G Model
CCLET 4312 No. of Pages 5
2
M. Zhou et al. / Chinese Chemical Letters xxx (2017) xxx–xxx
are evaluated with some racemic compounds under reversed-
phase conditions.
of carbon content from 7.0% to 13.0%. Surface coverage of CM-
b
-CD
on aminized silica gel was estimated to be about 0.28
using the following formula [16]:
m
mol/m2
FT-IR spectra were performed on a Thermo Fisher Nicolet 6700
FT-IR Spectrometer (Thermo Fisher, U.S.A.). EA was obtained with a
vario MICRO cube instrument (Elementar, Germany). High
resolution mass spectrometry (HRMS) was carried out on a
microTOF-Q II comprising a high-performance hybrid quadrupole
time-of-flight mass spectrometer (Bruker, Germany). TGA was
recorded on a TG/DSC 2 thermal analyzer (Mettler Toledo,
Switzerland). Evaluation of the new column was made on a
Shimadzu (Kyoto, Japan) LC-Solution equipped with a LC-20AT
binary LC pump, a Shimadzu SPD-20A UV–vis detector and a 7725i
% C
12 nc Â100
ꢀ
ꢁ
% C
S
1 À 12 n Â100 Â M
c
where %C is the increment of carbon content (6.02), nc is the
number of carbon atoms in the bonded moiety (48), M is the
molecular weight of the bonded moiety (1309) and S is the
aminized silica gel special surface (440 m2/g). The FT-IR data shows
a new intense absorption peak at 1647.0 cmÀ1 due to the amide
group of the modified silica gel. The loss of weight for CM-b-CD CSP
manual injector (Rheodyne Inc., U.S.A.) with a 20 mL sample loop.
was determined to be 29.9% from 35 ꢀC to 800 ꢀC under air
atmosphere at heating rate of 15 ꢀC/min, while the loss of weight
for aminized silica gel was only 9.6%, which could also confirm the
All solvents and reagents used in synthesis and mobile phases were
commercially available and directly used without further purifica-
tion.
successful bonding of the CM-b-CD CSP.
The synthetic route is shown in Scheme 1.
The CSP was slurry packed into stainless steel column
(150 mm  4.6 mm i.d.) using a slurry packing method with an
Alltech 1666 HPLC Slurry Packer. Carbon tetrachloride/1,4-diox-
ane = 2/1 (v/v) used as the slurry solvent and MeOH as the packing
solvent. Chromatographic performances of this CSP were evaluated
with 24 racemic compounds listed in Fig. 1 which were either
supplied from pharmaceutical companies or prepared in our
laboratory (compounds 6–9, Supporting information). All the
chromatograms were obtained at 1.00 mL/min and 254 nm unless
otherwise specified under reversed-phase mode. Mobile phases
17% Chloroacetic acid aqueous solution (9 mL) was added
dropwise to a solution of
b-CD (5.00 g) in aqueous 20% sodium
hydroxide solution (15 mL) on a water bath (60 ꢀC) over 10 min. The
resulting mixture was stirred for 4 h. Then hydrochloric acid was
added to adjust the pH to 6.0-7.0 while the reaction solution was
cooled to room temperature. The obtained product was precipi-
tated by addition of excess MeOH. After filtration, the well white
precipitate was washed with 3 Â 30 mL aqueous 50% MeOH
solution and dried in vacuo at 60 ꢀC to afford 3.32 g CM-
b-CD.
FT-IR (curve B, Fig. S1 in Supporting information) (KBr, cmÀ1):
3388.9, 2928.6, 1602.4, 1416.8, 1329.9, 1079.9, 1030.5, 579.5; HRMS
(Fig. S2 in Supporting information) (m/z, negative): 1249.3291,
1271.2849, 1307.3363, 1329.3159, 1365.3399, 1387.3190, 1409.2992
were filtered with 0.45 mm membranes and degassed with
sonication before use. All the working samples were prepared in
MeOH at a concentration of about 1.0–2.0 mg/mL, and the injected
volume was 3
In order to investigate the effect of mobile phase (salts, pH and
organic modifiers) on chiral separation of CM- -CD CSP, several
mL.
and 1489.2831. The FT-IR spectrum indicates that CM-b-CD exists
in the form of sodium CM-b
-CD. Two strong peaks at 1602.4 cmÀ1
and 1416.8 cmÀ1 are assigned to the asymmetrical stretching
vibration and symmetrical stretching vibration of carboxylic salts,
b
compounds were chosen for detailed study. Introduction of salt in
the mobile phases could greatly improve separation efficiency and
peak shape [17]. The influence of ammonium acetate (NH4Ac)
concentration on the chiral separation was investigated using a
neutral (compound 2), an acidic (compound 7) and a basic
(compound 17) compound as model analytes (Table 1). As shown
in Table 1, in the absence of NH4Ac in mobile phase, the neutral and
acidic compounds could be eluted while the basic compound could
not be eluted within 30 min. This is because there is an ion-pairing
formation between the protonated basic analyte and the free
respectively, which are not observed in native
b-CD. The HRMS
results further corroborate the success of the synthesis procedure.
Two peaks at 1249.3291 and 1271.2849 mean that two hydroxyl
groups on
1329.3159 indicate that three hydroxyl groups on
pated in reaction; Peaks at 1365.3399, 1387.3190, 1409.2992
suggest tetrasubstituted -CD, and 1489.2831 indicates five. The
b
-CD reacted with chloroacetic acid; Peaks at 1307.3363,
b
-CD partici-
b
average substitution degree of CM-b-CD is calculated to be 3
according to the HRMS results.
carboxyl on the CM-b-CD CSP. However, this interaction could not
This new CSP was obtained by chemically bonding CM-
b-CD
be aroused for the neutral and acidic compounds. It can be noted
that the retention factors (k) for all the three analytes are reduced
with the amount of NH4Ac in the mobile phase increasing from
0.50% (w/v, 0.0649 mol/L) to 2.00% (w/v, 0.260 mol/L), especially
for compound 17. Meanwhile, the selectivity factors (a) for all of
them are kept almost unchanged but the resolutions (Rs) decreases
slightly. The suitable amount of NH4Ac is 0.50%.
onto 3-aminopropyl silica gel. The CM- -CD (3.32 g), 1-ethyl-3-(3-
b
dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (3.51 g),
and N-hydroxysuccinimide (NHS) (2.03 g) were dissolved in
distilled water (70 mL), after which Platisil NH2 silica gel (3-
aminopropyl silica gel) (1.55 g) was added at 0 ꢀC and the reaction
was allowed to proceed for 24 h at room temperature, filtrated,
washed with water, MeOH, and then dried in vacuo at 60 ꢀC for 4 h.
In addition to NH4Ac, ammonium chloride (NH4Cl) and
monoammonium phosphate (NH4H2PO4) were tested as other
two ammonium salt additives, at identical salt concentration
(0.0649 mol/L) and MeOH content varied from 50% to 20%
(Tables S1–S3 in Supporting information), the representative
chromatograms are depicted in Fig. S4 (Supporting information).
The results indicate that anion species also affect the retention and
the enantioselectivity. NH4H2PO4 is found to be more effective in
reducing retention factors than NH4Cl for the three compounds.
However, NH4Ac has different effect in retention for different
natural compound. Acidic compound shows the lowest retention
in aqueous NH4Ac while basic compound exhibits the highest
retention. It’s not surprising because different salt solution has
different pH which might vary ionization of solute molecule with
acidic or basic functional groups. Among these salt additives,
CM-b-CD CSP was obtained as white powder (1.94 g, 125%). EA
found (%): C 13.02, H 2.68, N 2.93; FT-IR (curve C, Fig. S1 in
Supporting information) (KBr, cmÀ1): 3442.6, 2934.8, 1647.0,
1560.7, 1100.1, 468.5; TGA (curve b, Fig. S3 in Supporting
information): Weight loss was 29.9% (w/w). EA shows an increase
Scheme 1. Synthesis of CM-b-CD CSP. Reagents and conditions: (i) (a) chloroacetic
acid/sodium hydroxide/H2O/60 ꢀC, (b) hydrochloric acid; (ii) EDCI/NHS/3-amino-
propyl silica gel/H2O.
Please cite this article in press as: M. Zhou, et al., Preparation and chromatographic evaluation of a chiral stationary phase based on
b
Zhou, Min
