American Journal of Analytical Chemistry, 2012, 3, 669-674
http://dx.doi.org/10.4236/ajac.2012.39088 Published Online September 2012 (http://www.SciRP.org/journal/ajac)
Separation of Amino Acids Based on Thin-Layer
Chromatography by a Novel Quinazoline Based
Anti-Microbial Agent
Supriti Sen*, Sandipan Sarkar*, Pijush Kundu, Subrata Laskar
Department of Chemistry, The University of Burdwan, Golapbag, India
Received March 14, 2012; revised May 30, 2012; accepted June 9, 2012
ABSTRACT
A newly designed quinazoline based compound, 6-pyridin-2-yl-5,6-dihydro-benzo[4,5]imidazo[1,2-c] quinazoline
(PDBIQ) has shown the ability for the easy detection of nineteen amino acids on thin-layer chromatography plates as a
spray reagent. This new reagent enabled to produce various distinguishable colors with amino acids with different RF
values. The detection limits and the binding ability of PDBIQ with amino acids have been calculated. PDBIQ is also
able to detect aminoacids from hydrolised seed protein. The title compound also exhibited profound inhibitory action
against some gm (+ve) and gm (−ve) bacterial organisms. This paper deals with synthesis, spectroscopic application and
biological evaluation of the organic moity.
Keywords: Thin-Layer Chromatography; Amino Acid; Ninhydrin; Binding Constant; Antimicrobial Property
1. Introduction
The chemistry of Quinazolines class compounds are very
promising because it shows wide spectram of biological
activity like analgesic and anti-inflammatoryanti, antim-
icrobial, antihypertensive, anticancer [1-4] etc. activities.
Because of such enriched chemistry we are interested in
evaluation and application of quinazoline compound. The
detection or identification of amino acids is extremely
important in biomedical and biochemical analysis for the
evaluation of protein structure as the amino acids are the
monomeric units of proteins; these amino acids are used
by cells for protein biosynthesis, and also exist in the free
state in numerous natural products (seeds and leaves) and
as the C-terminal determination of degraded proteins. Sev-
eral specific and non-specific reagents have been reported
on thin-layer chromatography (TLC) plates [5-10]. Such
identification is the most well-known reagent is ninhy-
drin which is widely used for its remarkable high sensi-
tivity. But, it produces same purple/violet color with all
amino acids except proline and hydroxyproline. An attempt
has been established to overcome this color problem us-
ing 6-pyridin-2-yl-5,6-dihydro-benzo[4,5]imidazo[1,2-c]
quinazoline (PDBIQ)-ninhydrine as a new reagent which
affords distinguishable colors with twenty two protein ami-
no acids and, enables convenient and easy detection of such
compounds on silica gel “G” for TLC with very good sensi-
tivity (detection limit between 0.1 - 0.5 μg at cold condi-
tion and 0.05 - 0.2 μg after heating).
Herein we report an account on the systematic applica-
tion of a newly designed quinazoline based spraying re-
agent (PDBIQ) for the detection of amino acids at trace
level along with the equilibrium binding constant (k) with
different amino acids and bio-activity test against some
gm (+ve) and gm (−ve) bacterial organisms.
2. Experimental
2.1. Apparatus and Materials Used
Thin layer chromatography plates (20 × 20 cm, thickness
0.1 mm) were prepared using silica gel “G” (Merck, In-
dia) and a Unoplan coating apparatus (Shaudon, London,
UK). Sample solutions were spotted on to the plates by
means of a graduated micropipette (5.0 μL). Electronic
absorption spectra were recorded on a JASCO UV-Vis/NIR
spectrophotometer model V-570.
Pyridine-2-carboxylaldehyde and 2-(2-aminophenyl)
benzimidazole for the synthesis of the title compound
(PDBIQ) were purchased from Aldrich. Standard amino
acids and ninhydrin were procured from Sigma (USA)
and n-propanol from Merck (India). All other chemicals
and solvents were used as received. The spraying reagent,
6-pyridin-2-yl-5,6-dihydro-benzo[4,5]imidazo[1,2-c]quina-
zoline (PDBIQ) was synthesized in our laboratory as de-
scribed below.
*Corresponding authors.
C
opyright © 2012 SciRes. AJAC

S. SEN ET AL.
670
2.2. Synthesis of 6-Pyridin-2-yl-5,
6-dihydro-benzo[4,5]imidazo[1,2-c]
quinazoline (PDBIQ)
An ethanolic solution of 2-(2-aminophenyl)benzimida-
zole, (2.09 g, 10.0 mmol) was added to pyridine-2-carbo-
xylaldehyde (1.07 g, 10.0 mmol) in ethanol (25.0 mL) at
room temperature. Then this mixture was allowed to re-
flux for 4.0 h. The white colored crystalline precipitate of
the compound (PBBIQ) was obtained from the yellow col-
ored solution through slow evaporation of the solvent in
few days. The single crystals of L suitable for X-ray crys-
tallography were also obtained from the methanolic solu-
tion of the white colored product on slow evaporation at
room temperature. These single crystals have been used
in the experiments.
C19H14N4: Anal. Found: C, 76.56; H, 4.75; N, 18.49;
Calc.: C, 76.48; H, 4.73; N, 18.78. m.p. 231˚C ± 1˚C, MS:
[M + H]+, m/z, 299.34; IR (KBr, cm−1):
ν
N-H, 2950,
ν
C=N,
1477;. 1H NMR (δ, ppm in dmso-d6): 8.437 (d, 1H, j =
3.9); 7.906 (d, 1H, j = 7.2); 7.768 - 7.697 (m, 2H); 7.631
(d, 1H, j = 7.2); 7.351 - 7.096 (m, 7H); 6.853 - 6.769 (m,
2H); Yield: 90%.
2.3. Detection of Amino Acids on TLC Plates
Standard solutions (1 mg/ml) of amino acids were pre-
pared in 0.01 M phosphate buffer (pH 8.0) and spotted on
the TLC plates. Spotting volume was always 1 µL; the
solutions were diluted approximately when necessary. Pla-
tes were air-dried and subjected to TLC using n-propanol-
water, 70 + 30 (v/v) as mobile phase. After development
plates were dried and sprayed with 0.01% PDBIQ in ethyl
alcohol (Reagent 1) and again dried in air for complete
evaporation of solvent. The plates were then sprayed with
0.25% ninhydrin in acetone (Reagent 2), dried in air and
colors were noted Table 1. The plates were then heated at
110˚C for 10 min in an oven and the colors were recorded
again. Colors were always observed visually. Detection
limits for the amino acids [11] after use of ninhydrin alone
are also given in Table 1.
Table 1. Formation of color by amino acids using PDBIQ and ninhydrin reagents, de tection limits for these reagents and for
ninhydrin alone on silica gel with n-pr opanol-w a te r 70:30 as mobile phase.
Cold condition After final heating
Amino acids Observed colors Detection limit (μg)Observed colors Detection limit (μg)
Detection limit for ninhydrin
(μg) (RF)
Glycine Deep orange 0.5 Deep pink 0.1 0.01 (0.03)
Alanine Pinkish violet 0.1 Light pink/milky pink0.1 0.009 (0.22)
Valine Reddish pink 0.1 Reddish pink 0.05 0.01 (0.14)
Leucine Bluish violet 0.5 Violetish pink 0.1 0.01 (0.09)
Isoleucine Very light violet 1.0 Light violet 0.1 0.20 (0.32)
Serine Deep pink 0.1 Deep bluish pink 0.1 0.008 (0.38)
Threonine Yellowish orange/ivory 0.5 Yellowish pink/candy0.1 0.05 (0.28)
Aspartic acid Yellowish violet 0.2 Greyish violet 0.1 0.10 (0.12)
Aspargine Light yellow/pale cream 1.0 Greyish yellow 0.1 0.10 (0.45)
Glutamic acid Light violet 0.5 Light violet 0.1 0.04 (0.33)
Glutamine Light violet 0.5 Light violet 0.2 0.10 (0.38)
Lysine Reddish violet 0.2 Brick red 0.1 0.005 (0.42)
Histidine Yellowish violet 0.1 Yellowish pink/petal0.1 0.05 (0.18)
Arginine Light pink/mauve 0.5 Pink 0.1 0.01 (0.05)
Phenyl alanine Orangish violet 1.0 Greyish pink 0.2 0.05 (0.58)
Tyrosine Light violet 1.0 Light pink 0.1 0.03 (0.51)
Tryptophan Greyish violet 0.5 Pinkish violet 0.1 0.05 (0.55)
Cysteine Yellowish violet 2.0 Pinkish violet 1.0 0.02 (0.41)
Cystine Very light pink 2.0 Light pink 1.0 0.01 (0.35)
Methionine Lilac/bluish violet 0.5 Bluish violet 0.2 0.01 (0.48)
Proline Light yellow/off white 1.0 Grey/beige 0.2 0.10 (0.22)
Hydroxy proline Pinkish violet 0.2 Yellowish brown 0.1 0.05 (0.34)

S. SEN ET AL. 671
2.4. Determination of Equilibrium Binding
Constant
This experiment was carried out at pH 8.0 (phosphate buf-
fer) with a standard solutions (1 × 10−5 (M)) of aminoac-
ids. The solution of PDBIQ was prepared in ethanol with
a concentration of fifty times higher than that of the amino
acids [12]. Absorption titration experiment (as given in
Figure 1 as example) was performed varying the amino
acids concentration and concentration of the PDBIQ was
kept constant. In order to illustrate the binding strength of
PDBIQ with different amino acids, the equilibrium bind-
ing constant (k) was determined from the spectral titra-
tion data using the following equation.
[
]
()
[] ()()
Aminoacid
Aminoacid 1
af
bf
εε
εε
−
=−kbf εε

−

ε
f and
ε
b represent the extinction coefficients for the free
and fully bound amino acids complex.
ε
a is the extinction
coefficients during each addition of amino acids. The
[
]
()
Aminoacid εε −
af
plot against
[
]
Aminoacid gave
a linear relationship as shown in (Figure 2) for cysteine
for example. From these graphical plots, the slope of the
straight lines was determined to calculate the binding con-
stants (k) for the corresponding amino acids with PDBIQ.
2.5. Application—Detection of Amino Acids
Present in A. excelsa Seed Protein
The seed protein (5.0 mg) was hydrolysate with 8 mol·L−1
HCl in an evacuated sealed glass tube for 24 h at 110˚C
in a temperature controlled oven. Then the hydrolysate
was filtered and excess HCl was removed under reduced
pressure at 40˚C - 50˚C. Traces of HCl (if any) was re-
moved from the thin film of hydrochlorides of amino acids,
by placing it in a vacuum desiccator over solid anhydrous
KOH for 36 h. Finally, it was dissolved in 1 mL 10% n-
propanol. The finally obtained protein hydrolysate and
Figure 1. Electronic spectral titration of PDBIQ with cysteine.
[
]
Figure 2. Plot of ab Cysteine −εε vs.
[
]
Cysteine for the
absorption titration to deter mine binding constant.
amino acid standards were spotted on a TLC plate as above
said with n-propanol-water 70:30 (v/v) as mobile phase.
The plates were then dried, sprayed with a with 0.01%
6-pyridin-2-yl-5,6-dihydro-benzo[4,5]imidazo[1,2-c]qui-
nazoline (PDBIQ) in ethanol (Reagent 1),air dried, hea-
ted to 110˚C for 10 min, then sprayed with 0.25% ninhy-
drin solution (Reagent 2) in acetone. The plates were again
air dried and the colors were noted. Finally, the plates
were again heated to 110˚C for 10 min and colors noted
again. From the observed colors of the amino acids (both
seed protein hydrolysate and amino acid standards) and
also by comparing RF values with those of the amino acid
standards, it was possible to identify fourteen amino ac-
ids present in the seed protein of A. excelsa.
2.6. Inhibitory Test with PDBIQ Reagent
Antimicrobial testing was performed by cup plate method
[13]. All cultures were routinely maintained on NA (nu-
trient agar) and incubated at 37˚C. The inoculums of bac-
teria were performed by growing the culture in NA broth
at 37˚C for overnight. 27 mL of molten agar was added
to sterile Petri dishes and allowed to solidify for 1 h. The
bacterial suspensions (107 cell/mL) were spread uniformly
on the top of the agar medium by a sterilized glass spreader.
Six millimetre wide bores were made on the agar using a
borer. The solutions of PDBIQ (1000 mg/mL) were added
into each of the bores using a sterile tip with micropipette.
The plates were then incubated at 37˚C for 24 h. The
fungal strains were grown and maintained on Sabouraud
glucose agar plates. The plates were incubated at 26˚C
for 72 h. The degree of inhibition zone formed against
some gm (+ve) and gm (−ve) bacterial organisms. The
zone of the clearance around each bore after the incuba-
tion period confirms the antimicrobial activity. The clear
zones formed around each bore were measured and av-
erage diameter of the inhibition zone was calculated and

S. SEN ET AL.
672
expressed in millimeter.
3. Results and Discussion
3.1. Synthesis and Characterization of PDBIQ
The spraying reagent, 6-pyridin-2-yl-5,6-dihydro-benzo
[4,5]imidazo[1,2-c]quinazoline (PDBIQ) was synthesiz-
ed by refluxing 2-(2-aminophenyl)-benzimidazol and
pyridine-2-carboxaldehyde in equi-molar ratio in metha-
nol viz. Scheme 1. The rearranged white product (PDBIQ)
was obtained as the end-product. The structural analysis
by spectroscopic tools and the X-ray crystallographic tools
has confirmed this cyclic rearranged product. The solid
state structure of PDBIQ has already been reported in the
literature [9], and for that reason we are not describing
the structure here, and the solid state structure of PDBIQ
has been included in supplementary file as Figure S1.
3.2. Detection of Amino Acids
It is observed from Table 1 that detection limits obtained
after uses of PDBIQ are very low in both cases before
heating (0.1 - 2.0 μg) and after heating (0.05 - 1.0 μg) and
various distinguishable colors were produced. Sometimes
the detection limit is same before and after heating and in
other cases it is somewhat different. It should be noted
that identification of amino acids by ninhydrin is in prac-
tice difficult, in spite of the high sensitivity of ninhydrin.
So the new spray reagent unable to differentiate the amino
acids by color reaction. The mechanism of the color for-
mation is still uncertain, but we may assume that carbox-
ylic group of aminoacids first condensed with PDBIQ (hea-
ting at 90˚C for 10 min) to form a carbamide type inter-
mediates which form charge transfer complexes with nin-
hydrin.
3.3. Determination of Equilibrium Binding
Constant
The equilibrium binding constant of PDBIQ with differ-
ent amino acids was given in Table 2. The values are quite
high. This high value indicates that there are some inter-
action between PDBIQ and amino acids through charge
transfer transition. The Job’s plot Figure 3 shows the
maximum 1:1 adduct formation which confirms the ratio
of adduct formation between ligand and amino acids dur-
ing charge transfer transition.
NNH
NH2N
OHC MeOH N
N
H
N
N
+Reflux
Rearr angement
PDBIQ
Scheme 1. Synthetic strategy of the reage nt PDBIQ.
Table 2. Equilibrium bindi ng constants (k) of the molecular
complexes between organic reagent and amino acids at pH
8.0 (phosphate buffer) at 25˚C.
Amino acids k [dm3·mole−1]
L-Glycine 1.25 × 106
L-Alanine 1.32 × 106
L-Valine 0.89 × 106
L-Leucine 5.47 × 106
L-Isoleucine 3.94 × 106
L-Serine 2.56 × 106
L-Threonine 1.66 × 106
L-Aspartic acid 0.72 × 106
L-Aspargine 1.21 × 106
L-Glutamic acid 3.40 × 106
L-Glutamine 2.72 × 106
L-Lysine 1.72 × 106
L-Histidine 5.40 × 106
L-Arginine 3.34 × 106
L-Phenyl alanine 1.53 × 106
L-Tyrosine 0.86 × 106
L-Tryptophan 1.87 × 106
L-Cysteine 0.79 × 106
L-Cystine 8.52 × 106
L-Methionine 8.89 × 106
L-Proline 5.23 × 106
L-Hydroxy proline 4.04 × 106
3.4. Use of the Method for TLC Detection of the
Amino Acids Present in A. excelsa Seed
Protein
It was found that spraying with a 0.01% solution of 6-
pyridin-2-yl-5,6-dihydro-benzo[4,5]imidazo[1,2-c]quinazo-
line (PDBIQ) in ethanol combined with spraying with
0.25% ninhydrin solution in acetone enabled detection of
fourteen amino acids-arginine, isoleucine, glutamine, lysine,
asparagine, phenylalanine, serine, alanine, glutamic acid,
valine, aspartic acid, leucine, glycine, and proline even at
low concentration of the amino acids. The results were also
agreement with the respective RF values of the acids.
3.5. Inhibitory Test with PDBIQ
Antimicrobial test of PDBIQ was checked using cup plate
method and the observed results of the inhibitory test per-
formed with the reagent has been tabulated in Table 3.
Here, the diameter of inhibition zone for the organisms in
presence of PDBIQ is significantly greater than that of the
corresponding controlled replicate experiment. This study
indicates that the compound has profound activity against
the test organisms.

S. SEN ET AL.
673
Figure 3. Job’s plot analysis showing maximum emissionat
1:1 ratio [PDBIQ:Cysteine].
Table 3. Bioactivity test* of PDBIQ.
Organism Bacillus subtilis Bacillus sp. E. coli Salmonella sp.
Control (EtOH) 10 10 7 7
PDBIQ 14 15 11 10
*Values are diameter of inhibition zone (mm).
4. Conclusion
This newly designed quinazoline based spraying reagent,
PDBIQ has been established to detect twenty two amino
acids on thin-layer chromatography plates producing vari-
ous distinguishable colors with amino acids with a low de-
tection limits. The binding ability of PDBIQ with amino
acids has also been estimated by determining the binding
constants (k) spectroscopically and these values (k) are
significantly higher than those reported earlier. This reagent
has also shown the significant inhibitory action against
some gm (+ve) and gm (−ve) bacterial organisms.
5. Acknowledgements
Financial assistance from the Department of Science and
Technology (DST), New Delhi, India, is gratefully ackno-
wledged. S. Sen wishes to deeply acknowledge Dr. P.
Chattopadhyay, B.U. for his support and constant en-
couragement.
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674
Supplementary Information
X-Ray crystal structure analysis
Diffraction data were measured for 6-pyridin-2-yl-5, 6-
dihydro-benzo [4,5] imidazo [1,2-c] quinazoline (PDBIQ)
with MoKα (λ = 0.71073 Å) radiation at 293 K. The crys-
tals were positioned at 70 mm from the image plate and
95 frames were measured at 2˚ intervals with a counting
time of 2 min. Data analysis was carried out with the
XDS program. The structure was solved using direct met-
hods with the SHELXS97 program. The non-hydrogen
atoms were refined with anisotropic thermal parameters.
The hydrogen atoms bonded to carbon were included in
geometric positions and given thermal parameters equiv-
alent to 1.2 times those of the atom to which they were
attached. The hydrogen atoms attached to the water mole-
cules were located in difference Fourier maps and re-
fined with distance constraints. An empirical absorption
correction was carried out on 1 using the DIFABS pro-
gram. Refinement on all four structures was carried out
with a full matrix least squares method against F2 using
SHELXL97.
Figure S1. The structure of 6-pyridin-2-yl-5,6-dihydro-benzo
[4,5]imidazo[1,2-c]quinazoline (PDBIQ) with ellipsoids at
25% probability.