Characterization of binding affinities in a chromatographic system by suspended state HR/MAS NMR spectroscopy

Article abstract of DOI:10.1002/mrc.2551

In the current work a racemate of (R)-and (S)-benzylmandelate was separated with a stereoselective polysaccharide-based chiral stationary phase by HPLC. To elucidate the occurring chiral molecular recognition processes in the heterogeneous system used, NMR spectroscopy was chosen under high resolution/magic angle spinning (HR/MAS) NMR conditions in the suspended state. Therefore, and as a proof of concept, a combination of several NMR methods such as spin - lattice relaxation time (T₁) measurements (T₁), the saturation transfer difference, and the 2D experiment of the transferred nuclear overhauser enhancement spectroscopy technique were applied. With HR/MAS NMR it is feasible to combine NMR and chromatography to achieve further insights into the separation process. Copyright

Full text of DOI:10.1002/mrc.2551

Research Article
Received: 1 September 2009
Revised: 28 October 2009
Accepted: 30 October 2009
Published online in Wiley Interscience: 30 November 2009
(www.interscience.com) DOI 10.1002/mrc.2551
Characterization of binding affinities
in a chromatographic system by suspended
state HR/MAS NMR spectroscopy
Volker Friebolin, Silvia Marten and Klaus Albert∗
In the current work a racemate of (R)- and (S)-benzylmandelate was separated with a stereoselective polysaccharide-based
chiral stationary phase by HPLC. To elucidate the occurring chiral molecular recognition processes in the heterogeneous
system used, NMR spectroscopy was chosen under high resolution/magic angle spinning (HR/MAS) NMR conditions in the
suspended state. Therefore, and as a proof of concept, a combination of several NMR methods such as spin–lattice relaxation
time (T1) measurements (T1), the saturation transfer difference, and the 2D experiment of the transferred nuclear overhauser
enhancement spectroscopy technique were applied. With HR/MAS NMR it is feasible to combine NMR and chromatography to
c
achieve further insights into the separation process. Copyright ꢀ 2009 John Wiley & Sons, Ltd.
Keywords: HPLC; chiral stationary phase; 1H HR/MAS NMR; spin–lattice relaxation time (T1); saturation transfer difference; transferred
NOESY
Introduction
As stated previously, transient ligand–receptor complex for-
mation results in a different dynamic and relaxation behavior of
protons in the diastereomeric complex and free in solution.[6,7,13]
In this complex, ligand protons that interact with the CSP ma-
terial will probably gain the relaxation behavior of those of the
receptor due to restricted molecular motions yielding in a shorter
spin–lattice relaxation time T1.[5,14] Hence, as an indication of a
different binding affinity of both enantiomers toward the recep-
tor molecules, the measurement of T1 can be used for screening
purposes.
To study intermolecular interactions in host–guest complexes,
Meyer et al. used the saturation transfer difference (STD) NMR
method.[15] In our approach we adopted this technique to HR/MAS
and to the investigated suspension consisting of benzylmandelate
dissolved in an added solvent together with a CSP material. After
selective irradiation of CSP receptor protons the magnetization
spreads by effective spin diffusion over the whole macromolecule.
By cross-relaxation processes the saturation will be transferred
in the diastereomeric complex to the analyte, in our case to the
bound enantiomer. This results in a partial saturation of protons
of the enantiomer decreasing the signal intensities, especially
of those protons that are in close contact to the irradiated CSP
receptor.[9,16,17]
Throughout the last few decades, separations of racemic mixtures
in liquid chromatography or in capillary electrophoresis[1] have
been of increasing interest in the development of new drugs.
Today, a large variety of chiral stationary phases (CSPs) with
different synthetic, semi-synthetic, or naturally occurring chiral
selectors exist.[2,3] For instance, macrocyclic antibiotics (e.g.
vancomycin), pirkle-type selectors, molecular imprinted polymers,
or polysaccharide derivatives (cellulose or amylose) are commonly
used as CSPs selectors.[1,4]
The separation by HPLC is based on the formation of more or
less stable diastereomeric complexes of the enantiomers and the
CSP.[5] For the discovery of new enantioselective tailor-made col-
umn materials and to better understand the chromatographic
retention mechanism, knowledge about intermolecular inter-
actions forces like hydrogen bonding, π –π, dipole–dipole, or
electrostatic (Coulomb) forces is crucial. To study such spatial
interactions, several different NMR techniques have been ap-
plied. However, those experiments were commonly performed
in the liquid state, resulting in narrow lines of the recorded
resonances. In contrast, the investigated chromatographic sys-
tem consists of a suspension of a solid CSP based on silica
gel and an analyte solution, which is bound to the sorbent
material or dissolved in an added solvent. Hence, NMR spec-
tra of suspensions consist of broad signal resonances, due
to e.g. chemical shift anisotropy or dipole–dipole interac-
tions.
In addition, the nuclear overhauser effect (NOE) can be used
to detect through-space interactions. Principally, low molecular
weight compounds show a different NOE behavior compared to
macromolecules (e.g. CSP).[8,18–20] The NOE enhancement of large
receptor molecules builds up more quickly compared to small an-
1H high resolution NMR spectroscopy combined with magic
angle spinning (1H HR/MAS) reduces such line broadening effects
thus resulting in better resolved spectra, possibly contributing to
the elucidation of recognition processes at the binding site of
the CSP surface.[6,7] Additionally, a solvent added to the sample
mixture further decreases susceptibility effects leading to a better
analysis of the recorded spectra.[8–12]
∗
Correspondence to: Klaus Albert, University of Tuebingen, Institute of Organic
Chemistry, Auf der Morgenstelle 18, 72076 Tuebingen, Germany.
E-mail: klaus.albert@uni-tuebingen.de
University of Tuebingen, Institute of Organic Chemistry, Auf der Morgenstelle
18, 72076 Tuebingen, Germany
c
Magn. Reson. Chem. 2010, 48, 111–116
Copyright ꢀ 2009 John Wiley & Sons, Ltd.

V. Friebolin, S. Marten and K. Albert
Figure 1. Structure of the chiral selector on the silica surface upon which
the amylose tris (3,5-dimethylphenylcarbamate) CSP is based.
Figure 2. Chromatogram of the enantioseparation of (R)/(S)-
benzylmandelate (benzyl-2-hydroxy-3-phenylpropionate) on the amylose
tris (3,5-dimethylphenylcarbamate)-based CSP. For additional chromato-
graphic conditions, see Experimental Section.
alytes due to fast cross-relaxation and short spin–spin relaxation.
In the complex, the NOE effect of the bound analytes is dominated
by the NOE behavior of the receptor resulting in different signs of
the NOE cross-peaks in a 2D experiment. Although cross-peaks are
positive or weak negative for free analytes in solution, they alter
to strong negative by ligand–receptor complex formation. The
resulting effects depend on the size and lifetime of the complex,
the NOE buildup speed, represented by the mixing time τm in
the NOE experiment, and the correlation time τc of both free
and bound compounds.[20] Such changes of cross-peak signal
intensities caused by complexation and intermolecular magne-
tization transfer can be measured in a 2D transferred nuclear
overhauser enhancement spectroscopy (trNOESY) experiment
and give valuable information about the binding process.[8]
In the present approach, we used a combination of T1
measurements, STD, and the trNOESY NMR technique under
HR/MAS NMR conditions to characterize molecular recognition
processes in the investigated heterogeneous system consisting of
aCSPwithanamylosetris(3,5-dimethylphenylcarbamate)receptor
and (R)- and (S)-benzylmandelate enantiomers.
with a purity of more than 99.5% was obtained from Fluka AG,
Switzerland.
HPLC method
HPLC measurements were carried out on a Knauer Smartline Sys-
tem equipped with a Smartline Manager 5000 with degasser and
low pressure gradient unit, a Smartline Pump 1000, Smartline PDA
Detector 2800, and a Smartline Column Oven. The racemic sample
solution was introduced via the Smartline autosampler 3950, and
the chromatograms obtained were analyzed by ChromGate Soft-
ware. A separation of the benzylmandelate enantiomers was per-
formed isocratically by using the CSP with methanol acidified with
0.1% TFA at a flow rate of 1 ml/min. The separation was monitored
by UV at 210 nm at 25 ◦C (Fig. 2). All analytes were preliminarily dis-
solvedinmethanol.Ameasuredquantityof10 µlofthesamplewas
injected and peak identification in the chromatogram was realized
by single enantiomer injection under the same HPLC conditions.
Suppression of the benzylmandelate dissociation in the presence
ofTFAandtheminimizationofionicinteractionswithactivesurface
silanolgroupsoftheCSPsilicaweredetectedduetoaweakerreten-
tioncomparedtoanonmodifiedeluent.Theretentionfactorkꢁ that
represents a measure of the migration rate of the two compounds
was calculated by kꢁ = (tr − t0)/t0, wherein tr represents the reten-
tion time of the analyte and t0 the elution time of a nonretained
compound. Determination of t0 was realized by injecting TTBB, a
suitable marker for the determination of the void volume.[24] TTBB
has no retention on the investigated polysaccharide stationary
phase in normal phase mode or in polar organic mode. The elu-
tion time observed with TTBB shows comparable results of void
volume determined by the method of minor disturbance. The
Experimental Section
Materials
Both (R)- and (S)-benzylmandelate (benzyl-2-hydroxy-3-phenyl-
propionate) (Figs 3 and 4) with a purity of 99% as well as 1,3,5-
tris-tert-butylbenzene (TTBB) as HPLC dead volume marker were
obtained from Aldrich, Milwaukee, USA. Trifluoroacetic acid (TFA)
was purchased from VWR, Darmstadt, Germany. Methanol was
obtained from Merck, Darmstadt, Germany. The phenylcarbamate
derivative of amylose was prepared by the reaction of hydroxyl
groupsofamylosewi◦thanexcessof3,5-dimethylphenylisocyanate
indrypyridineat100 C.[21,22] ThepreparationoftheCSPconsistsof
a coating process via adsorption of the chiral selector on a suitable
silica phase.[23] The amylose tris(3,5-dimethylphenylcarbamate)
and silica based CSP used had a particle size of 3 µm and a pore
diameter of 1000 Å with an approximate surface area of 25 m2/g.
The coverage on the silica surface was about 20%. Figure 1 shows
the chemical structure of the chiral selector immobilized on the
stationaryphase.TheCSPwaspackedinaVertex250 mm×4.6 mm
(I.D.) stainless steel column from Knauer GmbH, Berlin, Germany.
For the NMR experiments, methanol-d4 with a HDO content
of less than 0.03% was received from Euriso-Top, France. TFA-d1
enantioselectivity α of the CSP, as a measure of the separation of
[5]
the two enantiomers, was obtained by α = kꢁ(S)/kꢁ(R)
.
HR/MAS NMR spectroscopy in the suspended state
1H HR/MAS NMR spectra of the benzylmandelate/CSP suspension
were recorded on a Bruker ARX 400 MHz spectrometer at a
spinning rate of 6 kHz in 4 mm double-bearing ZrO2 rotors. The
spectrometer was equipped with a deuterium lock setup, which
was set on the resonance frequency of methanol-d4. The 90◦ pulse
length was set to 10.5 µs. The spectra were recorded at 25 ◦C
c
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Magn. Reson. Chem. 2010, 48, 111–116

Binding affinities in a chiral chromatographic system
and multiplied by an exponential line broadening function of lb
= 0.5 Hz prior to Fourier transformation. Spectra processing was
performed using Bruker TOPSPIN 2.0 software. Broad background
signals of the stationary phase were reduced by using a weak
spin lock pulse (T1ρ filter) with a duration of 200 ms and an
attenuation of 30 dB to eliminate both transverse and longitudinal
magnetization of the receptor macromolecule. Resonances of the
free analyte were not affected considerably. Signal assignment
in the recorded spectra was accomplished using a 2D 1H–1H-
COSY–NMR spectrum under HR/MAS conditions. The rotor was
filled with 80 µl of the mobile phase with or without a 0.1 M
solution of the isolated enantiomers in methanol-d4 (with 0.1%
deuterated TFA) and 10 mg of the CSP material. The ratio between
the given analyte to receptor was determined using different
amounts (5–15 mg) of the column material, but the results were
not affected considerably.[8]
Spin–lattice relaxation times (T1) were measured with the inver-
sion recovery experiment. The time interval between the 90◦ and
180◦ pulses was varied from 0.01 to 20 s with a relaxation delay of
atleast60 sbetween16experiments. Themeasurementswerecar-
ried out several times and the deviation is estimated to be 0.1 s.
To obtain 1D STD NMR spectra, the selective saturation of the
target CSP material was achieved by a series of 40 shaped 270◦
Eburb-1 pulses of 50 ms length, separated from each other by a
30 ms delay yielding to a total saturation time of about 2 s.[9,25–27]
The STD parameters, including the length and power attenuation
of the T1ρ filter as well as the shape of the selective irradiation
pulses used were optimized to fit to the investigated system.
In total, 4k transients were recorded. Subtraction of a spectrum
with receptor saturation (ON-resonance) from one without (OFF-
resonance) leads to the STD spectrum only containing analyte
signals with binding affinities to the CSP material. This subtraction
was achieved internally by appropriate phase cycling after each
scan.TheirradiationintheON-resonancespectrumwithsaturation
of CSP signals was set to 340 Hz (∼1 ppm). The OFF-experiment
uses the same saturation pulses but with an irradiation more
than 1700 Hz away from any ligand resonances, thus avoiding e.g.
temperature differences due to different energy contents in the
systembetweentheON-andOFF-experiments.Thisfrequencywas
set to 32 kHz (80 ppm) yielding a direct magnetization of ligand
resonances of less than 0.2%. Due to the low signal intensities
of the STD spectrum (5–8% of the normal signal strength) and
for a better comparability of the saturation efficiency, the ratio of
the signal intensities ISTD/IOFF−Res was normalized to the strongest
STD signal of the protons HE/Eꢁ to 100%.[28]
the orientation of the analyte at the binding site of the stationary
phase can be determined principally via intermolecular trNOEs
between the analyte and the receptor. However, under HR/MAS
NMR conditions, it is hard to figure out steric features of the bound
analyte. In order to decide whether in our case the detected in-
teractions were due to the chiral selector on the silica surface,
experiments without selector were carried out. The error in the
NOESY/trNOESY integral values is considered to be 10%. Occasion-
ally, in an NOESY spectrum spin diffusion effects due to coherence
transfer between coupled spin systems can occur, which lead to
signal artifacts. To exclude this, an ROESY (rotating frame over-
hauser enhancement spectroscopy) experiment was performed.
This experiment allows a relatively clear distinction between mag-
netization transfer through spin diffusion and chemical exchange
effects. Spindiffusionprocessesleadtonegativecross-peaksinthe
ROESYspectrum, whereaschemicalexchangewillresultinpositive
cross-peaksinthesameexperiment.[19] Ourexperimentconfirmed
that the cross-peaks in the NOESY spectra have a negative signal
intensity and consequently correspond to NOE effects.
Results and Discussion
The separation of the benzylmandelate racemate into its
enantiomers by HPLC was accomplished with the derivatized
polysaccharide-based CSP (Fig. 1) and a mobile phase consisting
of methanol with 0.1% TFA. Due to the acidic nature of benzyl-
mandelate, the addition of TFA as a modifier to the mobile phase
in HPLC dramatically reduced the peak tailing for the polar organic
mode investigated. Figure 2 shows the HPLC chromatogram of
the separation in the polar organic mode in terms of resolution
per time unit. This chromatogram shows that the (R)-enantiomer
elutes prior to the (S)-enantiomer indicating that (S) interacts more
strongly with the stationary phase than (R). The retention factor k
which characterizes the interaction strength between a substance
andthecolumnpackingmaterialrepresentsanindependentvalue
of the HPLC system. For the HPLC parameters listed, it was de-
termined to be 0.28 for the (R)-enantiomer and to be 0.42 for
the (S)-enantiomer. The quality of the separation of the racemic
mixture is described by the separation factor α, in this case 1.5.
DespiteperformingHR/MASathighrotationfrequencies(6 kHz)
on a 400 MHz NMR spectrometer, it was not possible to resolve
signals of the single enantiomers in the racemic mixture in
the presence of the CSP (Fig. 3). For binding affinity screening
purposes, a comparison of spin–lattice relaxation times for all
protons is promising because these values allow an indication of a
different dynamic behavior of the (R)- or (S)-enantiomer bound to
the stationary phase. The T1 values obtained for each enantiomer
are longer in solution than in suspension with CSP, especially for
protons in the aromatic region. Probably, molecular motions in the
diastereomeric complex of both enantiomers are limited, yielding
to shorter correlation times τc (Table 1).
NOESY and trNOESY spectra were recorded with 2k (F2) × 512
(F1) data points for each experiment and a mixing time of 2 s and
200 ms, respectively. The mixing times τm for the NOESY/trNOESY
spectra were determined by a series of experiments with different
mixing times. The time at maximum cross-peak signal intensity of
the analyte signals in the early phase of the NOE experiment was
chosen as τm (200 ms) for the trNOESY. For the NOESY experiment,
a ten times higher τm (2 s) was chosen. Between τm = 0.8 and
2 s, the cross-peak signal intensities did not differ significantly. In
total, 16 transients and 16 dummy scans were performed. The
relaxation delay was set to 2 s. The spectra were phased to pure
absorption mode and the integration was performed in reference
to the aromatic region in the 2D NOESY/trNOESY. In general, one
can distinguish between intramolecular NOEs and intermolecular
transferredNOEs, whichcanbebuiltupfastduringthemixingtime
of an NOESY experiment. The structure of benzylmandelate can
be described in more detail by the intramolecular NOE, whereas
More detailed information about the specific binding site of the
analyte molecule can be achieved by STD NMR experiments. For
this purpose, the CSP material was selectively saturated at about
1 ppm in the aliphatic region. Ligand protons that closely interact
with the irradiated receptor receive magnetization via the NOE,
which will be transferred into solution after dissociation of the
ligand yielding to decreased signal intensities in a 1D spectrum.
Hence, the STD spectrum contains only signals corresponding to
analyte protons that were bound to the sorbent material which
permit the identification of protons or groups, which directly
c
Magn. Reson. Chem. 2010, 48, 111–116
Copyright ꢀ 2009 John Wiley & Sons, Ltd.
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V. Friebolin, S. Marten and K. Albert
Figure 3. 1H HR/MAS NMR spectrum of the racemic (R)/(S)-benzylmandelatetogether with the CSP recorded in acidified methanol-d4 (+0.1% TFA). Signal
assignment was performed with the help of 2D HR/MAS 1H–1H-COSY–NMR. S = solvent (methanol, water).
Figure 4. 1D 1H STD HR/MAS NMR spectrum of (S)-benzylmandelate in suspension with the CSP. The signal intensities are decreased by approximately a
factor of 10 compared to the OFF-resonance spectrum. S = solvent (methanol, water).
interact with the receptor. From the STD spectrum (Fig. 4), it
follows that all signals of the analyte can be detected but with
lower signal intensities (5–8%) compared to those of the OFF-
resonance spectrum. This suggests that all protons of the analyte
obtained saturation indicating binding affinities of all protons
toward the receptors which was additionally supported by the
decreased T1 values. The signal intensities normalized to the
strongest STD signal (HE/Eꢁ , 100%) are presented in the bar
diagram in Fig. 5. The values of the phenyl ring protons and
of HD of both benzylmandelate enantiomers are high, assuming
that obviously these protons or groups are important for the
molecular recognition process. Differences in the STD data for
(S) and (R) suggest that the proton at the stereogenic centre HD,
the methylene group -CH2-, and parts of the phenyl ring next to
the -CH2- group of the (S)-enantiomer interact stronger probably
with the carbamoyl moiety of the CSP due to hydrogen bonding
than those of to the (R)-enantiomer. The signal intensity for the
methyleneprotonsofthe(S)-enantiomerisnotasstrong(60–66%)
asmostvaluesofthephenylprotonsorHD.Hence,weproposethat
perhaps slight differences in the conformation at the stereogenic
centre can be responsible for the different binding process of both
enantiomers.
NOESY and trNOESY experiments were performed with (R)-
and (S)-benzylmandelate once without (Fig. 6) and once with
the CSP (Fig. 7) in order to confirm these assumptions. However,
a quantitative analysis of the recorded spectra is very hard to
achieve, therefore, only qualitative information about the bound
ligands were treated.[29]
Without CSP selector, only weak intra-ligand NOE cross-peaks
are detectable with positive intensity between the aromatic
protons HE/E and the proton HD as well as between HA/A
ꢁ ꢁ
and the methylene protons -CH2-. In the presence of the CSP
selector (Fig. 7 and Table 2), additional cross-peaks for both
(R)- and (S)-enantiomers with negative sign between the aryl
system (HB/B ,C and HF/F ,G) and HD and the -CH2- protons occur
ꢁ
ꢁ
respectively. This is expected for large molecules or receptor
complexes with short correlation time. No differences between
(R) and (S) were detectable, therefore, only NOESY spectra
recorded with the (S)-enantiomer are shown. Figure 8 shows
the trNOESY spectrum of the (S)-enantiomer/CSP mixture. The
(S)- as well as the (R)-enantiomer (data not presented) shows
in this spectrum an intense intermolecular negative cross-peak
between the aromatic benzylmandelate protons and those of the
aliphatic region of the stationary phase. The integral value of
c
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Magn. Reson. Chem. 2010, 48, 111–116

Binding affinities in a chiral chromatographic system
Figure 5. Bar diagram reflecting the normalized relative STD intensities for
(R)- and (S)-benzylmandelateenantiomers in the presence of CSP recorded
in methanol-d4 (+0.1% deuterated TFA). The relative STD data (%) arise
from the individual proton signal intensities of the STD spectrum in
comparison to the OFF-resonance 1H-HR/MAS NMR spectrum referenced
Figure 7. NOESY HR/MAS NMR spectrum of (S)-benzylmandelate in sus-
pensionwithCSPmaterial. Thebrokenlinesdenotepositiveintramolecular
to the methylene signal at 5.16 ppm.[16]
.
cross-peaks of the ligands HA/A and HE/E to the -CH2- group and to HD,
ꢁ
ꢁ
respectively. In addition, negative intramolecular cross-peaks from HB/B ,C
ꢁ
ꢁ
and HF/F ,G to the methylene group and to the chiral proton occur. S =
ꢁ
ꢁ
solvent (methanol, water).
Figure 6. NOESY HR/MAS NMR spectrum of (S)-benzylmandelate in
solution. Intramolecular cross-peaks occur with positive sign of the analyte
protons HE/E to the stereogenic centre (HD) and to the -CH2- group, and
ꢁ
Figure 8. trNOESY HR/MAS NMR spectrum of (S)-benzylmandelate in the
presence of CSP material. Besides the strong negative intra-ligand NOE
effects indicated by the broken lines, a negative intermolecular cross-
peak of the aromatic analyte moiety to the aliphatic region of the
stationary phase material occurred (displayed within the circle). S =
solvent (methanol, water).
HA/A to the methylene group. S = solvent (methanol, water).
ꢁ
this peak for (S) compared to (R) is stronger by approximately
a factor of 2 (Table 2). In sum the NOE intensities of this
system measured by trNOESY experiments are more negative
compared to the NOESY suggesting interactions toward the
CSP material (Table 2). The more negative integral values of
(S) compared to (R) are probably due to stronger interaction
forces of (S) with the CSP, which is in accordance with the other
NMR measurements and with the retention order detected in
chromatography.
Concluding Remarks
The separation of (R)- and (S)-benzylmandelate into its enan-
tiomers can be achieved by HPLC using amylose tris(3,5-
dimethylphenylcarbamate)-based silica CSP. The chiral recogni-
tion of both enantiomers relies on the formation of a transient
c
Magn. Reson. Chem. 2010, 48, 111–116
Copyright ꢀ 2009 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/mrc

V. Friebolin, S. Marten and K. Albert
Maria Campos for the support concerning this manuscript, and to
Dr Ing. Herbert Knauer GmbH for the supply of the HPLC system.
This work was financially supported through AL 298/14-1 provided
by the Deutsche Forschungsgemeinschaft (DFG) and the National
Science Foundation (NSF, CHE-0724218).
Table 1. Spin–lattice relaxation times (T1) for (R)- and (S)-
benzylmandelate enantiomers in solution and together with the CSP
material
T1 (s)
Signal
assignment
Chemical shift
(ppm)
R/S
(in solution)
R & CSP
S & CSP
References
HE/E
7.44
7.34
7.28
7.21
5.24
5.16
3.15
2.99
3.60
3.49
3.62
2.00
3.00
2.99
3.10
3.30
3.30
2.08
2.52
2.47
2.69
2.63
2.71
1.80
ꢁ
HF/F ,G
ꢁ
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Acknowledgements
Paul Schuler is acknowledged for valuable discussions and all
technical assistance in performing HR/MAS NMR measurements
in the suspended state. We would like to express our gratitude to
Table 2. Integral intensities of the observed NOESY (τm = 2 s) and trNOESY (τm = 200 ms) intra- and intermolecular NOE cross-peak signals of (R)-
and (S)-benzylmandelate in the presence or absence of unmodified silica or CSP (R/S and silica) material
NOESY (integral ×10−2
)
trNOESY (integral ×10−2
)
Cross-peaks
HE/E − H
R/S
R/S and silica
R and CSP
S and CSP
R/S
R/S and silica
R and CSP
S and CSP
−0.06
+2.2
–
+2.5
+2.0
–
−0.8
−2.4
–
−1.0
−2.3
–
−0.3
+0.2
–
+0.3
−0.02
–
−3.1
−2.6
−7.7
−2.8
−2.6
ꢁ
D
HA/A − CH
ꢁ
2
Phenyl-CSP
−15.4
c
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Magn. Reson. Chem. 2010, 48, 111–116