Journal of Colloid and Interface Science 224, 311–316 (2000)
doi:10.1006/jcis.1999.6686, available online at http://www.idealibrary.com on
In Situ Observation of Growth Process of α-L-Glutamic Acid
with Atomic Force Microscopy
Mitsutaka Kitamura∗,1 and Kazuo Onuma†
∗ Department of Chemical Engineering, Hiroshima University, 1-4-1, Kagamiyama, Hiroshima 739-8527, Japan; and †National Institute for Advanced
Interdisciplinary Research and National Institute of Materials and Chemical Research, Higashi 1-1-4, Tsukuba, Ibaraki 305-8562, Japan
Received August 13, 1999; accepted December 13, 1999
The growth kinetics of the (111) and (001) faces of an α-L-
glutamic acid crystal was investigated from the measurements of
the growth rates, coupling with in situ surface observation using
atomic force microscopy (AFM). The data of the growth rates were
examined by applying the theoretical equations of the BCF and NaN
models. The results indicate that the growth mechanism is not due to
the screw dislocation but to the two-dimensional nucleation, i.e., the
NaN model. It was confirmed by AFM observation that both the
(111) and (001) faces grew with the “nucleus above nucleus” (NaN)
mechanism. However, the difference of the two-dimensional nu-
cleation behavior was observed between the faces. The growing
surface of the (111) face was also observed in the presence of L-
phenylaline (L-Phe) and the pinning effect by L-Phe on the grow-
ing step on the (111) face was confirmed. This result supports
the mechanism of the additive effect, which was proposed pre-
viously. °C 2000 Academic Press
Key Words: atomic force microscopy; growth mechanism; amino
acid; additive effect.
INTRODUCTION
L-Glutamic acid (L-Glu) has two polymorphs of a metastable
α and a stable β, which have different lattice parameters of
the same space group of the orthorhombic P21 21 21 (1, 2). The
L-Glu molecule has two carboxylic groups and an amino acid
group, and it forms a zwitter ion in both aqueous solutions at
the isoelectric point (pH 3.22) and in the solid state. The con-
trol of the polymorph, crystal growth rate, and morphology of
L-Glu is a very important issue in amino acid industries, such
as agricultural and medical fields, since the crystal structure,
size, and morphology of crystals severely affects the properties
of products (3, 4). The kinetic data of each face of the crys-
tal are very important in determining the mechanism of the
morphological change. However, the growth kinetics and the
growth mechanism of L-Glu are not sufficiently clear. The growth
1 To whom correspondence should be addressed. E-mail: mkitamu@
ipc.hiroshima-u.ac.jp.
311
process is thought to be composed of the bulk diffusion of the so-
lute molecule and the surface reaction which contains adsorption
of the solute on the crystal surface, surface diffusion, and inte-
gration of the molecule into the crystal lattice. In previous work
(5, 6), it appeared that the growth rate was not influenced by the
flow rate of the solutions, indicating that the surface reaction pro-
cess controls the growth rate of L-Glu. For the surface reaction
process, mainly two kinds of mechanisms can be considered.
The one mechanism is the growth originating from the disloca-
tions with a spiral component (7) in which essentially the flat
crystal surface grows; i.e., it is called the BCF mechanism. The
other one was obtained from computer simulation in which the
growth progresses through the continuous two-dimensional nu-
cleation. This mechanism is known as the “nuclei above nuclei”
(NaN) or “birth and spread” mechanism (8, 9). In this work we
investigated the growth mechanism by two methods, i.e., appli-
cation of the growth equation of the BCF and NaN models to the
kinetic data and in situ observation by atomic force microscopy
(AFM). In situ observation of the growing surface is considered
the best way to directly determine the growth mechanism. Re-
cently, using AFM, the growth mechanism of crystals with slow
growth kinetics such as biominerals (10–12), proteins (13, 14),
virus crystal (15), and less soluble inorganic crystal (16) was in-
vestigated and resulted in new findings. In these crystals, AFM
can be utilized not only for the qualitative observation of the
growing surface but also for the measurements of the step ve-
locity, growth rate, etc. In soluble inorganic and organic crystals
(17–19), on the other hand, AFM has not been used for in situ
measurement since AFM could not follow the fast growth ki-
netics in real time. In this paper, the results of the growth rate
measurement of the (111) and (001) faces of α-L-Glu crystals
were coupled with in situ observations using AFM to discuss
the growth mechanism.
The influence of additives is also a very important issue in
the control of the crystallization process of α-L-Glu since the
additives change the ratio of the polymorphs, the growth rate,
and the morphology of the crystal (20, 21). In this work, the
growing step on the (111) face of α-L-Glu in the presence of L-
phenylalanine (L-Phe) as an impurity is also observed via AFM
to investigate the adsorption effect of L-Phe.
0021-9797/00 $35.00
Copyright °C 2000 by Academic Press
All rights of reproduction in any form reserved.
312 KITAMURA AND ONUMA

FIG. 1. Morphology of an α-L-Glu crystal.

EXPERIMENTAL

1. Growth Rate Measurements of α-L-Glu Crystals

The morphology of α-L-Glu crystal is shown in Fig. 1. The
growth rates of the (111) and (001) faces of α-L-Glu crystals
at 298 K were measured using the single crystal in the flowing
aqueous solutions by the same method as that shown elsewhere
(20); i.e., the displacement of each face was measured by the
microscope and the growth rate was calculated from the rela-
tionship between the displacement and time. The dependence of
the growth rates on the supersaturation was obtained to examine
the growth mechanism. The supersaturation of the solution, σ ,
was defined as σ = (C − Ce )/Ce , where C and Ce are actual and
FIG. 2. Relationship between the growth rate and supersaturation for the
equilibrium concentrations of α-L-Glu at 298 K.
(111) face of α-L-Glu. Solid line indicates the theoretical calculation by the NaN
model.
2. In Situ Surface Observation by AFM
In situ observation of a L-Glu crystal were carried out us- nuclei (NaN) or birth and spread mechanism) (8, 9). In this work
ing a Nanoscope III-a AFM (Digital Instruments, U.S.A.) in the the mechanism was examined by applying the equations to the
contact mode with a commercially available fluid cell. Fresh obtained data and calculating the parameters in each equation
pure solutions with the concentration 0.090 M, corresponding with the least-squares method.
to σ = 0.128, were introduced into the cell using a syringe at
the desired time interval for the observations of both the (001)
and (111) faces. The same concentrated solution but containing
7.7 × 10−4 M of L-Phe as the impurity was also used for the
observation of the (111) face. Oxide-sharpened silicon nitride
cantilevers with a spring constant of 0.58 N/m and a
J-type piezo scanner (100-µm scanning range) were used for
all the observations. The entire system was placed in a ther-
mostated room and was allowed to equilibrate prior to the ob-
servation. The observation temperature was fixed at 298 K to
avoid a thermal drift of the scanner, and to correspond the con-
dition with the measurement of the growth rate. All AFM im-
ages presented in this study were taken in the constant height
mode.

RESULTS

1. Growth Rate of Each Face

The growth rates of the (111) and (001) faces of α-L-Glu in
the pure solutions are shown in Figs. 2 and 3. It was confirmed
that the growth rate is controlled by the surface reaction process
(5, 6). For the surface reaction process mainly two kinds of
mechanisms can be considered, i.e., the BCF mechanism (7) FIG. 3. Relationship between the growth rate and supersaturation for the
and two-dimensional nucleation mechanism (the nuclei above (001) face of α-L-Glu.
 GROWTH PROCESS OF α-L-GLUTAMIC ACID
In the BCF model, the growth rate, G, is expressed as
G = E(σ 2 /F) tanh(F/σ ),
where E and F are given by Eqs. [2] and [3], which are the
constants dependent on the system:
±
E = ζDs n so Ä xs2 ,
F = 9.5γ a/skT xs .
In the NaN model, it can be written as
G = X σ 5/6 exp(−Y/σ ),
where X and Y are given by Eqs. [5] and [6], which are the
constants dependent on the system:
X = (2πC0 /3)1/3 (2xs /a)E 0
Y = (π/3)(γ /kT )2
It is found that by adoption of the BCF model to the growth
rate data of the (111) and (001) faces, negative values of F are
obtained by the least-squares method, which is contradictory to
the physical meaning of Eq. [3]. On the other hand, with the
NaN model, Eqs. [7] and [8] containing reasonable parameters
are obtained. The solid lines in Figs. 2 and 3 superimpose the
calculated values of these equations, and it can be seen that the
FIG. 4. Surface morphologies of the (111) face in the pure solution by in situ AFM. Arrows indicate the growth center. The height of each step from the
nucleated island corresponds to the minimum lattice spacing for the [111] direction.
313
obtained values coincide well with the experimental ones:
[1] G(111) = 1.25 × 10−7 σ 5/6 exp(−0.09/σ ), [7]
G(001) = 6.46 × 10−8 σ 5/6 exp(−0.05/σ ). [8]
This suggests that, in both faces, the crystal grows via the NaN
model; i.e., several nuclei spread on the surface simultaneously
[2] and each nucleus grows until it comes into contact with another
nucleus or the edges of the faces. The edge free energies of
[3] nucleated islands (γ in Eq. [6]) on both faces could be estimated
using the values in Eqs. [7] and [8], and the edge free energies
of 0.73 kJ/mol for the (111) face and 0.53 kJ/mol for the (001)
face were obtained.
[4]
2. Surface Observation of the (111) in the Pure
Solution Using AFM
To confirm the growth mechanism proposed by the growth
rate measurement, in situ observation of the growing (111) face
[5] is carried out. In Fig. 4a, a surface image with a 10-µm scanning
range is presented. Although a step pattern develops all over the
[6] surface, the step train does not show a regular morphology as
expected from the case of spiral growth. Several major sources
of steps can be seen, as indicated by arrows, and the step front of
each step is wavy. To observe the step source in detail, magnified
images are presented in Fig. 4b. It can be seen that many circular
islands develop on the surface and coalescence of the steps of
each island forms the steps with wavy fronts. The macroscopic
surface morphology is the result of the stacking of these bunched
steps. This feature is typical for the NaN growth mode. Indeed,
314
FIG. 5. Surface morphologies of the (001) face in the pure solution. Two-dimensional nucleation takes place at a certain position of the surface and repeatable
layers appear which are indicated as layers 1–3 in the figure.
spiral growth was never observed on the (111) face. The height of
step originating from each island is found to be 0.47 ± 0.02 nm,
which corresponds to the minimum lattice spacing for the [111]
direction, 0.485 nm.
3. Surface Observation of the (001) Face in the Pure Solution
Figure 5 shows the surface morphologies of the growing (001)
face. It is found that the (001) face also grows by the NaN
mode of two-dimensional nucleation, the same as the (111) face.
Although the (001) face mostly develops in the α-L-Glu crystal,
spiral growth was not observed in the present condition. This
observation is consistent with the analysis of the growth rate
measurement. The feature of the two-dimensional nucleation
is, however, different from that on the (111) face. On the (111)
face, two-dimensional islands randomly form all over the surface
and develop as seen in Fig. 4. On the (001) face, however, the
source of nucleation is very small. As presented in Figs. 5a–
5c, nucleation takes place at a certain position on the surface,
and repeatable steps are produced and stacked over each other.
The morphology of steps is irregular and does not show clear
crystallographic symmetry. The height of each step is measured
as 0.43 ± 0.02 nm, corresponding to the minimum height of the
step on the (001) face.
4. Surface Observation of the (111) Face
in the Presence of L-Phe
In a previous study (20), it was observed that the growth rate
of the α-L-Glu crystal in the [110] direction was retarded with the
addition of L-Phe and finally completely stopped. In the course
of the growth, the (110) face was found to newly appear. From
this result, the growth rate of the (111) face is also considered to
be suppressed by L-Phe. The kinetic effect of L-Phe on the growth
rate was explained by the proposed model which includes the
concept of the curved step; i.e., when the additive molecules
are adsorbed on the crystal surface, the advancing steps contin-
KITAMURA AND ONUMA
uously jut out like a bow between the additive molecules ad-
sorbed on the crystal surface (20). Figure 6a shows the surface
morphology of the (111) face after introduction of the solution
with L-Phe as the impurity. The picture was taken at a 4-µm
scanning range. It can be seen that a step front protrudes in the
left part of the photograph and the surface morphology becomes
irregular. Figure 6b is a low-magnification image, with a 10-µm
scanning range. By comparison of this image with Fig. 4a, it is
understood that the surface growing under the impure solution is
very rough, and the macroscopic step pattern seen in Fig. 6a be-
comes unclear, although the step from the nucleated island can
be observed microscopically. In Fig. 6b, several black hollows
can be identified as indicated by arrows. Although such hollows
can be seen in the case of a pure-solution-grown crystal as shown
in Fig. 4a, the number of hollows increases with time, and the
surface morphology drastically changes in the case of growth
with the impure solution. Figure 6c is the surface image taken
about 20 min after Fig. 6b. The surface morphology resembles a
cluster of grapes with many hollows. The macroscopic step pat-
tern has completely disappeared in this picture. The magnified
image of the hollow is presented in Fig. 6d. The morphology
of the hollow shows hexagonal symmetry, and the depth of the
hollow is found to be 10–20 nm with a point bottom. Numerous
small curved steps observed on the (111) surface indicate that
the growth of the face is influenced by L-Phe and supports the
model (20) that the adsorption of L-Phe pins the advancing steps.
DISCUSSION
Through the present study, the following are clarified:
(1) Both the (001) and (111) faces grew with the multiple two-
dimensional nucleation mode on the whole surfaces in the pure
solution and no spiral growth was observed. We confirmed this
growth feature by scanning at the many positions on the surface.
This result is consistent with the analysis of the growth rates us-
ing theoretical equations of the BCF spiral growth mechanism
 GROWTH PROCESS OF α-L-GLUTAMIC ACID 315

FIG. 6. Surface morphologies of the (111) face in the presence of L-Phe as the impurity. The surface gradually changes to irregular as time elapses. The step
front protrudes by adsorption of the impurity particle. Many black hollows are formed with time as shown in Fig. 6c.

and multiple two-dimensional nucleation mechanism. (2) The
growth rate was faster on the (111) face than on the (001) face.
(3) The edge free energy of the (001) face was smaller than that
of the (111) face, indicating that the two-dimensional nucleation
was easy to occur on the (001) face.
The most interesting phenomenon is the inconsistency be-
tween the growth rates of the (001) and (111) faces and edge
free energies of both faces. From the lower growth rate and
the morphology of crystals, the edge free energy of the (001)
face is expected to be larger than that of the (111) face. How-
ever, the calculated edge free energies were contradictory to
this prediction. We attribute this inconsistency to the heteroge-
neous two-dimensional nucleation effect on the (001) face by
adsorbed impurities. These impurities are not intentional, but
unavoidable when we prepare the solution. The adsorbed im-
purity particle may originate a heterogeneous nucleation with
decreasing free energy of the edge. The characteristic growth
feature in this case is that repeatable two-dimensional nucle-
ation occurs from the impurity adsorbed site as observed on
the (001) face. On the other hand, if the impurity particle has
no effect on the growth, homogeneous two-dimensional nucle-
ation occurs on the whole surface as observed on the (111)
face. This model was confirmed in aqueous-solution-grown in-
organic crystals. For example, Malkin et al. (22) pointed out
in the growth of the dislocation-free ADP (110) face that the
surface energy, which was proportional to the edge free energy,
showed an irregular change at critical supersaturation. Below the
critical supersaturation, the estimated surface energy is about
1
3
that above critical supersaturation. Malkin et al. (22) con-
cluded, from this result, that an impurity adsorbed on the surface
effectively acted as a nucleation center at low supersaturation
and decreased the surface energy in appearance. At high super-
saturation, on the other hand, homogeneous two-dimensional
nucleation took place over the surface. Exactly the same phe-
nomenon was reported by Maiwa et al. (23) for the (100) face
of the Ba(NO3 )2 crystal. The feature of the nucleation changed
from repeatable at a certain point on the face to random all
over the face with the increase of supersaturation and was again
316 KITAMURA AND ONUMA
repeatable originating at the edge of the crystal due to the Berg
effect at high supersaturation in this case. The surface energy
changed irregularly depending on the feature of the nucleation.
Maiwa et al. (23) also pointed out the possibility that edge dis-
location could act as a nucleation center at low supersaturation.
The feature of two-dimensional nucleation was repeatable in
this case. Not only in the growth of small molecular inorganic
crystals but also in the protein thaumatin (101) face, an irregular
change of surface energy due to the different origin of the nucle-
ation was reported by Malkin et al. (14). In the present condition,
we do not observe the irregular change of the edge free energy
on the (001) face. We think that the heterogeneous nucleation by
adsorbed impurity on the (001) face occurs in the whole range
of the supersaturation used in the present study since the growth
feature of the (001) face does not change in the whole super-
saturation range. If the supersaturation increases more than that
in the present study, homogeneous two-dimensional nucleation
will start to occur on the (001) face, and the edge free energy
of the (001) face will show irregular change, although three-
dimensional nucleation in the solution inhibits the observation.
Therefore, we can conclude that on the (111) face of α-L-Glu,
homogeneous two-dimensional nucleation occurs over the en-
tire surface. On the (001) face, repeatable nucleation is observed
originating from an impurity particle adsorbed on the surface,
although we do not add intentional impurity such as L-Phe, or
due to defects under the condition that the supersaturation is
considerably lower than that required for homogeneous two-
dimensional nucleation for this face.
Another interpretation between the growth rates and the edge
free energies is that the lateral step velocity of the nucleated
island is faster on the (111) face than on the (001) face. Since
the growth rate of multiple two-dimensional nucleation, R, is
expressed using the nucleation rate, J , and step velocity, v, as
R = h(π J 1/3 v 2/3 /3), where h is the step height, smaller step
velocity can result in the smaller growth rate of the (001) face,
even if the edge free energy is smaller on the (001) face, fol-
lowing the larger value of J . In this case, it is not necessary
to take into account the impurity effect to explain the lower
growth rate of the (001) face, despite the lower edge free en-
ergy. However, we think that this possibility is quite low since
the homogeneous two-dimensional nucleation should occur on
both faces in this case, which is obviously inconsistent with
the different growth features of two-dimensional nucleation as
shown in Figs. 3 and 4.
CONCLUSION
The growth mechanism was examined by applying the the-
oretical equations of the BCF and NaN models to the data of
the growth kinetics of the (111) and (001) faces of an α-L-Glu
crystal. The results indicate that the growth proceeds via two-
dimensional nucleation with the NaN model. The in situ observa-
tion of the growth was carried out by AFM and it was confirmed
that both the (111) and (001) faces grew according to the NaN
mechanism. However, the difference in the two-dimensional nu-
cleation behavior was observed between the faces. The growing
surface of the (111) face was also observed in the presence of
L-Phe. The pinning effect by L-Phe on the growing step on the
(111) face was confirmed, supporting the mechanism which was
proposed previously.
APPENDIX: LIST OF SYMBOLS
a lattice spacing
C0 equilibrium concentration of growth units in the solution
Ds surface diffusion coefficient
E0 given by Eq. [2] with ζ = 1
k Boltzmann constant
n so equilibrium value of the surface solute density
s number of co-operating spirals
T absolute temperature
xs mean surface diffusion distance
γ edge free energy
ζ retardation factor
σ relative supersaturation
Ä molecular volume in the crystal
REFERENCES AND NOTES
1. Hirayama, N., Shirahata, K., Ohsagi, Y., and Sasada, Y., Bull. Chem. Soc.
Jpn. 53, 30 (1980).
2. Hirokawa, S., Acta Crystallogr. 8, 637 (1955).
3. Hiramatsu, S., Nogei-kagakukaishi 51, 27 (1977).
4. Kitamura, M., J. Jpn. Assoc. Crystal Growth 16, 61 (1989).
5. Kitamura, M., ACS Symp. Ser. 667, 83 (1997).
6. Kitamura, M., and Ishizu, T., J. Crystal Growth, in press.
7. Burton, W. K., Cabrera, N., and Frank, F. C., Phil. Trans. R. Soc. London
243, 299 (1951).
8. Gilmer, G. H., and Bennema, P., J. Crystal Growth 13/14, 148 (1972).
9. Rosmalen, R., and Bennnema, P., J. Crystal Growth 29, 342 (1975).
10. Onuma, K., Ito, A., Tateishi, T., and Kameyama, T., J. Crystal Growth 154,
118 (1995).
11. Onuma, K., Ito, A., and Tateishi, T., J. Crystal Growth 167, 773 (1996).
12. Onuma, K., Ito, A., Tabe, I., and Tateishi, T., J. Phys. Chem. B 101, 8534
(1997).
13. Malkin, A. J., Land, T. A., Kuznetsov, Yu. G., McPherson, A., and DeYoreo,
J., J. Phys. Rev. Lett. 75, 2778 (1995).
14. Malkin, A. J., Kuznetsov, Yu. G., Glantz, W., and McPherson, A., J. Phys.
Chem. 100, 736 (1996).
15. Land, T. A., Malkin, A. J., Kuznetsov, Yu. G., McPherson, A., and DeYoreo,
J., J. Phys. Rev. Lett. 75, 2774 (1995).
16. Higgins, S. R., Jordan, G., and Eggleston, C. M., Langmuir 14, 4967
(1998).
17. De Yoreo, J. J., Land, T. A., and Dair, B., Phys. Rev. Lett. 73, 838 (1994).
18. Maiwa, K., Plomp, M., van Enckevort, W. J. P., and Bennema, P., J. Crystal
Growth 186, 214 (1998).
19. Ester, G. R., and Halfpenny, P. J., J. Crystal Growth 187, 111 (1998).
20. Kitamura, M., and Ishizu, T., J. Crystal Growth 192, 225 (1998).
21. Kitamura, M., and Funahara, H., J. Chem. Eng. Jpn. 27, 124 (1994).
22. Malkin, A. I., Chernov, A. A., and Alexeev, I. V., J. Crystal Growth 97, 765
(1989).
23. Maiwa, K., Tsukamoto, K., and Sunagawa, I., “Proceedings of the Fourth
Topical Meeting on Crystal Growth Mechanism, Tokyo, 1991.”