Properties of 2,4-dinitrophenyl derivatives of amino acids as analytical forms for high-performance liquid chromatography

Article abstract of DOI:10.1134/S1070427211060103

Dissociation constants of 2,4-dinitrophenyl derivatives of α-amino acids in micellar solutions of sodium dodecyl sulfate used as a micellar mobile phase in reversed-phase liquid chromatography were determined. The method of micellar liquid chromatography was used to determine the composition of polypeptide fractions of animal origin.

Full text of DOI:10.1134/S1070427211060103

ISSN 1070-4272, Russian Journal of Applied Chemistry, 2011, Vol. 84, No. 6, pp. 957–963. © Pleiades Publishing, Ltd., 2011.
Original Russian Text © A.P. Boichenko, O.S. Chernyshova, A.Yu. Kulikov, L.P. Loginova, 2011, published in Zhurnal Prikladnoi Khimii, 2011, Vol. 84,
No. 6, pp. 933–939.
PHYSICOCHEMICAL STUDIES
OF SYSTEMS AND PROCESSES
Properties of 2,4-Dinitrophenyl Derivatives
of Amino Acids as Analytical Forms
for High-Performance Liquid Chromatography
A. P. Boichenkoa, O. S. Chernyshovaa, A. Yu. Kulikova,b, and L. P. Loginovaa
a Karazin National University, Kharkov, Ukraine
b Ukrainian Scientific Pharmacopeic Center for Quality of Medicinal Preparations, Kharkov, Ukraine
Received September 3, 2010
Abstract—Dissociation constants of 2,4-dinitrophenyl derivatives of α-amino acids in micellar solutions of
sodium dodecyl sulfate used as a micellar mobile phase in reversed-phase liquid chromatography were
determined. The method of micellar liquid chromatography was used to determine the composition of
polypeptide fractions of animal origin.
DOI: 10.1134/S1070427211060103
Quantitative determination of amino acids occupies
an important place in chemical, biochemical, and
pharmaceutical analyses and is used to solve the
following problems: quantitative determination of
proteins, peptides, and medicinal preparations on their
basis; identity verification and structural analysis of
proteins or peptides; recording of atypical amino acids;
monitoring of peptide syntheses; detection of bio-
markers of various diseases [1–7]; and control over the
quality of pharmaceutical products and their
preservation [8, 9]. Although the first automated amino
acid analyzer was developed as far back as the mid-
XX century [10], the problem of a search for fast,
reproducible, and moderately expensive methods for
quantitative determination of amino acids remains
topical [5].
observed for tyrosine (absorption peak 275 nm),
tryptophan (279 nm), phenylalanine (257 nm), and
cystine (Cys)2 (240 nm), which can be used for their
determination [11]. Methods developed for deter-
mination of other acids employ pre- or post-column
derivatization to obtain detection-convenient analytical
forms. In the last case, additional special equipment is
to be used.
The search for reagents for derivatization of amino
acids is aimed to raise the hydrophobicity and suppress
dissociation of amino acids, introduce chromophore
groups, and improve the completeness of the reaction
between a reagent and an amino acid [6, 12]. One of
the first reagents for determination of amino acids was
dinitrofluorobenzene (DNFB), Sanger’s reagent, used
to determine the N-terminal amino acid in proteins
beginning at 1945 [13, 14]. The method of derivatiza-
tion of amino acids with DNFB remains important and
is continued to be used at present [15, 16]. However,
information about properties of 2,4-dinitrophenyl
derivatives (2,4-DNP) of amino acids, formed in the
process, is limited to data on their decomposition
under solar light and approximate values of dis-
sociation constants of some derivatives [17, 18].
The closest attention is presently focused on
development of procedures for amino acid analysis
with the standard chromatographic and electrophoretic
equipment [5]. However, this poses problems of
separation and detection, associated with the main two
difficulties: large difference in polarity between some
amino acids containing hydrophobic or hydrophilic
side groups with protolytic properties and small light
absorption coefficients of free amino acids in the UV
and visible spectral ranges. Among proteinogenic
amino acids, noticeable light absorption is only
Recently, several methods for separation and
concentration, based on use of liophilic nanosize dis-
persions of surfactants, have received wide
957

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BOICHENKO et al.
recognition: micellar chromatography, micellar extrac-
tion, and micellar electrokinetic chromatography [19–
22]. It has been shown previously that 2,4-dinitro-
phenyl derivatives of amino acids can be separated by
the method of micellar liquid chromatography with a
micellar mobile phase containing sodium dodecyl
sulfate (SDS) and additions of aliphatic alcohols or
carboxylic acids as modifiers [16].
The potentiometric cell comprised an ESL-63-07 glass
electrode and EVL-1M3 reference half-cell. In poten-
tiometric measurements in micellar solutions, an
additional salt bridge filled with a 1 M NH4NO3 solu-
tion in agar-agar gel was placed between a solution
under study and the reference solution to preclude
formation of the poorly soluble potassium dodecyl
sulfate. All the potentiometric studies were performed
at a temperature of 25.0 ± 0.1°C. The accidental error
in electromotive force measurements was 0.2 mV.
The goal of our study was to examine the protolytic
properties of 2,4-DNP-amino acids in micellar solu-
tions of sodium dodecyl sulfate and to assess the
influence exerted by the acidity of the medium and
light on the stability of the derivatives in micellar
solutions.
The method of potentiometric titration in micellar
0.10 M SDS solutions was used to determine the
dissociation constants of 2,4-DNP-amino acids at the
carboxy group and the corresponding α-amino acids;
the dissociation of the latter was also studied in
aqueous solutions with an ionic strength of 0.10 M
(NaCl). In micellar solutions, the ionic strength was
created with 0.10 M SDS.
EXPERIMENTAL
We used 2,4-DNP-derivatives of proteinogenic
amino acids of pure grade from the “Set of 2,4-di-
nitrophenyl derivatives” (Khimreaktivkomplekt) and
2,4-DNP-amino acids synthesized by the reaction of
free amino acids with 2,4-dinitrofluorobenzene
(Merck, Germany) at pH 9.18. The resulting deriva-
tives were purified by recrystallization from organic
solvents (acetone, ethanol, heptane). The structure of
the derivatives was confirmed by IR spectroscopic
data. We used glycine, leucine, threonine, valine, and
cysteine of pure grade and sodium dodecyl sulfate with
97% mass fraction of the main substance (Appli
Chem), additionally purified by recrystallization from
isopropyl alcohol [23]. NaOH solutions free of
carbonates were prepared by the known procedure
from a saturated NaOH solution [24] and standardized
against weighed portions of adipic acid, with phenol-
phthalein as indicator; solutions of HCl and acetic acid
were prepared by dilution of concentrated solutions of
HCl and acetic acid of chemically pure grade. The
solutions were prepared from carbonate-free twice
distilled water.
The concentration of free amino acids in a solution
being titrated was 1.0 × 10–2 M, and that of 2,4-DNP-
amino acids, 2.0 × 10–3 M. As titrants served sodium
hydroxide solutions of precisely known concentration,
which contained additions of NaCl or SDS maintaining
the ionic strength. The volume of a solution being
titrated was 20 ml. The titration curve was constituted
by 25 points; calculations were made with data
corresponding to extents to which a substance under
study was titrated in the range from 20 to 80%. The
pH-metric cell was calibrated against standard buffer
solutions with pH 1.68 and 4.01 before and after
titration; in this case, the calibration parameters were
found to be nearly invariable.
Chromatographic studies were carried out with a
Hewlett Packard 1050 liquid chromatograph (Agilent
Technologies, Waldbron, Germany) with an auto-
sampler, spectrophotometric detector with controllable
wavelength, and HP 3395 integrator. The separation
was made on a DeltaPak C18 column (5 μm, 150 ×
3.9 mm, Waters, United States; mobile phase, 0.040 M
solution of SDS; volume fraction of acetic acid, 15%;
column temperature 40°C; flow rate of the mobile
phase, 1 ml min–1; sample volume 30 μl).
Absorption spectra of 2,4-DNP-glycine were
measured using a KFK-3 concentration-type photo-
electric colorimeter (Zagorsk Optical-Mechanical Plant,
Russia) with 1-cm-long quartz cuvettes; the reference
solution contained all the components except that
under study. IR spectra of the synthesized amino acids
were measured with an Avatar 360 FTIR spectrometer
(Thermo Nicolet, United States) in pellets with
potassium bromide. Potentiometric measurements
were made by the compensation scheme (R 307
potentiometer, pH-121 pH meter as zero instrument).
The potentiometric titration data were processed
using CLINP 2.1 software (http://www.chemo.univer.-
kharkov.ua/kholin/clinp.html). The values of the loga-
rithms of the constants, obtained in parallel titrations,
were averaged using the previously suggested
approach [25]. The algorithm has been previously
implemented in the MATLAB 7.0 environment (http://
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PROPERTIES OF 2,4-DINITROPHENYL DERIVATIVES
959
A
A
(a)
(b)
τ, min
τ, min
Fig. 1. Absorption A at 360 nm of 2,4-DNP-glycine solutions vs. time τ at pH (a) 12.6 and (b) 1.4. (1) In the dark and (2) under
exposure to light.
mathworks.com) [26]. Values of pKa for 2,4-DNP-
amino acids in water were calculated by ACD/pKa
software (Advanced Chemistry Development, http://
www.acdlabs.com). Ancillary calculations were made
with Microsoft Excel (2002, Microsoft Corporation,
http://office.microsoft.com).
fast decomposition of all the 2,4-DNP-amino acids was
observed, becoming even stronger in an acid medium.
Hence follows that the most appropriate medium
for use of 2,4-DNP-amino acids is the acid medium in
which the acids are stable during a long time. Ho-
wever, all the studies should be performed without
exposure to solar light, and just this was done in all the
subsequent experiments.
An important factor in the case of pre-column
derivatization for a quantitative analysis is the stability
of the derivatives obtained. As test substances for a
study of the influence exerted by solar and UV light
and by the pH of the medium were chosen 2,4-DNP-
glycine, 2,4-DNP-leucine, 2,4-DNP-threonine, and
2,4-DNP-valine. The derivatives were dissolved in
micellar SDS solutions with a concentration of 0.10 M,
hydrochloric acid or sodium hydroxide were added to
adjust the pH, and the variation of the light absorption
in the course of time was monitored. The concentration
of the derivatives in the resulting solutions was about
8 × 10–5 M. For all the derivatives, similar time
dependences of the absorption by the solutions were
observed, and, therefore, kinetic dependences are
shown in Figs. 1a and 1b only for 2,4-DNP-glycine. In
micellar solutions with an alkaline medium, 2,4-DNP-
glycine is rather rapidly hydrolyzed both when
exposed to light and in the dark (Fig. 1a). When
solutions are stored in an acid medium in the dark, the
derivative is rather stable during a long time (Fig. 1b,
curve 1), whereas under exposure to light, it rapidly
decomposes (Fig. 1b, curve 2). Interestingly, the
decomposition rate of the derivative is even higher in
the last case than that in the alkaline medium (compare
Figs. 1a and 1b). The stability of 2,4-DNP-amino acids
in micellar solutions of SDS under artificial light from
a fluorescent lamp coincides with that of solutions
stored in the dark. Under UV light (365 nm), rather
The dissociation constants are important charac-
teristics of organic substances, necessary in analyses
for choosing an appropriate method and optimal
conditions of separation and assessing the possibility
of controlling the separation selectivity by changing
the acidity of the medium. In [27], an attempt was
made to determine the dissociation constants of 2,4-
DNP-amino acids. It was noted that the method of
potentiometric titration in aqueous solutions cannot be
used to determine the dissociation constants of 2,4-
DNP-amino acids because of their very low solubility
in water [27]. The spectrophotometric method based
on difference between the absorption spectra of
protonated and unprotonated forms of a substance
under study [24] was found to be the best suitable for
studies of 2,4-DNP-derivatives of α-amino acids [27].
As the number of carbon atoms separating amino and
carboxy groups becomes larger, the dissociation of the
latter affects to an increasingly low extent the state of
the chromophore 2,4-DNP-group, the absorption
ceases to depend on pH, and the spectrophotometric
technique for determining the dissociation constant
loses its information value. However, the difference
between the absorption spectra of forms with
undissociated and dissociated carboxy group is also
small for 2,4-DNP-derivatives of α-amino acids: the
maximum difference in the optical density for 2,4-
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BOICHENKO et al.
surfactants because of the solubilization [28]. It has
been found that, with 2,4-DNP-amino acids dissolved
in 0.10 M SDS, a derivative concentration sufficient
for potentiometric determination of the dissociation
constant can be reached [24]. The results obtained
when determining the dissociation constants of 2,4-
DNP-amino acids and the corresponding α-amino
acids at the carboxy group from potentiometric
titration data are listed in Table 1.
λ, nm
The dissociation constants of protolytes in aqueous
solutions are mixed constants because, due to the
calibration conditions of the pH-metric cell, they
include the activity of H+ ions. It is a common practice
to describe the dissociation of protolytes in micellar
solutions in terms of “apparent” dissociation constants
pKaiapp, which reflect the partial binding of conjugated
protolytic forms by the micellar pseudophase [29, 30]
and correspond to the following mass-action-law
expression:
Fig. 2. Absorption (ε) spectra of 8.3 × 10–5 M solutions of
2,4-DNP-glycine in the micellar medium of 0.1 M SDS. (λ)
Wavelength. pH: (1) 1.48 and (2) 11.91.
DNP-arginine in aqueous solutions was approximately
0.15 [27].
Figure 2 shows absorption spectra of the molecular
and dissociated forms of 2,4-DNP-glycine, obtained in
micellar 0.10 M solutions of SDS at pH 1.48 and
11.91, respectively. The absorption peak of the
undissociated form of 2,4-DNP-glycine is observed at
a wavelength of 355 nm, and that of the dissociated
form, at 365 nm. The maximum difference between the
optical densities in the spectra of two protolytic forms
does not exceed 0.08, which is insufficient for reliable
determination of the dissociation constants.
10−pH[HA]tot
pKaapp
,
=
[A]tot
where 10–pH is the activity of H+ ions, found poten-
tiometrically in the aqueous phase; [HA]tot = [HA]w +
[HA]m, total concentration of the protonated form in
the aqueous phase (subscript w) and in the micellar
pseudophase (subscript m); [A]tot = [A]w + [A]m, total
At the same time, the solubility of many com-
pounds substantially increases in micellar solutions of
Table 1. Dissociation constants of free amino acids and 2,4-DNP-amino acids in aqueous and micellar 0.10 M SDS solutions
Compound
Glycine
pKa, 0.10 M NaСl, P = 0.95, n = 3 рKaapp, 0.10 M SDS, P = 0.95, n = 3
ΔpKa=рKaapp – pKa
2.35±0.05 [31]
2.48±0.02
2.18 [31]
2.62±0.08 [31]
4.27±0.04
0.27±0.09
1.79±0.04
1.08±0.24
1.51±0.04
0.04±0.10
Leucine
Threonine
Valine
3.26±0.17 [31]
3.71±0.02
2.20±0.03
2,4-DNP-glycine
3.75±0.10a
2.8±0.1 [27]
3.79±0.01
2,4-DNP-leucine
2,4-DNP-threonine
2,4-DMP-valine
3.78±0.21a
5.03±0.12
4.04±0.10
4.79±0.14
1.25±0.24
0.69±0.14
1.02±0.24
3.35±0.10a
3.77±0.20a
3.1±0.1 [27]
2,4-DNP-cysteine
3.32±0.10a
4.46±0.10
1.14±0.14
a pKa values calculated by ACDLabs software.
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PROPERTIES OF 2,4-DINITROPHENYL DERIVATIVES
(a) (b)
961
τ, min
τ, min
Fig. 3. Separation chromatograms of derivatives of samples (a) 1 and (b) 2. (τ) Time.
concentration of the deprotonated form in the aqueous
phase and micellar pseudophase, related to the total
volume of the solution.
accordance with the error propagation rule. In-
terestingly, free amino acids and their 2,4-DNP-
derivatives are arranged in the same order of in-
creasing effect of the medium: ΔpKa grows in the order
glycine < threonine < valine < leucine (Table 1).
The data obtained in micellar solutions indicate that
introduction of a 2,4-DNP-substituent at the amino
group makes weaker the dissociation of the carboxy
group of amino acids, in agreement with published
data for aqueous solutions [27]. The difference
between the properties of an amino acid and its 2,4-
DNP-derivative in a micellar solution is the most
noticeable in the case of glycine. pKaapp is 2.62 for the
free amino acid and 3.79 for 2,4-DNP-glycine.
Previously, we have optimized the composition of
the micellar eluent that made it possible to separate a
mixture of 2,4-DNP-amino acids in the isocratic mode
by micellar liquid chromatography with new modifiers
of micellar eluents, aliphatic carboxylic acids [16]. In
doing so, we demonstrated that the best separation is
achieved at pH values of the mobile phase lower than 3
and assumed that 2,4-DNP-amino acids are in the
undissociated form at this acidity [16]. This
We assessed the effect of the micellar medium by
using the previously obtained data on dissociation
constants of amino acids in aqueous solutions and the
calculated dissociation constants of 2,4-DNP-amino
acids for aqueous solutions. It can be seen in Table 1,
that the strength of both free amino acids and 2,4-
DNP-amino acids decreases in micellar solutions of
SDS, which agrees with the known Hartley rule [32]
and is accounted for by the stronger binding of
undissociated forms of protolytes by micelles of the
anionic surfactant.
Table 2. Results of determination of the amino acid com-
position of samples of polypeptide fractions
Mass fraction, %
Amino acid
sample 1
1.6
sample 2
1.9
Threonine
Alanine, proline
2.0
1.5
Phenylalanine, leucine,
isoleucine
15.8
48.4
The difference between the dissociation constants
in micellar and aqueous solutions, ΔpKa = pKaapp–pKa,
is named the effect of the micellar medium [30]. The
confidence interval for ΔpKa was calculated in
Lysine
0.9
7.8
1.8
1.5
Unidentified components
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962
BOICHENKO et al.
assumption is fully confirmed by the results obtained
in the present study (Table 1).
(2) 2,4-Dinitrophenyl derivatives of amino acids are
sufficiently stable in the acid medium of micellar
solutions of sodium dodecyl sulfate in storage in the
dark and can be used as analytical forms for
quantitative determination of amino acids.
The data on the optimal composition of the micellar
eluent were used to determine the amino acid com-
position of two samples of a hydrolyzate of poly-
peptide fractions of animal origin. The acid hydrolysis
of the samples was performed by treatment with 6 M
HCl under heating on a water bath for 36 h, followed
by keeping at room temperature for 72 h. The resulting
mixture was neutralized with sodium carbonate,
methanol and then a buffer solution with pH 9.18 and
DNFB for derivatization were added, the mixture was
heated to 80°C on a water bath, and kept at this
temperature for 40 min.
(3) Use of micellar mobile phases with pH < 3 is
advisable as regards both the suppression of
dissociation of 2,4-dinitrophenyl amino acid deriva-
tives of amino acids to be separated, which favors their
retention, and the stability of analytical forms, because
the hydrolysis becomes faster with increasing pH.
(4) The strength of 2,4-dinitrophenyl derivatives of
amino acids decreases in micellar solutions of sodium
dodecyl sulfate, as also does that of free amino acids;
the effect of the micellar medium increases in the
order: glycine, threonine, valine, leucine.
The quantitative determination was made by the
external standard method. As the standard served a
mixture of amino acids derivatized under conditions
identical to those of the hydrolyzate sample under
study. In this way, we secured the same yield of the
product in the standard solution and a sample under
study.
(5) The micellar liquid chromatography with
mobile phases based on sodium dodecyl sulfate, used
to separate 2,4-dinitrophenyl derivatives of amino
acids, made it possible to determine the composition of
two polypeptide fractions of animal origin.
Peaks in chromatograms were identified by
comparison with chromatograms of solutions contain-
ing separate commercial preparations of 2,4-DNP-
amino acids: DNP-DL-α-aspartic acid, DNP-L-glu-
tamic acid, DNP-DL-threonine, DNP-glycine, DNP-
DL-proline, DNP-DL-methionine, DNP-D-valine,
DNP-D-tryptophan, DNP-leucine, (DNP)2-DL-tyro-
sine, DNP-DL-arginine, ε-DNP-DL-lysine, DNP-α-
alanine, α-DNP-histidine, and DNP-α-phenylalanine.
Before being introduced into the column, solutions
of derivatives were filtered through a paper filter.
Figures 3a and 3b show chromatograms of derivatives
of samples 1 and 2, respectively. According to the
identification results, the peak with a retention time of
about 6 min corresponds to a sum of alanine and
proline, and that with a retention time of about
23.5 min, to a sum of phenylalanine, leucine, and
isoleucine; also, the contents of threonine and lysine
were determined. The mass fraction of unidentified
components in terms of glycine was 7.8% for sample 1
and 1.5% for sample 2 (Table 2).
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