j........

ELSEVIER

CRYSTAL
G R O W T H

Journal of Crystal Growth 178 (1997/ 568-574

Effects of additives on the growth

of L-glutamic acid crystals (p-form)
Chiaki Sano a'*, Tatsuki Kashiwagi b, Nobuya Nagashima b, Tetsuya Kawakita a
a Technology & Engineering Laboratories, Ajinomoto Company, Inc., 1-1 Suzuki-cho. Kawasaki-ku. Kawasaki-shi 210, Japan
b Central Research Laboratories, Ajinomoto Company, Inc., 1-i Suzula'-eho, Kawasaki-ku, Kawasaki-shi 210, Japan

Received 6 December 1996

Abstract

To elucidate the mechanism of polymorph-selective crystallization of L-glutamic acid (L-Glu) crystals by the additives,
the effects of various L-amino acids, carboxylic acids, and a di-peptide, y-L-glutamyl-L-glutamic acid (y-Glu-Glu) on the
growth of the {1 0 1}, {0 1 0}, and {0 0 1}, three dominant faces of the B-form of L-Glu crystals have been investigated
experimentally and structurally, y-Glu-Glu and L-phenylalanine (L-Phe) showed nearly the same strong inhibitory
effects on the three faces of the 13-form in contrast to their different behavior on the two dominant faces of the a-form of
L-Glu. These phenomena were explained in terms of the characteristics of the hydrogen bonds of each face and it was
found that the B-form has less discriminating capacity than the a-form for the recognition of the molecules. Finally, the
cause of the polymorph-selective crystallization by the additives in the L-Glu is attributed mainly to the difference in the
inhibitory effects of the additives on the two dominant faces of the a-form.
PACS: 61.50.K; 61.66.Hq; 61.72.Ss; 61.72. - y; 81.10.Aj; 81.10.Dn; 84.30.Y
Keywords: L-glutamic acid; Polymorph; Transformation; Tailor-made additives; Amino acid

1. Introduction

L-glutamic acid (L-Glu) crystals have two polymorphs: granular a- and plate-like [3-forms [1]. T h e
crystal structures of the two p o l y m o r p h s were determined by X-ray [-1-4] and n e u t r o n [5, 6] diffractions. T h e a - f o r m (space g r o u p P 222222, a = 7.068,
b = 10.277, c - - 8 . 7 7 5 A E4]) is metastable and

* Corresponding author. Fax: + 81 44 211 7832.

transforms to the stable [3-form (space g r o u p

P 212221, a = 5.159, b = 17.30, c = 6.948 A [-6])
m e d i a t e d by water [-7, 8]. F r o m the viewpoint of
industrial crystallization, the a - f o r m is preferable in
shape to the [3-form because of its m u c h better
separability from the m o t h e r liquor [-4]. Additives
for the selective crystallization of these p o l y m o r p h s
have been investigated for the industrial production of m o n o sodium L - g l u t a m a t e (MSG). L-aa m i n o acids, such as L-phenylalanine (L-Phe) have
been found effective not only in crystallizing the

0022-0248/97/$17.00 Copyright 1997 Elsevier Science B.V. All rights reserved

PII 8 0 0 2 2 - 0 2 4 8 ( 9 7 ) 0 0 0 1 3 - 4

C. Sano et al. / Journal of Crystal Growth 178 (1997) 568-574

a-form [9] but also in inhibiting the transformation
from the a-form to the [3-form [-10]. Further, the
di-peptide y-L-glutamyl-L-glutamic acid (y-GluGlu) has been reported to be the only effective
y-L-glutamyl peptide among y-Glu-Val, y-GluLeu, and y-Glu-Glu-Glu for crystallizing the [3form [11]. The induction of one polymorph by
additives is thought to be closely related to the
growth inhibition of another polymorph. To elucidate the mechanism of polymorph-selective crystallization by additives, we have separately investigated the effects of L-Phe, 7-Glu-Leu, and 7-GluGlu on the growth of two dominant faces {1 1 1}
and {0 0 1} of the a-form [12]. Results showed that
the {1 1 1} faces of the a-form were strongly inhibited by L-Phe, y-Glu-Leu, and y-Glu-Glu, but the
{00 1} faces were inhibited only by y-Glu-Glu
[12]. However, the effects of these additives on the
[3-form have not been known. Here, the [3-form
crystals of L-Glu have been grown with additives
having partially similar chemical structures to
L-Glu (i.e., "tailor made additives" [13]) and
their effects on the J3-form crystals have
been experimentally and structurally investigated.

2. Experimental procedure
2.1. Seed crystals and determination of the indices
of the faces
The solubility of the [3-form of L - G l u was reported as log S = - 0.461 + 0.0159 x T, here S is
the solubility (g of solute per 100 g of water) and
T is temperature (C) [14]. The 323 K saturated
L-Glu aqueous solution was left at 300 K for one
day to crystallize [3-form seed crystals, which were
thin plate-like shape with 1.0 mm in length, 0.6 mm
in width, and 0.1 mm in thickness as shown in
Fig. 1.
The relation between crystal shape and crystal
axis were determined with X-ray diffraction that
the ,direction of the length, the width, and the thickness were related to the a-, c-, and b-axis, respectively. The index of each face was determined by
measuring the face angles. The faces appeared at
the end of the a-axis were identified a s {1 0 1}

569

1oi)
~a

c/

/~/ (101)

Fig. 1. Tlae morphology of the [3-form of L-glutamic acid

crystal.

according to the face angles of 53.4 or 126.6 with

the a-axis2 The {0 1 0} and {0 0 !} faces did not
always develop well.
2.2. The procedure of crystal growth
A seed crystal was attached to one end of a thin
glass fiber. The other ends of the two fibers were
fixed to the inside of the cap of a glass tube, then
these two seed crystals were dipped into 100 ml of
L-Glu supersaturated aqueous solution with or
without additives in the tube. These crystals were
grown for 15 h in the glass tubes immersed in
a shaking water bath controlled at 308 _+ 0.1 K.
The shaking speed and the amplitude were 30
strokes per minute and 4 cm, respectively. The concentration of L-Glu solution was 2.118% (wt/wt);
which corresponds to the equilibrium concentration at 323 K. The additives were added at 1 or
5 mol% to L-Glu. The p H of the growing solution
ranged between 3.0 and 3.4 when various additives
were dissolved. Since the saturated concentration
of L-Glu 13-form is 1.231% (wt/wt) at 308 K [14],
the starting supersaturation of the growing solution was ~r = 0.721. After 15h the weight of a
crystal increased 0.3 mg at most when nothing
was added. The decrease of the supersaturation
during the growth test can be estimated negligible.
The crystal sizes were measured by taking microphotographs after orientating the crystals either
horizontally or vertically to the c-axis before
and after the incubation. Six crystals (three tubes)
were used for each growth experiment, the results
were averaged, a n d standard deviations were calculated.

570

C. Sano et al. /Journal of Crystal Growth 178 (1997) 568-574

2.3. Materials

120
I tool% addition

Following additives have been tested; four Lamino acids (L-phenylalanine: L-Phe, L-lysine: LLys, L-aspartic acid: L-Asp, and L-alanine: L-Ala),
two D-amino acids (D-alanine: D-Ala, D-glutamic
acid: D-Glu), one y-L-glutamyl peptide (y-Lglutamyl-L-glutamic acid: y-Glu-Glu), and three
carboxylic acids (L-pyrrolidone carboxylic acid:
L-PCA, a-keto-glutaric acid: ~-KGA, y-amino
butylic acid: y-ABA). L-lysine was added as Llysine hydrochloride: L-LysHC1. All the amino
acids were medical grade prepared by Ajinomoto
Company Inc. D-amino acids and carboxylic acids
were purchased from Aldrich Chemical Company
Inc. By the amino acid analysis, less than 0.07%
(wt/wt) of L-Asp was detected in the L-Glu used in
this experiment.

100

[ ] {lol}

80

iiiiiii ............ z , ..........

T
]

....

V ......

7 GluGlu L-Phe L-Lys L-Ala L-Asp g-ABA

120
~-

10o
80

60

2.4. Amino acid and carboxylie acid analyses

40

The mother liquor was wiped off with a filter

paper from the seed crystals. Three o r four seed
crystals among six crystals were dissolved in the
distilled water and fed to the analyses of incorporated additives for each experimental condition. Lamino acids, D-Ala, y-Glu-Glu, and y-ABA were
analyzed by L-8500 amino acid analyzer (Hitachi
Limited) and L-PCA and ~-KGA were analyzed by
EYELA C-3000 carboxylic acid analyzer (Tokyo
Rikakikai Company Limited). Thus, the contents of
the additives occluded in the grown part of the
crystals were calculated by dividing the concentrations of the additives by the crystal weight increased during the growth test.

3. Results and discussions

3.1. Effects of additives on the growth of each

crystal face
The average growth rates and the standard deviations (in parenthesis) of 12 control crystals were
89.1 (9.3) gm/h for the {1 0 1} faces, 1.82 (0.21) gm/h
for the {0 1 0} faces, 3.76 (0.64) gm/h for the {00 1}
faces, respectively. While y-L-glutamyl peptide, Lamino acids and y-ABA showed inhibitory effects

20
0

7 GluGlu L-Phe L-Lys L-Ala

L-Asp y-ABA

Fig. 2. The effects of various additives on the growth of ]3-form

of L-glutamic acid crystals.

on the growth of the [3-form crystals, carboxylic

acids, L-PCA and ~-KGA, and D-amino acids did
not show any inhibitory effect. The effects of adding
1 or 5 tool% of y-Glu-Glu, L-Phe, L-Lys, L-Ala,
L-Asp, and y-ABA are shown in Fig. 2. The additives showed stronger inhibitory effects in the
{0 1 0} and {0 0 1} faces than in the {1 0 1} faces.
The degree of inhibitory effects of the additives
was in the decreasing order of y-Glu-Glu > LPhe > L-Lys > L-Asp > L-Ala, y-ABA at 1 mol%
addition. At 5 mol%, the effects of L-Phe and LAsp increased significantly. L-Lys especially suppressed the growth of the {0 1 0} faces. L-Phe
showed strong inhibition in the three faces and
especially in the {0 0 1} faces, y-Glu-Glu, L-Asp did
not show any large anisotropy on the growth inhibition of the ~-form.

C. Sano et al. / Journal of Crystal Growth 178 (1997) 568-574

It can be said that the [3-form has less capacity of
recognition of molecules than the u-form by the
following results: (1) additives having the moiety of
L-u-amino acid (NH + C H C O O - ) always showed
inhibition on the three dominant faces of the [~form, (2) only the additives having both the moiety
L-u-amino acid and 3,-CO or 3'-COOH group
could inhibit the growth of the {0 0 1} faces of the
u-form [12].

571

120

~" 100
80
~a

6o

.~ 40
z0

3.2. The amount of additives incorporated during

the growth of the E-form crystal
The distribution factor of the additives is defined as
D = Rc/Rs,
where Rc is the concentration of the additive in the
crystal and Rs is that of the additive in the solution.
The distribution factors [-15] shown in Fig. 3
contain significant errors when the relative growth
rates were suppressed to less than 10% by such
additives as y-Glu-Glu or L-Phe. Some of the additives neither showed any inhibitory effect nor detected in the crystals. Although 3'-Glu-Glu and L-Phe
indicated same level of strong inhibition, the detected distribution factor of 3'-Glu-Glu w a s much
higher than that of L-Phe at 5 mol% addition. This
suggests the different mechanism of inhibition
exists between the two additives. L-Asp has a high
distribution factor, but it caused weak retardation
in the growth rate at 1 mol%. This is explained by
its molecular structure; L-Asp is an acidic amino
acid similar to L-Glu with o n e C H 2 short side
chain and can be incorporated into the crystal
lattice of the f3-form substituting L-Glu when LAsp molecule takes the torsion angle of about '110
in C-C~-C~-Cv. However, when L-Asp is incorporated, the distance between both carboxyl carbons
will be about 0.7 A shorter than that of L-Glu. This
may cause a lattice distortion in the crystal of
L-Glu, and the accumulation of this distortion will
lead the growth retardation by L-Asp at 5 tool%.
3.3. The structural explanation
3,3.1. The hydrogen bonds of the fi-form crystal
L-Glu molecule is connected each other with
eight inter-molecular hydrogen bonds (four inde-

T GluGlu L-Phe L-Lys L-AIa L-Asp Y-ABA

Fig. 3. The distribution factors of the additives during the

growth process of the [3-formof L-glutamicacid crystal.

pendent hydrogen bonds) in the ]3-form crystals

[6]. These eight modes of the hydrogen bonds,
which are participated with three u-carboxyl, two
7-carboxyl, and three u-amino groups, are shown in
Table 1 with the positions attaching to L-Glu molecules.
Four types of L-Glu molecules are indicated as
A, B, C and D to be attached on the (0 1 0) face and
(00 1) face during the crystal growth process
(Fig. 4),
3.3.2. The anisotropy in the growth rates of the
fl-form crystal
The [3-form crystal of L-Glu showed an anisotropic shape; the growth rates of the {1 0 1} faces
were about 50 times larger than those of the {0 1 0}
faces. This extremely large difference in the growth
rates cannot be explained by PBC analysis [,-16].
According to Fig. 4, the {0 1 0} faces consist of
repeated stacks of two types of molecular layers
which consist of A, B and C, D along the b-axis.
However, it is noted that the molecular layers of A,
B and C, D have no hydrogen bonds in the direction of the c-axis. This means that a kink site has no
hydrogen bond in the step wall. Thereby, the kink
on the step behaves as if it is a one-dimensional
molecular row. The molecule thus attached on the
kink is unstable and remains to fluctuate. This
weak linkage of mlecules to the direction of the
c-axis explains the reason for t h e extremely low
growth rates of the {0 1 0} face than the {1 0 1}

572

C. Sano et al. / Journal o f Crystal Growth 178 (1997) 568-574

Table 1
Hydrogen bonds (HB) of the [3-form crystal of L-Glu
Mode of HB

Atoms of the [3-form

Molecules accept or donate HB

perticipate HB formation

Atoms

Postitions

#
#
#
#

1
2
3
4

a-carboxyl
a-carboxyl
e~-carboxyl
7-carboxyl

O1
02
02
03

7-carboxyl
a-amino
a-amino
a-amino

H(O4)
H(N1)
H(N2)
H(N3)

#
#
#
#

5
6
7
8

7-carboxyl
a-amino
a-amino
a-amino

H(O4)
H(N 1)
H(N2)
H(N3)

a-carboxyl
a-carboxyl
~-carboxyl
7-carboxyl

O1
O2
02
03

(-X+1, Y-1/2, -Z+3/2)

(-X+1/2,
-Y,Z-1/2)

(x - 1, Y, z)
( x - 1/2, - z + 3/2)

r + 1/2,

( - x + 1, Y + 1/2, - z + 3/2)
(--X+l/2,

--Y,Z+I/2)

(x + 1, Y, z)
(X+1/2, - - Y + 1 / 2 , - Z + 2 )

bond to the terrace. The precise energy calculation

will elucidate the growth anisotropy more quantitatively [17].

3.3.3. The effects of additives on the faces

of fi-form crystal

(olo

Fig. 4. The molecular packing of the [3-form of L-glutamic acid

crystal viewed down the a-axis.

face. Also, the small growth rate of the {0 0 1} faces

can be explained by the same reason mentioned
above, but in this case, a kink site has no hydrogen

The additives which did not show any growth

inhibition nor detected on the fi-form crystal were
D-amino acids, a - K G A and L-PCA. The sterical
hindrances brought about by their specific molecular structures are the cause of their effects; the
D-a-NH~- group of D-amino acids, the syn-planar
structure consists of a-CO and a - C O O H groups
for a-KGA, and the pyrrolidone ring for L-PCA
hindered these molecules to be adsorbed to the
[3-form crystals.
The additives which indicated inhibitory effects
are classified into four groups by their modes of
hydrogen bond formation as shown in Table 2. All
the molecules having the moiety of L-a-amino acid
can form hydrogen bonds to the three dominant
faces of the [3-form. This characteristic of hydrogen
bond formation of the [3-form explains its lower
capacity of molecular recognition than the a-form.
The similarity in the hydrogen bond formation of
additives to L-Glu is in the decreasing order of
L-Asp > 7-Glu-Glu > L-Phe, L-Lys, L-Ala > 7ABA. This order positively corresponded to the
order of distribution factors of the additives. The
strength of the inhibition depends on the multiplication of the two factors: (1) the stability on the
crystal face, i.e., the similarity in the hydrogen bond

C. Sano et al. / Journal of Crystal Growth 178 (1997) 568-574

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r-Coo
~

,.o

,.S

r-:

oo

oo
r-2 o e

oo

oo
t-:

,,~

,~

~d

t-2

r-

<

z ~

,-,f~'f,'~m ,,--gc.gc,-fm &,~:'~reg

oo

oo

t-.toe

t-~ o o

oo

o~

oo
oo

r-:

<R

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o~

c~

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<

P:

09

o2

-?

.o

oo

573

formation, (2) the bulk which is different in some

way hinders the next molecule to be adsorbed. The
difference of behaviors between L-Phe and L-Ala
supports this hypothesis.
The mechanism of inhibition caused by 3,-GluGlu should be different from that of L-Phe. One
7-Glu-Glu molecule can be incorporated as a pair
of L-Glu molecules as A B or C D. The incorporated y-Glu-Glu has a bending shape which is
formed such as from the moiety of L-a-amino
acid of the A molecule, via a part of hydrogen
bond between y=CO of the A molecule and
a-NH~ of the B molecule, to y-COOH of the
B molecule. Incorporated y-Glu-Glu causes the distortion of the crystal lattice and growth retardation. On the other hand, L-Phe is adsorbed on
a crystal surface and impedes the growth through
the sterical hindrances by its bulky phenyl side
chain. The smaller anisotropy in the growth inhibition of L-Asp and y-Glu-Glu than L-Phe supports
this interpretation.
The mechanism of the polymorph-selective crystallization by y-Glu-Glu (for the f3-form)and L-Phe
(for the a-form) can be attributed to predominant
inhibiting effects on the 0~-form of L-Glu crystals.
This is based on the two experimental results: (1) on
the p-form, both y-Glu-Glu and L-Phe showed
strong inhibition on the three dominant faces,
{1 0 1}, {0 1 0}, and {0 0 1}, (2) on the a-form, yGlu-Glu showed inhibition on both two dominant
faces, {1 1 1} and {0 0 1}, but L-Phe inhibited only
the {1 1 1} faces.

4. Conclusions

s
e.

cq
o

To elucidate the mechanism of polymorphselective crystallization of L-Glu crystals, the

effects of additives on the 13-form crystals have
been investigated. According to the results that
both y-Glu-Glu, the inducer of the ~-form, and
L-Phe, the inducer of the a-form, indicated
nearly the same strong inhibition on the growth of
the three faces of the ~3-form, the polymorph-selectire crystallization by the additives can be attributed to the difference in the inhibitory effects of
the additives on the two dominant faces of the
a-form.

574

C. Sano et aL /Journal of Crystal Growth 178 (1997) 568-574

Acknowledgements
The authors thank Professor Hiroshi Komatsu
of Tohoku University for a critical reading of the
manuscript.

[7]
[8]
[9]
[10]
[11]
[12]

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