ARTICLE IN PRESS

JOURNAL OF
FOOD COMPOSITION
AND ANALYSIS
Journal of Food Composition and Analysis 19 (2006) 112–117
www.elsevier.com/locate/jfca

Original Article

Efficiency improvements on ninhydrin method for

amino acid quantification
Shih-Wen Suna, Yi-Cheng Lina, Yih-Ming Wenga, Min-Jane Chenb,
a
Department of Food Science, National Chiayi University, 300 University Road, Chiayi (600), Taiwan, ROC
b
Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi (600), Taiwan, ROC
Received 22 September 2004; received in revised form 12 April 2005; accepted 13 April 2005

Abstract

The ninhydrin method was introduced for quantitative determination of amino acids in the late 1940s. During the past several
decades, considerable modifications including different heating times, heating temperatures, buffer systems, pH values of buffer
solutions and solvents for ninhydrin reagent have complicated this method. To simplify the diverse available protocols and to
enhance efficiency, the effects of heating time and pH values on the commonly used lithium hydroxide/acetic acid buffer system were
investigated. Furthermore, the suitability of using sodium hydroxide/acetic acid buffer to replace lithium hydroxide/acetic acid
buffer was also evaluated. The results show that relatively inexpensive sodium hydroxide/acetic acid buffer system or as simple as
sodium acetate solution could be used in the ninhydrin method. The results also showed that a shorter heating time of 10 min could
achieve a comparable degree of color development as the traditionally suggested heating time of 30 min. The improvements of the
ninhydrin method presented in the study make this method even more convenient, less expensive, and less time consuming for
quantification of compounds containing amino group.
r 2005 Elsevier Inc. All rights reserved.

Keywords: Ninhydrin method; Amino acid quantification

1. Introduction al., 2000; Amin et al., 2000), and quantification of

collagen-like polymer in microbial cell lysate (Yin et al.,
Ninhydrin method was introduced for quantitative 2002) have also been reported, indicating a continued
determination of amino acids in the late 1940s (Moore popularity of this method.
and Stein, 1948). Although originally developed for Although instrumental methods such as HPLC are
chromatographic elution from amino acid analyzer currently used for determining compounds containing
(Moore and Stein, 1948, 1954), this method has also amino group, the simple and convenient ninhydrin
been adapted for determination of amino group method still possess several advantages because no
containing compounds in foods as well as various types expensive equipment is required, and it is suitable for
of samples (Bailey, 1962; Hirs, 1967; Alexander et al., the routine analysis of large numbers of samples. To
1985; Hurst et al., 1995; Panasiuk et al., 1998). Recently, make this method more safe and convenient, several
the applications of ninhydrin method for the evaluation major modifications have been made over the past
of chitosan and deacetylation of chitin (Prochazkova et several decades. One of the more important improve-
al., 1999; Curotto and Aros, 1993), determination of ments was the replacement of flammable solvent
amino compounds in pharmaceutic products (Frutos et cellulose acetate with dimethyl sulfoxide (DMSO)
(Moore, 1968). However, considerable variations in
Corresponding author. Tel.: +886 5 2717611; fax: +886 5 2717901. experimental conditions involving different heating
E-mail address: [email protected] (M.-J. Chen). times (from 5 to 45 min), heating temperatures (from

0889-1575/$ - see front matter r 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2005.04.006
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S.-W. Sun et al. / Journal of Food Composition and Analysis 19 (2006) 112–117 113

85 to 100 1C), buffer systems (phosphate, sodium mixture was bubbled with nitrogen for at least 2 min,
acetate, lithium acetate, potassium acetate, sodium sealed and stored in a refrigerator (4 1C). Fresh
citrate, etc.), pH values (5–8) of buffer solutions and ninihydrin solution was prepared on each working day.
solvents (DMSO, absolute alcohol, acetone, ethylene Aqueous solutions of lithium acetate (4 N) or sodium
glycol, water) for ninhydrin have created rather acetate (4 N) were also used to mix with ninhydrin/
complicated situations (Moore and Stein, 1948; Hirs, DMSO to obtain the ninhydrin solution for determina-
1967; Curotto and Aros, 1993; Brewer et al., 1995; Hurst tion of amino acid concentrations.
et al., 1995; Fang and Liu, 2001; Laskar et al., 2001;
Starcher, 2001; Frutos et al., 2000; Yin et al., 2002).
2.3. Testing procedures and statistical analysis
Furthermore, in a few reports there are some question-
able components still appearing. For example, although
Amino acid solution (1 mL) and ninhydrin solution
stannous chloride had been removed from the reaction
(1 mL) were put in screw-capped test tubes and heated in
mixture (Moore and Stein, 1954), this compound was
a boiling water bath for a pre-determined period of time.
still used in some reagent solution systems (Starcher,
After heating, the tubes were immediately cooled in an
2001; Yin et al., 2002); toxic cadmium salt was used in
ice-bath. Then, 5 mL of 50% alcohol was added into
the ninhydrin test by some researchers (Fisher et al.,
each tube and thoroughly mixed with a Vortex mixer for
2001).
15 s. The absorbance (570 nm) of the reaction mixture
Although various combinations of experimental con-
was measured with a spectrophotometer (Model 340,
ditions are found in the literature, there is no
Sequoia-Turner Co., Taiwan). Two independent experi-
comprehensive research conducted to compare the
ments each with duplicate samples were conducted and
influence of those conditions on the ninhydrin analysis.
the reported values were the means of the two
To simplify the complicated experimental conditions
experiments. Linear regression was performed with
and to improve the analysis efficiency, the effects of
commercial statistical software on a personal computer.
heating time, pH of the buffers and the composition of
buffers on the color development in the ninhydrin test
were evaluated in this report.
3. Results and discussion

2. Materials and methods 3.1. The effects of heating time on the color development

2.1. Chemicals Since lithium hydroxide/acetic acid buffer (Moore,

1968) was introduced later than sodium hydroxide/
Amino acids (alanine, aspartic acid, glutamic acid, acetic acid buffer (Moore and Stein, 1954) for ninhydrin
histidine, isoleucine, lysine, methionine, tyrosine, and analysis, the lithium hydroxide/acetic acid buffer was
valine) were purchased from Aldrich Co. (Milwaukee, chosen to establish the kinetics of color development. In
WI, U.S.A). Hydrindantin, lithium hydroxide and the preliminary study, the amino acid/ninhydrin mixture
glucosamine were purchased from Sigma Co. (St. Louis, was heated for 30 min, which is a frequently reported
MO, U.S.A). DMSO was from Merck (Darmstadt, heating time for ninhydrin method in the literature
Germany). Ninhydrin was from Riedel-de Haen (Seelze, (Panasiuk et al., 1998; Prochazkova et al., 1999). Since
Germany). Glacial acetic acid and sodium hydroxide the absorbance of the amino acid/ninhydrin mixture
were purchased from Hayashi Pure Chemical Ltd. exceeded 1 (data not shown) for most of the amino acids
(Osaka, Japan). at the concentration of 50 mg/mL (the upper concentra-
tion limit used in this research), it indicated that the
2.2. Preparation of ninhydrin solution 30 min heating time might be longer than needed. As a
shorter heating time of 10 min was also reported
The lithium acetate buffer (4 N) was prepared by (Starcher, 2001), it was feasible to investigate the effects
adding glacial acetic acid into lithium hydroxide of shorter heating times on the color development.
solution as described in previous reports (Moore, The reaction mixture of amino acid and ninhydrin
1968; Prochazkova et al., 1999). For the preparation solution was heated in a boiling water bath from 5 to
of sodium acetate buffer (4 N), sodium hydroxide was 30 min, and the effects of heating time on the color
used to replace lithium hydroxide. If needed, sodium development of a typical amino acid (alanine) are
hydroxide (6 N) and hydrochloric acid (6 N) were used to illustrated in Fig. 1. While practically no difference
adjust the pH of the buffers. Ninihydrin (2 g) and could be detected for different heating times, linear
hydrindantin (0.3 g) were dissolved in 75 mL DMSO relationships (R2 40:97) between absorbance and amino
under a stream of nitrogen gas. After adding 25 mL acid concentration (up to 50 mg/mL) were found for all
lithium acetate buffer or sodium acetate buffer, the heating treatments. For this particular amino acid, a
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114 S.-W. Sun et al. / Journal of Food Composition and Analysis 19 (2006) 112–117

Fig. 1. Effects of heating time on the color development in ninhydrin Fig. 2. Relationships of amino acid concentration and absorbance in
method with lithium hydroxide/acetic acid buffer (pH 5.30). ninhydrin method with lithium hydroxide/acetic acid buffer (pH 5.30)
and heating time 10 min.

heating time of 5 min seems to be enough for color early researchers can thus be modified to a shorter
development. period of time.
Amino acids are frequently classified according to the
type of side chain, i.e., aliphatic, sulfur-containing, 3.2. The effects of buffer pH on the color development
aromatic, acidic, amide, or basic amino acids. To verify
the reaction conditions, alanine, aspartic acid, glutamic The pH of lithium hydroxide/acetic acid buffer for the
acid, histidine, isoleucine, lysine, methionine, tyrosine, conventional ninhydrin method has been suggested at a
and valine were chosen to represent amino acids relatively narrow range of 5.2–5.3 (Moore, 1968;
containing different side chains. Further testing with Prochazkova et al., 1999). Since the effects of the buffer
different amino acids showed that 5 min heating might pH on the color development in the ninhydrin method
not be enough for some amino acids (for example, have not been thoroughly investigated and different pH
aspartic acid, histidine, and tyrosine) at a concentration buffers have been used to carry out the ninhydrin
of 50 mg/mL to reach an absorbance near 1.00 (data not reaction, one of the objectives of the present research
shown). Therefore, the results of the absorbance for was designed to reveal the effects of pH. The pH values
reaction mixture heated in the boiling water bath for of lithium hydroxide/acetic acid buffer were adjusted to
10 min are shown in Fig. 2. While different amino acids approximately 8, 7, and 6. While the absorbance for pH
showed different degrees of color development, the 7 and pH 8 was about the same (Fig. 3), the highest
linear relationships (R2 40:98) between absorbance and absorbance was obtained at pH 6 rather than at pH 5.3.
amino acid concentrations were still observed for all The linear relationships (R2 40:98) between absorbance
amino acids. Based on the current results, it is suggested and amino acid concentrations were found for all four
that a 10-min heating period is appropriate for pH values tested. Thus, the results indicate that a wider
ninhydrin method in quantification of amino acids. pH range could be used in the ninhydrin test. It suggests
Compared with previously published reports which that the pH value could be more flexible while
used longer heating time to obtain required absorbance, conducting the ninhydrin analysis.
one of the possible reasons for the lower intensity of
color development in those previous reports might be 3.3. Sodium hydroxide/acetic acid as the buffer system
the purity of the chemicals used (Fruchter and Crest-
field, 1965; Moore, 1968). Industrial grade instead of Although lithium hydroxide/acetic acid (Moore, 1968;
analytical grade dimethyl sufloxide was used in Moore’s Brewer et al., 1995; Prochazkova et al., 1999) as well as
report (1968). Other chemical residuals resulting from sodium hydroxide/acetic acid (Moore and Stein, 1954;
protein hydrolysis and column chromatography might Hirs, 1967) have been used as the buffer systems for
also interfere with the color development (Fruchter and ninhydrin method, there are no specific preferences cited
Crestfield, 1965). The 30-min heating time suggested by in the literature. From an economic point of view,
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S.-W. Sun et al. / Journal of Food Composition and Analysis 19 (2006) 112–117 115

Fig. 3. Effects of pH on the color development in ninhydrin method Fig. 4. Effects of heating time on the color development in ninhydrin
with lithium hydroxide/acetic acid buffer. Heating time: 10 min. method with sodium hydroxide/acetic acid buffer (pH 5.32).

lithium hydroxide is not a common chemical in

laboratory and is much more expensive. On the other
hand, sodium hydroxide is inexpensive and readily
available in almost all laboratories. Therefore, the
influences of lithium hydroxide/acetic acid buffer and
sodium hydroxide/acetic acid buffer on the color
development were studied to evaluate the feasibility of
replacing lithium acetate with sodium acetate as the
buffer system for the ninhydrin method. Although with
slightly decreased absorbance (Fig. 4), the color devel-
opment in sodium hydroxide/acetic acid buffer system
was almost the same as the color development in lithium
hydroxide/acetic acid buffer system (Fig. 1). While
linear relationships (R2 40:98) between absorbance
and amino acid concentrations were observed for all
different heating times, it should be noted that heating
time of 30 min resulted in a slight decrease in the
absorbance compared to other shorter heating times
(5–25 min). The feasibility of replacing lithium hydro-
xide with sodium hydroxide was further confirmed in Fig. 5. Relationships between absorbance and amino acid concentra-
tion in ninhydrin method with sodium hydroxide/acetic acid buffer
quantification analysis of other amino acids (Fig. 5).
(pH 5.32) and heating time 10 min.
With heating time set at 10 min for comparison purpose,
the linear relationships (R2 40:97) between absorbance
and amino acid concentrations were still observed for all 8.99, respectively, the quantification of amino acid was
amino acids used in this study. The results suggest that it conducted with 4 N lithium acetate or sodium acetate
is feasible to use sodium hydroxide/acetic acid as the replacing lithium hydroxide/acetic acid buffer for the
buffer system in the ninhydrin method. preparation of ninhydrin solution. The absorbance of
reaction mixture resulting from amino acid and ninhy-
3.4. Lithium acetate or sodium acetate as the salt drin is shown in Fig. 6. The linear relationships
solutions (R2 40:98) of absorbance against amino acid concentra-
tion up to 50 mg/mL were established for all tested
Since the ninhydrin method could be carried out in a amino acids when lithium acetate (4 N) was used as the
wider range of pH (5–8) and the pH values of 4 N lithium salt solution. Similar results with R2 40:99 were
acetate and 4 N sodium acetate solutions are 8.22 and obtained when sodium acetate (4 N) was used as the salt
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116 S.-W. Sun et al. / Journal of Food Composition and Analysis 19 (2006) 112–117

could achieve the comparable degree of color develop-

ment as the mixture heated for as long as
30 min. Furthermore, the linear correlation is improved
together with the upper limit of application of the
method when salt solutions were used. The modifica-
tions presented in this study make ninhydrin method
even more convenient, less expensive, and less time
consuming for quantification of compounds containing
amino group.

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