Download cyclodextrin polymer for adsorption of aromatic amino acids

JOURNAL OF MOLECULAR RECOGNITION
J. Mol. Recognit. 2006; 19: 39–48
Published online 2 November 2005 in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/jmr.756
Application of cross-linked b-cyclodextrin polymer
for adsorption of aromatic amino acids
Shouwan Tang, Liang Kong, Junjie Ou, Yueqi Liu, Xin Li and Hanfa Zou*
National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, People’s
Republic of China
b-Cyclodextrin (b-CyD) was cross-linked by hexamethylene diisocyanate and the polymer was investigated
for adsorption of aromatic amino acids (AAA) from phosphate buffer. High adsorption rates were observed
at the beginning and the adsorption equilibrium was then gradually achieved in about 45 min. The
adsorption of AAA decreased with the increase of initial concentration and also temperature. Under the
same conditions, the adsorption efficiencies of AAA were in the order of L-tryptophan (L-Trp) > Lphenylalanine (L-Phe) > L-tyrosine (L-Tyr). Much higher adsorption values, up to 52.4 and 43.0 mg/g
for L-Trp and L-Phe, respectively, at 50 mmol/L and 3.2 mg/g for L-Tyr at 2 mmol/L, were obtained
with the b-CyD polymer at 37 C. It was shown that the adsorption of AAA on the b-CyD polymer was
consistent with the Freundlich isotherm equation. The adsorption of mixed aromatic amino acids and
branched-chain amino acids (BCAA) showed that AAA were preferentially adsorbed with adsorption
efficiencies 10–24%, while those of BCAA were lower than 2%. It seems that the structure and
hydrophobicity of amino acid molecules are responsible for the difference in adsorption, by influencing
the strength of interactions between amino acid molecule and the polymer. Copyright # 2005 John Wiley &
Sons, Ltd.
Keywords: b-cyclodextrin polymer; adsorption; aromatic amino acids; branched-chain amino acids
Received 19 May 2005; revised 16 August 2005; accepted 23 August 2005
INTRODUCTION
Molecular recognition plays an important role in nature, for
instance, receptors, enzymes, transporters, and so forth.
Developing artificial receptors, in other words constructing
molecular recognition sites artificially, is one of the major
goals in chemistry. To date, various synthetic molecular
clefts and cavities (e.g., crown ethers, cyclodextrins, and
cyclophanes) have been developed by utilizing interactions
such as hydrogen bonding, ionic interaction, van der Waals
interaction and hydrophobic effect. The cyclodextrins
(CyDs) are a series of cyclic oligosaccharides with a
hydrophilic exterior and a hydrophobic interior capable of
binding small hydrophobic structures. -CyD and their
*Correspondence to: Prof. Dr Hanfa Zou, National Chromatographic Research
and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian 116023, People’s Republic of China.
E-mail: [email protected]
Contract/grant sponsor: Natural Science Foundation of China; contract/grant
number: 20475054.
Contract/grant sponsor: Knowledge Innovation Program of DICP.
Abbreviations used: AAA, aromatic amino acids; BCAA, branched-chain
amino acids; CyD, cyclodextrin; DMF, dimethylformamide; DNFB, 2,4-dinitrofluorobenzene; DNP-OH, 2,4-dinitrophenol; (DNP)2O, diDNP ether; FT-IR,
Fourier transform infrared; Gly, glycine; HMDI, hexamethylene diisocyanate;
HPLC, high performance liquid chromatography; Ile, isoleucine; Leu, leucine;
Phe, phenylalanine; MIPs, molecularly imprinted polymers; SEM, scanning
electron microscopic; Trp, tryptophan; Tyr, tyrosine; Val, valine.
Copyright # 2005 John Wiley & Sons, Ltd.
derivatives are well known for their ability to form inclusion
complexes with a variety of compounds. -CyD dimers and
trimers have been prepared and investigated as binders for
a series of compounds containing t-butylphenyl groups,
dimeric and trimeric amino acid amides, respectively
(Breslow et al., 1995; Leung et al., 2001). Recently, molecular imprinting as a technique to generate recognition sites
of predetermined selectivity has received considerable attention (Andersson and Nicholls, 1997). Molecularly imprinted polymers (MIPs) based on methacrylic acid and 4vinylpyridine, Cu(II)-N-(4-vinylbenzyl) iminodiacetic acid,
etc. have been used for chiral recognition of aromatic amino
acids (AAA) (Huang et al., 2003, 2004; Vidyasankar et al.,
1997). MIPs based on -CyD have been extensively examined for efficient recognition of steroids, peptides, Dphenylalanine (D-Phe), amino acid derivatives, antibiotics,
etc. (Akiyama et al., 2001; Asanuma et al., 1997, 1998,
1999, 2001, 2004; Hishiya et al., 1999, 2002; Piletsky et al.,
1998, 1999; Sreenivasan, 1998; Sreenivasan and
Sivakumar, 1998; Zhong et al., 2001). Highly enantioselective polymers for L-Phe, D- or L-tryptophan methyl ester
were prepared by the so-called ‘surface molecular imprinting technique’ (Nakamura et al., 1999; Yoshida et al.,
2000a,b). Pilestsky et al. (1998, 1999), demonstrated the
rational use of hydrophobic and electrostatic ligand–
polymer interactions for recognition of D-Phe in a novel
MIP system. MIPs with element of CyDs were also found
to have enhanced adsorption capacity in addition to the
40
S. TANG ET AL.
selectivity acquired by the imprinting, which was attributed
to the presence of -CyD moieties in the copolymer
(Sreenivasan, 1998; Sreenivasan and Sivakumar, 1998).
In this study, we developed an alternative sorbent—
cross-linked -CyD polymer for adsorption of AAA based
on cavity inclusion effect. Polymeric sorbents have been
extensively tested for amino acid adsorption (Doulia et al.,
2001; Dı́ez et al., 1998; Grzegorczyck and Carta, 1996a,b).
The -CyD polymer has been shown to be a good sorbent
for bilirubin in our previous study (Zheng et al., 2004). In
the present article, we report our preliminary results related
to the AAA adsorption behavior of this polymer in phosphate buffer.
structures of amino acids used in this paper are shown in
Figure 1. Acetonitrile of HPLC grade was purchased from
Merck (Darmstadt, Germany). Double distilled water purified by Milli-Q (Millipore Co., Miford, MA, USA) was
utilized throughout the experiments. Other reagents were
analytical grade.
Instruments
V-550 UV/VIS spectrophotometer was from Jasco (Tokyo,
Japan). HPLC system consisted of two LC-10ADvp pumps
(Shimadzu, Kyoto, Japan), a SPD-10Avp UV-Vis detector
(Shimadzu, Kyoto, Japan), a column (250 4.6 mm I.D.)
packed with 5 mm Kromasil C18 (Akzo Noble AB, Nacka,
Sweden), a TC-100 temperature controller (Autoscience,
Tianjin, China) and a WDL-95 chromatographic workstation (National Chromatographic R&A Center, Dalian,
China).
EXPERIMENTAL
Materials
-Cyclodextrin (-CyD) of chemical grade (Shanghai
Chemical Reagent Co., Ltd, Shanghai, China) was recrystallized three times and dried under vacuum at 110 C for
24 h before use. Pyridine of analytical grade (Tianjin
Kermel Chemical Development Center, Tianjin, China)
was dried with molecular sieve 4A, refluxed with CaH2
for 2 h, and then distilled for use. L-Tryptophan (L-Trp), Ltyrosine (L-Tyr), L-phenylalanine (L-Phe), 2,4-dinitrofluorobenzene (DNFB), and hexamethylene diisocyanate
(HMDI, 99% pure) were obtained from Acros (New Jersey,
USA). L-Valine (L-Val), L-isoleucine (L-Ile), and L-leucine
(L-Leu) were obtained from TCI (Tokyo, Japan). The
Preparation of the b-CyD polymer
The -CyD polymer was prepared according to the procedures described elsewhere (Hishiya et al., 1999) with a few
modifications. -CyD (5 g) was dissolved in dry pyridine
(50 ml) and then HMDI (4.5 ml) was added. After being
magnetically stirred at 65 C for 2 h, the mixture was poured
into acetone to obtain the cross-linked polymer. Then the
polymer was washed with hot water and hot ethanol to
remove completely unreacted cross-linker and free -CyD.
O
H2N
CH
O
C
OH
H2N
CH2
CH
C
O
H2N
OH
CH
C
OH
CH2
CH2
HN
OH
(a)
(b)
O
H2N
CH
C
CH2
CH
CH3
(c)
O
OH
H2N
CH
C
CH
CH3
O
OH
H2N
CH
C
CH
CH3
OH
CH2
CH3
CH3
(d )
(e)
CH3
(f )
Figure 1. Structures of amino acid molecules. (a) Tryptophan; (b) phenylalanine;
(c) tyrosine; (d) leucine; (e) isoleucine; (f) valine.
Copyright # 2005 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2006; 19: 39–48
ADSORPTION OF AROMATIC AMINO ACIDS
Finally, the polymer was dried under vacuum at 70 C for
24 h before use.
Sample preparation
Preparation of single amino acid samples. Stock solutions (2.0 mmol/L) of L-Trp, L-Phe or L-Tyr were prepared
by dissolving them in 10 mmol/L phosphate buffer (pH 7.4)
separately. AAA solutions (0.01–0.40 mmol/L) were obtained by dilution of the stock solutions with 10 mmol/L
phosphate buffer (pH 7.4).
Preparation of mixed amino acid samples. Stock solutions of mixed AAA (2.0 mmol/L for each amino acid) and
aromatic amino acids with branched-chain amino acids
(BCAA) solution (2.0 mmol/L for each AAA and
1.0 mmol/L for each BCAA) were prepared by dissolving
L-Trp, L-Phe and L-Tyr or L-Trp, L-Phe, L-Tyr, L-Val, L-Leu
and L-Ile in 10 mmol/L phosphate buffer (pH 7.4), respectively. Mixed AAA solutions (0.02–0.40 mmol/L for each
AAA) or AAA-BCAA solution (0.2 mmol/L for each AAA
and 0.1 mmol/L for each BCAA) were obtained by dilution
of the corresponding stock solution above with 10 mmol/L
phosphate buffer (pH 7.4).
Determination of the concentration of amino acids
Adsorption of AAA from phosphate buffer with the -CyD
polymer was studied batchwise. Five milliliters of amino
acid solution was incubated with 100 mg of the -CyD
polymer and shaken for a certain time. For single amino
acid samples, UV-Vis spectrophotometer was used to
determine the concentration of amino acids after adsorption.
The detection wavelengths of L-Trp, L-Phe, L-Tyr were
280, 257, 275 nm, respectively. For mixed amino acid
samples, they were first derivatized with DNFB (Robert
and Gerhard, 1988) and then separated and detected by
HPLC.
41
0.5 mol/L NaHCO3 (pH 9.0) and 50 ml of acetonitrile containing 1% DNFB (v/v) were added and mixed. Derivatization was carried out at 60 C for 1 h while shielded from
light. Finally, 600 ml of 50 mmol/L KH2PO4 (pH 7.0) was
added after the mixture cooled down to room temperature.
Chromatographic analysis of mixed amino acids
A 250 4.6 mm I.D. column packed with 5 mm Kromasil
C18 was used for analysis of the derivatized amino acids
by keeping column at 33 C. Mobile phase A was acetonitrile-water (50:50, v/v) and B was 100 mmol/L sodium
acetate buffer (pH 6.4), containing 1% dimethylformamide
(DMF) (v/v). Gradient elution was carried out as follows:
0–0.3 min, 84%B; 0.3–4 min, 84%B–70%B; 4–7 min, 70%
B–66%B; 7–12 min, 66%B–57%B; 12–22 min, 57%B–45%
B; 22–25 min, 45%B; 25–40 min, 45%B–0%B, at a flow
rate of 1.0 ml/min. Detection was by UV at 360 nm.
Characterizing the b-CyD polymer
The polymer dried under vacuum at 70 C for 24 h was
placed on a sticky carbon foil which was attached to a
standard aluminum specimen stubs. The carbon foil was to
increase conductivity. The sample was vapor-deposited with
gold (Eiko IB3 ion coater). Scanning electron microscopic
(SEM) images of the -CyD polymer were obtained with a
JSM-6360 LV scanning electron microscope (Jeol, Tokyo,
Japan).
-CyD was dried under vacuum at 110 C for 24 h. The
IR spectra for -CyD and the -CyD polymer were
obtained with an FT-IR spectrophotometer (Perkin-Elmer,
Boston, MA, USA) as KBr tablets in the 4000–400 cm1
region.
RESULTS AND DISCUSSION
Characterization of the b-CyD polymer
Derivatization procedure
The dinitrophenylation of mixed amino acids was according
to the following procedure: to 100 ml of sample, 250 ml of
The morphology of the polymer was closely related to the
polymerization conditions. The scanning electron micrographs for the internal structure of the -CyD polymer are
shown in Figure 2.
Figure 2. Scanning electron micrograph of the -CyD polymer. (a) 1,000 ; (b) 10,000 .
Copyright # 2005 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2006; 19: 39–48
42
S. TANG ET AL.
From the FT-IR (KBr) of -CyD and the -CyD polymer
as shown in Figure 3, it can be seen that: for -CyD, 3600–
3000 ((O-H)), 2928 cm1 ((C-H)); while for the -CyD
polymer, 3600–3000 ((O-H)), 2933 ((C-H)), 1701
((C ¼ O)) and 1541 cm1 ((N-H)). The existence of
1701 ((C ¼ O)) and 1541 cm1 ((N-H)) is the evidence
of the polymer being cross-linked by HMDI.
Adsorption of single AAA
Figure 4 gives the adsorption rate of different AAA. The
-CyD polymer was incubated with the samples for 5, 15,
30, 45, and 60 min at 37 C. As shown in Figure 4, the
adsorption process was completed within about 45 min and
this value can be considered as the equilibrium time for
AAA adsorption. Relatively high adsorption rates of AAA
were observed at the beginning and more than 82% of the
equilibrium amount was adsorbed within 5 min for AAA.
Under the same conditions, the adsorption efficiencies of
AAA were in the order of L-Trp > L-Phe > L-Tyr. This is due
to the complementarity of the structure and hydrophobicity
of AAA molecules to the cavity of the -CyD polymer.
Since the polymer was obtained by cross-linking of hydro-
xyl groups on the primary faces of two or more -CyDs, the
hydrophobic cavity was little affected (the cavity volume of
-CyD [Å3], 262). As shown in Table 1, Trp is the biggest
(volume [Å3], 227.8) and most hydrophobic among AAA
molecules. So, the interactions between L-Trp and the
-CyD polymer are the strongest among AAA, resulting
in the highest adsorption efficiency. As shown in Figure 1,
one hydroxyl group distinguishes L-Tyr from L-Phe, but the
adsorption efficiency of L-Phe was 3–4% higher than L-Tyr
in the adsorption process studied. This is probably due to the
hydroxyl group decreasing considerably the hydrophobicity
of the molecule while increasing the volume of the molecule
slightly. For the reason above, the volume of Tyr is slightly
bigger than Phe, but the hydrophobicity of Phe is much
higher than Tyr as shown in Table 1. From these two
parameters—molecular volume and hydrophobicity, the
interactions between L-Phe and the -CyD polymer are
stronger than those of L-Tyr. Thus the adsorption efficiency
of L-Phe is higher than L-Tyr.
Figure 5 gives adsorption efficiency versus initial concentration of AAA. The -CyD polymer was incubated with
AAA samples containing different amounts of AAA (0.02–
0.40 mmol/L) at 37 C for 1 h. The adsorption efficiency of
AAA decreased with increasing AAA initial concentration.
Figure 3. FT-IR spectra of -CyD and the -CyD polymer. (a) -CyD; (b) the -CyD polymer.
Copyright # 2005 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2006; 19: 39–48
ADSORPTION OF AROMATIC AMINO ACIDS
obtained. But from the trend of adsorption capacity with
respect to concentrations as shown in Figure 6(c), the
adsorption was far from saturation.
Adsorption of AAA from phosphate buffer onto the
surface of the -CyD polymer can be explained by the
Freundlich isotherm using the following equation
30
Adsorption efficiency (%)
43
25
q ¼ kC 1=n
20
ð1Þ
where q is the amount of substance adsorbed per unit mass
of sorbent at equilibrium (mg/g), C is the equilibrium
concentration of the solute being adsorbed (mmol/L), while
k and n are empirical constants dependent on the nature of
sorbent and solute and the temperature. By taking logarithms of both sides of equation (1), we obtain
15
1
lg q ¼ lg k þ lg C
n
10
0
10
20
30
40
50
60
Adsorption time (min)
Figure 4. Adsorption rates of L-Trp (~), L-Phe (*) and L-Tyr (&).
Experimental conditions: 5 ml of 0.2 mmol/L L-Trp, L-Phe or L-Tyr
incubated with 100 mg of the -CyD polymer for 5, 15, 30, 45,
60 min, respectively; temperature, 37 C.
When the concentration of AAA increased from 0.02 to
0.40 mmol/L, the adsorption efficiencies of L-Trp, L-Phe,
and L-Tyr decreased from 35.55, 34.31, and 32.05% to
24.27, 18.81, and 12.17%, respectively. Also the adsorption
efficiencies remained in the same order, L-Trp > L-Phe >
L-Tyr.
The effect of temperature was also studied. The AAA
adsorption at 25, 37, and 50 C, representing the amount of
AAA adsorbed with respect to the AAA initial concentrations, is shown in Figure 6. The amount of adsorbed AAA
per unit amount of the polymer decreased with increasing
temperature. Note that the adsorption values of L-Trp and
L-Phe reached 52.4, and 43.0 mg/g polymer, respectively, at
50 mmol/L, while 3.2 mg/g polymer for L-Tyr at 2 mmol/L
especially at 37 C. Because of low solubility of L-Tyr in
water (solubility at 25 C, 0.46 g/kg H2O) (Lide, 2003),
the adsorption values at higher concentrations were not
ð2Þ
and hence a plot of lg q versus lg C should be linear with
slope equal to 1/n and intercept equal to lg k. The adsorption
isotherms of L-Trp, L-Phe, and L-Tyr adsorbed on the -CyD
polymer are presented in Figure 6, and the corresponding
Freundlich adsorption isotherms are shown in Figure 7. The
empirical constants k, 1/n and correlation coefficients obtained from Figure 7 are listed in Table 2. Plots in Figure 7
with correlation coefficients varying from 0.9901 to 0.9985
indicate that the adsorption of AAA on the -CyD polymer
was consistent with the Freundlich isotherm equation,
which indicates that the adsorption mechanism is a monolayer adsorption.
Adsorption of mixed amino acids
In order to study the adsorption of mixed amino acids on the
-CyD polymer, HPLC method was established for quantitative determination of amino acids as described in experimental section. Amino acid analysis is usually carried out
by ion exchange chromatography followed by postcolumn
derivatization with ninhydrin or fluorescent reagents
(Benson and Hare, 1975). These methods are limited by
the fact that the equipment is elaborate and dedicated to the
sole purpose of amino acid analysis. An alternate approach
involves the general method of precolumn derivatization
Table 1. The physical properties of amino acids
Amino
acids
Volume
(Å3)a
Residue non-polar
surface area (Å2)bc
Trp
Phe
Tyr
Val
Ile
Leu
227.8
189.9
193.6
140.0
166.7
166.7
37 þ 199
39 þ 155
38 þ 116
135
155
164
Estimated hydrophobic effect
for residue burial (kcal/mol)b
4.11
3.46
2.81
3.38
3.88
4.10
Estimated hydrophobic effect
for side chain burial (kcal/mol)bd
2.9
2.3
1.6
2.2
2.7
2.9
a
Values from Zamyatin, 1972.
Values from Karplus, 1997.
All surfaces associated with main- and side-chain carbon atoms were included except for amide, carboxylate and guanidino carbons. For aromatic
side chains, the aliphatic and aromatic surface areas are reported separately.
d
The values are obtained from the previous column by substracting the value for Gly (1.18 kcal/mol) from each residue.
b
c
Copyright # 2005 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2006; 19: 39–48
44
S. TANG ET AL.
(a)
60
Adsorption capacity (mg/g)
Adsorption efficiency (%)
35
30
25
20
40
20
0
0
10
15
20
30
40
50
40
50
Concentration (mmol/L)
0.0
0.1
0.2
0.3
0.4
Concentration (mmol/L)
Figure 5. Effect of concentration on adsorption of L-Trp (~), LPhe (*) and L-Tyr (&). Experimental conditions: L-Trp, L-Phe or LTyr at a concentration of 0.02–0.40 mmol/L incubated with
100 mg of the -CyD polymer for 1 h. Other conditions are the
same as in Figure 4.
with chromophores followed by separation of the amino
acid derivatives. The derivatization procedures in current
usage include o-phthalaldehyde (Jones et al., 1981), dansylation (De Jong et al., 1982) and dabsylation (Chang et al.,
1981). However, these types of amino acid modification
procedures may lead to problems, such as the inability of the
reagent to react with all amino acids, fluorescence quenching by the buffers used for amino acid elution, poor detection of some amino acids and instability of certain amino
acid derivatives. A dinitrophenyl derivative, which is frequently overlooked, is formed by reaction with DNFB
(Robert et al., 1988). These derivatives have extremely
high extinction coefficients and are also very stable. Dinitrophenylation thus seems to be well suited for precolumn
derivatization of amino acids.
The representative chromatogram of amino acid derivatives with DNFB as shown in Figure 8 clearly indicates that
baseline resolution of 2,4-DNP-amino acids can be achieved
in just over 45 min. Dinitrophenylation is quantitative as
demonstrated by the calibration equation with correlation
coefficient in the range of 0.9991–0.9998 as shown in
Table 3. This establishes that for a given sample of derivatized amino acid the precise number of moles can be
calculated from the integrated peak areas. Table 3 gives
the analytical parameters of this method. In the range of
0.01–0.40 mmol/L good linearities were found for L- Trp,
L-Phe and L-Tyr, while 0.005–0.20 mmol/L for L-Val, L-Ile
and L-Leu. The RSDs of the peak areas for the calibration
curves were between 1.53 and 4.71% for the tested amino
acids.
Figure 9 gives the adsorption rate of mixed AAA at 37 C.
Solutions at a concentration of 0.20 mmol/L for each AAA
were used. The characteristics of adsorption rates of mixed
AAA were almost the same as single AAA as shown in
Figure 4, that is, the adsorption efficiencies of AAA were in
the order of L-Trp > L-Phe > L-Tyr and the adsorption equilibrium was gradually achieved in about 45 min.
The -CyD polymer was incubated with mixed AAA
samples containing different amounts of AAA (0.02–
Copyright # 2005 John Wiley & Sons, Ltd.
40
20
0
0
10
20
30
Concentration (mmol/L)
(c)
4
Adsorption capacity (mg/g)
10
Adsorption capacity (mg/g)
(b)
3
2
1
0
0.0
0.5
1.0
1.5
2.0
Concentration (mmol/L)
Figure 6. Adsorption isotherms of AAA at 25 C (^), 37 C (*)
and 50 C (!). (a) L-Trp; (b) L-Phe; (c) L-Tyr. Experimental conditions: L-Trp, L-Phe or L-Tyr at various concentrations incubated
with the -CyD polymer for 1 h. Other conditions are the same as
in Figure 5.
0.40 mmol/L each) at 37 C for 1 h. Figure 10 shows the
adsorption efficiencies with respect to initial concentrations
of mixed AAA. Compared with single amino acid as shown
in Figure 5, there are similarities and also differences. The
adsorption efficiencies of AAA remained in the same order
as L-Trp > L-Phe > L-Tyr, but were lower than when loaded
individually under the same conditions. For L-Trp, the
adsorption behaviors were almost the same in mixed and
single AAA solutions. For L-Phe, the adsorption efficiency
declined slightly (3.33%) in the concentration range studied
in mixed AAA solutions, while dramatically (14.27%) in the
range of 0.02–0.10 mmol/L and slightly (2.02%) in the
range of 0.10–0.40 mmol/L in single AAA solutions. For
L-Tyr, the adsorption efficiency decreased slightly (3.91%,
including 3.05% from 0.10 to 0.20 mmol/L) when concentration increased from 0.02 to 0.40 mmol/L in mixed AAA
solutions, and more importantly (15.29%) before and
slightly (4.49%) after 0.10 mmol/L in single AAA solutions.
The structure of AAA is one of the key factors that affects
J. Mol. Recognit. 2006; 19: 39–48
ADSORPTION OF AROMATIC AMINO ACIDS
45
Table 2. The values of different parameters of the
Freundlich adsorption isotherms for L-Trp, L-Phe and
L-Tyr at 25, 37, and 50 C
Amino
acids
L-Trp
L-Phe
L-Tyr
Temperature
( C)
k
1/n
Correlation
coefficient (r)
25
37
50
25
37
50
25
37
50
4.00
3.29
3.16
3.92
3.78
3.88
1.51
1.47
1.32
0.69
0.72
0.64
0.67
0.65
0.62
1.07
1.04
0.94
0.9952
0.9970
0.9961
0.9946
0.9970
0.9901
0.9938
0.9979
0.9985
Figure 8. Chromatogram for HPLC separation of DNFB derivatives of amino acids. Column, Kromasil C18, 5 mm, 250 4.6 mm
I.D., maintained at 33 C. Mobile phase A, acetonitrile–water
(50:50, v/v) and B, 100 mmol/L sodium acetate buffer (pH 6.4),
containing 1% DMF (v/v). Gradient elution was carried out as
described in experimental section at a flow rate of 1 ml/min.
Detection: UV at 360 nm. Peak identification: (1) DNP-OH; (2) Val;
(3) Ile; (4) Leu; (5) Trp; (6) Phe; (7) (DNP)2O; (8) Tyr.
competed for the interaction sites of the -CyD
polymer, resulting in lower adsorption efficiencies than
when loaded individually. With the strongest interactions
with the -CyD polymer, the adsorption behavior of L-Trp
was slightly affected by the presence of the other AAA,
whose interactions with the -CyD polymer were weaker
than L-Trp, showing almost the same behavior as in single
AAA solutions. But for L-Phe and L-Tyr, which interact
weakly with the -CyD polymer, their adsorption behaviors
were greatly influenced by the presence of L-Trp in mixed
AAA solutions. In the case of L-Tyr, the presence of L-Phe
may also affect the adsorption behavior. Certainly, the
existence of AAA with weaker interactions with the CyD polymer also has some limited influence on the
L-Tyr
Figure 7. The Freundlich isotherms of AAA adsorbed on the CyD polymer at 25 C (^), 37 C (*) and 50 C (!). (a) L-Trp; (b) LPhe; (c) L-Tyr.
the stability of the complex, which is decisive for the
strength and positioning of the AAA–polymer interactions.
As described above, the interaction strength between AAA
and the -CyD polymer was in the order of L-Trp >
L-Phe > L-Tyr. When loaded together, L-Trp, L-Phe and
Copyright # 2005 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2006; 19: 39–48
46
S. TANG ET AL.
Table 3. Analytical parameters for the determination of derivatized amino acids by HPLC
Amino
acids
Calibration range
(mmol/L)
Calibration
equationa
Correlation
coefficient(r)
0.01–0.40
0.01–0.40
0.01–0.40
0.005–0.20
0.005–0.20
0.005–0.20
Y ¼ (1.90E-6 2.41E-8) X þ (0.00235 0.00166)
Y ¼ (1.67E-6 2.38E-8) X þ (0.00306 0.00185)
Y ¼ (1.69E-6 3.34E-8) X þ (0.00290 0.00257)
Y ¼ (1.61E-6 3.17E-8) X þ (-0.0137 0.00155)
Y ¼ (1.56E-6 2.72E-8) X þ (0.00104 0.00114)
Y ¼ (1.60E-6 3.90E-8) X þ (0.00185 0.00158)
0.9998
0.9997
0.9991
0.9994
0.9995
0.9991
L-Trp
L-Phe
L-Tyr
L-Val
L-Ile
L-leu
a
b
Repeatability
(RSD,%)b
2.63
1.53
1.67
2.11
2.14
4.71
Y, the concentration of amino acid before derivatization (mmol/L); X, the peak areas of amino acids. n ¼ 5.
n ¼ 3.
Adsorption efficiency (%)
25
20
15
10
0
10
20
30
40
50
60
Adsorption time (min)
Figure 9. Adsorption rates of L-Trp (~), L-Phe (*) and L-Tyr (&)
in mixed AAA solutions. Experimental conditions: 5 ml of
0.20 mmol/L L-Trp, L-Phe and L-Tyr incubated with the -CyD
polymer. Other conditions are the same as in Figure 4.
Adsorption efficiency (%)
35
30
25
adsorption behaviors of AAA with stronger interactions with
the -CyD polymer. So, the different AAA adsorption
behaviors in mixed and single AAA solutions are the
consequence of different interaction strength between
AAA and the -CyD polymer, which is determined by the
complementarity of the structure and hydrophobicity of
AAA molecules to the cavity of the -CyD polymer.
To clarify if the -CyD polymer was a specific sorbent for
AAA adsorption, the effect of BCAA on AAA adsorption
was investigated. The -CyD polymer was incubated with
mixed AAA-BCAA solution at 37 C for 1 h. The pre- and
post-samples were collected, derivatized with DNFB and
then analyzed by HPLC as described in experimental section. Table 4 gives the results for adsorption of mixed amino
acids. It can be seen that AAA were preferentially adsorbed
on the -CyD polymer with adsorption efficiencies in the
range of 10.40–23.88%, while those of BCAA were lower
than 2%. The adsorption efficiencies of BCAA were in the
order of L-Leu > L-Ile > L-Val, which is also consistent with
the volume and hydrophobicity of BCAA molecules shown
in Table 1. The structure of molecules is the key factor for
the big difference between AAA and BCAA in adsorption
efficiencies, by influencing the formation and strength of
interactions between amino acid molecules and the polymer.
Compared with BCAA molecules, AAA molecules contain
a benzene ring, making them more suitable to the cavity of
the -CyD polymer. Since the BCAA molecule is too small
to allow it to fit well into the cavity of the polymer, it leads to
low adsorption efficiency. The ratio of the number (as
mmol) of AAA molecules to BCAA molecules adsorbed
20
Table 4. Adsorption of AAA and BCAA on the b-CyD
polymer in the mixed solution
15
Amino Before adsorption
acids
(mmol/L)
10
L-Val
0.0
0.1
0.2
0.3
0.4
Concentration (mmol/L)
Figure 10. Effect of concentration on adsorption of L-Trp (~), LPhe (*) and L-Tyr (&) in mixed AAA solutions. Experimental
conditions: the concentration of mixed AAA varied from 0.02 to
0.40 mmol/L for each AAA. Other conditions are the same as in
Figure 5.
Copyright # 2005 John Wiley & Sons, Ltd.
L-Ile
L-Leu
L-Trp
L-Phe
L-Tyr
0.103
0.096
0.101
0.201
0.206
0.202
After adsorption
(mmol/L)
0.102
0.095
0.099
0.153
0.164
0.181
Adsorption
efficiency (%)
0.97
1.04
1.98
23.88
20.39
10.40
100 mg of the -CyD polymer was incubated with 5 ml of mixed AAABCAA at 37 C for 1 h.
J. Mol. Recognit. 2006; 19: 39–48
ADSORPTION OF AROMATIC AMINO ACIDS
on the sorbent was about 27 in this case. It indicates that the
-CyD polymer was a specific sorbent for AAA adsorption,
while being non-specific for BCAA.
CONCLUSION
In this article, we studied the adsorption behavior of AAA
on the -CyD polymer in phosphate buffer. High adsorption
rates were observed at the beginning and the adsorption
equilibrium was then gradually achieved in about 45 min.
The adsorption of AAA decreased with the increase of
initial concentration and also temperature. Under the same
conditions, the adsorption efficiencies of AAA were in the
order of L-Trp > L-Phe > L-Tyr. Much higher values up to
52.4, 43.0 (at 50 mmol/L), and 3.2 (at 2 mmol/L) mg/g
polymer for L-Trp, L-Phe and L-Tyr, respectively, were
obtained at 37 C. The ratio (as mmol) of AAA molecules
47
to BCAA molecules adsorbed on the sorbent was about 27,
indicating that AAA molecules were preferentially adsorbed
on the -CyD polymer. It was found that the structure and
hydrophobicity of amino acid molecules were responsible
for the difference in adsorption, by influencing the strength
of interactions between amino acid molecule and the polymer. These results together with those obtained previously
for adsorption of bilirubin (Zheng et al., 2004) indicate that
this inexpensive sorbent may be an important alternative to
existing sorbents used in the therapy of liver patients
showing elevated bilirubin and AAA.
Acknowledgments
Financial supports from the Natural Science Foundation of China
(No. 20475054) and the Knowledge Innovation Program of DICP to Prof.
Hanfa Zou are gratefully acknowledged.
REFERENCES
Akiyama T, Hishiya T, Asanuma H, Komiyama M. 2001.
Molecular imprinting of cyclodextrin on silica-gel
support for the stationary phase of high-performanceliquid-chromatography. J. Incl. Phenom. Macro. 41:
149–153.
Andersson HS, Nicholls IA. 1997. Molecular imprinting: recent
innovations in synthetic polymer receptor and enzyme
mimics. Recent Res. Develop. Pure Appl. Chem. 1:
133–157.
Asanuma H, Akiyama T, Kajiya K, Hishiya T, Komiyama M. 2001.
Molecular imprinting of cyclodextrin in water for the recognition of nanometer-scaled guests. Anal. Chim. Acta. 435:
25–33.
Asanuma H, Hishiya T, Komiyama M. 2004. Efficient separation
of hydrophobic molecules by molecularly imprinted cyclodextrin polymers. J. Incl. Phenom. Macro. 50: 51–55.
Asanuma H, Kajiya K, Hishiya T, Komiyama M. 1999. Molecular
imprinting of cyclodextrin in water for the recognition of
peptides. Chem. Lett. 665–666.
Asanuma H, Kakazu M, Shibata M, Hishiya T, Komiyama M.
1997. Molecularly imprinted polymer of -cyclodextrin for
the efficient recognition of cholesterol. Chem. Commun.
1971–1972.
Asanuma H, Kakazu M, Shibata M, Hishiya T, Komiyama M.
1998. Synthesis of molecularly imprinted polymer of
-cyclodextrin for the efficient recognition of cholesterol.
Supramol. Sci. 5: 417–421.
Benson JR, Hare PE. 1975. Ortho-phthalaldehyde—fluorogenic
detection of primary amines in picomole range—comparison with fluorescamine and ninhydrin. Proc. Nat. Acad. Sci.
USA 72: 619–622.
Breslow R, Halfon S, Zhang BL. 1995. Molecular recognition by
cyclodextrin dimers. Tetrahedron. 51: 377–388.
Chang JY, Knecht R, Braun DG. 1981. Amino-acid-analysis at the
picomole level—application to the c-terminal sequenceanalysis of polypeptides. Biochem. J. 199: 547–555.
De Jong C, Hughes GJ, Van Wieringen E, Wilson KJ. 1982.
Amino acid analyses by high-performance liquid chromatography: an evaluation of the usefulness of precolumn Dns
derivatization. J. Chromatogr. 241: 345–359.
Dı́ez S, Leitão A, Ferreira L, Rodrigues A. 1998. Adsorption of
phenylalanine onto polymeric resins: equilibrium, kinetics
and operation of a parametric pumping unit. Sep. Purif.
Technol. 13: 25–35.
Copyright # 2005 John Wiley & Sons, Ltd.
Doulia D, Rigas F, Gimouhopoulos C. 2001. Removal of amino
acids from water by adsorption on polystyrene resins.
J. Chem. Technol. Biotechnol. 76: 83–89.
Grzegorczyck DS, Carta G. 1996a. Adsorption of aminoacids on
porous polymeric adsorbents I-equilibrium. Chem. Eng. Sci.
51: 807–817.
Grzegorczyck DS, Carta G. 1996b. Adsorption of aminoacids on
porous polymeric adsorbents II. Intraparticle mass transport.
Chem. Eng. Sci. 51: 819–826.
Hishiya T, Akiyama T, Asanuma H, Komiyama M. 2002. Molecular imprinting of cyclodextrins leading to synthetic antibodies. J. Incl. Phenom. Macro. 44: 365–367.
Hishiya T, Shibata M, Kakazu M, Asanuma H, Komiyama
M. 1999. Molecularly imprinted cyclodextrins as selective
receptors for steroids. Macromolecules 32: 2265–2269.
Huang XD, Zou HF, Chen XM, Luo QZ, Kong L. 2003. Molecularly
imprinted monolithic stationary phases for liquid chromatographic separation of enantiomers and diastereomers.
J. Chromatogr. A 984: 273–282.
Huang XD, Qin F, Chen XM, Liu YQ, Zou HF. 2004. Short columns
with molecularly imprinted monolithic stationary phases
for rapid separation of diastereomers and enantiomers.
J. Chromatogr. B 804: 13–18.
Jones BN, Paabo S, Stein S. 1981. Amino-acid-analysis and
enzymatic sequence determination of peptides by an improved ortho-phthaldialdehyde pre-column labeling procedure. J. Liq. Chromatogr. 4: 565–586.
Karplus PA. 1997. Hydrophobicity regained. Protein Sci. 6: 1302–
1307.
Leung DK, Atkins JH, Breslow R. 2001. Synthesis and binding
properties of cyclodextrin trimers. Tetrahedron Lett. 42:
6255–6258.
Lide DR (ed.). 2003. CRC Handbook of Chemistry and Physics
(84th edn.) CRC Press LLC: Boca Raton, London, New York,
Washington, D.C.; 7–1.
Nakamura S, Takeuchi H, Nakamura H, Maeda M, Takagi M.
1999. Molecularly imprinted resin for recognition of amino
acids using self-assembly at o/w interface. Trans. Mater. Res.
Soc. Jpn. 24: 453–456.
Piletsky SA, Andersson HS, Nicholls IA. 1998. The rational use of
hydrophobic effect-based recognition in molecularly imprinted polymers. J. Mol Recogn. 11: 94–97.
Piletsky SA, Andersson HS, Nicholls IA. 1999. Combined hydrophobic and electrostatic interaction-based recognition in
J. Mol. Recognit. 2006; 19: 39–48
48
S. TANG ET AL.
molecularly imprinted polymers. Macromolecules. 32:
633–636.
Robert CM, Gerhard EG. 1988. Amino acid analysis by dinitrophenylation and reverse-phase high-pressure liquid chromatography. Anal. Biochem. 170: 220–227.
Sreenivasan K. 1998. Synthesis and evaluation of a beta cyclodextrin-based molecularly imprinted copolymer. J. Appl.
Polym. Sci. 70: 15–18.
Sreenivasan K, Sivakumar R. 1998. Evaluation of a methyl betacyclodextrin based molecularly imprinted copolymer as
novel absorbent for steroids. Macromol.—New Front.,
Proc. IUPAC Int. Symp. Adv. Polym. Sci. Technol. 2: 594–596.
Vidyasankar S, Ru M, Arnold FH. 1997. Molecularly imprinted
ligand-exhange adsorbents for the chiral separation of underivatized amino acids. J. Chromatogr. A 775: 51–63.
Copyright # 2005 John Wiley & Sons, Ltd.
Yoshida M, Uezu K, Goto M, Furusaki S. 2000a. Surface imprinted polymers recognizing amino acid chirality. J. Appl.
Polym. Sci. 78: 695–703.
Yoshida M, Hatate Y, Uezu K, Goto M, Furusaki S. 2000b.
Chiral-recognition polymer prepared by surface molecular imprinting technique. Collids Surf. A 169:
259–269.
Zamyatin AA. 1972. Protein volume in solution. Prog. Biophys.
Mol. Biol. 24: 107–123.
Zheng CJ, Huang XD, Kong L, Li X, Zou HF. 2004. Cross-linked
-cyclodextrin polymer used for bilirubin removal. Chin. J.
Chromatogr. 22: 128–130.
Zhong N, Byun H-S, Bittman R. 2001. Hydrophilic cholesterolbinding molecular imprinted polymers. Tetrahedron Lett. 42:
1839–1841.
J. Mol. Recognit. 2006; 19: 39–48