SHORTCOMMUNICATIONS 525

An improved colloidal stabilisor for use in the nephelometric determination of the sulphate ion

Durmg the course of an mvestigation of the ncphelometric determination of the nitrate ion1 by means of the N-substituted naphthylmethylamine reagentsz, considerable difficulties were experienced in stablllsmg the suspensions of the organic amme nitrates for nephelomctric measurement in the way described for +amino-q’- chlorodphenyl (CAD) sulphate, that IS, with peptone and gum ghattl3. The addition of peptone to the precipitant helps considerably m the formation of uniformly sized particles of the precipitate but unfortunately its prcscnce has the effect of decreasing the sensrtivlty of the preclpltatlon reaction which, m turn, limits the sensrtrvrty of the nephelometric procedure.

In later attempts to produce small evenly-sized particles of CAD sulphate, a mechanical process was investigated The hot sulphate solution was treated with the reagent solution and the mixture was stlrrcd thoroughly during rapid cooling to room temperature In this way, finely-divrded precipitates of the amme sulphate were obtained with as little as 0.0125 mg of sulphate ion m IO-ml volumes (1.25 p.p.m. of sulphate).

The suspensions given by these precrpltates were, however, unstable; rapid coagulatron of the particles occurred as shown by the decrease m light scattering observed with time, and the addition of some form of protective collold was essential if these suspensions were to be apphcd m any nephelometric procedure. A recognised substance which acts m this way is gum ghattl, this is an exudate from the plant, Ano&sszcs htzfolza, and chemically is a polysaccharlde contaming L-arabinose, D-XylOSC, u-galactose, n-mannosc and glucuromc acid, belongmg to the class of hydrophilic gums. A solution of this gum was found to be reasonably effective for the formation of stable CAD sulphate suspensions in the previously described method. Although th.is method is satisfactory, rt has the disadvantage of most nephelometrrc procedures that measurements must be made at a defuute time after precipitation of the light-scattering particles; the presence of gum ghatti is essential for reproducible measurements. However, when the gum ghattl solution was added to the suspensions of amine sulphate prepared as described above, only partial stabllisatton of the suspensions was observed, and coagulation of the particles, although slowed down, still occurred, and made nephelometric measurements rmpracticable. The gum ghatti solution had been prepared by dissolving the powdered mater-la1 in hot (70”) 0.05 N hydrochlorrc acid; tins process could result m an appreciable degradation of the labile long-chwn sugar molecules as a result of hydrolysis. Obviously, it was desirable to mimmise the extent of this reaction, because a larger positively-charged colloidal molecule should be a more effective stablliser for suspensions of amine salts formed in an acidic medium. The precipitate of CAD sulphate (and other amme salts spanngly soluble in water) might be expected to form particles wrth the negative ions (the most hydrophrhc part of the molecules) on the outside of the particle and shielding the organic cation. The low solubility of CAD sulphate would favour a highly sym- metrrcal arrangement of the dphenyl nuclei in the crystal lattice of the salt (Fig. I).

Suspensions of CAD sulphate in dilute acid should then be partly stablltsed by HeO+ ions and partly by positively charged molecules of polysaccaride, in this case, gum ghattr.

Anal Chrm.Acla,3g (x967)525-528

526 SHORT COMMUNICATIONS

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The longer the sugar cham of the gum ghattl molecule, the more effcctlve it should bc a\ a protective collo~cl, and attention was given to the preparation of solutions with lmprovcd colloidal properties Of several solutions examined, three rn particular appeared to have some of the desired propertlcs. Solution A was prepared by forming aslurry of 2 g of finely powdcrcd gum ghatti with a little water and diluting to 200 ml with warm (50”) water. Solution B was prcparcd from a slurry of 2 g of the gum m 5 ml of 0.05 N hydrochloric acid followed by gradual clllutlon with 200 ml of the same acid at 50” Solution C was slmllar to solutron B except that clllutlon was with 200 ml of water at 50” All three solutions were considerably more viscous than the dllutc gum I;hatti solution used previously. Only solutions A and C retained this vlscostty for more than 24 h when stored m a rcfngerator at 3”. It was necessary to allow each solution to stand for some hours for the undlssolvcd matcrlal to settle out, the clear supcrnatant gum solution was then decanted off, and stored m ice-water. The mdivrdual solutions were then cxamincd with suspensions of ammc sulphatc, prepared by rapid cooling of the reactants as clescrlbcd above Ncphelomctrlc mcasure- mcnts were carried out on these suspensions at time intervals in the range IO mm to 48 h. Only solution B gave stable suspensions, these lasting for at least 48 h In fact, there was not much change cvcn after g6 h but after this, the particles showed signs of rapid coagulation as a result of the bursting of the protective “skin” of gum ghattl surrounding each particle

Unfortunately, this gum solution was stable for only 24 h after preparation, even at 3’ ; it was lmpractlcal to prepare it each day, because some hours storage time wa5 required for the undissolved matter to settle out Solution A, however, appeared to be perfectly stable for several clays at 3”, but attempts to use this neutral solution and sufflclent o,o5 N hydrochloric acid to maintain suitable conditions of acidity m the suspensions, were unsuccessful. It was evldcnt from these experiments that the acid must be added wtth the gum ghattl solution; only m this way would the molecules of the polysaccharide acquire a sufflclent positive charge to stablhse the suspensions of the ammc salt. The lnstablllty of the gum solution was overcome by preparing a stock solution of solution C above This solution alone was ineffectlve as a stabillser, but was itself stable for prolonged periods if stored at 3”. When this solution was treated with hydrochlorrc acid (2.5 ml of 2 N acid/Ioo ml), it had all the desirable properties of an effective stabihser, although the solution could not be kept for periods greater than 12 h. When this mixture was used m place of the gum solution recommended m the original procedure, m conjunction with the mo&fled process for formation of the precipitate, it proved possible to develop a considerably slmphfied procedure for the nephelometrlc determination of the sulphate ion.

Anal Chm Ada, 39 (x967) 525-528

SHORT COMMUNICATIONS 527

ExPeramentaZ CA D-lrydrocltlovzde sol&on Dissolve I g of +ammo-4’-chlorodlphenyl hydro-

chlorrdc in 400 ml of o 05 N hydrochloric acid. Stock gum glmttt soltrtzon. MIX IO g of finely powdered gum ghattl (Hopkin &

Willrams reagent) with 25 ml of o 05 N hydrochlorrc acid To this slurry, add I lure of water at 50-55”, slowly and wrth constant strrring throughout the addrtron. Falter the warm solutron through glass wool to remove the coarser undissolved partrclcs and store the solutron at 3O, untrl the remammg msoluble matter has settled out Decant as much of the clear liquid as possrble and store this permanently m a refrigerator. Thts solutron IS stable for at least two months (the maxtmal time examined)

Aczd gum ghattt sol&on Just before use, add 2 5 ml of 2 N hydrochlorrc acid to IOO ml of the stock solution of the gum Store in rcc-water throughout its period of use and drscard after 12 h

Procedure. Add with gentle stlrrmg, 12 ml of hot (70”) CAD reagent to the hot (70”) IO-ml neutral test sample contamcd in a so-ml beaker Allow the mrxturc to cool to about so”, stlrrrng gently m the meanwhile, and continue the coolmg and stirring m Ice-water, until about 2 mm after the preciprtate has formed Add 0.5 ml of the cold (o”) acid gum ghattr solutron and star for 15 see Transfer the suspenston to a so-ml volumetrrc flask with the ard of suffrcrent water to give a volume of about 45 ml in the flask. Add a further o 5 ml of the acid gum ghattr solutron and dilute the suspension to 50 ml wrth water. Leave for 30 mm, and transfer suffrcicnt of the well-mixed suspension to a 2” dram nephelometcr tube to fill it completely Stopper the nephelo- meter tube and mvcrt once or twrcc before taking the nephelomctcr reading Prepare standard samples and a reagent blank in the same way, with the scnsrtrvlty control of the instrument set to give a maxrmal deflcctron of xoo umts wrth the most concentrated standard, and record the galvanometer deflectron with the appropriate perspex standard provided with the instrument.

ResrrEts atrd dzscztsszon I The effect of the modified gum ghattl solution on the stability of the suspen-

srons of CAD sulphate is quite remarkable and lends support to the supposrtion that posltlvely charged molecules of the polysaccharule are the stabihsmg species. Figure 2

shows the light-scattering properttes of the freshly prepared suspensions compared with the same suspensions 48 h later. The degree of comcldence of these measurements IS extraordinanly hrgh and has an important bearing on the general analytical procc- durc.

Once a calibration graph has been prepared using a partrcular solution of gum ghattr, rt is no longer necessary to carry out a dally rccahbration. Only the most concentrated standard suspension need be prepared, m order to set the sensltlvrty of the instrument to give the maximal galvanomcter deflectron. Then, if the reading with the permanent perspex standard IS the same as that obtained when the cahbra- tion graph was constructed, rt IS permrssrble to proceed directly to the determinatron of sulphate m the test samples without further preparations.

The present method has several advantages over the method previously descrlbeda. The range of concentratron of sulphate ion now measurable is approxrmate- ly 1-25 p p.m., that is a z-fold increase rn sensitivity at the lower end of the range. The accuracy of the method is not significantly different from that of the orrginal

Anal. Cham. Ada, 39 (1967) 525-528

528 SHORT COMMUNICATKONS

method, although the reproducibihty IS appreciably better This compares very favourably with the spectrophotometric procedure for sub-mrcrogram amounts of sulphate described by JONES AXD LETHA~I 4 The complexity and number of reagent solutions are reduced to a mmlmum, and only one solution of the protective collard

005 010 015 020 025 000 003 010 015 020

Mg ot sulphote In IOml test solution

025

l3g 2 Ncphclomctrlc dctcrmmatlon of sulphatc -Mcasurcmcnts mntlc 30 mm nftcr prcaprtatlon . . Mcilvurcmcnts mxlc 2 clays nftcr prccqxtatton on bnmc suspensions

which has a long storage life is used. None of the reagent solutions possesses ltght- scattering properties which may change from day to day, and measurements arc reproducible at least for as long as the same solution of gum ghattl is in use. An im- portant practical advantage is that nephelomctrlc measurements need not be made at a particular time after formation of the suspension. this IS useful when a number of samples has to be analysed, and creates less &ram on the operator to maintain the exact experimental conditions with respect to the time factor.

The effect of other anions on the experimental procedure has not been mvesti- gated Thts should be no more pronouncecl than m the original method, so that moder- ate amounts of fluoride ion (up to 25 p.p m ) could be tolerated The procedures for the treatment of acuhc samples and for the removal of the phosphate ion should be duectly applrcable to the present method

Department of Chemistry, J. M. MARTIN

The Univevszty, W. I. STEPHEN

P 0. Box No. 363, Birntznglram 15 (Great Bvatatn)

I J M MARTIN, M SC 2 /resrs, Umvcrsityof Bu-mmgham, rgGG 2 I< Cl HUTTON, S. A SALAn! AND w I. STEPHEN, J Ckent Sot , A (1966) 1573 3 J M MARTIN AND W. I STEPHEN, Anal Clttttt AGCR, 39 (1967) 175 4 A S JONES AND D S. LZTHAM, Annlyrl, 81 (rggG) 15

(Received May 3xst, 1967)

Anal Chaws Ada, 39 (x967) 525-528