LC-NMR And Applications Of NMR Spectroscopy

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 TABLE OF CONTENTS
CONTENTS
• Introduction
• History LC-NMR
• Principle of LC & NMR
• Instrumentation of LC-NMR
a) LC Components
b) Interface Components
c) NMR Components
• Modes of LC-NMR
• Technology to improve sensitivity of LC-NMR Method
LC Methods
NMR Method
Solvent suppression method
Applications Of NMR
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 Introduction
• NMR is one of the most powerful techniques for the structural
elucidation of organic compounds.
• Separation and isolation of the individual components of a sample mixture
are the normal steps before analyzing their structures by NMR.
• Prior to isolation, LC/MS is used routinely to analyze the components of
the mixture to evaluate the need to isolate the compound(s) of interest.
• In many cases, however, NMR is necessary for the identification of
ambiguous structures.
• Even though hyphenated LC-NMR has been known since the late 1970s,
the technique was not implemented widely until the last two decades.
• For a more complete structural analysis, LC-MS and LC- NMR have
been combined (LC-NMR-MS), with its efficacy demonstrated
successfully.

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 History of NMR
• NMR is one of the most powerful techniques for the structural
elucidation of organic compounds.
• The first on-line LC–NMR experiments were performed in the
late 1970s by Watanabe and Niki who demonstrated stopped-flow
measurements of a mixture of known compounds.
• The conventional NMR probe was transformed to a flow-through
probe by the introduction of a thin-walled Teflon capillary within
a standard NMR tube and spectra were recorded with sample rotation .
• The first real sample analyzed by LC–NMR was military jet fuel using
normal phase column and deuterated chloroform and Freon
• In theory, the physical coupling of LC with NMR could save a lot of
time and was already proposed over 20 years ago.
• However, a successful and practical LC–NMR coupling has been
achieved only in the last decade.
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 Principle of LC (HPLC)
• High performance liquid chromatography (HPLC) is basically a
highly improved form of column liquid chromatography.
• Instead of a solvent being allowed to drip through a column under
gravity, it is forced through under high pressures of up to 400
atmospheres.
• That makes it much faster.
• All chromatographic separations, including HPLC operate under the
same basic principle;separation of a sample into its constituent
parts because of the difference in the relative affinities of
different molecules for the mobile phase and the stationary
phase used in the separation.
Types of HPLC
1. Normal phase HPLC 3. Size-Exclusion HPLC
2. Reverse phase HPLC 4. Ion-Exchange HPLC
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 Principle of NMR
• The principle behind NMR is that many nuclei have spin and all
nuclei are electrically charged.
• If an external magnetic field is applied, an energy transfer is
possible between the base energy to a higher energy level
(generally a single energy gap).
• The energy transfer takes place at a wavelength that corresponds
to radio frequencies and when the spin returns to its base level,
energy is emitted at the same frequency.
• The signal that matches this transfer is measured in many ways and
processed in order to yield an NMR spectrum for the nucleus
concerned.

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 above, relates to spin-½ nuclei that include the most commonly used NMR
nucleus, proton (1H or hydrogen-1) as well as many other nuclei such as 13C, 15N
and 31P.
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Instrumentation of LC-NMR

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Instrumentation of LC-NMR

Diagrammatic Representation of LC-NMR

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Instrumentation of LC-NMR

Representation of LC-NMR 10
 LC Unit

• As shown in the schematic diagram in Figure above, HPLC

instrumentation includes a pump, injector, column, detector and
data acquisition and display system.
• The heart of the system is the column where separation occurs.
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 LC Unit
1) Solvent Reservoir : Mobile phase contents are contained in a glass
reservoir. It is usually a mixture of polar and non-polar liquid
components whose respective concentrations are varied depending on
the composition of the sample.
2) Pump : A pump aspirates the mobile phase from the solvent
resorvoir and forces it through the system’s column and detector.
Depending on a number of factors including column dimensions,
particle size of the stationary phase, the flow rate and composition
of the mobile phase, operating pressures of up to 42000 kPa
(about 6000 psi) can be generated.
3) Sample Injector : The injector can be a single injection or an
automated injection system. An injector for an HPLC system should
provide injection of the liquid sample within the range of 0.1-100 mL of
volume with high reproducibility and under high pressure (up to 4000
psi).
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 LC Unit
4)Columns : Columns are usually made of polished stainless steel, are
between 50 and 300 mm long and have an ID( 2 and 5 mm).They are
commonly filled with a stationary phase with a particle size of 3–10
µm. Columns with internal diameters of less than 2 mm are often
referred to as microbore columns. Ideally the temperature of the
mobile phase and the column should be kept constant during an
analysis.
5) Detector : The HPLC detector, located at the end of the column
detect the analytes as they elute from the chromatographic column.
Commonly used detectors are UV-spectroscopy, fluorescence, mass-
spectrometric and electrochemical detectors.
6) Data Collection Devices : Signals from the detector may be
collected on chart recorders or electronic integrators .The
computer integrates the response of the detector to each component
and places it into a chromatograph that is easy to read and interpret.
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•
LC-NMR Interface
1) Direct coupling: It include direct flow of LC effluent in to
NMR flow cell and continuous recording of spectra
 post-column splitter
 valve-switching interface i.e BNMI (Bruker NMR-Mass
Spectrometry Interface)

2) Indirect coupling:
intermediate storage loop
which transfer outlet of lc to
NMR flow cell at specified
time interval
SPE unit

fig: 36 Loop Cassette 14

 NMR Unit Bo
Basically NMR instrumentation
involves the following units.
1. A magnet to separate the nuclear
spin energy state.
2. Two RF channels, one for the N S Magnet
field/frequency stabilization and one to
supply RF irradiating energy.
B1
3. A sample probe containing coils
for coupling the sample with the RF
field; Recorder
it consists of Sample holder, RF Frequency
oscillator, Sweep generator and RF Generator
receiver.
4. A detector to process the NMR Detector
signals.
5. A recorder to display the spectrum.

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16
 MAGNETS
• It is used to supply the principal part of the field Ho, which
determines the Larmor or precessional frequency of any nucleus.
• The stronger the magnetic field, the better the line separation of
chemically shifted nuclei on the frequency scale.
• The relative populations of the lower energy spin level increases
with the increasing field, leading to a corresponding increase in
the sensitivity of the NMR experiment.
Features
1. It should give homogeneous magnetic field i.e.; the strength and
direction of the magnetic field should be constant over longer periods.
2. The strength of the field should be very high at least 20,000 gaus.
TYPES OFMAGNETS
1. PERMANENT magnets
2. ELECTRO magnets and
3. SUPER CONDUCTING magnets 17
 SUPERCONDUCTING MAGNETS (scm):
A Big Stainless Steel Dewar
• use persistent superconducting magnets to
generate the B0 field;
• at low temperatures (less than 6 K, typically)
the resistance goes to zero – that is the
wire(eg.Nb alloy) is superconducting;
• To maintain the wire in its superconducting
state the coil is immersed in a bath of liquid
helium (4 K, expensive);
•“heat shield” kept at 77 K by contact with a
bath of liquid nitrogen (cheap) to reduces the
amount of liquid helium boils off;
• vacuum flask so as to further reduce the
heat flow.
Advantages (scm):
1. Strongest Magnet; 2. Stable & homogeneous magnet field Bo;
3. Low running cost.
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 MAGNETIC COILS
• It is not easy or convenient to vary the magnetic field of large stable
magnets, however this problem can be overcome by superimposing
a small variable magnetic field on the main field.
• Using a pair of Helmholtz coils(A Helmholtz coil is a device for
producing a region of nearly uniform magnetic field, named
after the German physicist Hermann von Helmholtz) on the
pole faces of the permanent magnet does this.
• These coils induce a magnetic field that can be varied by varying
the current flowing through them.
• The small magnetic field is produced in the same direction as the
main field and is added to it.
• The sample is exposed to both fields, which appear one field to the
nucleus.
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 THE PROBE UNIT
• It is a sensing element of the spectrophotometer system.
• It is inserted between the pole faces of the magnet in X-Y plane of the
magnet air gap an adjustable probe Holder.
• So the sample in NMR experiment experiences the combined effect of two
magnetic fields ie Ho and RF (EMR).
• The usual NMR sample cell is generally made up of the glass, which is strong
and cheap. It consist of a 5 mm outer diameter and 7.5 cm long glass tube
containing 0.4 ml of liquid.
• The sample tube in NMR is held vertically between the poles faces of the
magnet.
• The probe contains a sample holder, sweep source and detector coils,
with the reference cell.
• The detector and receiver coils are orientated at 90 to each other.
• The sample probe rotates the sample tube at a 30-40 revolutions on the
longitudinal axis.
• Each part of the sample tube experiences the same time average the field.

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 THE RADIOFREQUENCY GENERATOR
• Using an RF oscillator creates the radio frequency radiation, required to
induce transition in the nuclei of the sample from the ground state to
excited states.
• The source is highly stable crystal controlled oscillator.
• It is mounted at the right angles to the path of the field of wound around
the sample tube perpendicular to the magnetic field to get maximum
interaction with the sample.
• The oscillator irradiates the sample with RF radiation.
• Radio frequencies are generated by the electronic multiplication of natural
frequency of a quartz crystal contained in a thermo stated block.
• To achieve the maximum interaction of the RF radiation with the
sample, the coil of oscillator is wound around the sample container.
• The RFO coil is installed perpendicular (90 ºC) to the applied magnetic
field and transmits radio waves of fixed frequency such as 60,100,200 or 300
MHz to a small coil that energies the sample in the probe.
• This is done so that the applied RF field should not change the effective
magnetic field in the process of irradiation
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 Shim Coils
• High resolution NMR requires linewidths of 1 Hz or less
• Magnetic field across the sample must be homogeneous so
that the corresponding variation in the Larmor frequency is small.
• Surround the sample with a set of shim coils, each of which
produces a tiny magnetic field with a particular spatial profile to
canceling out the small residual inhomogeneities in the
main magnetic field.
• Modern spectrometers might have up to 40 different shim coils
labeled according to the field profiles they generate, such as x, y, z,
z2, z3, z4, z5, xy, xz, yz, x2-y2, etc…
• Shimming, the process to optimize the shims, requires skill and
experience because various shims will interact with each other.

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 The Transmitter Channel
• Synthesizer: RF source which produces
a stable frequency which can be set
precisely.
• RF amplifier: boost this small signal to
a power of 100 W or more to provide
enough energy to excite the NMR
active nuclei in the sample.
• Attenuator: altering the RF power level
in units of decibels (dB)
• All under computer control

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Modes of LC-NMR
1.Continuous Flow(on flow):- Eluent sampled in “real-
time” as flowing through NMR Detection Coil
2.Stopped Flow:- Pump is stopped at desired location and data acquired
3.Time Slices:- Regions, or “time-slices” of interest are
analyzed
4.Peak Parking:- Peaks of interest are “parked” in off-line sample loops
5.Peak Trapping:- Solid Phase Extraction cartridges are
used to “re-concentrate” samples

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 Modes of LC-NMR
1) 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
Equipment: - Any HPLC system, which delivers a stable pulse free flow.
- LC-NMR Probe
- LC-NMR interface not required
- With any of the LC-NMR interface this working mode is also possible,
however they are not required.

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 Modes of LC-NMR
2) Stopped Flow method
• The outlet of the to the LC-detector is connected directly 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 system
- LC-NMR Probe
- Controlling station
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 Modes of LC-NMR
3) Time slice method
• It include to stop the flow at short interval over the chromatography
peak to time slice different part of chromatography run.
• It is useful if there is poor chromatography separation or if
compound under study have poor or no UV chromophore or if the
exact chromatography retention time is unknown.
• The data from such a time slice experiment referred as a total NMR
chromatogram (tNMRc)

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 Modes of LC-NMR
4) Peak Parking method
• 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 for transfer
- LC-NMR Probe
-BPSU-12 (Bruker Peak Sampling Unit – 12)
- Controlling station 28
 Modes of LC-NMR
5) Peak Trapping method
• The outlet of the LC-detector is connected to the SPE unit.
• A LC-detector ( normally UV ) is used to detect peaks eluting from the
column.
• A detected peak is moved trapped on a SPE cartridge without
interrupting the chromatography.
• When the chromatography is completed, the chromatography
solvents are removed and the peak is transfer with fully deuterated
solvents into the NMR probe
Equipment :- Any HPLC system
- pump under control for transfer
- LC-NMR Probe
- SPE system
- Controlling station 29
Technology to improve sensitivity of LC-NMR method
1) LC method
a) On line SPE method
b) On line column trapping method
c) Use of semi micro column

2) NMR method
a) high strength magnetic field
b) high sensitivity probe

3) Solvent suppression method

a) presaturation
b) soft pulse multiple irradiation
c) WET method
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 1) LC method
a) On line SPE method
• It is important to eliminate unnecessary fractions by efficient
pretreatment, introducing only the targeted component to the
column and controlling overloading.
• The SPE cartridge absorbs the desired peak.
• After the sample is dried with N2 gas and the contents are finally
eluted from the cartridge into the NMR flow probe.
SPE principle
• Solid phase extraction involves the separation of components of
samples in solution through their selective interaction with and
retention by a solid, particulate sorbent.
• The specific hydrophobic organic functional moieties are chemically
bonded intimately to a solid surface, such as powdered
chromatographic grade silica.
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Contd…
• These groups will interact with hydrophobic organic compounds by
Vander Waals forces and extract them from an aqueous sample in
contact with the solid surface.
• SPE uses the affinity of solutes dissolved or suspended in a liquid
(known as the mobile phase) for a solid through which the sample is
passed (known as the stationary phase) to separate a mixture into
desired and undesired components.
• The result is that either the desired analytes of interest or
undesired impurities in the sample are retained on the stationary
phase.
• The portion that passes through the stationary phase is collected or
discarded, depending on whether it contains the desired analytes
or undesired impurities.

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Contd…
• If the portion retained on the stationary phase includes the desired
analytes, they can then be removed from the stationary
phase for collection in an additional step, in which the stationary
phase is rinsed with an appropriate eluent.

SPE DEVICES
Several SPE configurations are used which are as following:
o Cartridge
o Disk
o Micropipette tip
o 96-well plate
o Coated fiber

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 Contd…
CARTRIDGE
• The most popular SPE configuration is the cartridge.
• It is carried out using a small packed bed of sorbent with a nominal
particle size of 50-60 µm contained in a cartridge made from a
polypropylene syringe barrel, fitted with luer tip, so that a needle
can be affixed to direct the effluent to a small container or vial.
• The sorbent being retained in position by use of fits.
• Frits are made of polytetrafluoroethylene (PTFE), polypropylene
or stainless steel with a porosity of 10 to 20 µm and thus offer little
flow resistance.
• The sorbent generally occupies only the lower half of the cartridge,
leaving space above to accommodate several milliliters of the
sample solution or washing and solvents.

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 Contd…
Advantages of catridges (SPE)
1. Highly economical as
nondeuterated solvents and HPLC
buffers are used
2. The final transfer volume of
200–500 μl is deuterated
3. SPE uses less solvent than
liquid-liquid extraction (LLE)
4. SPE is faster (at least 5 times)
5. High capacity
6. Total SPE costs are considerably
less than LLE
7. High selectivity: broad choice
of bonded phases and solvents
8. Automation much easier 35
 1) LC method
b) On line column trapping
• In this method, after separation using a conventional column,
concentration is first done in a trap column, and the sample is
separated again using a semi-micro column then introduced to
NMR.
• Concentration by this technique is highly effective.
• Once sufficient sample has been collected on the trap, the flow
reversed and the solute is transported to the NMR for further
analysis
c) Use of semi micro column
• The highest sensitivity is provided when all of the components
separated by HPLC are introduced to the flow-cell of NMR.
• However, the peak volume separated by HPLC is greater than the flow-
cell capacity (normally about 30 μL to 120 μL) therefore, only part of
the component is actually the target of measurements
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 On line column trapping.
• The method of using columns with an internal diameter of around
2 mm, known as semi-micro columns, is a peak concentration
method suited to LC-NMR.
• The volume of a semi-micro column is around 1/5 of a conventional
column, and since the required amount of solvent is reduced in
proportion to the elution, highly concentrated sample solutions
can be introduced to LC-NMR 37
 2) NMR methods
a) High strength magnetic field
• NMR detection sensitivity is proportional to the magnetic field
strength to the 3/2 power, and the stronger the external
magnetic field is, the higher the sensitivity.
• Currently, the magnetic field strength has reached 1000 MHz.
• Magnetic fields being generated by modern instruments
employing cryomagnets, field homogeneity is high and as a
consequence the sample need not be rotated.
b) high-sensitivity probe
• It is also known as a cryogenic probe that reduces the heat noise
arising during NMR signal detection by cooling the coil using
superconductor materials.
• This will eliminates the thermal electronic noise associated with
the initial stages of signal detection and increases the coil quality
factor.
• This leads to an improvement in the S/Nratio by a factor of 3-4. 38
Contd…

Conventional NMR probe Continuous flow NMR probe

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 Contd…
Flow Cells – Active Volume

a) 3mm - 60µL
b) 4mm - 120µL
c) 5mm - 240 µL

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 3) Solvent supression methods
a) Presaturation
• The most widespread solvent suppression in use is the so-called
presaturation technique
• It depend on the phenomenon that nuclei which are unable to
relax because their population in ground state and exited state
is same, do not contribute to free induction decay after pulse
irradiation.
• Before the data acquisition, a highly selective low power pulse
irradiates the desired solvent signal for 0.5 to 2 s.
• This leading to saturation of solvent signal frequency.
• During data acquisition, no irradiation should occur.
• This method is used for stopped flow mode.

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 3) Solvent supression methods
b) soft pulse multiple irradiation
• Here, presaturation is performed with the use of shaped pulse
which has a broader excitation profile. This method is better
suitable for suppression of multiplets
• Advantage:
1. Easy to implement
2. Multiple presaturation can be possible
• Disadvantage:
1. Spin with resonance close to solvent frequency will also be
saturated and 2D cross peak will be absent

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 3) Solvent supression methods
c) WET method
• This technique contains NMR difference probe.
• This difference probe consists of a dual coil probe that contains
two sample coils in a resonant circuit that switches between parallel
excitation and serial acquisition to cancel common signals, such
as solvent and solvent impurities.
• Essentially, this technique is based on a dual beam background
subtraction, where the reference signal and sample signal that are
collected simultaneously are subtracted from each other automatically.
• No software manipulation, pulse sequence modification, or
spectrometer alteration is necessary.
• Hence the technique does not lengthen the pulse sequence but it
reduces experimental time.
• It takes 50-100 ms, So it is used for on flow method.
• This method is used for on flow mode. 43
Contd…
v Other possible steps for solvent supression
• Using eluents that have as few 1H NMR resonances as
possible, e.g. H2O, ACN, or MeOH.
• Using at least one deuterated solvent,
e.g., D2O (approx. $290/L), ACN-d3 (approx. $1600/L), or
MeOD (approx. $3000/L).
• Using buffers that have as few 1H NMR resonances as
possible, e.g., TFA or ammonium acetate.
• Using ion pair reagents that have as few 1H NMR
resonances as possible,
e.g., ion pairs with t-butyl groups create an additional resonance

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 Advantages of LC-NMR
• The information between the two (three) techniques is so orthogonal;
HPLC methods resolve “complexity of a mixture” by separation,
whereas NMR resolves virtually any structure question (especially
with different experiments).
• The NMR can determine if the LC peak impure.
• LC-NMR/MS is “THE” ultimate instrument.
• NMR data can be taken without complete separation of mixture.
• It is nondestructive technique.
• Sample can be stored for analysis by another method.

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Disadvantages of LC-NMR
• high costs.
• Capital equipment costs; long experiment times; partial use of
2H solvents.
• operator training requirements.
• Doing LC-NMR/MS requires a unique set of skills.
• Difficulty in solvent selection.

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 Application of LC-NMR
• Degradation Products.
• Impurities.
• Trace Analysis.
• Analysis of Mixtures.
• Tautomer Kinetics.
• Unstable Products.
• Natural Products.
• Proteins/Peptides.

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References:
1) LC-NMR: A powerful tool for analyzing and characterizing complex
chemical mixtures without the need of chemical separation,
Bhumika. D. Sakhreliya*, Swati Kansara Department of Quality
Assurance, A-One Pharmacy College, Anasan Ahmedabad, Gujarat,
India.
2) LC-NMR-Expanding-the-Limits-of-Structure-Elucidation, Nina C.
Gonnella.
3) [Applications of NMR Spectroscopy Volume 2] Choudhary, M. Iqbal_
Rahman, Atta-ur - Applications of NMR Spectroscopy. Volume 2 (2015,
Bentham Science Publishers Ltd)
4) Klaus Albert - On-line LC-NMR and Related Techniques (2002,
Wiley)

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