ARTICLE:
A MODIFIED NINHYDRIN REAGENT
FOR THE PHOTOMETRIC
DETERMINATION OF AMINO ACIDS
AND RELATED COMPOUNDS
Stanford Moore and William H. Stein
J. Biol. Chem. 1954, 211:907-913.
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BY STANFORDMOORE AND WILLIAMH. STEIN
(From the Laboratories of The RockefellerInstitutefor MedicalResearch,
New York $1, New York)
(Received for publication, July 6, 1954)
The photometric ninhydrin method described previously (1) for use in
the chromatography of amino acids has been the subject of further study
during the development of the Dowex 50-X4 procedure (2). The adjust-
ment of the pH of the effluent fractions prior to analysis required by the
earlier method has been largely eliminated by increasing the strength of
the buffer in the reagent 5-fold. A second modification involves the elimi-
nation of the stannous chloride which was previously added to form re-
duced ninhydrin (hydrindantin) in the reagent solution. Hydrindantin
itself is now added directly, and this change avoids the precipitation of tin
salts which occurs when the previous reagent is used to analyze samples
of the phosphate buffers frequently employed in the chromatography of
proteins (3). The effective concentration of hydrindantin has been raised
to 0.3 per cent. As a result, the antioxidant effect of 1 ml. of the modified
reagent is sufficient to prevent interference from the dissolved oxygen in a
2 ml. sample submitted to analysis. With 1 ml. fractions, 0.5 ml. of the
ninhydrin reagent is adequate. The quantity of reagent required is thus
reduced to one-half or one-quarter the amount formerly recommended (1).
Troll and Cannan (4) have recently described conditions under which the
yield of blue color (diketohydrindylidenediketohydrindamine)in the re-
action of ninhydrin with amino acids is raised to 100 per cent of theory in
most cases. For this purpose, the reaction is performed in a predominantly
organic solvent (phenol-pyridine-alcohol) having a maximal water content
of about 20 per cent. The method, when applicable, has the fundamental
advantage of giving maximal color yields. The present procedure, in
which the reproducible color yield from leucine is 95 per cent of theory,
appears to be more convenient for the analysis of the aqueous effluent frac-
tions obtained in ion exchange chromatography.
Reagents
Ninhydrin-Severalcommercial sources provide ninhydrin sufficiently
pure to be used without recrystallization (1). The reagent employed in
this study was purchased from Dougherty Chemicals, 87-34 134th Street,
Richmond Hill 18, New York.
907 Downl from http://w.org/ b on Jun2014
908 PHOTOMETRIC NINHYDRIN METHOD
Hydrindantin-Thecompound was prepared by the reduction of ninhy-
drin with ascorbic acid (cf. Abderhalden(5), West and Rinehart(6)).
To 80 gm. of ninhydrin in 2 liters of water at 90”, add with stirring a solu-
tion of 80 gm. of ascorbic acid (Merck) in 400 ml. of water at 40”. Crys-
tallization of hydrindantinstarts immediatelyand is allowed to proceed
for 30 minutes without further heating.During the next hour the solu-
tion is cooled to room temperatureunder running tap water.The
hydrindantinis filtered off, washed well with water, and dried to constant
weight over PzOb in a vacuum desiccator protected from light.Yield, 75
gm. The compound should be stored in dark glass.The crystallization
is carried out rapidly to give small crystals which will dissolve readily
in methyl Cellosolve.The use of the anhydrousform rather than the
air-dried dihydratealso increases the rate of solution in the organic sol-
vent.(Hydrindantinthus prepared is available from DoughertyChemi-
cals.)
4~ Sodium Acetate Bu$er- (pH 5.5)-To2 liters of water, add 2720 gm.
of NaOAc.3HzO(reagent grade) and stir on a steam or-water bath until
solution is complete.Cool to room temperature,add 500 ml. of glacial
acetic acid, and make up to a volume of 5 liters.The solution (undiluted)
should be at pH 5.51 f 0.03. If final adjustment of the pH is necessary,
5 gm. of NaOH correspond to about 0.04 pH unit. The buffer can be
stored at 4” without a preservative.
Reagent Solution-Dissolve20 gm. of ninhydrin and 3 gm. of hydrindan-
tin in 750 ml. of methyl Cellosolve.’The stirring should not incorporate
air bubbles into the solution.Add 250 ml. of the buffer of pH 5.5 and im-
mediately transfer the resulting reddish reagent solution to a 1 liter dark
glass reservoir bottle arranged to permit the solution to be stored under ni-
trogen, as previously described (1) .2 The second and third bottles of the
storage assembly are of 2 liter size. The 250 ml. dropping funnel used be-
fore (1) has been replaced by an inlet, tube bearing a 6 mm. bore stop-cock
and a small semiball joint, normally closed by a correspondingstopper.
In refilling the reservoir, a bent glass tube reaching to the bottom of a
beaker of the reagent is attached to the inlet tube through the ground joint
connection.Slight air pressure is then applied to the third bottle to drive Downl from http://w.org/ b on Jun2014
1 The solvent(monomethylether of ethyleneglycol)is obtainablefrom the manu-
facturer(Carbideand CarbonChemicalsCorporation,30 East 42nd Street,New York
17) or supplier(e.g., AmendDrugand ChemicalCompany,117 East 24th Street,New
Each lot is tested for peroxidecontentbyYork10) relativelyfree from peroxides.
adding2 ml. of the solventto 1 ml. of freshlyprepared4 per cent aqueousKI.A
colorlessto light straw-yellowtest is satisfactory.
2 Evaporationfrom the concentratedsalt solutionmay cause crystallizationof so-
dium acetateon the syringeof the pipettingmachine(1).The exposedsectionof the
plungershouldbe wiped free of any appreciabledepositto preventstrainon the syr-
inge assembly.
S. MOORE AND W. H. STEIN 909
reagent remaining in the reservoir into the filling tube and to displace the
air therein.The air pressure is replaced by suction to draw the reagent
solution into the reservoir.Care should be taken not to introduce air into
the assembly at the end of the filling operation.
The solution is not as stable on storage as the previous reagent.After
1 month, the blank readings have about doubled, and, unless the bottle
has been stored in the dark, the color yields can run low by as much as 5
per cent. For this reason, not much more than a 1 week’s supply of re-
agent should be prepared at a given time.
Procedure (Cf. (1))
For the analysis of 1 ml. or 2 ml. effluent fractions from ion exchange
chromatograms(2), 1 ml. of the ninhydrin solution is used. The buffer is
sufficiently strong so that preliminary adjustment of the pH of the samples
is seldom necessary.If, however, more than 1 ml. of 0.1 N HCl or 0.1 N
NaOH should be required to bring the effluent fractions to pH 4 to 6, ad-
dition of acid or alkali may be necessary to bring the pH of the samples
to the optimum for color development.The capped tubes are shaken
briefly (
for 15 minutes (accurately timed) in a covered boiling water bath (1). It
is convenient to use 50:50 ethanol-wateras a diluent rather than n-pro-
panol-water(1). After dilution, the tubes (kept out of direct sunlight)
are wiped dry and transferredto a dry rack, cooled to below 30” in front
of an electric fan, and thoroughly shaken (about 30 seconds) before being
read at 570 mμ rnp for proline and hydroxyproline).Because the
modified reagent contains a higher concentrationof hydrindantin,more of
the reddish color of this compound remains at the end of the heating period.
In order to reduce the blank to the desired level, therefore, the tubes should
be shaken sufficiently so that the major portion of the residual hydrindan-
tin is oxidized by air. For this reason, the tubes are shaken uniformly in a
rack, rather than individually.With the modified reagent, the blank read-
ings for 2 ml. samples and 1 ml. of ninhydrin solution have been 0.05 to 0.10
on the optical density scale. If there is evidence that high blank readings
are being caused by uptake of NH3 during the period that the tubes are
standing on the fraction collector, the cover of the machine should be lined
with cloth impregnatedwith citric acid (7).3 If effluent fractionsare
stored prior to analysis, they should be stoppered with corks treated with
citric acid (7) and wrapped in aluminum foil.
The absence of tin salts in the reagent solution not only avoids the pre-
cipitation of tin phosphate when samples containing phosphate are ana-
3 If ammoniainterferes,it can be removedby renderingthe samplesalkalineand
placingthe tubes in a vacuumdesiccatorfor a few hours before the ninhydrinanaly-
ses (2). Downl from http://w.org/ b on Jun2014
910 PHOTOMETRIC NINHYDRIN METHOD
Calculations
A standard curve is plotted in the manner previously described (1) by
utilizing 1 ml. samples of leucine (0.05 to 0.2 InM) in 0.1 M citrate buffer
at pH 5. For use with chromatograms,two direct reading tables are pre-
pared in the format of Table I previously given (I), one for 1 ml. samples
and 1 ml. of ninhydrin solution, and the second for 2 ml. samples and 1 ml.
of the reagent.It is convenient to have twice the millimolar concentra-
tions listed in the table for the 2 ml. samples.In this way both tables
yield micromoles of amino acid directly, without the use of factors (( 1) Table V), when the values correspondingto the points of a given peak on an ef-
fluent curve are added. Fractions that are off the scale of the table (above
an optical density of 1.0 after dilution with 15 ml. of diluent) must be
diluted further by hand before being read. The values taken from the
final column of the table must then, of course, be multiplied by the appro-
priate dilution factor.When samples require neutralizationor, in pre-
parative chromatography,when aliquots from larger fractions are pipetted
(1): direct reading tables can be prepared for the special case, or appropriate volume corrections can be applied to the values read from a table for a
different fraction size. In computing the volume corrections, no factor is
required for the negligible loss by evaporation which occurs during the
heating of the aqueous samples (1).
The color yields obtained with a number of ninhydrin-positivesubstances likely to be encountered in biological systems are listed in Table I. The
values, as well as the color yields from peptides and proteins, are similar in
most cases to those obtained with the previous reagent (1, 3). The yields
were determined on 2 ml. samples of the compounds dissolved in the buffers in which they emerge from columns of Dowex 50-X4 in the current chro-
matographicprocedure (2). Although the color yields are reproducible
to f2 per cent in a given laboratory,the exact values should be checked
under the user’s experimental conditions if maximal accuracy is desired.
4 Transfer pipettes No. 283-R,Microchemical Company, 1834 Univer-sity Avenue, Berkeley 3, California. Specialties Downl from http://w.org/ b on Jun2014
MOORE AND W. H. STEIN 911
The molecular weight divided by the color yield provides the factor by
which the chromatographicresults (expressed in leucine equivalentsin
micromoles) should be multiplied to give micrograms of the substance in
TABLE I
Color Yields from Amino Acids and RelatedCompounds Basis Relative to
Leucine on Molar
Determinedon 2 ml. samplesof 0.1 mM solutionsin the buffers (pH 2.2 to 5) in which the compoundsemergein the Dowex50-X4 chromatographic procedure(2);heatingtime 15 minutes;read at 570 mu.(The urea and creatinine
were 3.0 mm.)
-
Compound Color yield Compound Color yield
Asparticacid 0.94 &NHz-isobutyricacid 0.44 Threonine 0.94 Carnosine
Serine 0.95 0.93
Citrulline 1.04
Proline(440 rnfi) 0.225* Creatinine 0.027 Glutamicacid 0.99 Cysteicacid 0.99 Glycine 0.95 Diaminopimelicacid(per 1.24 Alanine 0.97 2 NH2 groups)
Valine 0.97 Ethanolamine
Half cystine 0.55 Felinine
Methionine 1.02 Glutamine 0.91Isoleucine 1.00 Glucosamine
0.95
Leucine 1.00 Glutathione(oxidized,half)
Tyrosine 1.00 0.99
Glycerophosphoethanol- 1.03
Phenylalanine 1.00 amine
0.93Ammonia 0.97 Hydroxylysine
0.50Lysine 1.10 Hydroxyproline(440 rnp)
Histidine 1.02
Methioninesulfone
Tryptophan 0.94 “
Arginine 1.01
a-NHz-adipicacid sulfoxide
0.96 1-Methylhistidine 1.12
@-Alanine 0.50 3-Methylhistidine 0.077*Anserine 0.78 Ornithine
1.02
Asparagine 0.95 Phosphoethanolamine 0.98 a-NHz-n-butyricacid 1.02 Sarcosine 0.88 r-NHz-butyricacid 1.01
Taurine 0.86
Urea 1.12
0.43
0.28
0.88
0.0314
* The readingstakenat 440 rnp are first convertedto “leucineequivalents”by
using the same conversiontable that is employedfor the otheraminoacids meas-
ured at 570 rnp, and the concentrationsof the iminoacids are subsequentlyobtained
by dividingby the abovecolor yields.
question.Each of the first eighteen amino acids (Table I) was checked for
purity by elementary analysis and by chromatographyon Dowex 50. The
other compounds were commercial or research samples possessing the cor- rect elementary composition. Downl from http://w.org/ b on Jun2014
912 PHOTOMETRIC NINHYDRIN METHOD
DISCUSSION
The proportion of methyl Cellosolve in the reagent solution has been
raised from 50 per cent to 75 per cent in order to keep the increased quantityof hydrindantin in solution. The increase in the strength of the buffer has
required the use of sodium acetate (cf. Boissonnas (lo)), which is more sol-
uble in methyl Cellosolve, in place of sodium citrate. With the stronger
buffer, the pH at which maximal color yields are obtained in the ninhydrin
reaction is very similar to that employed previously.Although the 4 N
sodium acetate buffer is initially at pH 5.5, the pH drops to 5.1 when the
buffer is diluted to 1 N, which is the actual normality employed in the
reagent. With the previous reagent, maximal color yields were obtained
at pH 5.0 with a 0.2 M citrate buffer.
Satisfactory recoveries of proline from chromatograms were obtained
with use of the modified reagent (1) despite the low value of the color
yield. With hydroxyproline, however, which has a much lower color yield,
this would not be the case. Greater sensitivity for proline is exhibited by
the procedure of Chinard (1 l), which utilizes the red color formed with
ninhydrin in acid solution, and this method has been employed by Harfen-
ist (12) to analyze the effluent from Dowex 50-X8 chromatograms. The
special procedures of Neuman and Logan (13) and Troll and Cannan (4)
(cf. Rogers et al. (14)) provide more sensitive tests for hydroxyproline.
The authors wish to acknowledge the technical assistance of Mrs. Ger-
trude C. Carey and Miss Renate Mikk in the performance of these experi-
ments. Downl from http://w.org/ b on Jun2014
S. MOORE AND W. H. STEIN 913
SUMMARY
An extension of earlier studies on the determinationof amino acids and
related compounds with ninhydrin has led to the development of a modi-
fied reagent composed of 2 per cent ninhydrin and 0.3 per cent hydrindantin
The strengthN sodium acetate buffer (pH 5.5).in 3 : 1 methyl Cellosolve-
of the buffer eliminates or reduces the need for preliminaryadjustment of
the pH of the samples when the method is used for the analysis of effluent
fractions obtained in ion exchange chromatography,and the higher hy-
drindantin concentrationof the reagent solution permits economies in the
use of ninhydrin.
BIBLIOGRAPHY
1. Moore,S., and Stein, W. H., J. Biol. Chem., 176, 367 (1948).
2.
Moore,S., and Stein, W. H., J. Biol. &em.,211, 893 (1954).
3. Hirs, C. H. W., Moore,S., and Stein, W. H., J. Biol. Chem., 200, 493 (1953).
4. Troll,W., and Cannan,R. K., J. Biol. Chem., 200, 803 (1953).
5.
Abderhalden,R., 2. physiol.Chem.,262, 81 (1938).
6. West, E. S., and Rinehart,R. E., J. BioZ. Chem., 146, 105 (1942).
7. Moore,S., and Stein, W. H., J. BioZ. Chem., 178, 53 (1949).
8. Schram,E., Moore,S., and Bigwood,E. J., Biochem.J., 6’7, 33 (1954).
9.
10. Crokaert,R., Moore,S., and Bigwood,E. J., Bull. Sot. chim. biol., 33, 1209 (1951).
Boissonnas,R. A., HeZv. chim. acta, 33, 1975 (1950).
11. Chinard,F. P., J. BioZ. Chem., 199, 91 (1952).
12. Harfenist,E. J., J. Am. Chem. Sot., 76, 5528 (1953).
13. Neuman,R. E., and Logan,M. A., J. BioZ. Chem., 184,299(1950).
14. Rogers,C. J., Kimmel,J. R., Hutchin,M. E., and Harper,H. A., J. BioZ. Chem.,
206, 553 (1954). Downl from http://w.org/ b on Jun2014
ARTICLE:
A MODIFIED NINHYDRIN REAGENT
FOR THE PHOTOMETRIC
DETERMINATION OF AMINO ACIDS
AND RELATED COMPOUNDS
Stanford Moore and William H. Stein
J. Biol. Chem. 1954, 211:907-913.
Access the most updated version of this article at
http://www.jbc.org/content/211/2/907.citation
Find articles, minireviews, Reflections and
Classics on similar topics on the JBC Affinity
Sites .
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tml#ref-list-1 Downl from http://w.org/ b on Jun2014
BY STANFORDMOORE AND WILLIAMH. STEIN
(From the Laboratories of The RockefellerInstitutefor MedicalResearch,
New York $1, New York)
(Received for publication, July 6, 1954)
The photometric ninhydrin method described previously (1) for use in
the chromatography of amino acids has been the subject of further study
during the development of the Dowex 50-X4 procedure (2). The adjust-
ment of the pH of the effluent fractions prior to analysis required by the
earlier method has been largely eliminated by increasing the strength of
the buffer in the reagent 5-fold. A second modification involves the elimi-
nation of the stannous chloride which was previously added to form re-
duced ninhydrin (hydrindantin) in the reagent solution. Hydrindantin
itself is now added directly, and this change avoids the precipitation of tin
salts which occurs when the previous reagent is used to analyze samples
of the phosphate buffers frequently employed in the chromatography of
proteins (3). The effective concentration of hydrindantin has been raised
to 0.3 per cent. As a result, the antioxidant effect of 1 ml. of the modified
reagent is sufficient to prevent interference from the dissolved oxygen in a
2 ml. sample submitted to analysis. With 1 ml. fractions, 0.5 ml. of the
ninhydrin reagent is adequate. The quantity of reagent required is thus
reduced to one-half or one-quarter the amount formerly recommended (1).
Troll and Cannan (4) have recently described conditions under which the
yield of blue color (diketohydrindylidenediketohydrindamine)in the re-
action of ninhydrin with amino acids is raised to 100 per cent of theory in
most cases. For this purpose, the reaction is performed in a predominantly
organic solvent (phenol-pyridine-alcohol) having a maximal water content
of about 20 per cent. The method, when applicable, has the fundamental
advantage of giving maximal color yields. The present procedure, in
which the reproducible color yield from leucine is 95 per cent of theory,
appears to be more convenient for the analysis of the aqueous effluent frac-
tions obtained in ion exchange chromatography.
Reagents
Ninhydrin-Severalcommercial sources provide ninhydrin sufficiently
pure to be used without recrystallization (1). The reagent employed in
this study was purchased from Dougherty Chemicals, 87-34 134th Street,
Richmond Hill 18, New York.
907 Downl from http://w.org/ b on Jun2014
908 PHOTOMETRIC NINHYDRIN METHOD
Hydrindantin-Thecompound was prepared by the reduction of ninhy-
drin with ascorbic acid (cf. Abderhalden(5), West and Rinehart(6)).
To 80 gm. of ninhydrin in 2 liters of water at 90”, add with stirring a solu-
tion of 80 gm. of ascorbic acid (Merck) in 400 ml. of water at 40”. Crys-
tallization of hydrindantinstarts immediatelyand is allowed to proceed
for 30 minutes without further heating.During the next hour the solu-
tion is cooled to room temperatureunder running tap water.The
hydrindantinis filtered off, washed well with water, and dried to constant
weight over PzOb in a vacuum desiccator protected from light.Yield, 75
gm. The compound should be stored in dark glass.The crystallization
is carried out rapidly to give small crystals which will dissolve readily
in methyl Cellosolve.The use of the anhydrousform rather than the
air-dried dihydratealso increases the rate of solution in the organic sol-
vent.(Hydrindantinthus prepared is available from DoughertyChemi-
cals.)
4~ Sodium Acetate Bu$er- (pH 5.5)-To2 liters of water, add 2720 gm.
of NaOAc.3HzO(reagent grade) and stir on a steam or-water bath until
solution is complete.Cool to room temperature,add 500 ml. of glacial
acetic acid, and make up to a volume of 5 liters.The solution (undiluted)
should be at pH 5.51 f 0.03. If final adjustment of the pH is necessary,
5 gm. of NaOH correspond to about 0.04 pH unit. The buffer can be
stored at 4” without a preservative.
Reagent Solution-Dissolve20 gm. of ninhydrin and 3 gm. of hydrindan-
tin in 750 ml. of methyl Cellosolve.’The stirring should not incorporate
air bubbles into the solution.Add 250 ml. of the buffer of pH 5.5 and im-
mediately transfer the resulting reddish reagent solution to a 1 liter dark
glass reservoir bottle arranged to permit the solution to be stored under ni-
trogen, as previously described (1) .2 The second and third bottles of the
storage assembly are of 2 liter size. The 250 ml. dropping funnel used be-
fore (1) has been replaced by an inlet, tube bearing a 6 mm. bore stop-cock
and a small semiball joint, normally closed by a correspondingstopper.
In refilling the reservoir, a bent glass tube reaching to the bottom of a
beaker of the reagent is attached to the inlet tube through the ground joint
connection.Slight air pressure is then applied to the third bottle to drive Downl from http://w.org/ b on Jun2014
1 The solvent(monomethylether of ethyleneglycol)is obtainablefrom the manu-
facturer(Carbideand CarbonChemicalsCorporation,30 East 42nd Street,New York
17) or supplier(e.g., AmendDrugand ChemicalCompany,117 East 24th Street,New
Each lot is tested for peroxidecontentbyYork10) relativelyfree from peroxides.
adding2 ml. of the solventto 1 ml. of freshlyprepared4 per cent aqueousKI.A
colorlessto light straw-yellowtest is satisfactory.
2 Evaporationfrom the concentratedsalt solutionmay cause crystallizationof so-
dium acetateon the syringeof the pipettingmachine(1).The exposedsectionof the
plungershouldbe wiped free of any appreciabledepositto preventstrainon the syr-
inge assembly.
S. MOORE AND W. H. STEIN 909
reagent remaining in the reservoir into the filling tube and to displace the
air therein.The air pressure is replaced by suction to draw the reagent
solution into the reservoir.Care should be taken not to introduce air into
the assembly at the end of the filling operation.
The solution is not as stable on storage as the previous reagent.After
1 month, the blank readings have about doubled, and, unless the bottle
has been stored in the dark, the color yields can run low by as much as 5
per cent. For this reason, not much more than a 1 week’s supply of re-
agent should be prepared at a given time.
Procedure (Cf. (1))
For the analysis of 1 ml. or 2 ml. effluent fractions from ion exchange
chromatograms(2), 1 ml. of the ninhydrin solution is used. The buffer is
sufficiently strong so that preliminary adjustment of the pH of the samples
is seldom necessary.If, however, more than 1 ml. of 0.1 N HCl or 0.1 N
NaOH should be required to bring the effluent fractions to pH 4 to 6, ad-
dition of acid or alkali may be necessary to bring the pH of the samples
to the optimum for color development.The capped tubes are shaken
briefly (
for 15 minutes (accurately timed) in a covered boiling water bath (1). It
is convenient to use 50:50 ethanol-wateras a diluent rather than n-pro-
panol-water(1). After dilution, the tubes (kept out of direct sunlight)
are wiped dry and transferredto a dry rack, cooled to below 30” in front
of an electric fan, and thoroughly shaken (about 30 seconds) before being
read at 570 mμ rnp for proline and hydroxyproline).Because the
modified reagent contains a higher concentrationof hydrindantin,more of
the reddish color of this compound remains at the end of the heating period.
In order to reduce the blank to the desired level, therefore, the tubes should
be shaken sufficiently so that the major portion of the residual hydrindan-
tin is oxidized by air. For this reason, the tubes are shaken uniformly in a
rack, rather than individually.With the modified reagent, the blank read-
ings for 2 ml. samples and 1 ml. of ninhydrin solution have been 0.05 to 0.10
on the optical density scale. If there is evidence that high blank readings
are being caused by uptake of NH3 during the period that the tubes are
standing on the fraction collector, the cover of the machine should be lined
with cloth impregnatedwith citric acid (7).3 If effluent fractionsare
stored prior to analysis, they should be stoppered with corks treated with
citric acid (7) and wrapped in aluminum foil.
The absence of tin salts in the reagent solution not only avoids the pre-
cipitation of tin phosphate when samples containing phosphate are ana-
3 If ammoniainterferes,it can be removedby renderingthe samplesalkalineand
placingthe tubes in a vacuumdesiccatorfor a few hours before the ninhydrinanaly-
ses (2). Downl from http://w.org/ b on Jun2014
910 PHOTOMETRIC NINHYDRIN METHOD
Calculations
A standard curve is plotted in the manner previously described (1) by
utilizing 1 ml. samples of leucine (0.05 to 0.2 InM) in 0.1 M citrate buffer
at pH 5. For use with chromatograms,two direct reading tables are pre-
pared in the format of Table I previously given (I), one for 1 ml. samples
and 1 ml. of ninhydrin solution, and the second for 2 ml. samples and 1 ml.
of the reagent.It is convenient to have twice the millimolar concentra-
tions listed in the table for the 2 ml. samples.In this way both tables
yield micromoles of amino acid directly, without the use of factors (( 1) Table V), when the values correspondingto the points of a given peak on an ef-
fluent curve are added. Fractions that are off the scale of the table (above
an optical density of 1.0 after dilution with 15 ml. of diluent) must be
diluted further by hand before being read. The values taken from the
final column of the table must then, of course, be multiplied by the appro-
priate dilution factor.When samples require neutralizationor, in pre-
parative chromatography,when aliquots from larger fractions are pipetted
(1): direct reading tables can be prepared for the special case, or appropriate volume corrections can be applied to the values read from a table for a
different fraction size. In computing the volume corrections, no factor is
required for the negligible loss by evaporation which occurs during the
heating of the aqueous samples (1).
The color yields obtained with a number of ninhydrin-positivesubstances likely to be encountered in biological systems are listed in Table I. The
values, as well as the color yields from peptides and proteins, are similar in
most cases to those obtained with the previous reagent (1, 3). The yields
were determined on 2 ml. samples of the compounds dissolved in the buffers in which they emerge from columns of Dowex 50-X4 in the current chro-
matographicprocedure (2). Although the color yields are reproducible
to f2 per cent in a given laboratory,the exact values should be checked
under the user’s experimental conditions if maximal accuracy is desired.
4 Transfer pipettes No. 283-R,Microchemical Company, 1834 Univer-sity Avenue, Berkeley 3, California. Specialties Downl from http://w.org/ b on Jun2014
MOORE AND W. H. STEIN 911
The molecular weight divided by the color yield provides the factor by
which the chromatographicresults (expressed in leucine equivalentsin
micromoles) should be multiplied to give micrograms of the substance in
TABLE I
Color Yields from Amino Acids and RelatedCompounds Basis Relative to
Leucine on Molar
Determinedon 2 ml. samplesof 0.1 mM solutionsin the buffers (pH 2.2 to 5) in which the compoundsemergein the Dowex50-X4 chromatographic procedure(2);heatingtime 15 minutes;read at 570 mu.(The urea and creatinine
were 3.0 mm.)
-
Compound Color yield Compound Color yield
Asparticacid 0.94 &NHz-isobutyricacid 0.44 Threonine 0.94 Carnosine
Serine 0.95 0.93
Citrulline 1.04
Proline(440 rnfi) 0.225* Creatinine 0.027 Glutamicacid 0.99 Cysteicacid 0.99 Glycine 0.95 Diaminopimelicacid(per 1.24 Alanine 0.97 2 NH2 groups)
Valine 0.97 Ethanolamine
Half cystine 0.55 Felinine
Methionine 1.02 Glutamine 0.91Isoleucine 1.00 Glucosamine
0.95
Leucine 1.00 Glutathione(oxidized,half)
Tyrosine 1.00 0.99
Glycerophosphoethanol- 1.03
Phenylalanine 1.00 amine
0.93Ammonia 0.97 Hydroxylysine
0.50Lysine 1.10 Hydroxyproline(440 rnp)
Histidine 1.02
Methioninesulfone
Tryptophan 0.94 “
Arginine 1.01
a-NHz-adipicacid sulfoxide
0.96 1-Methylhistidine 1.12
@-Alanine 0.50 3-Methylhistidine 0.077*Anserine 0.78 Ornithine
1.02
Asparagine 0.95 Phosphoethanolamine 0.98 a-NHz-n-butyricacid 1.02 Sarcosine 0.88 r-NHz-butyricacid 1.01
Taurine 0.86
Urea 1.12
0.43
0.28
0.88
0.0314
* The readingstakenat 440 rnp are first convertedto “leucineequivalents”by
using the same conversiontable that is employedfor the otheraminoacids meas-
ured at 570 rnp, and the concentrationsof the iminoacids are subsequentlyobtained
by dividingby the abovecolor yields.
question.Each of the first eighteen amino acids (Table I) was checked for
purity by elementary analysis and by chromatographyon Dowex 50. The
other compounds were commercial or research samples possessing the cor- rect elementary composition. Downl from http://w.org/ b on Jun2014
912 PHOTOMETRIC NINHYDRIN METHOD
DISCUSSION
The proportion of methyl Cellosolve in the reagent solution has been
raised from 50 per cent to 75 per cent in order to keep the increased quantityof hydrindantin in solution. The increase in the strength of the buffer has
required the use of sodium acetate (cf. Boissonnas (lo)), which is more sol-
uble in methyl Cellosolve, in place of sodium citrate. With the stronger
buffer, the pH at which maximal color yields are obtained in the ninhydrin
reaction is very similar to that employed previously.Although the 4 N
sodium acetate buffer is initially at pH 5.5, the pH drops to 5.1 when the
buffer is diluted to 1 N, which is the actual normality employed in the
reagent. With the previous reagent, maximal color yields were obtained
at pH 5.0 with a 0.2 M citrate buffer.
Satisfactory recoveries of proline from chromatograms were obtained
with use of the modified reagent (1) despite the low value of the color
yield. With hydroxyproline, however, which has a much lower color yield,
this would not be the case. Greater sensitivity for proline is exhibited by
the procedure of Chinard (1 l), which utilizes the red color formed with
ninhydrin in acid solution, and this method has been employed by Harfen-
ist (12) to analyze the effluent from Dowex 50-X8 chromatograms. The
special procedures of Neuman and Logan (13) and Troll and Cannan (4)
(cf. Rogers et al. (14)) provide more sensitive tests for hydroxyproline.
The authors wish to acknowledge the technical assistance of Mrs. Ger-
trude C. Carey and Miss Renate Mikk in the performance of these experi-
ments. Downl from http://w.org/ b on Jun2014
S. MOORE AND W. H. STEIN 913
SUMMARY
An extension of earlier studies on the determinationof amino acids and
related compounds with ninhydrin has led to the development of a modi-
fied reagent composed of 2 per cent ninhydrin and 0.3 per cent hydrindantin
The strengthN sodium acetate buffer (pH 5.5).in 3 : 1 methyl Cellosolve-
of the buffer eliminates or reduces the need for preliminaryadjustment of
the pH of the samples when the method is used for the analysis of effluent
fractions obtained in ion exchange chromatography,and the higher hy-
drindantin concentrationof the reagent solution permits economies in the
use of ninhydrin.
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