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Spectrophotometricdetermination of organiccarbon in soilI. Walinga a , M. Kithome a , I. Novozamsky a , V.J. G. Houba a & J. J. van der Lee aa Department of Soil Science and PlantNutrition, Wageningen Agricultural University,PO Box 8005, Wageningen, 6700 EC, TheNetherlandsPublished online: 11 Nov 2008.

To cite this article: I. Walinga , M. Kithome , I. Novozamsky , V. J. G. Houba &J. J. van der Lee (1992): Spectrophotometric determination of organic carbon insoil, Communications in Soil Science and Plant Analysis, 23:15-16, 1935-1944

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COMMUN. SOIL SCI. PLANT ANAL., 23 (15&16), 1935-1944 (1992)

SPECTROPHOTOMETRIC DETERMINATION OF ORGANICCARBON IN SOIL

I. Walinga, M. Kithome1, I. Novozamsky, V. J. G. Houba, andJ. J. van der Lee2

Wageningen Agricultural University, Department of Soil Science and PlantNutrition, PO Box 8005, 6700 EC Wageningen, The Netherlands

ABSTRACT: A detailed study was made of the well-known organic carbon

determination that is based on measuring the absorbance of the green chrom-

ium(III) complex generated when organic matter is oxidized by potassium

dichromate in acidic medium. The green chromic colour proved to be unstable,

turning slowly to a violet colour. It was also found that chromic sulphate exerts a

catalytic influence on the decomposition of excess dichromate. Based on this

work, a modified method was developed which is simple, rapid, and very accurate

when working under carefully controlled conditions.

INTRODUCTIONOrganic matter influences the physical and chemical properties of soils far out

of proportion to the small quantities present, and is thus one of the important

constituents of soils. Therefore, the determination of organic matter content is a

routine procedure in many soil testing laboratories.

Although soil organic matter has been and still is a subject of considerable

interest, most methods determine carbon that is a constituent of organic matter, or

rather the reducing power of the soil organic matter, such as the "Kurmies" or

"Walkley-Black" method. These methods must ultimately be calibrated by some

1. University of Nairobi, Faculty of Agriculture, Department of Soil Science, PO Box 30197,Nairobi, Kenya.

2. Corresponding author.

1935

Copyright © 1992 by Marcel Dekker, Inc.

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1936 WAL1NGA ET AL.

reference method based on carbon dioxide evolution (e.g. "Allison", or elemental

analysis).

The "Walkley-Black" method (2) has found widespread use because of its

speed, but the method suffers from low precision. The "Kurmies" method (1)

gives far more constant yields, that also approach 100%, but the method takes

more time and effort to complete. Both methods employ in the procedure a

titrimetric step, a technique that requires some experience to identify the end point.

The alternate colorimetric measurement may be more useful, but it does not always

give consistent results.

The fact that such an apparently established analysis method, on which a

sizable amount of research has been done for more than a half century, still can

give an unsatisfactory result, brought us to evaluate in some detail the extensive

literature on this subject. Our attention was focused particularly on the "Kurmies"

method (1). It was found that the key to the problem might lie in the fact that all

authors neglect the complex chromium chemistry on which the method is based.

Most striking was the observation that the green colour of the reduced dichromate

turns violet, thus causing an appreciable error in the absorbance measurements.

Moreover, an autocatalytic effect of trivalent chromium has been reported (4)

which results in high results at low organic matter levels.

Therefore, the "Kurmies" method (1) was studied a new with particular

emphasis on the above identified observations in order to establish the best

conditions for a rapid and confident method.

MATERIAL AND METHODS

Spectrophotometric absorption measurements were conducted using a

Shimadzu UV- 200 double beam spectrophotometer, to which a Kipp and Zonen

BD-8 flat-bed recorder was coupled. Spectral band width was adjusted at 1 to 2

nm.

Potentiometric titrations were conducted using an Orion model 701 digital pH

meter equipped with a platinum indicator electrode and a saturated calomel

reference electrode. A 25-mL piston burette was applied, with the solution

magnetically stirred.

For reference purposes, the "Allison" method as described by Houba et al. (5)

was applied.

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DETERMINATION OF CARBON IN SOIL 1937

Potassium Dichromate Solution (0.333 mol/L): 98.06 grams K2Cr2O7

were dissolved in a mixture of 300 mL water and 100 mL concentrated

H2SO4, and then diluted with distilled water to 1-litre in a volumetric flask.

I rondl ) Ammonium Sulphate Solution (0.22 mol/L): 86.24 grams

(NH4)2SO4-FeSO4-6H2O were dissolved in a mixture of 300 mL water and

100 mL of concentrated H2SO4, and then diluted with distilled water to 1-litre

in a volumetric flask.

Potassium Permanganate Solution (0.0200 mol/L): 6.32 grams KMnO4

were dissolved in 2 litres of hot distilled water. The mixture was boiled for 5

minutes, cooled and filtered through a glass frit. The resulting solution was

standardized against sodium oxalate.

Three substances of exact known carbon (C) content were used as standards,

sucrose (C12H22O11), potassium hydrogen phthalate (KHC8H4O4), and sodium

oxalate (Na2C2O4).

Soils; The soils used in this investigation were normal Dutch soils (with the

exception of soil number 4 which is Kenyan). They were selected on basis of their

organic carbon content, which ranged from 0.1 to 7.4% as per oven-dry soil

(Table 1). The same soils had previously been used in our Department for the

determination of various soil parameters, and as far as known, they contained no

appreciable carbonate content and no significant amounts of other possibly

interfering substances, such as chlorides or charcoal. Sub-samples of the soils

were air-dried and ground with a pestle and mortar to pass a 0.5-mra sieve.

Results are reported on an air-dry basis.

"Kurmies" Procedure: An amount of soil containing not more than 150 mg

of organic matter was precisely weighed, and then carefully transferred to a dry

250-mL volumetric flask. A 25-mL aliquot of 0.333 M potassium dichromate

(K2Cr2O7) was added and the flask swirled a few times during a period of ten

minutes to wet the soil completely. While cooling, 40 mL concentrated sulphuric

acid was added. The flask was heated in a boiling water bath for 1.5 hours, during

which period it was swirled gently every 15 minutes. Thereafter, the flask was

cooled in a sink and the volume made up to 250 mL with distilled water. After

mixing thoroughly, the soil suspensions were allowed to settle to the bottom of the

flask.

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1938 WAL1NGA ET AL.

Table 1. Soils used in the present study.

Number

123456789

1011

Type

claysandy claypeat soilred soilsea claysandy soilsea clayclay loamsandy soilsandy soilclay soil

%C

1.91.27.43.00.53.11.00.11.70.81.2

Remarks

from Kenya

"WalkleV'Black" Procedure: For comparison, the conventional "Walkley-

Black" method (2) was applied to all the soils using the titrimetric method to

measure the unexpended chromate.

"Allison" Procedure: As a reference, organic carbon was also determined by

the "Allison" method (3).

Potentiometric Titration: In all soils, organic carbon was determined by

means of titration either with 0.0200 M KMnCM or with 0.01667 M K2Cr2O7,

followed potentiometrically.

Spectrophotometric Determination: Solutions containing chromic (Cr3*) ion

were obtained by oxidizing sodium oxalate with K2Cr2O7 as well as by dissolving

an amount of KCr(SO4)2-12H2O. The absorption spectra were scanned over the

range from 700 to 350 nm.

To verify adherence to Beer's Law, a standard series of Cr3"1" was generated

by oxidizing different amounts of sodium oxalate, and its absorbance measured at

a wavelength of 590 nm.

In case of the soil samples, the oxidized sample was centrifuged for 15 min at

1750 g, and then the transmittance of the clear soil-free solution measured at 590

nm. By comparing the sample's absorbance with that of the standard series, the

Cr3+ content of the sample, and thus the organic carbon content, was calculated.

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DETERMINATION OF CARBON IN SOIL 1939

RESULTS AND DISCUSSION

Optimization of Conditions

The conditions for an optimum determination of organic carbon, using the

oxidation methods involving concentrated sulphuric acid and aqueous dichromate

solutions, have been studied by a large number of authors. In spite of (or maybe

due to) these extensive investigations, there are many differences in the recom-

mended procedures.

One of these is the order of addition of the reagents. Some authors advocate

adding the sulphuric acid before the dichromate, however, without a really com-

pelling argument. In our study, both procedures were compared on standard

substances as well as on soils. No significant effect on the organic C recovery was

found using sucrose, sodium oxalate, and the soils identified in Table 1.

Another point of disagreement is the use of the titrant, either permanganate or

dichromate. In our study, both have been used for titrating sucrose and sodium

oxalate standards, and soils. No significant differences were found. From a

practical point of view, the dichromate solution is preferred because it is easier to

prepare and does not need standardization. Moreover, it may be prepared from the

oxidation solution.

When adopting the spectrophotometric measurement procedure, the choice of

the measuring wavelength is important because both the dichromate ion and the

chromic ion absorb in the visible region. However, at the absorption maximum of

Cr3* (590 nm), there is no absorption from Cr2O72-. In the past, some authors

used varying wavelengths for the measurement of Cr3+ (or even measured the

remaining dichromate concentration), which led to erroneous results (6,7). In our

study, 590 nm was the wavelength used.

Adherence to Beer's Law of the absorbance by the Cr3+ ion at 590 nm was

verified indirectly using organic C containing standard substances: sucrose

(C12H22O11); sodium oxalate (Na2C2O4); and potassium hydrogen phthalate

(KHC8H4O4), which generated known concentrations of Cr3* upon oxidation

with aqueous K2Cr2O7 in acidic medium. Adherence to Beer's Law of the

absorbance by the Cr3"1" ion at 590 nm was also verified directly by using pure

Cr3"1" generated from KCr(SO4)2-12H2O. In both cases, a linear relationship with

a highly signi- ficant correlation coefficient (r = 1.000) was found between Cr3+

concentration and absorbance (Table 2).

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1940 WALINGA ET AL.

Table 2. Adherance to Beer's Law of the absorbance by the Cr3"1" ion at 590 nm.

Substance

KCr(SO4)2-H2O

Sucrose, C12H22O11

Na-oxalte, Na2C2O4

KH-phthalate, KHC8H4O4

Regression Line

y = 0.02204 x

y = 0.01835 x +0.01387

y = 0.01996 x+0.01240

y = 0.01966 x +0.01123

CorrelationCoefficient

1.00

0.9996

1.000

1.000

In our investigation, three standard substances containing organic carbon were

compared: sucrose (C12H22O11), sodium oxalate (Na2C2O4), and potassium

hydrogen phthalate (KHC8H4O4). Using different amounts of each substance, it

was observed that recoveries very close to 100% were obtained with larger

amounts. When applying small amounts, however, the recoveries were raised

above the theoretical 100%. Additional experiments confirmed this initial

observation, and it was found that increasing the ratio of dichromate to standard

substance increased the recovery. As a tentative explanation, it was assumed that

dichromate is subject to autodecomposition which is proportional to the amount of

excess dichromate remaining after the reaction. However, the zero standard

solutions, though having the largest excess of dichromate, showed very little

effect due to decomposition. This was also found by Walkley (8). Therefore, it

was assumed that the reaction products might catalyze the auto-decomposition of

the dichromate.

Further experiments showed that chromium(III) sulphate influences the

autodecomposition of the.dichromate, the effect becoming less and less with

increasing amounts of chromium(III) sulphate as if a state of saturation were

reached. These findings are in agreement with the work of Snethlage (4,9), who

has shown that the kinetics of the decomposition of chromic acid are not as simple

as may initially be expected, but are rather complex, and is best represented by

assuming that two reactions, one mono-molecular and the other bi-molecular,

occur simultaneously.

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DETERMINATION OF CARBON IN SOIL 1941

Table 3. Effect of period of standing on absorbance readings for standardsubstances.

Standard Number

01234

01234

01234

Dayl Day 2

KCr(SO4)2'

0.0000.0680.1330.2000.270

0.0000.1000.2000.2970.400

0.0110.1130.2110.3080.410

0.0000.0690.1380.2070.274

Day 3 Day 22

12H2O (not heated)

0.0000.0700.1380.2080.278

0.0000.0780.1530.2320.311

KCr(SO4)2-12H2O (heated)

0.0000.0980.1940.2900.389

0.0000.0930.1900.2820.378

0.0000.0800.1600.2390.320

Na2C2O4 (heated)

0.0110.1120.2090.3020.400

0.0110.1100.2020.2950.390

0.0110.0950.1730.2520.332

Day 45

0.0010.0800.1600.2380.317

0.0000.0800.1600.2370.317

0.0100.0900.1700.2470.323

In the course of our experimental work, small differences were observed in

results obtained on different days. This raised a question about the stability of the

green-coloured chromic ion. Therefore, absorbance measurements were done on

heated (water bath) and non-heated solutions of KCr(SO4)2-12H2O, and

compared with a known procedure (oxidation of oxalate by dichromate). The

results showed significant differences in absorbance values between the heated

and the non-heated KCr(SO4)2-12H2O series (Table 3). These differences were

expected, since the cold solution of KCr(SO4)2-12H2O is coloured violet, and

turns green on heating. However, on prolonged standing (up to 45 days), the

absorbance of the non-heated solutions increased, whereas the absorbance of the

heated solutions decreased. At the same time, it was observed that the non-heated

(violet) solutions slowly turned green, whereas the heated (green) solutions

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1942 WALINGA ET AL.

slowly turned violet. These observations were supported by the spectra measured

after 45 days, which were found to be virtually identical.

A search in the older literature (10,11) revealed that this phenomenon has been

known in solutions containing both sulphate and trivalent chromium, and is

explained by assuming an equilibrium of the reaction:

[Cr2CH2O)12]6+ + 3SO42- ^ *• [Cr2(H2O)6(SO4)3] + 6H2Oviolet green

As a practical consequence, it can be concluded that the determination of

organic carbon by this procedure should be standardized with respect to time and

temperature, and that the final absorption measurements be done on the same day

as the oxidation (or one day after) with fresh standard solutions.

Since potassium dichromate might decompose while heating, the optimum

period of heating was also investigated. The temperature chosen was 100'C

(boiling water bath), which appears to be low enough to avoid thermal decom-

position, yet high enough for complete and rapid reaction. For this purpose, soils

and standards were heated for 0.5 - 3 hours at 100*C. It turned out that heating

beyond 1 hour gave no worthwhile gain in oxidation yield. To be sure, 1.5 hours

was adopted as a part of the routine determination.

Application to Soils

The final spectrophotometric measurement result was compared with that

obtained by a titrimetric measurement on the soils described in Table 1 following

the same oxidation procedure. The linear relationship found is described by the

equation: y = 1.00656x - 0.013, with a correlation coefficient of 0.9999.

The proposed spectrophotometric method was also compared with the

heat-of-dilution method according to Walkley and Black (2), employing the

titrimetric technique as the final measurement procedure. As expected, the results

from the Walkley-Black method (2) were low and varied widely, with organic C

recoveries ranging from 60 to 90% as compared to results obtained when the

spectrophotometric method was used.

Finally, the proposed spectrophotometric method was compared with

Allison's reference method (3). The mean recovery for all soils listed in Table 1

was 98-99%, ranging for most soils from 93 to 104%.

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DETERMINATION OF CARBON IN SOIL 1943

Thus, our final conclusion is that the proposed spectrophotometric procedure

as we have described in the Appendix has proven to be a simple, rapid, economic,

and very accurate method for the determination of organic carbon in soils.

REFERENCES:

1. Kurmies, B. 1949. Determination of humus by the dichromate methodwithout potassium iodide. Z. Pflanzenern. Dueng. Bodenk. 44:121-125.

2. Walkley, A. and T.A. Black. 1934. An examination of the Degtjareff methodfor determining soil organic matter, and a proposed modification of thechromic acid titration method. Soil Sci. 37:29-38.

3. Allison, L.E. 1935. Organic matter determination by reduction of chromicacid. Soil Sci. 40:311-320.

4. Snethlage, H.C.S. 1936. On the catalytic influence of chromic sulphate on thespeed of decomposition of CrO3 by heat when dissolved in H2SO4 of varyingstrengths. Rec. Trav. Chim. Pays-Bas 55:874-880.

5. Houba, V.J.G., J.J. van der Lee, I. Novozamsky, and I. Walinga. 1989.Soil and Plant Analysis, a series of syllabi. Part 5. Soil Analysis Procedures.Department of Soil Science and Plant Nutrition, Wageningen AgriculturalUniversity, The Netherlands.

6. Graham, E.R. 1948. Determination of soil organic matter by means of aphotoelectruc colorimeter. Soil Sci. 65:181-183.

7. Carolan, R. 1948. Modification of Graham's method for determining soilorganic matter by colorimetric analysis. Soil Sci. 66:241-247.

8. Walkley, A. 1947. A critical examination of a rapid method for deter-mining organic carbon in soils - effect of variations in digestion condi-tions and of inorganic soil constituents. Soil Sci. 63:251-264.

9. Snethlage, H.C.S. 1936. On the speed of decomposition of chromic acid insolutions of sulphur trioxide in water by heat and on the relation between thisreaction and the oxidising power. Rec. Trav. Chim. Pays Bas 55:712-722.

10. Holleman, A.F., E.H. Buchner, and E.H. Wiebenga. 1953. Leerboek derAnorganische Chemie. Wolters, Groningen, The Netherlands (in Dutch).

11. Udy, M.J. 1965. Chromium (Vol. 1). Chemitsry of Chromium Com-pounds. American Chemical Society. Monograph Series. Reinhold, NewYork, NY.

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APPENDIX:

Proposed Spectrophotometric Procedure for Routine Determinationof Organic Carbon Contents of Soils

Reagents:

Sulphuric acid: c(H2SCto) = 18 mol/L.Potassium dichromate solution (0.333 mol/L): Dissolve 98.06 grams of

K2Cr2O7 in a mixture of 300 ml water and 100 mL concentrated H2SO4.Dilute with distilled water to 1-litre in a volumetric flask.

Sodium oxalate (Na2C2O4).

Oxidation Procedure:

Weigh precisely an amount of soil, containing organic matter in the range of 80 -150 mg, and transfer it carefully to a dry 250-mL volumetric flask. Weighaccurately 1005 mg of sodium oxalate and transfer this also carefully to a dry250-mL volumetric flask. Include also two blanks. Add to each flask 25.0 mL0.333 M potassium dichromate solution. Swirl a few times during the next fiveminutes. Then put the flasks in a sink and add very carefully, while cooling, 40mL concentrated sulphuric acid (use a measuring cylinder), and swirl the flasksgently while adding the acid. Thereafter, put the flasks in a boiling water bath for1.5 hours, swirling gently every 15 minutes. Then cool in the sink and make up tovolume with distilled water. Mix the contents of the flask thoroughly and let thesoil settle.

Preparation of Soil Samples for Measurement:

Pour a portion of the soil suspension from the 250-mL volumetric flask into aplastic centrifuge tube. Centrifuge for 10 minutes at 1750 g. Decant the clearsupernatant into glass test tubes.

Preparation of Standard Series:

Pipette from the 250-mL flask containing sodium oxalate standard, respectivevolumes of 0, 25.0, 50.0, 75.0 and 100.0 mL solution into 100-mL volumetricflasks. Make up to the mark with die blank and mix. Pour portions of therespective, standard solutions into glass test tubes. This standard seriescorresponds to 0,5,10,15, and 20 mmol Cr3"1" per litre of solution.

Spectronhotometric Measurements:

Measure the absorbance of the standard solutions and the sample supernatants in a4-cm cuvette at a wavelength of 590 nm within 24 hours after oxidation. Plot theabsorbance values for the standard solutions against the calculated Cr3+ concen-trations, and read the Cr3+ concentrations for the soil sample supernatants.Calculate the percentage organic carbon in the soil samples by multiplying the Cr3"1"concentrations found by 0.2250/w, where w is the weight of the oven-dry soilsample.

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