Liquid

Chromatography -
NMR
 NMR coupling different kinds of chromatographic
separation systems can be coupled to a flow probe
of an NMR spectrometer.
 All Separation techniques like
 RP ( Reversed Phase )
 NP ( Normal Phase )
 GPC ( Gel Permeation Chromatography ) and
 SEC ( Size Exclusion Chromatography ) are applicable.
With special equipment also SFC ( Supercritical Fluid
Chromatography ).
 LC-NMR experiments can be performed in three modes.
 On-flow mode
 Direct stopped flow mode
 Loop storage mode
 On-flow mode:
 The outlet of the LC-detector is connected directly to the NMR probe.
 While the peaks are eluting, NMR spectra are continuously acquired.
 The chromatographic system is used to move the samples/peaks through
the NMR cell.
 Ex: Any HPLC system, which delivers a stable pulsation free flow
 LC-NMR Probe
 LCNMR interface not required
 With any of the LC-NMR interface ( BPSU / BSFU ) this working
mode is also possible, however they are not required.
 Direct stopped flow mode:
 Outlet of the LC-detector is connected directly to the NMR
probe.
 A LC-detector ( normally UV ) is used to detect peaks
eluting from the column.
 When a peak is detected, the flow continues until the peak
arrives in the NMR cell.
 At this time, the chromatography ( pump, data acquisition,
gradient ) stops and the NMR experiments are performed.
 Once the NMR experiments are completed, the
chromatography resumes until the next peak is found.
 This process can be repeated several times within one
chromatogram.
 Equipment:
 HPLC under control BRUKER Chromatography software
 LC-NMR Probe
 BSFU or BPSU-O
 Controlling station with BRUKER Chromatography
software
 With the LCNMR interfaces BPSU-O/BPSU-36 or
BPSU-12 this working mode is also possible,
however they are not required.
 Storage mode
 The outlet of the LC-detector is connected to the sample
loops of the BPSU-36 or BPSU-12.
 A LC-detector ( normally UV ) is used to detect peaks
eluting from the column.
 A detected peak is moved into one of the sample loops
without interrupting the chromatography.
 When the chromatography is completed, the HPLC
pump is used to transfer the peaks from the loops into
the NMR probe.
 Equipment:
 Any HPLC system
 pump under control of BRUKER Chromatography
software for transfer
 LC-NMR Probe
 BPFU-36, BPSU-12
 Controlling station with BRUKER Chromatography
software.
LC-NMR interface

 For LCNMR work an interface is

necessary.
 This interface has to control the flow
path during the experiments.
 It has to divert the flow from the pump
and the detector to the desired targets.
 It also is used to position and keep the
peaks/fractions at the desired positions
for storage or measurement.
The BRUKER Peak Stop Flow Unit ( BSFU )

 It contains a motor driven, computer controlled

valve.
 It is designed to allow LCNMR experiments in
 on-flow and stop-flow mode
 The software controlling the device is HyStar® or
 Chromstar in combination with BSFU program.
BPSU-12 HP
 The Bruker Peak Sampling Unit with 12 loops
 High Performance model for SPE and micro LC) (BPSU-12
HP)
 allows –on flow, stop-flow, loop-sampling, loop-transfer
and chromatography only mode.
 It has 12 build in sample loops.
 A manual injector and a column holder is also included.
 sample is directly submitted to the NMR
spectrometer or intermediately stored in sample loops.
 operation is totally under control of HyStar®
 Manual injection (without auto sampler HPLC). The twelve
sample loops hold a sample volume of 240μl each.
 Fractions from a chromatographic separation can be stored
an transferred to the NMR later.
 The BPSU-12 HP uses high pressure valves and capillaries.
 compatible with the LC-SPE-NMR interface
LC-NMR Automation

 The BRUKER chromatography software HyStar or Chromstar with BPSU-

12 or BSFU is able to perform automatic peak detection.
 Also the NMR software contains programs enabling automated acquisition
of the LC-NMR spectra.
 The combination of those programs is able to run LC-NMR in full
automation.
 The chromatography software detects peaks, moves them into the NMR
probe and signals the NMR-software that the sample is ready.
 All information about the sample ( retention time etc. ) is transferred to the
NMR.
 The NMR-software carries out the selected NMR experiments.
 When the experiments for the a sample are completed, the next sample is
requested from the chromatography software.
 By using an autosampler in the chromatographic system it is possible to run
several chromatograms without any user interaction.
 The automation used for LCNMR-software is similar to the automation used
for the BACS automatic sample changer.
Applications

1. Identification of drug impurities

2. combinatorial chemistry, phytochemical analysis, drug
discovery
3. Characterization of isomers and vitamin A derivatives
4. Charact erization of endogeneous and xenobiotics
metabolites directly from biological fluid
5. able to identify 2-hydroxyibuprofen, carboxyibuprofen,
and unmetabolized ibuprofen molecules from a small urine
sample after a therapeutic dose of ibuprofen.
6. Combination of LC-NMR and LC-MS
7. LC-NMR allowed the differentiation of isomers and
identification without reference compounds
8. LC-NMR MS have identified analogoes of vitamin E of palm
oil extract