, rea.etion. \ \ ' h en tha t is done, the bev, i!,_~ering prchIem of urinar3 ealeuli and their relaticm to. m'i.,> :x l.e~mtion will be Jn a. fair way to being solved.

Mcth.od of Employing DMicator Papers. }:': ,m the eonsiderations briefly alluded to., it will be seen

th:< :he onh- sat isfactory method of employing indieator pa.F,:!~> is to dip the paper into. the bulk o.f the solution ttl" ]~l examinat ion and leave it, there for a short time.

31!>- eonlmon eastern o,f put t ing a drop of the solution rm ~h:~ test. paper almost invariably leads to inaeeurate eon- (,lt~,}0ns. The test paper is liable to extraet more or le~s ~,f ~l~e solute from the drop of solutioa, a. partit ion effeet res~:]~s, and the indieator dye will give a result either too ]ti.:;)i 3': too lc.w, aeeording to whether it interacts with the e(-.;~:'antra.ted solute on the surfaee c,.f the paper or the ,dilute s~]~.'te in the bulk of the drop.

206 D U B L I N JOURNAL OF ~IEDICAL SCIENCE

With litmus paper, the latter condition is what, generally occurs.

With many organic solutions it mas" be neeessars' to keep the test. paper immersed for some time before the final equilibrium tint is l~aehed.

This can be decided by dipping one paper in the solution Yc~r ten minutes; then dipping a similar strip ef paper in for one minute, withdra.wing both strips, and comparing the tint by placing them side by side on a, white surface.

If the shades are the same, one may conclude that. the. equilibrium tint has been reached.

This is of special interest in the determination of the reaction of milk, a subject which will be considered later.

The errors introduced in indicator work by the presence ,~ large quantities of " neutral salts " are probably examples of selective adsorption, referred to previously. The so- called " buffer " or regulator action o.f certain salts may be sho,wn by the folIowing exper iment , :~

To a test-tube half-full of water add ~ couple <~ drops of ~,trong ammonia, then a drop of phenol phthalein solu- tion (usually made up to 1 per cent. in aqueous alcohol). The alkalinity of the solution due to the ammonia will exceed pH 10, and the phenol phthalein wilt give a deep red solu- tion.

Now add to the solution an excess of solid ammonium ~ulphate crystals, so as to saturate the solution, and lea.re a layer a,t the bottom of the. test-tube.

There is at fimt no change; then, above the ammonium sulphate las-er a clear ring will be seen to develop in the solution. This is due to hydrolytic dissociation of the am- monium sulphate into ammonia, and free sulphuric acid. The nmnber of hydrox2~'l-ions due, to the, wea.k base anlmonia. will be less than the number of hydrogen-ions due to the strong sulphurie acid, consequently the hydrogen-ion con- centration in the region above the solid ammonium sulphate ia increased, the P~ falls below 8.3 (since the prr method of expressing react.ion varies inversely with the I-I-ion concentration, as explained in the first paper on ~he preseng subject., Dublin Jour. Med. Set., D e c . , 1920), the phenol t.,hthalein is colourless below P~ 8.3, and, consequently, a elea," zone appears in the liquid.

ACIDS AND ALKALIES. 207

Rothera.'s Test~. This li~tte experiment is. of medical interest, since it

affords the probable explanation r the part played by the ammonium sulphate in ]lothera's. valuable test for acetone and aceto-acetic ,,cid.

Without committing oneself to much as regards the chemistry of the reaction--which has not ye t been worked ou t - - t he mechanism appears to be .as follows : -

Sodium nitroprusside, Na 2 Fe (CN)~, NO, is readily re- duced in alkaline solution with the formation of a. purple- red unstable pigment.

Creatinine is able to bring about this reduction in the presence of strong alkalies, such as Na..OH and K.OH, but not in the presence of weak alkalies, such as NH4.0H.

Acetone and aceto-acetic acid are able to bring about the reduction of nitroprusside in both strong and weak alkalies.

If Na..OH and sodium nitroprusside be added to urine containing creatinine and aceto-acetic acid, the purple colour formed will be ambiguous.

If, on the other hand NH4.0H and nitroprusside be added to urine, the appearance of a purple colour indicates acetone or aceto-acetic acid

Sirme creatinine does not react, under these conditions, unle.ss a large excess of NH~.OH is employed, this is an important point-- in testing for acetone, or a ceto-aeetic acid in urine th6 NH~.OH added should not be more than one part in ten of the total liquid under examination.

The purple pigment is soon destroyed in alkaline solution, especially in the creatinine test~ where, there is an excess of alkali. I t is more stable in the neutral or faintly acid zone that forms above the layer of ammonium sulphate crystals in Rothera 's modification of .*he test. This pro- bably accounts for the great delicacy of the test, which was shown by I turt ley (1913, " Lancet.," April 26th), to. indicate one part of a ceto-acet,ic acid in 400,000 in 5 minutes. In his original paper (J. Physiol., 37), t lothera has stated that most of the salts of ammonia have this intensifying effec~ on the reaction, with the exception of ammonium oxalate, an observation which is of considerable significance in con- nection with the doubtful constitution of many of the am- monium salts.