Effect of additives on eremomycin sorbent selectivity in separation of salbutamol enantiomers using supercritical fluid chromatography

Article abstract of DOI:10.1134/S0036024417140011

A regime is found in which chiral stationary phase based on macrocyclic glycopeptide eremomycin allows separation of salbutamol sulfate enantiomers in supercritical fluid chromatography. Enantioseparation occurs only when two dynamic modifiers are used simultaneously: isopropylamin + trifluoroacetic acid or isopropylamin + ammonium acetate. Amine molar concentration in mobile phase has to be higher than acid molar concentration, otherwise enantiomers coelute. We suppose that with amine excess a mechanism of enantiorecognition is realized which involves ionic sorbent-sorbate interactions. Such mechanism is well-known for glycopeptide chiral selectors in liquid chromatography, but for supercritical fluid chromatography it is reported for the first time.

Full text of DOI:10.1134/S0036024417140011

ISSN 0036-0244, Russian Journal of Physical Chemistry A, 2017, Vol. 91, No. 11, pp. 2288–2290. © Pleiades Publishing, Ltd., 2017.
Published in Russian in Zhurnal Fizicheskoi Khimii, 2017, Vol. 91, No. 11, pp. 1999–2002.
SHORT
COMMUNICATIONS
Effect of Additives on Eremomycin Sorbent Selectivity
in Separation of Salbutamol Enantiomers
1
Using Supercritical Fluid Chromatography
a,b,
b
b
a,b
a,b
O. I. Pokrovskiy *, A. S. Kayda , O. I. Usovich , O. O. Parenago , and V. V. Lunin
Kurnakov Institute of General and Inorganic Chemistry of Russian Academy of Sciences, Moscow, Russia
a
b
Moscow State University, Chemical Department, Moscow, Russia
*
e-mail: oleg.pokrovskiy@supercritical.ru
Received July 24, 2017
Abstract—A regime is found in which chiral stationary phase based on macrocyclic glycopeptide eremomycin
allows separation of salbutamol sulfate enantiomers in supercritical fluid chromatography. Enantioseparation
occurs only when two dynamic modifiers are used simultaneously: isopropylamin + trifluoroacetic acid or isopro-
pylamin + ammonium acetate. Amine molar concentration in mobile phase has to be higher than acid molar con-
centration, otherwise enantiomers coelute. We suppose that with amine excess a mechanism of enantiorecognition
is realized which involves ionic sorbent-sorbate interactions. Such mechanism is well-known for glycopeptide chi-
ral selectors in liquid chromatography, but for supercritical fluid chromatography it is reported for the first time.
Keywords: supercritical fluid chromatography, enantioselectivity, eremomycin, salbutamol, ionic interac-
tions, polar ionic mode
DOI: 10.1134/S0036024417140011
1
. INTRODUCTION
Supercritical fluid chromatography (SFC) has
become very popular in preparative chiral separations in
the last several decades [3]. Several examples of the use
of MGA-based chiral stationary phases in SFC are
described in literature [4–8], but, unlike in HPLC,
enantiorecognition mechanisms on these selectors in
SFC mode are not well understood. Recently, a number
of groups reported the possibility of ionic interaction
realization in SFC [9–12]. This make us have a closer
look on the perspectives of MGA chiral selectors in
SFC, especially in separation of ionic compounds.
Macrocyclic glycopeptide antibiotics (MGA) are a
class of chiral selectors used in chromatography for
enantiomer separation [1]. Stationary phases based on
silicagels with covalently bonded MGA are able to
interact with sorbates by various mechanisms, and as
such they are exploited in high performance liquid
chromatography (HPLC) both in reverse phase and
normal phase modes. Moreover, there is an additional
separation regime known for MGA in HPLC called
polar ionic mode (PIM) which is used for chiral sepa-
rations of ionic compounds. Mobile phase (MP) in
PIM consists only of polar organic solvent, most fre- salbutamol sulfate enantiomer separation on eremom-
quently methanol, and ionizable additives, or dynamic ycin-based sorbent being, to our knowledge, the first
modifiers (DM)–carboxylic acids, amines or ammo- example of polar ionic mode in SFC.
nium salts [2]. MGA have amine, phenol and carbox-
ylic functional groups in their structure with different
pK values which can persist in neutral or ionized form
In this short communication, we report a case of
2. MATERIALS AND METHODS
a
depending on MP conditions. Additives composition
in PIM is optimized in such a manner so as to allow
the required input of ionic MGA-analyte interactions
into enantiorecognition. Most frequently it is per-
formed by adding two DMs into MP, for instance, a
primary amine and a carboxylic acid. In this case
MGA charge management is achieved via fine-tuning
the amine-acid concentration ratio [2].
Materials
Salbutamol sulfate racemate was provided by JSC
“
5
Altaivitaminy” (Biysk, Russia). Nautilus E 250 × 4.6
μm column with eremomycin chiral selector manu-
factured by JSC “Biochimmak ST” (Moscow, Russia)
was used as a stationary phase. Food grade CO
2
(99.5%) was purchased from “Linde Gas Rus” (Bala-
shikha, Russia), HPLC grade methanol–from
“Chimmed,” isopropylamine, trifluoroacetic acid and
1
The article was translated by the authors.
2288

EFFECT OF ADDITIVES ON EREMOMYCIN SORBENT SELECTIVITY
2289
arb. units
(a)
0.100
0.080
0
.060
.040
0
0.020
0
0
0
0
2
4
6
8
10 12 14 16 18 20 22 24 26 28 30
τ, min
0
.100
(b)
0
.080
0
.060
.040
0
0
.020
0.5
1.0
1.5
2.0
2.5
c)
3.0
3.5
4.0
4.5
5.0
τ, min
0
.040
(
0
0
0
0
.035
.030
.025
.020
0
0
.015
.010
0
.005
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
τ, min
Fig. 1. Three scenarios of salbutamol sulfate elution from eremomycin-based sorbent in SFC using additives. (a) Additive is IPAm
or AA. Excessive retention, enantioselectivity is registered, severe peak tailing. (b) Additive is IPAm + TFA, С
≤ С
TFA
. Rea-
IPAm
TFA
sonable retention, symmetrical peak shape, no enantiomer separation. (c) Additive is IPAm + TFA, С
> С
. Reasonable
IPAm
retention, symmetrical peak shape, enantiomers are separated.
ammonium acetate from “Galachem.” All chemicals A sample was the solution of salbutamol sulfate in
were used without additional purification.
methanol at 1 mg/mL concentration. Injection vol-
ume was 10 μL. Methanol was used as a cosolvent in
all experiments, additive composition was varied. For
each experiment with the new additive composition a
fresh solution of additives in methanol was prepared,
and the chromatograph system was flushed with the
MP containing the newly prepared cosolvent for at
Supercritical Fluid Chromatography
All experiments were performed using Waters Corp
Investigator supercritical fluid chromatography sys-
tem. All experiments were conducted at back pressure
1
4
00 bar, column over temperature 25°C, MP flowrate least 1 h. Each experiment was performed at least
mL/min, cosolvent volume fraction 30%. Mono- thrice, retention time values were average of three
chromatic UV detection was performed at 276 nm. repeatable experiments.
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 91 No. 11 2017

2290
POKROVSKIY et al.
Table 1. Selectivity coefficients (α) and resolution factors rated when AA is used as a sole additive is in favor of
(
R ) of salbutamol enantiomer separation depending on
s
this assumption. If a sorbent and a sorbate are involved
into ionic interactions, then the presence of a compet-
itive cation in the MP would be required for the elu-
tion of salbutamol from the column. In case of
IPAm + TFA two-component additive isopropylam-
monium cation resulting from acid-base interaction
can act as such; in case of AA single additive ammo-
nium cation will play that role.
IPAm : TFA molar concentration ratio in MP
Concentration ratio
Selectivity, α
Resolution, Rs
IPAm : TFA
1 : 1
≤
1
0
3
3
4
5
: 2
: 1
: 1
: 1
1.11
1.12
1.14
1.10
0.66
0.62
0.73
0.60
To the best of our knowledge, such separation
mode is reported for supercritical fluid chromatogra-
phy for the first time. Further research focuses would
be a direct confirmation of the occurrence of ionic
3. RESULTS AND DISCUSSION
interactions in SFC as well as the investigation of CO
2
No elution of salbutamol sulfate is achieved when influence on acid-base equilibriums involving glyco-
MP consists only of CO and methanol without any peptide chiral selectors.
2
additives. Eluting for 2 h did not lead to any detectable
peaks. Three types of results achieved when using
ACKNOWLEDGMENTS
This work was financially supported by Ministry of
additives are depicted in Fig. 1. Adding either isopro-
pylamine (IPAm) or ammonium acetate (AA) into
MP at 50 mM concentration two separated peaks are Education and Science of Russian Federation, project
registered, but elution takes much time at these condi- no. 03.G25.31.0273.
tions and peaks exhibit severe tailing, practically
smearing to the baseline (Fig. 1a). Using trifluoroace-
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Vol. 91
No. 11
2017