ON THE COLORIMETRIC DETERMINATION OF CREATININE

BY THE JAFFE REACTION*

BY ROY W. BONSNES AND HERTHA H. TAUSSKY
(From the Department of Obstetrics and Gynecology, Cornell University Medicat College
and the New York Hospital, New York)

(Received for publication, February 5, 1945)

Creatinine has been determined calorimetrically by the Jaffe reaction

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(1) for many years. Yet there are no published data which indicate why
the particular concentrations of reagents are used. Folin (2) in the first
paper which describes the determination of creatinine in urine by this
reaction simply states: “Als Resultat vielfacher Untersuehungen beztiglich
der Reaktion des Kreatinins mit alkalischer Pikrinsiiurelosung hat sich
folgendes ergeben . . .” and Shaffer (3) similarly states, “As a result of
several hundreds of determinations under different conditions we con-
cluded that the optimum conditions are . . .” No further details are given
in either instance. Nor does subsequent work indicate what trend might
be expected in the color developed if the concentration of picric acid or
alkali, the volume, etc., are altered. The following experiments were
undertaken, therefore, to obtain such information with particular reference
t,o the determination of creatinine in very dilute solution.

EXPERIMENTAL

The color densities were measured throughout with a Klett-Summerson

photoelectric calorimeter (4). Filter 54 obtained with the instrument was
used. By actual measurement in a Beckman spectrophotometer (5)
t,his filter exhibits a maximum absorption at about 525 rnp. This is
about the best wave-length at which to measure this color since picric
acid absorbs strongly above 510 rnp and the color due to creatinine does
not absorb much light above 540 rnp.
The concentrations of creatinine used were such that the readings ob-
tained would fall within the most sensitive range of the photoelectric
calorimeter. The volumes of the sample of the creatinine solution and
t,he reagents were selected arbitrarily so that the determination could be
carried out in a Klett-Summerson calorimeter tube or in Pyrex or other
test-tubes which could be adapted to the instrument, and so that ordi-
nary volumetric transfer pipettes may be used.
The standard creatinine solutions were prepared fresh daily by the
* This study was aided by a grant from the John and Mary R. Markle Foundation.
581
582 DETERMIXaTION OF CREATININE

proper dilution of a stock standard containing 0.5 mg. of creatinine per ml.
of 0.1 M HCl.
The picric acid solutions were prepared by diluting the saturated stock
solution. These dilutions were carried out at 25” =t 1”.
The sodium hydroxide was similarly prepared by diluting a 50 per cent
solution by weight to either 10 or 20 per cent. These solutions were then
diluted to the desired strength,
E$ect of Varying Concentrations of Pick Acid and Sodium Hydroxide
upon Color Developed-The first experiments were carried out at a constant

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CONCENTRATlON OF SODIUM HYDROXIDE AWED - %

FIG. 1. Color developed with 20 y of creatinine as a function of the concentration of

sodium hydroxide added at the following concentrations of 2 ml. of picric acid: Line
1, saturated picric acid; Lines 2,3, and 4, one-half, one-third, and one-fifth saturated
picric acid respectively; Line 5, average of values.

creatinine concentration as follows: 2 ml. of the picric acid solution and

1 ml. of the sodium hydroxide solution to be studied were added to 2 ml. of
a solution containing 20 y of creatinine. The mixture was allowed to
stand until the maximum color had developed. A blank determination
in which water was used in place of the creatinine solution was prepared
simultaneously in all cases. The color developed in both the standard
solution and in the reagent blank \vas determined. The zero adjustment
of the calorimeter was made with a tube containing distilled water.
The total color developed with different concentrations of picric acid
at a constant creatinine concentration is plotted in Fig. 1 as a fun&ion
of the concentration of the 1 ml. of sodium hydroxide added. The color
 R. W. BONSNES AND H. H. TAUSSKY 583

obtained (as read with the calorimeter adjusted to zero with distilled water)
with 2 ml. of saturated picric acid is described by Line 1. Similarly, the
color obtained with 2 ml. of one-half saturated picric acid is shown by
Line 2; that with 2 ml. of one-third saturated picric acid by Line 3; and
that with 2 ml. of one-fifth saturated picric acid by Line 4. These data
seem to indicate that the color is a function of both the concentration of
the picric acid and of the alkali. However, if the values obtained at the
same concentration of creatinine and of added alkali but at different con-
centrations of picric acid are corrected for their own reagent blanks, the
resulting values are essentially identical. The values so obtained in these

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160
c

2 4 16 20 22 24
&lo b4 0oN%I(&I0N '*,s:."

FIG. 2. Total color developed with 30 y of creatinine as a function of the picric acid
concentration. Total volume 5 ml.; 1 ml. of 3 per cent sodium hydroxide used in all
cases.

experiments have been averaged and are plotted as Line 5 in Fig. 1. The
vertical lines indicate the range of values.
These data illustrate that the color obtained in the Jaffe reaction is
independent of the concentration of picric acid, at least over a fairly wide
range, and that it is dependent upon the concentration of the alkali
employed. It is also apparent that as the concentration of the sodium
hydroxide is increased the fraction of the total color attributable to cre-
atinine is decreased, until at the higher concentrations of alkali nearly all
t,he color is from the reagent blank.
Additional data are presented in Fig. 2 which show that the total color
minus the reagent blank is independent, over a wide range, of the picric
acid concentration at a constant alkali concentration. Since this graph
includes data from experiments in which 3 ml. of creatinine solution and
584 DETERMINATION OF CREATININE

1 ml. of picric acid were used as well as data from the experiments in which
2 ml. of picric acid were used, the picric acid concentrations are expressed
in terms of moles of picric acid.
Rate of Color Development-The rate at which the color develops is
inversely proportional to the concentration of both the picric ac?d and the
sodium hydroxide; this is shown in Fig. 3.
Proportionality of Color Developed to Coneentration of Creatdnine-The
relationship between the color developed and the concentration of cre-
atinine was studied with several different concentrations of picric acid
and alkali. As could be predicted from the previous data, the maximum
color was obtained with 1 per cent alkali at all concentrations of creatinine.

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FIG. 3. The rate of color development as a function of the concentration of the

sodium hydroxide added. 20 y of creatinine. Line 1, saturated picric acid added;
Line 2, one-third saturated picric acid added; Line 3, one-fifth saturat,ed picric acid
added.

Progressively less color was also obt,ained at all concentrat,ions of creatinine

as more concentrated alkali was used. These data are shown in Fig. 4
where Lines 1, 2, 3, and 4 were drawn to act as visual guides to the data
obtained at 10 to 50 y of creatinine when the alkali concentration (in the
1 ml. of sodium hydroxide added) was 1,2,3, and 4 per cent respectively.
The concentration of picric acid used again seemsto make little difference,
although we have not studied this factor at all concentrations of alkali.
However, the dat,a indicated by Line 3 are the average results obtained
in experiments in which 1 ml. of five different concentrations (saturated,
one-half, one-third, one-fift,h, and one-tenth saturat,ed) of picric acid were
used.
It is also apparent from these data that the color developed is not pro-
 R. W. BONSNES AND H. H. T.4USSKY 585

portional to the concentration of creatinine when the total volume is 5 ml.

To substantiate t.his qualitative observation we have calculated the ap-
proximate molar extinction coefficients from the mean calorimeter readings
obtained at creatinine concentrations of from 1 to 10 and at 20, 30, 40,
and 50 y per 5 ml. with the creatinine method to be described below. These
molar extinction coefficients are shown in Table I. The Lambert-Beer
law seems to apply at creatinine concentrations below 10 y per 5 ml. (Fig. 4).

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FIG. 4. Color developed as a function of the creatinine concentration. Lines .I,

2,3, and 4 show color obtained with 1,2,3, and 4 per cent NaOI-I, respectively; Lines
5 and 6, result of diluting solutions from Line 3 to 2 and 5 times their original volumes
with water; Line 7, t.he Folin-Wu method.

Above this concentration, however, the calculated extinction coefficients

show a gradual decrease. The Lamhert-Beer law, t,herefore, does not
apply at these concentrations.
However, the proportionality between the color and the concentration
can seemingly be improved by diluting the reaction mixture after the color
has developed or by allowing the color to develop in a more dilute solution.
For example, if the solution obtained in the creatinine method described
below is diluted with an equal volume of water (i.e., to a total volume of
10 ml.), the proportionality is improved somewhat. The color developed
before dilution, in terms of calorimeter readings, is about the same as t,hat
586 DETERMINATION OF CREATININE

indicated by Line 3, Fig. 4. The calorimeter readings obtained after the

solution is diluted with an equal volume of water are indicated by Line 5.
If this reaction mixture is diluted still further to a total volume of 25 ml.,
the apparent proportionality is still better (Line 6) and the calculated
molar extinction coefficients are approximately constant (Table I).
TABLE I
Molar Extinction Coe.ficients (Approximate) *
-
Molar extinction coefficients, X 101
Creatinine Creatinine
per volume per liter I Diluted
analyzed Undiluted - Folin method
1:l 1:s
-

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Y micromoles
1 1.8 5.65
2 3.5 5.65
3 5.3 6.00
4 7.1 5.95
5 8.9 5.65
6 10.6 5.65
7 12.4 5.50
8 14.2 5.50
9 16.9 5.40
10 17.7t 5.65 6.65 7.90 5.45
20 35.4 5.35 6.20 7.35 5.80
30 53.1 5.05 6.10 7.40 5.40
40 70.8 4.95 6.00 7.50 5.40
50 88.5 4.75 5.85 7.25 5.30
- -
* The calorimeter reading on the Klett-Summerson instrument multiplied by 2
X 10-s gives a value which approximates D = (2 - log G). If one assumes that the
cylindrical tube used in this instrument is approximately equivalent to a solution
thickness of 1 cm., D becomes approximately equal to the extinction, Ic, or more
properly k’, where the prime indicates the inclusion of unknown correction factors.
This L’ divided by the concentration of creatinine in micromoles per liter is the
approximate molar extinction coefficient described.
t This and the subsequent figures in this column must be divided by 6 for the
Folin method.

Also, when the color is allowed t,o develop in a more dilute solution, as
in the Folin-Wu method for blood (6), the proportionality between the
color and the concentration is good (Line 7, Fig. 4) and the molar extinction
coefficients are approximately constant (Table I). It is, however, im-
mediately apparent that there is a decrease in both total color and in sensi-
tivity when the Lambert-Beer law seems to apply.
Application to Determination of Creatinine and Creatine in Diluted Urine
and in Blood Filtrates-We have selected for our own use one procedure from
the many possibilities indicated above. The reasons which motivated
 H. \V. BONSNES AKD H. H. TACSSKY 587

this particular selection are presented in the discussion. The values

obtained by this procedure have been compared with the values
obtained by the Folin methods for the determination of creatinine in
urine (7) and in blood filtrates (S).l
The procedure employed by us follolvs: 3 ml. of the unknown solution
(diluted urine or blood filtrate) containing up to 50 y of ereatinine are
used. To this solution is added 1 ml. of 0.04 III picric acid follo\ved by 1 ml.
of standardized 0.75 N (3 per cent) sodium hydroxide. The color is allowed
to develop for 15 minutes. It is stable for at least another 30 minutes.
The creatinine content of the solution is calculat.ed from a standard curve
obtained by averaging the results obtained in six different experiments

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at 1 to 10 and at 20, 30, 40, and 50 y of creatinine.
Added creatinine may be recovered quant,itat,ively from the urine. Some
typical results are shown in Table II.

Creatinine added Creatinine found Creatinine recovered

__-__ ___-. _---_
Y Y *er CLm
0 9.5
5 14.4 98
10 19.8 103
20 29.7 101
30 40.0 102

In a series of thirty urines analyzed for creatinine by this procedure

the average deviation from the values obtained by the Folin method (7)
was -0.4 per cent,, with a maximum range of f10 per cent. However,
the majority of the determinations deviated less than =t5 per cent from
the value obtained with the Folin method.
Ten whole blood filtrates from patients who might be expected to have
normal blood creatininc levels were analyzed by both methods. These
bloods contained an average of 1.5 mg. per cent (range 1.4 to 1.7) of creati-
nine by the Folin method and 1.4 mg. per cent (range 1.2 to 1.5) by the
procedure described above.
Creatine is 80 per cent converted to creatinine at these concentrations
by heating the creatine-cont.aining solution with picric acid in a boiling
water bath for 45 minutes. This percentage conversion is of the same order
of magnitude as that obtained in the Folin autoclave method. These
observations confirm those of Albanesc and Wnngerin (8) who found tha,t
1 The color density in both these methods was measured with the photo-
electric calorimeter. Standard curves were also used in both instances.
588 DETERMINSTION OF CREATININE

creatine is not converted quantitatively to creatinine by the Folin auto-

clave method.
The percentage conversion is fairly constant and reaches a maximum
value at about 80 per cent conversion when creatine is heated with picric
acid in a boiling water bath, as described below. No creatinine is de-
stroyed by this procedure. It seems, therefore, that for many purposes
the procedure described below might serve as a method for the determina-
tion of creatine in dilute solution in that the procedure does not require an
autoclave and requires no attention if a constant level water bath is a-
vailable.
Creatine in urine is determined by the procedure described above for
creatinine after heating 3 ml. of the creatine-containing solution with 1 ml.

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of 0..04 M picric acid in an unstoppered tube graduated at 5 ml. for 45
minutes in a vigorously boiling water bath.’ After the solution has cooled
to room temperature, 1 ml. of alkali is added. The volume is then adjusted
to 5 ml. with water. To calculate creatine as creatinine the value ob-
tained by difference in the usual manner is multiplied by 1.25. The values
for total creatinine obtained by this method upon dilut,ed urine were
essentially the same as those obtained by the Folin autoclave method.
If a quantitative conversion is required, the method described by Peters
(9) might be applied, or the method described by Benedict (10) can be
used before the urine is diluted. These procedures, however, have not
been extensively studied by us. No attempt has been made to determine
creatine in blood filtrates.
It appears, therefore, that the method described above for the determina-
tion of creatinine and creatine yields essentially the same values for cre-
atinine and creatine in dilut,ed urine and for creatinine in blood filtrates
as do the Folin methods cited for the determination of these substances.
This method has now been in use in our laboratory for the past several
months for the routine determination of creatinine.

DISCUSSION

The ideal conditions for the determination of any substance by a color-

imetric procedure are those which yield a color which is directly pro-
portional to the concentration of the substance to be determined. The
data presented above indicate that this ideal cannot be reached with this
particular reaction regardless of the conditions chosen. However, one
may approximate this ideal condition over the range of from 10 to 50 y
of creatinine if procedures such as are described above (i.e. dilution) or
the Folin blood method is used. But, it is doubtful whether dilution of

2 The water bath must be boiling vigorously.

 II. W. DONSNES ASD 11. H. ThUSSKY 589

the reaction mixture actually improves the proportionality. For when

these reaction mixtures are diluted 5 times with water, the slope of the
line describing the color as a function of the creatinine concentration is
decreased to such an extent that the variations which occur probably fall
within the resolving power of t.he instrument, since the measurements arc
made, perforce, at a constant solution thickness. So what appears to be
an improvement in proportionality may be merely an artifact. Further-
more, when such procedures are followed, there is a great loss in sensitivity.
For instance, when the Folin method is used, a difference of 40 y of cre-
atinine results in a change of 31 scale divisions or 0.78 scale division per
microgram. On the other hand, when the procedure described herein
is used (assuming a linear relationship), a similar change in creatinine con-

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centration results in a change in 161 scale divisions or approximately 4.0
scale divisions per microgram. It would appear, therefore, that if urines
are diluted sufficiently so that the creatinine can be determined by the
Folin blood method, as suggested by Peters (9), the low sensitivity of the
method might lead to a relatively large error in the result. However,
when blood filtrates are analyzed, one must choose between a method with
a low sen&ivity or a procedure which has greater sensitivity but which
gives lower calorimeter readings.3
The assumption which has been made above that a linear relationship
holds in the method described is obviously fallacious. However, it is of
interest to calculate the actual percentage error which results if this as-
sumption is made. For these calculations the mean values obtained at
10,20,30,40, and 50 y of creatinine are compared with the values obtained
from the best straight lint which fits the data as calculated by the method
of least squares. The values which are obtained from the straight line
are then 6 per cent high at 10 y, 2 per cent low at 20 y, 1.5 per cent low at
30 y, 1.1 per cent low at 40 y, and 1.4 per cent high at 50 y. If the mean
values are compared with the values obtained from the best straight line
which fits the data between 10 and 40 y, the values obtained from the
straight line are 2 per cent high at 10 y, 2 per cent low at 20 y, 0.75 per cent
low at 30 y, and 0.58 per cent high at 40 y. It is possible, therefore, to use
a formula4 to calculate the creatinine concentration without introducing
a very great error, even though a linear relationship is not found to exist.
3 Similar results have been obtained when the Evelyn photoelectric calorimeter
(11) has been used. To use this calorimeter all volumes are doubled. Filter 520 is
used at Aperture 6.
4 The mean values, the equations, and the formulas which may be derived from
them are not presented because they are relative and not absolute values. It is not
expected that identical numerical results can be obtained by others because of the
variations which exist in filters.
590 DETERMIXATION OF CREATININE

However, one must be cognizant of the fact that errors are involved in
such a procedure.
The particular method which w-e have outlined above was selected
because it fits in best with our present analytical routines. The 0.04 M
picric acid and the 0.75 N (3 per cent) alkali are used because the color
develops sufficiently rapidly at these concentrations of picric acid and
alkali and the blank color is still at a minimum. At the concentration of
alkali at which the maximum color develops the rate of color development
is too slow even with saturated picrjc acid for our purposes.
It is possible that at the concentration of alkali used more color may
be produced from non-creatinine substances such as glucose t.han at

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lower concentrations of alkali. We have tested this point experimentally
wit.1~ respect to glucose. The color remains constant for 30 minutes after
the maximum color has been attained with Folin-Wu blood filtrates and
in urines to which 1 per cent glucose has been added. There may be
an increase in color after 40 minutes. There is no difference in the cre-
atinine values obtained in urine before and after the addition of glucose.
The presence of glucose, therefore, does not seem to int.erferc with the
determination.
Many questions concerning the mechanism of the Jaffe reaction are
raised by the data. We feel, hoiyever, that there is no justification for a
discussion of these points until more data are available.

SUMMARY

The effect of varying the concentration of picric acid and of alkali upon
the color formed in the Jaffe reaction used for the determination of creat-
inine has been studied in solutions which c0ntai.n 1 to 50 y of creatinine per
5 ml. The amount of colored creat,inine compound formed as measured
by the color developed is independent of the concentration of the picric
acid added above a low limiting concentration of picnic acid. The amount
of colored creatinine compound formed is greatest at low concentrations
of alkali and progressively decreases as the concentration of alkali is in-
creased. The rate at which the color develops is inversely proportional
to the concentration of both the picric acid and the sodium hydroxide.
The color formed is not, directly proportional to the concentration of
we&nine except at very low concentrations in the 5 ml. of solution em-
ployed. By diluting this solution the linearity seems to improve. This
apparent improvement, however, is considered an artifact.
A procedure for the determination of creatinine in blood filtrates and in
diluted urine is described which is based upon the data obt,ained.
 R. TV. BOKSNES AND H. H. ThUSSKY

BIBLIOGRAPHY

1. Jaffe, X, Z. physiol. Chen~., 10,391 (1886).

2. Folin, O., Z. physiol. Chem., 41,223 (1964).
3. Shaffer, P. A., J. Biol. Chem., 18,525 (1914).
4. Summerson, W. H., J. Biol. Chem., 130,149 (1939).
5. Cary, II. H., and Beckman, A. O., J. Optical Sot. An&., 31,682 (1941).
6. Folin, O., and Wu, H., J. Biol. Chem., 38,81 (1919).
7. Folin, O., J. BioZ. Chem., 1’7,469 (1914).
8. Albanese, A. A., and Wangerin, D. M., Science, 199, 58 (1944).
9. Peters, J. H., J. Biol. Chem., 146, 179 (1942).
10. Benedict, S. R.,J. Biol. Chem.,18,191 (1914).
11. Evelyn, K. A., J. BioZ. Chem., 116.63 (1936).

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 ON THE COLORIMETRIC
DETERMINATION OF CREATININE BY
THE JAFFE REACTION
Roy W. Bonsnes and Hertha H. Taussky
J. Biol. Chem. 1945, 158:581-591.

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