Journal of Chemical Engineering of Japan, Vol. 53, No. 11, pp.

708–712, 2020 Research Paper

Effect of Additives on the Crystal Growth of L-Aspartic Acid

Kiyoka Maruyama, Masaaki Yokota, Saki Yoshida and
Norihito Doki
Department of Chemistry and Bioengineering, Faculty of Engineering, Iwate University,
4-3-5 Ueda, Morioka-shi, Iwate 020-8551, Japan

Keywords: L-Aspartic Acid, Crystal Growth, Additives

We examined the effect of the amino acid asparagine (Asn) as a tailor-made additive to control the face-selective crys-
tal growth rate of L-aspartic acid (L-Asp). The growth rate in the c-axis direction decreased in the presence of L-Asn and
D-Asn. The Kubota–Mullin growth model was found to describe the decreased crystal growth in the c-axis direction well.
Different factors were found to be responsible for the effectiveness of L-Asn and D-Asn. The different growth–decreasing
effects of L-Asn and D-Asn on the L-Asp crystal in the c-axis direction were due to their rates of adsorption on the active
sites, which were related to the different configurations of the two additives. Based on AFM observations, a pinning
mechanism was proposed as the origin of the decreased growth rate in the presence of L-Asn. We attributed this pinning
mechanism to the repulsion between the lone pair of the nitrogen atom of the L-Asn NH2 group, which was present on
the crystal surface due to the adsorption of L-Asn, and the oxygen atom of the L-Asp carboxyl group.

do not move, and that the speed of advancing steps de-

Introduction
Crystallization is a very important technique used in a
wide range of industrial processes, such as those in the phar-
maceutical, food, and agricultural industries. Amino acids
are generally produced by fermentation and separated from
the crude fermentation solutions via crystallization. The
crude solutions contain a variety of impurities, including
microbial enzymes, media components, and microbial me-
tabolites, in addition to the desired amino acid. The amino
acids isolated and purified from the crude liquid must have
a purity of 98.5% or more for feed use and of 99.8% or more
for pharmaceutical use. Therefore, isolation and purification
of the amino acids using the differences between the physi-
cal and chemical properties of the amino acids and the im-
purities is crucial (Hasegawa and Fuke, 2008). While amino
acids are commonly isolated via crystallization, impurities
with similar molecular structures may inhibit crystal growth
because of their strong interactions with the target amino
acid. Specifically, the adsorption of the impurities on the
crystal surface inhibits growth by reducing the step advance
speed (van der Eerden and Muller-Krumbhaar, 1986). When
the advancing step encounters the adsorbed impurity on the
crystal surface, it cannot move forward at the point of the
impurity, but can continue to advance at other positions.
Thus, the steps are forced to curve. The speed of the step
then decreases as if the step were pinned by the impurities.
This is known as the Cabrera–Vermilyea pinning mecha-
nism (Burton et al., 1951). The Cabrera–Vermilyea mecha-
nism states that once adsorbed on a terrace, the impurities
Received on May 28, 2020; accepted on August 26, 2020
DOI: 10.1252/jcej.20we091
Correspondence concerning this article should be addressed to N. Doki
(E-mail address: doki@iwate-u.ac.jp).
creases when the steps encounter impurities. In recent years,
Kubota and Mullin (Kubota et al., 1997) have modified the
Cabrera–Vermilyea model. They assumed the adsorption
of impurities on the step, not the terrace, and expressed the
adsorption equilibrium using the Langmuir adsorption iso-
therm. As a result, the crystal growth rate in the presence of
an impurity can be represented as a function of the impurity
concentration and degree of supersaturation.
However, impurities often adsorb selectively on a specific
crystal face based on their molecular structures. This selec-
tive adsorption can change the crystal habit dramatically,
and impurities are sometimes deliberately added to control
the crystal habit. Such impurities are referred to as tailor-
made additives (Weissbuch et al., 2001). When the impuri-
ties have molecular structures similar to that of the crystal,
they can change the functional group present on the crystal
surface by replacing the crystal molecules; in this way,
crystal growth on a specific crystal face can be inhibited.
Therefore, studies of the effects of such similar impurities on
crystal growth are required.
Previous studies have reported the crystal growth be-
havior of L-Asp in the presence of L-Asn as a tailor-made
additive (Sato et al., 2016). L-Asn has a molecular structure
similar to that of L-Asp (Figure 1) and can adsorb on L-Asp
via its amino acid group (Sato et al., 2016). In this study, we
examined the effect of L-Asn on the crystal growth of L-Asp
along each of its axes. D-Asn was also used for the first time
as an additive to control the crystal growth in the c-axis di-
rection. The effects of L-Asn and D-Asn on crystal growth in
the c-axis direction were compared. To elucidate the effects
of L-Asn on the directional growth of L-Asp in more detail,
we compared atomic force microscopy (AFM) images of the
L-Asp crystal growth surface in the presence and absence of
the additive.

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The Society
of Chemical
of Chemical
Engineering
Engineers,
of Japan
 Fig. 1 Molecular structures of (a) L-Asp and (b) L-Asn
Fig. 2 Optical micrograph of an L-Asp crystal
1. Materials and Methods
1.1 L-Asp crystals
L-Asp crystals have two polymorphs, α and β. The α
form crystallizes as plates in the monoclinic system, with
the space group P21 (Derissen et al., 1968; Zhang et al.,
2006; Bendeif and Jelsch, 2007; Li et al., 2009), and the β
form crystallizes in the orthorhombic system with the space
group P212121 (Suresh and Vijayan, 1983). We obtained only
α-form crystals in our crystallization experiments. Figure
2 shows an optical microscope image of the L-Asp crystal.
The crystal has a plate morphology, and the c-axis shows
acute edges (Sato et al., 2016). The plane index in each axial
direction was determined via single-crystal X-ray structure
analysis (Figure 3).
1.2 Preparation of the L-Asp seed crystals
L-Asp seed crystals were prepared using the following
procedure. L-Asp (2.54 g) and distilled water (300 mL) were
added to a 300 mL glass beaker, which was sealed to avoid
solvent evaporation. The mixture was stirred and dissolved
in a thermostatic bath at 45°C to obtain a saturated solution
of L-Asp at 40°C. The solution was transferred to a crystal-
lizing dish and cooled to 25°C at 4°C/h. The solution was al-
lowed to stand at 25°C for 2–4 days, after which well-shaped
crystals were collected for use as L-Asp seed crystals.
1.3 Measurement of the L-Asp crystal growth rate
The L-Asp crystal growth rate was measured along each
axis using the following procedure (Figure 4). The seed
crystal was attached to a Petri dish. 20 mL of a 35°C satu-
rated aqueous solution of L-Asp was added to the dish,
which was then placed in a jacketed crystallizer. When the
solution became stable at the measurement temperature of
25°C, the crystal growth rate in the absence of additives (G0)
was measured for 60 min using an optical microscope. The
additive-free solution was then replaced with a saturated
aqueous solution of L-Asp at 35°C containing L-Asn (0, 1,
Vol. 53  No. 11  2020
Fig. 3 Axis directions and plane indices of the L-Asp crystal
Fig. 4 Experimental setup (a) thermostatic bath, (b) optical micro-
scope, (c) jacketed crystallizer, (d) stage, (e) Petri dish, (f)
cover to prevent evaporation
Fig. 5 L-Asp crystal growth over time (a- and b-axes). The rate in the
presence of 3 mol% L-Asn is shown for each axis direction
3, 5, 7, or 10 mol%), and the crystal growth rate (G) of an
identical seed crystal in the presence of L-Asn was measured
in the same manner. The degree of relative supersaturation,
σ, was 0.4. No nucleated crystals were observed in the Petri
dish after measurement by visual inspection.
The individuality of a seed crystal (surface state) affects
the growth rate (Sato et al., 2016). Therefore, the L-Asp crys-
tal growth rate ratio (G/G0) was evaluated to eliminate the
effect of the differences among L-Asp seed crystals and to
discuss the effect of L-Asn quantitatively.
2. Results and Discussion
2.1 Effect of L-Asn on the crystal growth of L-Asp along
the a- and b-axes
The G/G0 values at different concentrations of L-Asn (0,
3, 5, 7, and 10 mol%) were evaluated under identical condi-
tions. Figure 5 depicts the changes in L-Asp crystal growth
along the a- and b-axes over time in the presence of 3 mol%
L-Asn. The slope of the blue line corresponds to G0, and that
of the red line to G. The effect of L-Asn on the L-Asp on the
crystal growth rate along the a- and b-axis directions was
small, as G/G0 was approximately equal to 1.
709
 Fig. 7 Molecular structure of D-Asn

Fig. 6 L-Asp crystal growth over time (c-axis). The rate in the pres-
ence of 3 mol% L-Asn is shown

2.2 Effect of L-Asn on the crystal growth of L-Asp along

the c-axis
Figure 6 shows the changes in the crystal growth of L-Asp
along the c-axis over time. As shown, the growth rate along
the c-axis decreased with the addition of L-Asn.

2.3 Effect of D-Asn on the crystal growth of L-Asp along

the c-axis
Subsequently, crystal growth of L-asp along the c-axis di-
rection in the presence of D-Asn was investigated. Figure 7
shows the molecular structure of D-Asn. Figure 8 shows the
relationship between the concentration of L-Asn or D-Asn
(c) and the L-Asp crystal growth rate ratio (G/G0) in the
c-axis direction. The relationship between G/G0 and c was
fitted well by the Kubota–Mullin growth model.
2.4 Effectiveness of the additives
Table 1 shows the calculated effectiveness factor α for
each additive. The Langmuir constant K is the ratio of
the rate constant of the adsorption process, ka, to the rate
constant of the desorption process, kd (K=ka/kd). K was
determined to be 1.15 [mol%]−1 for the two additives. The
value of K varies greatly depending on the adsorption forces
between the crystal surface and the additive molecules. Ad-
ditives with different bonding modes (for example, ionic
bonding vs. hydrogen bonding) are expected to have large
differences between their K values. In the case of the L-Asp
crystal and the Asn additives, it can be assumed that these
tailor-made additives are adsorbed via the similar molecu-
lar structure, namely, their amino acid groups. The growth
of the L-Asp crystal in the c-axis direction occurs via the
formation of intermolecular hydrogen bonds in the crystal.
Such hydrogen bonds could also be formed when the Asn
additives are adsorbed. Therefore, the bonding mode (hy-
drogen bonding) between the L-Asp crystal and the Asn
additives was considered to be the same for both L-Asn
and D-Asn; the number of hydrogen bonds formed per
additive molecule (via the amino acid groups) would also
be the same. Therefore, it was assumed that the L-Asn and
D-Asn additives would exhibit almost identical K values.
Using K=1.15 [mol%]−1, the additive effectiveness factor α
Fig. 8 Relationship between the L-Asn or D-Asn concentration (c)
and the crystal growth rate ratio of L-Asp (G/G0) in the c-axis
direction. The Asn concentration is expressed as the mole ratio
of Asn to Asp. The mean values and error bars of three mea-
surements are shown
Table 1 Effectiveness factor of additive α (K=1.15)
L-Asn D-Asn
α [—] 0.993 0.380
was calculated to be 0.993 in the L-Asn system 0.380 in the
D-Asn system, as shown in Table 1; i.e., the α value for L-Asn
was approximately three times that of D-Asn. This means
that the rates of adsorption on the active sites of the L-Asn
and D-Asn systems differed by a factor of ∼3. Therefore,
the different inhibitory effects of L-Asn and D-Asn on the
growth of the L-Asp crystal in the c-axis direction was attrib-
uted to the difference in the rates of adsorption on the active
sites of the two additives.
2.5 AFM observations
Figure 9 shows AFM images of the c-axis direction crys-
tal growth plane in the presence of 0, 3, and 5 mol% L-Asn.
In the absence of the additive, a flat surface was observed,
while in the presence of the additive, arc-shaped bumps
were observed on the surface. The width and number of
bumps were measured at each additive concentration; as

710 Journal of Chemical Engineering of Japan

Fig. 9 AFM image of growth surface in the c-axis direction (AFM Z-height image) and histograms depicting the relationship between the width and
number of bumps in the presence of (a) 3 mol% L-Asn, and (b) 5 mol% L-Asn

the additive concentration was increased, the width of the

bumps decreased and their number increased. This result
suggested that the additive decreased the growth rate in the
c-axis direction via a pinning mechanism in which the addi-
tive adsorbs onto the step, causing the step to curve (Burton
et al., 1951; Sato et al., 2016).

2.6 Crystal structure of L-Asp, L-Asn, and D-Asn

Furthermore, we considered the effects of the additives
L-Asn and D-Asn on the crystal growth in the L-Asp c-
axis direction based on structures determined using single-
crystal X-ray diffraction. Figure 10(a) shows the molecular
structure along the L-Asp c-axis direction, and (b) shows its
structure when L-Asn is adsorbed. L-Asn is adsorbed onto
L-Asp crystals via the formation of hydrogen bonds between
the CO group of L-Asn and the OH and NH groups of L-Asp
(Kubota et al., 1997). When L-Asn is adsorbed, its NH2
group is present on the crystal surface. The nitrogen atom of Fig. 10 Molecular structure of L-Asp. (a) shows the crystal structure
the NH2 group and the oxygen atom of the L-Asp carboxyl along the L-Asp c-axis direction, and (b) shows the crystal
structure along the L-Asp c-axis direction when the additive
group experience repulsion due to the lone pair, thereby
L-Asn is adsorbed
causing pinning and inhibiting the crystal growth of L-Asp
in the c-axis direction. When D-Asn is used as the additive,
the differences in its configuration relative to its enantiomer
L-Asn must be considered. Figure 11 shows a schematic
diagram of the adsorption of Asn during the forward move-
ment of the step of the L-Asp crystal in the c-axis direction.
The additive L-Asn can adsorb on the surface of the L-Asp
crystal, but D-Asn cannot due to the steric hindrance of
NH3. Therefore, the D-Asn system would have a longer ad-
sorption active point interval and a smaller additive effect
than the L-Asn system.

Conclusions
Fig. 11 Schematic diagram of the adsorption of the asparagine addi-
In conclusion, the additive L-Asn had no effect on the tives during forward movement of the step of the L-aspartic
growth of L-Asp in the a- and b-axis directions, but inhib- acid crystals in the c-axis direction
ited growth in the c-axis direction. In addition, the Kubota–
Mullin growth model was found to describe the decreased

Vol. 53  No. 11  2020 711

crystal growth in the c-axis direction well. D-Asn had a
smaller effectiveness factor and growth decreasing effect in
the c-axis direction than L-Asn. The different effects of L-Asn
and D-Asn on the growth of the L-Asp crystal in the c-axis
direction were proposed to be due to rates of adsorption
on the active sites, which originates from the different con-
figurations of the two additives. In AFM images, arc-shaped
bumps were observed on the crystal surface in the presence
of the additive. The width of the bumps decreased and their
number increased as the additive concentration was in-
creased. Based on this result, the additive was proposed to
decrease the growth rate in the c-axis direction via a pinning
mechanism. We hypothesized that this pinning mechanism
was due to the repulsion between the lone pair of the nitro-
gen atom of the adsorbed L-Asn NH2 group on the crystal
surface and the oxygen atom of the L-Asp carboxyl group.
Further investigations of the effects of similar additives may
have potential applications in separation processes.
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