ADSORPTION OF TWENTY BIO-AMINO ACIDS BY NATURAL ALLOPHANE ANDIMOGOLITE
Hashizume Hideo
National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan
Fax: +81-29-860-4667
E-mail: HASHIZUME.Hideo@nims.go.jp(Received: July 30, 2014, Accepted: September 30, 2014)
Abstract
Adsorption of 20 bio-amino acids at pH 4, 6, and 8
was carried out using short-range order soil minerals,
viz. allophane and imogolite. The initialconcentration of the amino acid solution was fixed at
2.5 mmol⋅dm-3. We compared the extent ofadsorption of the 20 amino acids by allophane andimogolite. Both allophane and imogolite adsorbed
cysteine, aspartic acid, glutamic acid, lysine, arginine,and histidine considerably well at all three pH values,
while they poorly adsorbed glycine, alanine, valine,
leucine, isoleucine, methionine, threonine, tryptophan,and proline. The dissociation of each amino acidaffects the adsorption behavior as well as the surface
charges of allophane and imogolite. The amino acids
that were easily adsorbed by allophone and imogolite
harbored a carboxyl or amino group on their side
chain. The extent of adsorption of allophane wasquite similar to that of imogolite, because the surface
charge of allophane is almost the same as that of
imogolite at pH 4, 6, and 8. In addition, weinvestigated whether allophane and imogolite wereable to differentiate between D- and L-amino acids.
Our results suggest that such discrimination is not
feasible.
Keywords:
imogolite

adsorption,

amino

acid,

acids, and montmorillonite could adsorb basic aminoacids but not acidic amino acids [8]. Hashizume
showed that serpentine poorly adsorbed 10 different
amino acids. These poorly adsorbed amino acids
presumably existed on early earth, and the amount ofserpentine-adsorbed amino acids was similar to thatabsorbed by kaolinite [8,9]. Acidic and basic aminoacids would be similarly preferred by serpentine andkaolinite owing to the same crystal structures.
The clay minerals allophane and imogolite are
obtained from the weathering of volcanic ash.
Allophane is hollow and spherule-shaped, with adiameter of ~5 nm. Imogolite is tubular-shaped andhas a diameter of ~2 nm. Its length varies fromnanometers to several micrometers. The outer andinner layers of allophane and imogolite are composedof aluminum hydroxide (gibbsite structure) andsilicate, respectively [10]. The Al/Si ratio is 1~2 forallophane and 2 for imogolite. Allophane andimogolite possess a surface charge. A positive,
apparently neutral, or negative charge is under, about,or over pH 6, respectively [11]. They presumably
existed on the early earth, and were most probably
synthesized from volcanic ash with relative ease.
They might have affected the syntheses andconcentration as well as the polymerization of
bio-organic molecules during the early stages ofearth.
In order to gain an insight into the interactions
between allophane/imogolite and various amino acids,
we studied the allophane-/imogolite-mediatedadsorption of 20 bio-amino acids at pH 4, 6, and 8.
We also investigated the relationship between theadsorption of clay minerals and the type of aminoacids involved, the dependence of adsorption on pH,
and the difference between the adsorption mediatedby allophane and imogolite.

allophane,

1. Introduction
Interactions between clay minerals and organic
molecules have been studied extensively, because
clay minerals exhibit adsorptive and catalytic
activities towards hydrophilic and hydrophobicorganic molecules [1,2]. Clay minerals candifferentiate between organic compounds because of
their adsorptive activities. Certain clay minerals canpolymerize or decompose organic molecules via theircatalytic activities. A complex formed by the
association of a clay mineral with an organic
molecule can also exhibit new characteristics inaddition to those displayed by the uncomplexed clay
mineral [3]. Various characteristics of clay minerals
might have played an important role during chemical
evolution. These include accumulation of organic
molecules through the process of adsorption andpolymerization of organic molecules via theircatalytic activities [4]. For example, montmorillonite
prefers basic amino acids such as lysine, histidine,
and arginine to acidic or non-polar amino acids [5].
Largerpeptidesarebetteradsorbedbymontmorillonite or allophane than are smaller
peptides or monomers [6,7]. Hedge and Hare showedthat kaolinite could adsorb acidic and basic amino

Viva Origino 42 (2014) 18 - 22
© 2014 by SSOEL Japan

2. Materials and methods
Allophane and imogolite were separated from theallophanic soil obtained from the Kitakami region in
Iwate Prefecture, Japan. The isolation of allophanefrom the allophanic soil was carried out according to
the method adopted by Hashizume and Theng [12].
Allophonic soil shows the presence of gelatinous
imogolite, which was picked up and collected using
tweezers. It was gently washed and dispersed indeionized water with pH 3.5~4 using an ultrasonicwasher. The suspension was then removed to a
volumetric cylinder. Particles under 2 µm (i.e.,
imogolite particles) were collected by gravitational
sedimentation. After coagulating imogolite with 0.1
mol⋅dm-3 of NaCl, the pH was increased from 6.5 to
7.0. The coagulated solution was dialyzed using

18Viva Origino 42 (2014) 18 - 22deionized water, until all the salt was eliminated. The
suspension was then dispersed in deionized water at
an acidic pH (pH 3.5–4), again using an ultrasonicwasher. The series of treatments performed to obtain
imogolite was conducted twice to obtain pure
imogolite. Before the dialysis, the suspension was
treated with water containing 10% H2O2 at 100 °C toremove the natural organic matter. After the dialysis,
the suspension was dried at 60 °C in an oven.
Allophane and imogolite used were confirmed using
XRD and TEM (Fig. 1). The XRD patterns (Fig. 1(a))
and TEM images (Fig. 1(b)) of allophane are quite
similar to those reported by Hashizume and Theng[13]. The imogolite peaks in Fig. 1(c) are broader
than the typical XRD peaks of imogolite reported in
the Handbook of Clays and Clay Minerals [10],because we did not order the imogolite. The TEM
image of imogolite shows the presence of long strings(Fig. 1 (d)). The 20 bio-amino acids used during thisstudy have been listed in Table 1. All the amino acids
purchased were of reagent grade. D- and L-glutamine
were used in this study instead of DL-glutamine,
because the latter could not be obtained.
The adsorption treatment was carried out at 25 ± 1ºC. For the pH dependence, a 2.5 mmol⋅dm-3 aminoacid solution was prepared by using solutions
maintained at pH 2, 4, or 12, and pH-adjusted with
100 mmol⋅dm-3 of HCl and NaOH. After the

Intensity / Arbitrary unit

50nm%
(b)

(a)

10

20

30

40

50

60

Intensity / Arbitrary unit

2θ /° Cu Kα

(d)

(c)

10

20

30

40

50

100 nm602θ /° Cu KαFig. 1% (a) XRD pattern of allophane. (b) TEM image ofallophane. (c) XRD pattern of imogolite. (d) TEM imageof imogolite.

Table 1 Disociation constans and solubilities of 20 bio-amino acids.
Amino acidside chain

Dissociation constant [15]pK1!-COOH

pK2

Solubility [16](g/100g)

pK3
side chain

Glycine

(Gly)

H

2.35

!-NH3+
9.78

Alanine

(Ala)

CH3

2.35

9.87

13.55

Valine(Val)

CH(CH3)2

2.29

9.74

6.38

Leucine(Leu)

CH2CH(CH3)2

2.33

9.74

0.93

Isileucine(Ile)

CH(CH3)CH2CH3

2.32

9.76

2.079

*

Phenyalanine

(Phe)

CH 2-

2.16

9.18

1.290

*

Tyrosine

(Tyr)

-OH

2.20

9.11

Serine

(Ser)

CH2OH

2.19

9.21

Threonine(Thr)

CH(OH)CH3

2.09

9.10

Cysteine

(Cyc)

CH2SH

1.92

10.78

Methionine(Met)

CH2CH2SCH3

2.13

9.28

Aspartic acid

(Asp)

CH2COOH

1.99

9.90

3.90

Glutamic acid(Glu)

CH2CH2COOH

2.00

9.47

4.07

Aspargine(Asn)

CH2CO(NH2)

2.1

8.84

Glutamine(Gln)

CH2CH2CO(NH2)

2.17

9.13

2.5

?, L-, #Lysine(Lys)

CH2CH2CH2CH2NH2

2.16

9.18

10.79

($-NH3+)10?, L-, #Histidine

(His)

CH2 -

1.80

9.33

6.04

(imodazol)

5?, L-, #Arginine

(Arg)

1.82

8.99

12.48

(guanidine)

14.87*, L-, #

2.43

9.44

1.041

*, L-

2.95

10.65

5

?, L-, #

CH 2-

20.0

10.13

8.33

(phenol)(Sulfhydryl)

0.0294

*

4.11

*

9

*, L-, #0.1096

L-

2.908

*

("-COOH)

0.629*(#-COOH)

1.693

*

2

*, L-, #N
N
H
CH2CH2CH2NHC(NH)(NH2)CH2
Tryptophan(Trp)

Proline

(Pro)

N
H
H2
C CH2

HOOC
C
H

CH2
N
H

Solubility is measured at 25 C except * and ?. * is 20 C. ? is not shown. L- is an L-amino acid.
# is reference [17] without reference [16]19

Viva Origino 42 (2014) 18 - 22

adsorption treatment, the pH shifted from 2 to about 4,
from 4 to about 6, and from 12 to about 8. A glass
bottle with a stopper was used to mix and shake
60–100 mg of allophane and imogolite. In addition, 6
cm3 of an amino acid solution was shaken for 40 h.
The equilibrium pH was in the range 3.5–4.5, 5.5–6.5,
or 8–9, for the solutions with an initial pH of 2, 4,
and 12, respectively. After separating the supernatantfrom the suspension by centrifugation, the carbon
contents of the supernatant and an initial solution
were measured using a total organic carbon analyzer(Shimadzu TOC-5000A). The supernatant and theinitial solution were analyzed eight times using TOC.
We chose at least five analytical values out of eight inorder to ensure a smaller CV value (i.e. the ratio of
the standard deviation to the mean value). The
concentrations of the supernatant and the initialsolution were also determined. The extent ofadsorption was estimated from the concentrations of
the supernatant and the initial solution by obtaining
their carbon contents, as shown in the equation
below.
(1)
A = V・(C0-C) / W
where A, the adsorption; V, the volume added; C0,
the initial concentration of the adsorbate; C, theconcentration of the adsorbate after the adsorption
treatment; and W, the weight of the adsorbent.
Experiments involving the adsorption of amino acidsby allophane and imogolite were conducted morethan twice. The extent of adsorption of the aminoacids was reported as the mean value of several
adsorption experiments.

bio-amino acids are shown in Table 1. The aminoacids possessing alkyl groups (e.g., Ala, Val, Leu,
and Ile) were not adsorbed well, and those possessing
aromatic or cyclocarbon groups (e.g., Phe, Tyr, Trp,
and Pro) were not preferred by allophane. Pro
behaved similarly to the amino acids possessing analkyl chain. The sulfur-containing side-chain of Met
showed no effect on adsorption. Therefore, theadsorption of Met was similar to that of other aminoacids possessing alkyl groups. Asp and Glu contain anegatively charged carboxyl group in their side-chain,
whereas Lys and Arg contain a positively charged
amino group in their side-chain. Allophane shows apositive or negative charge at various pH values, andadsorbs Asp, Glu, Lys, and Arg to a significant extent.
Ser and Thr contain a hydroxide group. Asn changes
the hydroxide group in the carboxyl side chain of Asp
to an acid amide (β-monoamide). Similarly, Gln isthe γ-amide of Glu. The side-chains of Ser, Thr, Asn,and Gln are uncharged and polar, and their polarity
does show some effect on allophane adsorption. Gly
contains a hydrogen atom in the side-chain, whereas
His contains an imidazole group in its side- chain.
His is similar in nature to Lys and Arg, but theadsorption of His is not greater than that of Lys or
Arg. The cyclic structure of the imidazole groupprobably affects the adsorption of His. The
adsorption of Gly is similar to that of the amino acids
containing an alkyl side-chain.
We showed that the adsorption varied at various pH
values. The dissociation constant of each amino acidis mentioned in Table 1. When the pH changed froma lower to a higher value, the charge on each aminoacid and that on the surface of allophane change
according to the pH. At pH 4, allophane shows apositive charge. Asp and Glu were adsorbed better
than the other amino acids. Because Asp and Glu
contained negatively charged –COO- groups insteadof the neutral –COOH groups, the positively charged
allophane could easily adsorb them. The adsorptionof Asn or Gln was slightly greater than that of allother amino acids, except Cys, Asp, and Glu. The

Adsorption, mmoll/g

3. Results and discussion
3. 1 Allophane
Figure 2 shows the extents of adsorption of the 20
bio-amino acids by allophane at pH 4, 6, and 8,
respectively. Cys, Asp, Glu, Lys, and Arg wereadsorbed more than the other amino acids.
Adsorption was seen to a lesser extent in the case ofAla, Val, Leu, Ile, Phe, Tyr, Thr, Met, Trp, and Pro,
than the other amino acids. The side-chains of the 20

0.2

pH4
pH6
pH8

0.15
0.1

0.05
Pro

Arg

Trp

His

20

Lys

% 

D-Gln

Fig. 2 Adsorption of 20 amino acids by allophane at pH 4, 6, and 8.

L-Gln

Amino acid

Asn

Glu

Asp

Met

Cys

Thr

Ser

Tyr

Phe

Ile

Leu

Val

Ala

Gly

0

Viva Origino 42 (2014) 18 - 22

Adsorption, mmoll/gpolarity of the side chains of Asn and Gln might have
had some effects on its adsorption behavior. Most
amino acid besides Asp, Glu, Asn, and Gln containcharged –COO- and –NH3+ groups in their α-aminoacid structures. These amino acids were not adsorbed
well by allophane, because the positive charge on theamino groups of these amino acids repelled thepositive surface charge of allophane. At pH 6, theallophane surface is neutrally charged. As thenegative charge of allophane increased, theadsorption sites of the two –COO- groups of Asp and
Glu showed a corresponding decrease owing to therepulsion between two negative charges. The aminogroup of Lys and the guanidine group of Arg are
positively charged, whereas the imidazole group of
the side chain of His is neutral or shows a slight
positive charge. Lys, Arg, and His were adsorbed
better by allophane, because of the increase in thenegative charge of allophane. At pH 8, the aminoacids containing hydrogen or alkyl group side-chains(e.g., Gly, Ala, Val, Leu, and Ile) were adsorbed
slightly better than the same amino acids at pH 4 and6, respectively. Because the allophane surface isnegatively charged and because the amino groups ofcertain amino acids are positively charged, such
positively charged amino acids were adsorbed wellby allophane. The adsorption of Phe, Tyr, Ser, andThr could be explained using the same reasoning. In
the case Asp and Glu, the extent of adsorption
decreased, because the allophane surface wasnegatively charged and because the two carboxylgroups on Asp and Glu also carried a negative charge.
The amino groups of Asp and Glu are either neutral
or carry a slight positive charge. Because the aminogroups of Lys and Arg are positively charged, andbecause the allophane surface shows a negative
charge, Lys and Arg were easily adsorbed byallophane. The imidazole group of His being neuralin nature, the extent of adsorption of His decreased at
pH 8, as compared to its adsorption at pH 4 and 6,
respectively.
Cys was adsorbed well by allophane at a pH of 4, 6,
and 8, respectively. The adsorption of Cys hardly
showed an effect on the corresponding dissociation

0.2

constants at various pH values. Greathouse et al.
investigated the adsorption mechanism of methyl,
ethyl, and n-propyl thiols on Al(OH)3, FeO(OH), andcarbon surfaces [14]. They showed that the hydrogenand sulfur atoms of the thiol bonded with an oxygen
and a hydrogen of Al(OH)3 via van der Waals
interactions. The outer surface of allophane iscovered with aluminum hydroxide. A sulfur and ahydrogen atom of the sulfhydryl group of Cys might
possibly bind with the hydrogen and oxygen atoms of
the aluminum hydroxide present on the allophanesurface. Cys is adsorbed well by allophane regardlessof the pH.
3.2 Imogolite
The adsorption of 20 bio-amino acids by imogolite isshown in Fig. 3. The surface charge of imogolite is
positive, almost neutral, and negative at pH 4, 6, and8, respectively. The change of the surface charge ofimogolite is almost similar to that of allophane.
The adsorption of amino acids on the imogolite
surface is similar to that on the allophane surface.
The adsorption of Cys, Asp, Glu, Lys, His, and Arg
on the imogolite surface is higher than that of any
other amino acids on the same surface. The aminoacids containing alkyl, aromatic, and heterocyclic
groups in the side-chain are poorly adsorbed byimogolite. The pH dependence of the adsorption
tendency of imogolite is similar to that of allophane.
In general, the adsorption by the imogolite surface
was higher than that by the allophane surface, as
evident from the comparison of Fig. 3 with Fig. 2.
3.3 Solubility
Solubility affects the adsorption behavior as well as
the kinetics of dissociation. Generally, materials
showing high solubility are not adsorbed well by theadsorbent, and materials showing low solubility areadsorbed well by the adsorbent. For example, theadsorption from Gly to Tyr in Figs. 2 and 3 isinversely correlated with the solubilities of theseamino acids mentioned in Table 1. The adsorption ofLys, His, and Arg is not strongly correlated with theircorresponding solubilities, presumably because their

pH4
pH6
pH8

0.15
0.1

0.05
Pro

Arg

Trp

21

His



Lys

%

D-Gln

Fig. 3 Adsorption of 20 amino acids by imogolite at pH 4, 6, and 8.

L-Gln

Amino acid

Asn

Glu

Asp

Met

Cys

Thr

Ser

Tyr

Phe

Ile

Leu

Val

Ala

Gly

0

Viva Origino 42 (2014) 18 - 22large positive charges completely outweigh the effectof their corresponding solubilities. The adsorption ofLys, His, and Arg, as expected from their solubilities,
is in the order His > Lys > Arg. However, Figs. 2 and3 show a slightly different order (Lys > Arg > His).
At pH 4, the extent of adsorption of these aminoacids on the allophane surface was nicely correlatedwith their corresponding solubilities (i.e., His > Lys >Arg).
The neutral amino acids showed a good inverse
correlation with their corresponding solubilities.

2.
3.
4.

5.

4. Summary
The adsorption of 20 bio-amino acids by allophane
and imogolite was studied at a concentration of 2.5 m
mol⋅dm-3 of the initial solutions (Figs. 2 and 3). The
extent of adsorption varied according to the pH. In
other words, it depended on the dissociation of theamino acid and the corresponding surface charge onallophane or imogolite at that particular pH. The
surface charge of allophane is quite similar to that of
imogolite. The tendency of adsorption of various
amino acids on the allophane surface is therefore
similar to that on the imogolite surface. The aminoacids with methyl or heterocyclic group side-chainswere not adsorbed well by allophane and imogolite.
Asp and Glu, with a carboxyl group in the side-chain,and Lys, His, and Arg with amino or imidazole
groups in the side-chain were adsorbed well byallophane and imogolite.
We also investigated the ability of allophane andimogolite to discriminate between D- and L-aminoacids. The supernatants were analyzed using an
HPLC instrument attached with a CD-detector.
However, we could not detect a clear difference thebetween D- and L-amino acids on the chromatograph.
Hedge and Hare studied the adsorption of sixteen
amino acids on kaolinite and montmorillontie
surfaces [8]. Asp and Lys were adsorbed well bykaolinite. Montmorillonite adsorbed Lys well, but
adsorbed Asp poorly. Thr amino acids with alkyl
group side-chains were not adsorbed bymontmorillonite and kaolinite. These results are quite
similar to the results obtained by us. The structure ofkaolinite reveals a stratification of the aluminumhydroxide and silicate sheets. Allophane andimogolite contain only one layer that stratifies thealuminum hydroxide and silicate sheets. Because of
the similarities in the crystal structures of kaolinite,
allophane, and imogolite, the adsorption shown by
allophane and imogolite would be similar to thatshown by kaolinite.

6.
7.

8.
9.
10.
11.
12.
13.
14.

15.

16.
17.

5. Acknowledgement
I thank Mr. Kurashima for studying the samples
using TEM. I thank Dr. Tomura in AIST for
providing me with the allophonic soil from the
Kitakami region. In addition, I also thank thereviewers for their useful comments.
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