Chromic oxide (Cr2O3) is the most com-monly used index substance in digestibilitydeterminations. However, a subcommitteeof the Animal Nutrition Research Councilfound that there was poor agreementbetween laboratories in the determination ofCr2O3 and indicated the need for improve-ment in the analytical procedures (Carew19'73).

From the existing methods (Hill andAnderson 1958; Brisson 1956; Czarnocki etal. 1961) we have developed a procedure(method A) which improves accuracy andallows an output of at least 40 determinationsper day. In addition, two other methods (Band C) which were minor modifications ofmethod A were also studied in order to testthe advantages or disadvantages of takingspectrophotometric readings at 375 nm as insodium fusion methods or at 350 nm as

recommended by Czarnocki et al. (1961).In method A, samples of feed or feces

containing 20-60 mg Cr2O3 were weighedinto 30-ml pyrex beakers and ashed

overnight in a muffle furnace at 450'C. Aftercooling, 15.0 ml of a digestion mixture (10 gof sodium molybdate dihydrate dissolved in500 ml of a 150: 150:200 mixture of distilledwater-concentrated sulfuric acid-707o per-chloric acid) was added to each sample andheated on a hot plate (surface temperature upto 300'C) until a yellowish or reddish colordeveloped. The sdmples were heated for a

further 10-15 min, removed from the hotplate and allowed to cool. The digests werethen quantitatively transferred to 200-mlvolumetric flasks with distilled water andmade to volume. Approximately l0 ml of thediluted digest was poured into a tube(disposable polystyrene culture tube (17 x

Can. J. Anim. Sci. 59: 631-63 (Sept. f979)

NorES 631

AN IMPROVED PROCEDURE FOR TIM DETERMINATIONOF CHROMIC OXIDE IN FEED AND FECES

A spectrophotometric procedure was developed using ashing at 450"C followed byacid digeslion in beakers and reading the diluted digests at 440 nm. Errors were

minimiied by use of blanks and maintenance of a constant acid concentlation in the

diluted digests.

100 mm) with polYethYlene cap) and

centrifuged for 5 min at 700 g. The opticaldensity (OD) was measured in a l-cmcuvette vs. distilled water at 440 nm.

In method B a 5-ml aliquot from the

200-ml dilution of method A was transferredto a 100-ml volumetric flask. Approximately40 ml of 2 N NaOH was added and made tovolume with distilled water. After centrifu-gation the OD vs. distilled water was read at

375 nm. In method C, a 10-ml aliquot fromthe 200-ml dilution of method A was

transferred to a 100-ml volumetric flask and

made to volume with distilled water. Aftercentrifugation the OD vs. distilled water was

read at 350 nm.Standard curves were prepared by weigh-

ing several 5- to 60-mg portions of pure

CrzOs and carrying them through each

method. The OD was Plotted against

milligrams of CrzOs. Blank correctioncurves were prepared by weighing severalportions of feed or feces samples containingno Cr2O3 and carrying them through each

method. The OD was plotted against gramsample.

To determine the amount of CrzOe in a

sample, the equations OD" : agtb for the

standards and ODn : azw *b for the blankswere solved where OD, : OD of the sample(or standard) vs. distilled water, x:mgCr2O3, w:gram sample, ar : slope of the

standard curvo, ?z : sloPe of the blankcurve, b : intercept (the same value for boththe standard and blank curves) and ODo :OD of the blank vs . distilled water. Thus the

corrected sample OD was OD" - ODb 'Therefore for a given samPle

x:((ODs-azw -2b)lay)mg Cr2O3 per gram samPle.

The disestion mixture oxidizes Cr2O3 to

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632 CANADIAN JOURNAL OF ANIMAL SCIENCE

the soluble CrO3. In solution, CrO3 is knownas chromic acid (CrO3 ' H2O or FlCrOa). Thelatter readily polymerizes by loss of water toform dichromic acid, trichromic acid andhigher polymers (Udy 1956). The effect ofacid concentration on the OD at 440 nm of a0.25-mglml solution of sodium dichromatewas tested in the range of 2.4 to 4.8 mlsulfuric acid per 200 ml and 1.6 to 6.4 mlperchloric acid per 200 ml. The sulfuric acidcaused decreasing OD readings of 0.010 ODunits/ml while perchloric acid causeddecreasing OD readings of 0.001 ODunits/ml. The greater effect of sulfuric acidmust be a result of its greater dehydratingpower causing the equilibrium to shifttowards the higher polymers which do nothave absorption bands at the samewavelength as the dichromate ion. Due tothis equilibrium condition it was importantto keep the acid concentration of thedilutions for methods A and C as constant aspossible. The temperature of the digest at theend of the digestion period was 200'C whichis the temperature of the constant boilingazeotrope of perchloric acid. Since theboiling point of sulfuric acid is 290'C, littlesulfuric acid would be lost in the absence oforganic matter. Small variations in volumeofthe digest at the end ofthe digestion periodwere the result of differential rates of loss ofthe perchloric azeotrope but as perchloricacid concentration had a minimal effect onOD little error would be introduced.

After digestion and dilution of feed andfeces samples, an insoluble residue waspresent which had to be eliminated in orderto obtain meaningful OD values. Otherworkers (Hill and Anderson 1958) allowedan overnight settling period but in the presentstudy this was found inadequate as centrifu-gation resulted in OD values as much as 57olower for some samples. In these samples avery fine suspension was visible after sittingovernight. When making basic dilutions(method B) the residue dissolved but aprecipitate tended to form later, especially infeed samples.

Table 1 gives the slopes and intercepts for

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NOTES 633

Table 2. Blank conection curves for chromic oxide determination in a feed and feces sample where OD : aw -lb and

lr : grams feed or fecestf

Feed

Method

b (intercept)a (slope)$

0 0 .006.00179 .00239 .00754(.00014)// (.00084) (.00079)

0.00158

(.00108)

0 .006.00182 .02241(.00060) (.00261)

tUsing a Beckman-DBG spectrophotometer with l-cm quartz cells.tFeed was a swine ration (107o crude fiber) with alfalfa as a supplementary protein source

$Mean of nine values.//Standard deviation of a.

the standard curves obtained from eachmethod. The slopes of the curves were notsignificantly affected by the addition of feedor feces but method A gave an appreciablylower standard deviation than the othermethods. The intercept became significantlylarger upon addition of feed or feces whichindicated the need for blanks.

Table 2 gives the slopes and intercepts forthe blank correction curves for feed andfeces. As the wavelength was decreasedfrom 440 nm (method A) to 350 nm (methodC) the slopes of the correction curvesincreased. Since the standard deviations forthese slopes were quite large (i.e. there wasconsiderable variability in the blanks) itwould appear desirable to use the methodthat had the least slope for the correctioncurve in order to minimize the correction tothe sample OD. Clearly then, method A wasthe preferable method because it had theslope with the smallest standard deviation(Table 1), gave the correction curves withthe smallest slopes (Table 2) and had theadded advantage of requiring the fewestprocedural manipulations. Some furtheradvantages over other procedures were:ashing and digestion in a single smallcontainer; elimination of a predigestion stepwith nitric acid; use of volumetric flasksrather than self-calibrated Kjeldahl flasks;elimination of the need for micro-Kjeldahldigestion units; the number of samples thatcould be digested was only limited by thesize of the hot plate orthe number of samplesthe analyst could safely look after at one

time; and the removal of the insolublesuspension by centrifugation.

The equation for calculation of mg Cr2O3per gram sample allowed correction of thesample OD for any given weight of samplefrom the blank correction curve. This type ofcorrection was most convenient, as thesample size was the only parameter thatcould be adjusted to bring the OD into theproper analytical range. In our laboratoryover one thousand ashings and digestionshave been carried out using method Awithout any problems. The digestionmixture boils gently, if at all, and does notbump. The elimination of organic materialby ashing removes some of the dangers ofusing perchloric acid but the analyst mustkeep in mind that if nearly all the perchloricazeotrope should evaporate there is a dangerof explosion due to anhydrous perchloricacid (Steere 1967).

BRISSON, G. J. 1956. On the routinedetermination of chromic oxide in feces. Can. J.

Agric. Sci. 36: 2lO-212.CAREW, L. B. 197 3. Establishing standardizedprocedures for metabolizable energy determina-tions. Feedstuffs March 19. pp.25-26.CZARNOCKI. J.. SIBBALD.I. R. andEVANS.E. V. 1961. The determination of chromic oxidein samples of feed and excreta by acid digestionand spectrophotometry. Can. J. Anim. Sci. 41:16',7-1'79.HILL, F. W. and ANDERSON, D. L. 1958.Comparison of metabolizable energy and produc-tive energy determinations with growing chicks.J. Nutr. 64: 587-603.

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634 CANADIAN JOURNAL OF ANIMAL SCIENCE

STEERE, N. V. 1967. Handbook of laboratorysafety. The Chemical Rubber Co., Cleveland,Ohio. pp. 205-216.UDY, M. J. 1956. Chromium. Volume 1.

Chemistry of chromium and its compounds.Reinhold Publishing Corp., New York, N.Y.

T. W. FENTON and MIRJANA FENTON

Department of Animal Science, University ofAlberta. Edmonton. Alberta T6G 283.Received 5 Apr. 1979, accepted 6 June1979.

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