Online coupling of enantioselective capillary gas chromatography with proton nuclear magnetic resonance spectroscopy

Article abstract of DOI:10.1002/chir.20840

The hyphenation of enantioselective capillary gas chromatography and mass spectrometry is not always sufficient to distinguish between structural isomers, thus requiring peak identification by NMR spectroscopy. Here the first online coupling of enantiosel

Full text of DOI:10.1002/chir.20840

CHIRALITY 22:808–812 (2010)
Online Coupling of Enantioselective Capillary Gas
Chromatography with Proton Nuclear Magnetic
Resonance Spectroscopy
¨
MAXIMILIAN KUHNLE, DIANA KREIDLER, KARSTEN HOLTIN, HARRI CZESLA, PAUL SCHULER,
*
*
VOLKER SCHURIG, AND KLAUS ALBERT
Institute of Organic Chemistry, University of Tu¨bingen, Tu¨bingen D-72076, Germany
ABSTRACT
The hyphenation of enantioselective capillary gas chromatography and
mass spectrometry is not always sufficient to distinguish between structural isomers,
thus requiring peak identification by NMR spectroscopy. Here the first online coupling
of enantioselective capillary gas chromatography with proton nuclear resonance spec-
troscopy is described for the unfunctionalized chiral alkane 2,4-dimethylhexane resolved
on octakis(6-O-methyl-2,3-di-O-pentyl)-g-cyclodextrin at 608C. NMR allows constitutional
and configurational isomers (diastereomers and enantiomers) to be distinguished.
Enantiomers display identical spectra at different retention times, which enable an
indirect identification of these unfunctionalized alkanes. The presented method is still
at an early development stage, and will require instrumental optimization in the future.
C
VV
Chirality 22:808–812, 2010.
2010 Wiley-Liss, Inc.
KEY WORDS: chiral alkane; enantiomers; cyclodextrin; enantioselective online-coupling
GC-NMR
6,7
´
INTRODUCTION
by Koscielski in 1983 with packed columns containing
Celite coated with a-cyclodextrin in formamide. With the
advent of high resolution capillary gas chromatography
employing permethylated ß-cyclodextrin in a semipolar
polysiloxane matrix unfunctionalized 1,2-dialkylcyclohex-
anes could be enantioseparated in 19898 followed by the
enantioseparation of simple chiral alkanes later on.9–14
Nuclear magnetic resonance (NMR) spectroscopy,
which is the most powerful analytical method for the struc-
tural elucidation of organic compounds, can be envisioned
as an ideal detection method to identify stereoisomers pre-
separated by gas chromatography. For combining the very
high separation power of gas chromatography with the
high information capacity of NMR spectroscopy two
approaches can be found in the literature. Marriott and co-
workers described quite recently two model applications
for the identification of volatile compounds out of complex
matrices using an offline coupling of microscale-prepara-
tive multidimensional gas chromatography (MDGC) and
NMR spectroscopy.1,15 In one of their applications they
isolated 8.6 lg geraniol from a complex essential oil matrix
In chromatography, unequivocal chemical assignment
requires structure elucidation of a pure compound by
spectrometric and/or spectroscopic techniques such as
mass spectrometry and NMR spectroscopy.1 Unfunctional-
ized saturated hydrocarbons, i.e., alkanes, consist of a
wide range of constitutional and configurational isomers
(including enantiomers), which are difficult to distinguish
by mass spectrometry. The number of constitutional and
configurational isomers is dependent on the total number
of carbon atoms and is increased from 24 isomers for C8
to 3395.964 for C20,2 which requires high separation per-
formances3 and powerful detection systems to achieve a
correct structural assignment. Due to the nonexistent
functionality, the gas chromatographic enantioseparation
of simple chiral alkanes cannot be achieved by hydrogen-
bonding chiral selectors (e.g. Chirasil-Val)4 or coordina-
tion-type chiral selectors (Chirasil-Metal).5 Thus, the chro-
matographic separation and identification of small unfunc-
tionalized configurational isomers represents a great chal-
lenge in separation science. Due to the absence of suitable
functionalities, enantioseparation can only be achieved via
weak Van der Waals forces by employing highly enantio-
selective selectors such as modified cyclodextrins. These
selectors display unique enantioselectivities for small chi-
ral compounds because of the possibility to include the
analyte in their molecular cavities. The size of the cavities
can be controlled by different substitution patterns or the
number of sugar molecules in the cyclodextrin ring. The
enantioseparation of configurational isomers of terpenoic
1
using MDGC and recorded remarkable H NMR spectra
Contract grant sponsor: Deutsche Forschungsgemeinschaft (Graduierten-
kolleg ‘‘Chemie in Interphasen’’); Contract grant number: 441/3
*Correspondence to: Klaus Albert, Institute of Organic Chemistry, Univer-
sity of Tu¨bingen, Auf der Morgenstelle 18, Tu¨bingen D-72076, Germany.
E-mail: klaus.albert@uni-tuebingen.de or Volker Schurig, Institute of
Organic Chemistry, University of Tu¨bingen, Auf der Morgenstelle 18,
D-72076 Tu¨bingen, Germany. E-mail: volker.schurig@uni-tuebingen.de
Received for publication 24 August 2009; Accepted 20 December 2009
DOI: 10.1002/chir.20840
Published online 12 April 2010 in Wiley Online Library
cycloalkanes and cycloalkenes was for the first time solved (wileyonlinelibrary.com).
C
VV
2010 Wiley-Liss, Inc.

ENANTIONSELECTIVE CAPILLARY GC WITH NMR SPECTROSCOPY
809
with a 800 MHz NMR spectrometer equipped with a cry- row 64 transients with 1.5 k time domain points and a spec-
oprobe within an acquisition time of 1 hr.15 The second tral width of 4595 Hz were accumulated with a relaxation
direct approach was published also quite recently and delay of 10 ms. During the separation 128 rows with a ac-
demonstrated the feasibility of an online coupling of gas quisition time of 13.5 s/row were recorded.
chromatography and NMR spectroscopy16,17 based on ear-
Gas chromatography. The enantioseparation was
performed on a Fractovap Series 2350 (Carlo Erba Stru-
lier reports of the direct combination of packed column
and NMR instrumentation.18,19 In a recent work, the suc-
mentazione, Milan, Italy) gas chromatograph employing
cessful capillary gas chromatographic separation and
undiluted octakis(6-O-methyl-2,3-di-O-pentyl)-g-cyclodex-
online identification of the stereoisomers of E,Z-2-pentene
trin as the stationary phase (30 m 3 250 lm i.d., 0.50 lm
and of E,Z-2-hexene using a Chirasil-b-Dex stationary
film thickness). The carrier gas was dinitrogen 5.0 (West-
phase and a 400 MHz NMR spectrometer equipped with a
falen AG, Muenster, Germany) with a calculated flowrate
custom-built, double resonant solenoidal microprobe was
described.17
of 0.5 mL/min. The oven temperature was set to 608C dur-
ing the whole separation and 0.3 lL of racemic 2,4-di-
methylhexane was injected in the splitless injection mode.
Both methods benefit from newer developments in the
field of NMR such as microprobes16,17,20,21 or cryop-
robes22–25 and stronger magnets, which dramatically
improved the sensitivity of the relatively insensitive spec-
troscopic method so that the largest sample amounts,
which can be separated by capillary GC are getting closer
to the detection limit of NMR spectroscopy. Thus it is to
be expected that many applications using a combined
method of GC and NMR (offline as well as online) will
emerge in the future especially if a high purity isolation is
required for an unequivocal identification.
To the best of our knowledge, the present work reports
on the first separation of enantiomers by gas chromatogra-
phy with a continuous online NMR detection and it adds
to the enormous potential of GC-NMR hyphenation. More-
over, the novel enantioselective online GC-NMR experi-
ment has been probed for a most difficult enantiosepara-
tion task, i.e., that of the resolution of an unfunctionalized
racemic alkane.
Data Processing
All recorded NMR spectra show a significant back-
ground signal. To obtain more significant information, a
special baseline correction procedure had to be per-
1
formed. For the correction of the 1D spectra one H NMR
spectrum with exactly the same parameters but without
any sample was recorded. This spectrum was subtracted
from the previously recorded stopped-flow spectrum. For
the correction of the rows in the 2D plots one of the first
rows that show no eluting analyte was chosen. This row
was subtracted from each other row in the 2D plot.
RESULTS
The gas chromatographic enantioseparation of 2,4-di-
methylhexane, 2,4-dimethylheptane and 2,4-dimethyl-hep-1-
ene on octakis(6-O-methyl-2,3-di-O-pentyl)-g-cyclodextrin
(Lipodex G) is shown in Figure 1. The thermodynamic
Gibbs-Helmholtz parameters of enantiorecognition of 2,4-
dimethylhexane by Lipodex G have been reported previ-
ously.3 The enantioseparation factor increases from a 5
1.16 at 658C to a 5 1.53 at 258C.3
Figure 2 depicts the contour plot of the online GC-NMR
enantioseparation of racemic 2,4-dimethylhexane. For this
experiment a mixture of 0.3 lL racemic 2,4-dimethyl-
hexane was injected into the gas chromatograph, which
was directly connected to the NMR spectrometer.
MATERIALS AND METHODS
Materials
Racemic 2,4-dimethylhexane was obtained from Fluka
Chemie GmbH (Buchs, Switzerland). Octakis(6-O-methyl-
2,3-di-O-pentyl)-g-cyclodextrin (Lipodex G)12 was pur-
chased from Cyclolab Ltd. (Budapest, Hungary).
Instrumentation
NMR. All NMR spectra were recorded on a Bruker
ARX 400 spectrometer (Bruker BioSpin GmbH, Rheinstet-
ten, Germany). The outlet of the gas chromatographic cap-
illary column was connected to a unheated custom-built,
double resonant solenoidal microprobe with an active
detection volume of 2 ll by using a 2 m fused silica trans-
fer capillary (250 lm i.d.). For all capillary connections uni-
versal glass connectors were used (Klaus Ziemer GmbH,
Langerwehe, Germany). The NMR spectrometer was oper-
ated by an O2 workstation (Silicon Graphics) and XWIN-
NMR software (Bruker Biospin GmbH, Rheinstetten, Ger-
many). Data analysis was executed with XWIN-NMR 3.5.
The 908 1H flip angle was adjusted to 10 ls (16 dB attenua-
tion/50 W amplifier). Data acquisition parameters for
The contour plot shows the chromatographic retention
1
time versus the H chemical shift axis. The flow rate can
be calculated from the residence time s of the analytes in
the detection cell and its known size. The residence time s
in the detection cell can be calculated as follows:
Wflow ¼ Wstationary þ 1=s
W ¼ signal width at half-height
The residence time s in the detection cell obtained by
stopped-flow-experiments: 60 k transients (overnight) with this calculation was 0.25 s leading to a line broadening of
10 k time domain points and a spectral width of 5580 Hz 4 Hz and a corresponding flow rate of 0.5 mL / min.
were accumulated with a relaxation delay of 500 ms. Data
The published enantioselectivity factor of a 5 1.5 at
acquisition parameters for the 2D-experiment: For each 308C on octakis(6-O-methyl-2,3-di-O-pentyl)-g-cyclodextrin
Chirality DOI 10.1002/chir

¨
KUHNLE ET AL.
810
Fig. 1. Gas chromatographic enantioseparation of 2,4-dimethylhexane, 2,4-dimethylheptane, and 2,4-dimethyl-hep-1-ene on octakis(6-O-methyl-2,3-di-
O-pentyl)-g-cyclodextrin (Lipodex G)12,13 (50 m x 250 lm i.d. fused silica capillary, film thickness 0.25 lm, carrier gas: 100 kPa dihydrogen, temperature: 368C).
for the single GC experiment3 could not be achieved for temperature).26 However, the increased temperature is
this GC-NMR setup. The reduction of the enantioselectiv- necessary to reach a higher concentration of the analyte in
ity is due the higher oven temperature of 608C, which the detection volume to compensate for the insensitive
reduces the enantioselectivity of the stationary phase in NMR detection. Furthermore, the higher temperature
the enthalpy-controlled region (below the enantioselective leads to a faster separation increasing peak heights. In
comparison to the uncoupled GC-FID experiment, the flow
had to be reduced in the GC-NMR experiment, which
decreases the resolution factor Rs due to the loss of effi-
cency away from the van Deemter optimum. However,
higher flow rates would lead to shorter residence times
resulting in very broad NMR signals.
Figure 2 is a novel proof of principle to detect the enan-
tiomers at different chromatographic retention times. For
the identification of the enantiomers the quality of the
NMR spectra from the 2D-plot is not sufficient. More
detailed information has to be obtained by recording
stopped-flow spectra. Due to the very low concentration of
the analyte in the detection cell, the background signal of
the detection cell can no longer be disregarded (Fig. 3). It
seems that the background signal is caused by residual
protons in the glass ware of the detection cell. Also
unknown contaminants, which may adhere directly to the
surface of the detection cell might be responsible for the
observed background noise. The problem can be solved
by recording a stopped-flow spectrum of the empty detec-
tion cell, which has to be subtracted from the stopped-flow
spectrum of the analyte as described in Materials and
Methods. Another alternative to the differential spectra
approach could be using a T1q filter to suppress the broad
Fig. 2. Contour plot of a GC-NMR enantioseparation of racemic 2,4-
background signals with short relaxation times. Neverthe-
less the comparison of the stopped flow difference spectra
of the two peaks is advanced in Figure 4. Apart from a dif-
ferent signal-to-noise ratio, two identical NMR spectra are
dimethylhexane on octakis(6-O-methyl-2,3-di-O-pentyl)-g-cyclodextrin.
Data acquisition parameters: For each row 64 transients with 1.5 k time
domain points and a spectral width of 4595 Hz were accumulated with a
relaxation delay of 10 ms. During the separation 128 rows with an acquisi-
tion time of 13.5 s/row were recorded.
Chirality DOI 10.1002/chir

ENANTIONSELECTIVE CAPILLARY GC WITH NMR SPECTROSCOPY
811
Fig. 3. Comparison of the 1H NMR spectra (a) of the stopped-flow gas
phase spectrum of 2,4-dimethylhexane (enantiomer of the second eluted
peak) (b) of the empty GC-NMR probe with 1H NMR background signals.
obtained from the two peaks which indirectly proves the
presence of incongruent enantiomers.
Fig. 5. Comparison of the stopped-flow gas phase 1H NMR spectrum
of (a) one enantiomer of 2,4-dimethylhexane and (b) the off line high reso-
lution 1H NMR reference spectrum of 2,4-dimethylhexane.
Compared to the high resolution reference spectra it
can be shown that the spectral quality of the stopped flow
difference spectra are sufficient to achieve the correct
structural assignment with the aid of a reference spectrum
(Fig. 5), which are for many compounds commonly acces-
sible by spectral data bases. Other helpful information
such as integration results or coupling constants could not
yet been obtained for this experiment due to the very low
sample amount in the detection cell. Therefore an
uncoupled offline system that is able to collect the differ-
ent peaks of several runs would be necessary as was
demonstrated by Marriott and coworkers.1
The preliminary results are promising as a combined
approach employing enantio-GC and NMR methodology.
Hitherto all online GC-NMR experiments have been per-
formed at relatively low field strength of 400 MHz. The
signal-to-noise ratio could be improved by a factor of four
by using magnets with a proton resonance frequency of
800 MHz. Also shielded magnets would lead to a more ef-
ficient GC-NMR system due a shorter transfer line and the
corresponding increased peak intensity caused by a
decrease of diffusion effects.
CONCLUSIONS
In this work, it is demonstrated that even extremely
challenging separation problems such as enantiosepara-
tions of small molecules can be investigated by a hyphen-
ated GC-NMR system. Employing an online capillary GC-
NMR system the successful separation and identification
of the enantiomers of a volatile unfunctionalized chiral
alkane via a continuous NMR detection was achieved.
Stopped-flow experiments enabled the identification of
the enantiomers with the aid of reference spectra, which
are for many compounds accessible by spectra data bases.
The sample amount of a successful GC-NMR experiment
was about 100–300 lg at 400 MHz using a solenoidal
microprobe, but instrumental improvements are antici-
pated to further decrease this amount.
Although yet at its infancy, this first enantioselective
GC-NMR experiments holds promise in the future in line
with instrumental refinement. The GC-NMR approach
may complement the well established GC-MS hyphenation
in the realm of stereochemical analysis.
Fig. 4. Comparison of the stopped-flow gas phase difference spectra of
(a) the first eluted enantiomer of 2,4-dimethylhexane (b) the second
eluted enantiomer of 2,4-dimethylhexane.
Chirality DOI 10.1002/chir

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KUHNLE ET AL.
812
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