A MODIFIED

PHOTOMETRIC
NINHYDRIN
METHOD
THE ANALYSIS
OF AMINO
AND IMINO
ACIDS
BY

(From the Department

WALTER

TROLL*

of Chemistry,

New

AND

R. KEITH

York University
York)

FOR

CANNAN
College of Medicine,

New

York, New

(Received for publication,

June 18, 1952)

* Present address, May Institute

Association, Cincinnati, Ohio.

for Medical
803

Research of the Jewish Hospital

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Primary amino acids may be quantitatively deaminated by ninhydrin

with the formation of carbon dioxide and an aldehyde. Under some conditions, the ammonia appears as such, but, under others, it condensesto a
greater or lesser extent with the reagent to form diketohydrindylideneThe familiar violet color which is associdiketohydrindamine (DYDA).
ated with the reaction of amino acids with ninhydrin is attributed to the
anion of this compound. Ruhemann (l-3) was of the opinion that the
first step in the reaction is the oxidative deamination of the amino acid
with the formation of a molecule of ammonia and a molecule of the reductant of ninhydrin, diketohydrindol, in the form of the meriquinone, hydrindantin. The ammonia then condenses with the hydrindantin to form
DYDA.
This scheme is consistent with the fact that ammonia forms
DYDA with ninhydrin only in the presence of a reducing agent capable
of producing some hydrindantin.
It is inconsistent, however, with the
fact that ammonia reacts more slowly and less completely than do amino
acids under the sameconditions. One must conclude that the deamination
and the condensation are coupled in some fashion.
Moore and Stein (4) have recently developed the reaction into a rapid
and convenient photometric method for the estimation of amino acids.
They showed that the low and variabIe yields of color usually obtained
were due to the autoxidation of some of the diketohydrindol formed as a
result of the oxidative deamination. Introduction of a reducing agent into
the system materially increased the amount of color and made it possible
to obtain consistent and reproducible results, Even so, in the procedure
of Moore and Stein, the yield of color per molecule varies with the amino
acid, and, in no case, is as great as that of a molecule of DYDA.
Seeking an explanation for the apparent lack of stoichiometry of the
reaction, we were impressed with the observation of Moore and Stein that
the colors formed in their procedure slowly faded on standing at room
temperature over a period of hours. Since the colors are developed by
exposure of the reaction system to 100 for 20 minutes, it was reasonable

804

NINHYDRIN

METHOD

to infer that some destruction of DYDA occurred in this step. We, therefore, sought conditions under which the rate of formation of DYDA was
increased relative to its rate of destruction.
Organic solvents such as
alcohol, dioxane, methyl Cellosolve, pyridine, and phenol were found to
accelerate the development
of color to varying degrees. Ultimately
a
phenol-pyridine
system was adopted as the most effective solvent.
In this
system containing 5 per cent water the majority of the amino acids gave
quantitative
yields of color in 20 minutes at room temperature (Table I).

Glycine. ...................................
Alanine ....................................
Valine .....................................
Leucine ...................................
Isoleucine .................................
Phenylalanine .............................
Tyrosine ..................................
Tryptophan ...............................
Serine .....................................
Threonine .................................
Methionine ................................
Glutamic acid .............................
Aspartic
.............................
Histidine ..................................
Arginine ...................................

10.00
21.2
21.5
21.7
21.4
10.3
12.2
14.2
20.5
21.2
21.5
21.7
9.25
14.6
21.8

46.5
98.4
99.5
100.5
99.0
48.0
55.5
65.7
95.0
98.4
99.5
100.5
42.7
68.0
101 .o

Exposure to 100 for 3 to 5 minutes gave quantitative yields of color for all
the amino acids except tryptophan and lysine (Table II) in a system containing 20 per cent water.
The imino acids react with ninhydrin in an entirely different fashion.
Carbon dioxide is evolved and yellow colors are formed very rapidly, even
at room temperature and at pH 7. Reducing agents are without influence.
Deamination does not occur and the imino acid residue condensesdirectly
with the ninhydrin to form the pigment.
Grassmann and von Arnim (5) have isolated red derivatives of proline
and hydroxyproline and have identified them as di(diketohydrindylidene)pyrroles. We have prepared these compounds and have observed that in

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TABLE
I
Color Recoveries Obtained at Room Temperature
0.01 ml. of amino acid, 0.002 M, 2 ml. of 80 per cent phenol in alcohol, 0.5 ml. of
pyridine, 500 mg. of ninhydrin, and 100 mg. of hydrindantin
are allowed to react for
20 minutes at room temperature, and diluted to 10 ml. with 60 per cent alcohol and
1 per cent formaldehyde.
Amino acid
IX extinction
(570 mp 1 Per cent yield of DYDA

1%. TROLL

AND

R.

K.

805

CANNAN

aqueous solution they readily change to yellow products possessing a broad

absorption with a maximum at 440 rnp. They are not well suited to
photometry because of the relatively large absorption of ninhydrin in this
region of the spectrum,
The red compounds, on the other hand, exhibit
intense absorption in the region 550 to 570 rn~.
TABLE

Results
Amino

with

Recommended

acid

Glyeine....................................
Alanine ....................................
Valine .....................................
Isoleucine .................................
Phenylalanine .............................
Tyrosine

..................................

Tryptophan ...............................
Serine .....................................
Threonine .................................
Methionine ................................
Glutamic acid .............................
Aspartic
.............................
Histidine ..................................
Arginine ...................................
Lysine .....................................
Sarcosine
..................................
Ammonia
..................................
Leucylglycine.
.............................

Glycylglycylleucine.

.......................

Alanylglycylglycylleucine ..................
Urea, creatinine,
aniline, p-aminobenzoic
acid, hippuric
acid .......................

-.

for

Amino Acids

/I

nud extinction

21.1
22.0
21.6
21.8
21.6
21.8
21.3
16.3
21.4
22.1
22.0
21.4
21.3
22.0
21.1
23.8
5.2
6.3
20.6
19.3
18.3

(570 mp) I I er cent yield

of DYDA

98
102.0
100
100.1
100
100.1
98.8
75.4
99.0
102.6
102.0
99.0
98.6
102.0
98.0
110.5
24.6
29.2
95.5
89.5
85.0

0.0
__-

It is the yellow products of the imino acids that are formed when the
procedure of Moore and Stein is applied to a protein hydrolysate.
We
have demonstrated, however, that the red compounds are fugitive intermediates. It has been possible to obtain quantitative recovery of the red
derivative of hydroxyproline by continuous extraction of an appropriate
reaction system with benzene. Under the conditions to be described, the
red pigment of proline forms more slowly and is converted to the yellow
product more rapidly. A small amount of it is extracted with benzene.
Based on these observations a method has been devised for the photometric
determination of hydroxyproline in amino acid mixtures.

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Leucine...................................

II
Method

806

NINHYDRIN

METHOD

EXPERIMENTAL

Amino

At%&

Recommended Method for Primary

Amino

Acids

ReagentsI. Ninhydrin
solution.
500 mg. of ninhydrin are dissolved in 10 ml. of
absolute alcohol.
2. 80 per cent phenol solution.
80 gm. of reagent grade phenol are
dissolved in 20 ml. of absolute alcohol, with gentle heating.
The solution
is shaken with 1 gm. of Permutit for about 20 minutes to remove traces of
ammonia and then decanted.
2 ml. of a 0.01 M solution of KCN are diluted
3. KCN-pyridine
reagent.
to 100 ml. with ammonia-free pyridine, prepared by shaking 100 ml. of
pyridine with 1 gm. of Permutit for about 20 minutes.
4. 60 per cent alcohol (by volume).

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Preliminary
Observations-The
reaction of alanine with ninhydrin under
varying conditions was first explored.
The conclusion of Moore and Stein
that the optimum pH for the reaction was at 5 was confirmed.
At room
temperature the reaction was very slow and, on completion, fell far short
of giving the theoretical amount of color. The reaction was speeded up
considerably
in the presence of excess of alcohol, both rate and yield
increasing progressively
as the concentration of alcohol rose from 80 to 98
per cent. Other water-miscible
organic solvents gave similar results.
Pyridine was particularly
effective (6), as was a concentrated solution of
phenol in alcohol. Ultimately we chose an 80 per cent solution of phenol
buffered with pyridine as the most effective solvent.
In the early experiments ascorbic acid was used as the reducing agent or
In the search for a
hydrindantin
itself was added to the reaction mixture.
more stable and soluble reducing agent we observed that potassium cyanide
led to the formation of hydrindantin
when added to a solution of ninhydrin,
This was inferred from the observation that a red color was formed in
The spectra
borate buffers and a blue color in dilute sodium hydroxide.
of these two colors were indistinguishable
from those of hydrindantin in the
same two solvents.
The stability of DYDA in the phenol-pyridine
system was examined.
In the absence of ninhydrin the color faded rapidly at loo, but in the
presence of ninhydrin there was no detectable loss of color after heating for
20 minutes.
The explanation of this protective effect of ninhydrin is not
known.
On the basis of the above observations
the procedure described below
was devised and tested on seventeen amino acids and a few peptides.

W.

TROLL

AND

R.

K.

CANNAN

807

Imino

Acids

Preliminary Observations-In
the met,hod which has been described, proline and hydroxyproline
formed yellow colors with a wide absorption maximum in the 440 rnp region. This reaction was more rapid in water-miscible
organic solvents, but phenol did not show any advantage over other solvents.
Reducing agents were without influence.
In reaction systems buffered between pH 4 and 8 with aqueous buffers containing water-miscible
organic solvents such as methyl Cellosolve, hydroxyproline
passed through
The red product was
an intense dark red stage before becoming yellow.
isolated by extracting the system with benzene and was found to have an
absorption spectrum identical with that of the isolated di(diketohydrindylidene)pyrrole
compound prepared according to Grassmann
and von
Arnim (5).
The latter compounds are readily prepared by treating 1 mole of the
imino acids with 2 moles of ninhydrin in phosphate buffer at pH 7 for
1 hour at 40-60.
The partially crystalline
precipitate is dissolved in

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All reagents appear to be stable for at least a month at room temperature.

The phenol solution tends to absorb ammonia from the air and should be
shaken weekly with Permutit.
Procedure-O.4
to 0.5 ml. of aqueous amino acid solution, of any pH
between 1 and 8, containing from 0.05 to 0.5 PM of amino acid, is heated
with 1 ml. of KCN-pyridine
reagent and 1 ml. of 80 per cent phenol reagent in a boiling water bath. When the mixture has reached the temperature of the water bath, 0.2 ml. of the ninhydrin solution is added, the
reaction vessel is stoppered, and the reaction is allowed to proceed for 3 to
5 minutes.
The solution is cooled and made up to 10 ml. with 60 per cent
alcohol, and the optical density at 570 rnp is determined.
0.4 to 0.5 ml. of
ammonia-free water, subjected to the same procedure, is used as a reagent]
blank.
Results-All
the amino acids tested, except tryptophan
and lysine, gave
colors equivalent to 97 to 102 per cent of that of pure DYDA in the same
solvent,
Tryptophan gave about 70 per cent yield, and lysine 110 per cent
(Table II).
The absorption spectra of the colors obtained from amino
acids in this procedure were identical with that of DYDA (Fig. 1). The
peptides tested did not react quantitatively,
but gave yields of color diminThe
ishing from 95 to 85 per cent in passing from a di- to a tetrapeptide.
reaction of ammonia was less than one-third complete, while urea, uric
acid, creatine, and creatinine did not, give any observable color. It is of
interest to note that, although proline and hydroxyproline
do not form
DYDA, the imino acid, sarcosine, did react t.o an extent comparable with
the reaction of ammonia.

808

NINHYDRIN

METHOD

benzyl alcohol, and the compound is allowed to crystallize from the filtered solution at O-5 after the addition of 4 volumes of ethyl alcohol.
Both the proline and hydroxyproline
derivatives crystallize as microscopic
dark purple needles with a green fluorescence.
The nitrogen contents of
our products agreed with the analytical data reported by Grassmann and
von Arnim (5).

o-color

given

by leucine in procedure

420

460

500

WAVE LENGTH

540

580

620

660

IN MILLIMICRONS

1. Comparison of the absorption spectra of isolated DYDA and of the color

given by an equimolar concentration
of leucine when carried through the procedure
for primary amino acids.
FIG.

When the isolated proline and hydroxyproline derivatives were added to

a system containing water and ninhydrin, they formed the same yellow
colors as do the amino acids themselves. It is concluded that the compounds of Grassmann and von Arnim (5) are intermediat,es in the formation
of the yellow colors. The red compounds may be extracted with benzene.
This solvent does not remove the yellow pigments nor does it extract
DYDA.
It is therefore possible to apply the process directly to protein
hydrolysates. Conditions have been found under which a quantitative
recovery of the derivative of hydroxyproline may be obtained, accompanied

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W.

TROLL

AND

R.

K.

809

CANNAN

by only a small yield of the corresponding derivative of proline.

The
absorption spectra of the proline and the hydroxyproline derivatives show
If readings of the optical densimaxima at 550 and 570 rnl.r, respectively.
ties at these two wave-lengths are made, one may correct for the amount
of proline present with the aid of constants derived from the absorption
spectra of the pure compounds (Fig. 2). The molecular extinction coefficients are about 3 times as great as that of DYDA.
84807672686456-

52-

48

w 44
$j 40

I/

32-

=!

28-

2420.
16

-HYDROXYPROLINE
8
2

3612;J/
4
440

FIG.

2. Absorption

,i,
460

spectra

480

500

520

540

560

WAVE LENGTH IN MILLIMICRONS

of the isolated
proline
and

Recommended Procedure for Determination

580

600 620

hydroxyproline

compounds

of Hydroxyproline

Reagents-

1. Ninhydrin solution.
0.1 M ninhydrin in methyl Cellosolve.
2. Phosphate buffer, pH 7.0, 0.1 M.
3. Benzene, c. p.
Procedure-O.1 ml. of the sample, containing 1 to 15 y of hydroxyproline,
0.1 ml. of phosphate buffer, and 2.5 ml. of benzene are placed in a FolinWu tube. The tube is attached to a rapid vibrator, the bulb dipping into
a water bath maintained at 75 f 1. The vibrator is started and 0.2 ml.
of ninhydrin solution is added. The tube is withdrawn after 5 minutes

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60$
F

810

NINHYDRIN

METHOD

and the benzene layer is made up to 10 ml. in a volumetric flask.

optical densities of the solution at 550 and 570 rnp are determined
Beckman spectrophotometer.
TABLE
Recovery

of Hydroxyproline

from

The
in a

III

Mixtures

of

Amino

Acids and Urine

0.1 ml. of hydroxyproline

of the concentration
indicated
added to 0.1 ml. of solutions of 0.01 M amino acid mixture
containing
glycine,
alanine,
valine,
leucine,
isoleucine,
tyrosine,
tryptophan,
phenylalanine,
methionine,
glutamic
acid, aspartic
acid,
histidine,
arginine,
lysine,
serine,
threonine,
and sarcosine,
each 5.25 per
cent; proline
10.5 per cent of amino
nitrogen.
0.1 ml. of hydroxyproline
added to
0.1 ml. of normal
urine samples.
Molar concentrat~dio,ocf hydroxyproline

Apparent contribution of hydroxyproline to OD at 570 w

acid

0
2
4
6
8
1

x
x
X
x
x

10-b
10-d
1O-4
10-4
10-S

per cent

mixtures
0.010
0.142
0.284
0.415
0.535
0.691

103
102
101
97
100.5

0.024
0.160
0.302
0.430

102
99.5

Urine
0

2 x 10-4
4 x 10-d
6 X 1OP

line

* Calculated
compound.

from

the millimolar

extinction

99.5

at 570 rnp of the isolated

hydroxypro-

The contribution of hydroxyproline to the color in the benzene layer is

calculated from the following equation:
HOs,,,

= 1.46 OD,,,

0.592

0055,~

where OD650is the optical density at 550 rnp, 00670 the optical density at
570 rnp, and HO570 the contribution of hydroxyproline to the optical density
at 570 rnp.
Optical standards are established with 0.1 ml. of solutions containing
0.01 to 0.1 PM (1.3 to 13 7) of hydroxyproline.
Reagent blanks are determined by substituting 0.1 ml. of water for the sample solution.
Results-Recovery experiments were carried out by adding 0.1 ml. of
solutions of hydroxyproline in phosphate buffer to 0.1 ml. of a 0.01 M

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Amino

Recovery,*

W.

TROLL

AND

R.

K.

CANNAN

811

SUMMARY

1. A modified procedure is described for the photometric determination

of amino acids with ninhydrin.
Ten of the naturally occurring amino
acids give theoretical yields of color at room temperature.
At 100 all
amino acids, except tryptophan and lysine, react quantitatively.
2. A method for the photometric estimation of hydroxyproline
in amino
acid mixtures is described.
It is based on the extraction of a red derivative
from the reaction system with benzene. This pigment is a precursor of the
yellow compound which is the final product of the reaction with ninhydrin.
BIBLIOGRAPHY

1.
2.
3.
4.
5.
6.

Ruhemann, S., J. Chem. Sot., 97, 1438 (1910).

Ruhemann, S., J. Chem. Xoc., 99, 792 (1911).
Ruhemann, S., J. Chem. Sot., 99, 1491 (1911).
Moore, S., and Stein, W. H., J. Biol. Chem., 176, 367 (1948).
Grassmann, W., and von Arnim, K., Ann. Chem., 609, 288 (1934).
Harding, V. J., and MacLean, R. M., J. Biol. Chem., 26,337 (1916).

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solution of a mixture of sixteen amino acids, together with sarcosine and

proline.
In six experiments, with amounts of hydroxyproline
ranging from
Similar
0.02 to 0.1 PM, recoveries of 97.8 to 103 per cent were obtained.
recoveries were obtained when hydroxyproline
was added to urine (Table
III).
The absorption spectra of the colors obtained in these recovery
experiments could be entirely accounted for as being due to the hydroxyproline and proline derivatives.
The method was then applied to protein hydrolysates
and to urine.
Hydrolysates
of crystalline lactoglobulin, crystalline ricin, and gelatin were
Aliquots containing 0.1 mg. of
adjusted to pH 7 with phosphate buffer.
protein per ml. (gelatin) or 5 mg. of protein per ml. (lactoglobulin and
ricin) were analyzed.
The amount of hydroxyproline
found in lactoglobulin and ricin was less than 0.1 per cent. The amount found in gelatin was
12.83 per cent. A series of normal urines was brought to pH 7 by adding
solid dibasic potassium phosphate, and 0.1 ml. samples were submitted to
The benzene layer was diluted to 5 rather than 10 ml. because
analysis.
of the small amount of color present.
The normal excretion of hydroxyproline appears to be of the order of 0.5 to 2.5 mg. of hydroxyproline
per
day (four samples).

ARTICLE:
A MODIFIED PHOTOMETRIC
NINHYDRIN METHOD FOR THE
ANALYSIS OF AMINO AND IMINO
ACIDS
J. Biol. Chem. 1953, 200:803-811.

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Walter Troll and R. Keith Cannan