PROPAGATION OF BIOCHIRALITY
769
MATERIALS AND METHODS
Experiments
Chemicals
Solubility values. About 10 mg of aspartic acid and alanine samples
were weighed in a 20 mL scintillation vial. Drops of water were titrated
carefully into the vial by a micropipette with intermittent shaking until
all sample solids were just dissolved. The solubility value of samples in
water at a given temperature was calculated as the weight of sample in
a vial divided by the total volume of water added to a vial. The solubility
values of aspartic acid and alanine samples in water were determined at
25º and 40°C. All temperatures were maintained and controlled by a
water bath. Although the gravimetric method appeared to have an
inherent inaccuracy of about 10%, its advantages were its robustness,
simplicity, without the need of performing any calibration, and without
the concern of any hydrate formation and any racemic conglomerate-racemic
compound transformation. All measurements were repeated at least three
times.2
d-(ꢀ)-aspartic acid (C4H7NO4, purity 99%, mp = 300°C, MW = 133.1, ee
(GLC): 98%, lot 03610DJ), dl-( )-aspartic acid (C4H7NO4, purity 99%, mp =
280°C, MW = 133.1, lot 029K0053), l-(+)-aspartic acid (C4H7NO4, purity
98%, mp = 300°C, MW = 133.1, ee (GLC): 98%, lot 066K0184), (l-asp)4
(C16H22N4O13, purity of 97% , MW = 478.4, lot 089K1083), d-(ꢀ)-alanine
(C3H7NO2, purity 98%, mp = 291°C, MW = 89.09, lot 107 K1536), dl-( )-ala-
nine (C3H7NO2, purity 99%, mp = 295°C, MW = 89.09, lot 108K0115), l-(+)-
alanine (C3H7NO2, purity 98%, mp = 314.5°C, MW = 89.09, lot
0001440397) and succinic acid (C4H6O6, purity 99%, mp = 185º to 188°C,
MW = 118.09, lot 058K0706) were all obtained from Sigma-Aldrich (St.
Louis, MO).
Solvents
Acetone (CH3COCH3, purity 99.5%, bp= 56°C, MW= 58.09, lot AA-1101)
was purchased from Cecho (Miaoli, Taiwan). Reversible osmosis (RO)
water was clarified by a water purification system (model Milli-RO Plus)
bought from Millipore (Billerica, MA).
Crossovers. All crossover experiments were conducted at 40°C for
10 min. A standard 6-h, 40°C vacuum oven drying protocol was used to
evaporate water and to generate solids from all aqueous solutions. The
characteristic IR assignments2 and the PXRD diffraction peaks2 (i.e., 2θ
= 11.8º, 25.5º, and 28.2º for racemic conglomerate aspartic acid, and 2θ
= 13.2o and 19.5º for racemic compound aspartic acid) were used to distin-
guish racemic conglomerates from racemic compounds. However, from
the previous literature,13–15 IR spectra have shown discriminative peaks
for the functional groups, such as -COO- (in-plane bending), -COOH
(in-plane bending), -CH2 (rocking), -CN (stretching), -CH2 (twisting)
for aspartic acid, and -NH2 (scissoring), -NH2 (puckering) for alanine.
Instead of employing PXRD, IR characterization was mainly used to
identify racemic conglomerate and/or racemic compound, and conve-
niently determine the stability for racemic conglomerate.2
Instrumentations
Fourier transform infrared spectroscopy (FTIR). Transmission Fou-
rier transform infrared (FTIR) spectroscopy13–15 was used to distinguish
between a racemic conglomerate and a racemic compound of all dried
solids based on IR assignments. IR spectra were recorded on a Perkin
Elmer Spectrum One spectrometer (Perkin Elmer Instruments, Shelton,
CT). The KBr sample disk was scanned with a scan number of 8 from
400 to 4000 cm-1 having a resolution of 2 cm-1.
Powder X-ray diffraction (PXRD). Diffractograms of the powder
samples were collected by the Bruker D8 Avance X-ray diffractometer
(Germany). The source of PXRD was Cu Kα (1.542 Å) and the diffractometer
was operated at 40 kV and 41 mA. The X-ray was passed through a 1-mm slit
and the signal was passed through a 1-mm slit, a nickel filter, and another
0.1-mm slit. The detector type was a scintillation counter. The scanning rate
was set at 0.05° 2θ/sec ranging from 5° to 35°. The quantity of sample used
was around 20 to 30 mg.
Since the solubility values of dl-aspartic acid, d- or l-aspartic acid,
dl-alanine and d- or l-alanine in water at 40°C were about 10.1, 6.7,
200.7, and 169.9 mg/mL, respectively,2,16 the clear solutions obtained
after the 10-min incubation right before drying were unsaturated and
should not contain any undissolved seeds.
Experimental procedures of Crossovers I between racemic conglomer-
ate and racemic compound aspartic acid are described in Table 1, Cross-
overs II between racemic conglomerate and racemic compound alanine in
Table 2, Crossovers III between l-aspartic acid and racemic compound
aspartic acid in Table 3, Crossovers IV between l-alanine and racemic
compound alanine in Table 4, and Crossovers V between (l-asp)4 and ra-
cemic compound aspartic acid in Table 5. Since (l-asp)4 was very expen-
sive, it was used with a relatively small amount as templates or seeds
only.
Optical microscopy (OM). An optical microscope (SZII; Olympus,
Tokyo, Japan) equipped with a CCD camera (SSC-DC50A; SONY, Tokyo,
Japan) was used to take the images of the crystal habits.
Scanning electron microscopy (SEM). A scanning electron micro-
scope (SEM) (Hitachi S-3500N, Tokyo, Japan) was used to observe the
morphology of the crystals. Both secondary electron imaging (SEI) and
backscattered electron imaging (BEI) were used for the SEM detector
and the magnification was 15 to 300,000-fold. The operating pressure
was 10-5 Pa vacuum and the voltage was 15.0 kV. All samples were
mounted on a carbon conductive tape (Prod. No. 16073, TED Pella, Inc.,
Redding, CA) and then sputter-coated with gold (Hitachi E-1010 Ion
Spotter, Tokyo, Japan) with a thickness of about 6 nm. The discharge
current used was about 0 to 30 mA and the vacuum was around 10 Pa.
Although FTIR spectra and XRD patterns of l-aspartic acid and racemic
conglomerate aspartic acid looked identical, we inferred that a mixture of
l-aspartic acid and racemic conglomerate aspartic acid instead of pure l-
aspartic acid or pure racemic conglomerate aspartic acid was obtained
based on the component balance of d- and l-enantiomers.
Crystallization kinetics. Recrystallization of aspartic acid was carried
out in a 250 cm3 three-neck, round-bottom flask by antisolvent addition
at 25°C. A known amount of aspartic acid was first dissolved in 60 mL
of water. To further ensure a complete dissolution and to eliminate the
invisible seeds, the unsaturated solution was warmed to 40°C for
30 min. It was then cooled back down to 25°C and stirred by a magnetic
spin bar at 250 rpm for 1 h. Then 140 mL of acetone with or without seeds
were added as an antisolvent. The total volume of 140 mL of acetone + 60
mL of water gave 195 mL of water–acetone solution. The electrical
conductance of the resultant solution was monitored as a function of time.
All experiments were run for at least three times to test for
reproducibility.
Electrical conductance. Electrical conductivity meter (CONSORT
K611, Conductivity Instruments, Turnhout, Belgium) was used to moni-
tor the conductivity of aspartic acid in a water–acetone system where
acetone was added in the aqueous solution as an antisolvent. The electri-
cal conductivity meter was calibrated with 0.01 M of KCl each time before
use with an extrapolated conductivity of 1413 μS 1 M of KCl at 25°C. The
purchased racemic aspartic acid was used to establish the calibration
standards because it was inexpensive and the electrical conductivity
values were independent from the chirality of aspartic acid. The linear
relationship between electrical conductance and concentration: Conduc-
tivity (μS) = 2830.02 × Concentration (mol/L) + 3.32 with a correlation
coefficient of 0.99, was established based on nine various concentrations
of 5.8 × 10-4 M, 1.1 × 10-3 M, 2.3 × 10-3 M, 3.5 × 10-3 M, 4.6 × 10-3 M, 5.8 × 10-
3 M, 6.9 × 10-3 M, 8.1 × 10-3 M, and 9.3 × 10-3 M of racemic aspartic acid in
the solution of 60 mL of water + 140 mL of acetone at 25°C.
Racemic compound aspartic acid.. A known aqueous solution of
racemic compound aspartic acid with four different concentrations of
4.0, 3.7, 3.3, and 3.1 mg/mL was prepared and stirred by a magnetic spin
bar at 250 rpm for 1 min. Because the solubility of racemic compound
aspartic acid in the water–acetone solution was 0.30 mg/mL (i.e.,
C* = 2.2 × 10-3 M) at 25°C, the initial concentrations, C0, would become
Chirality DOI 10.1002/chir