LC-NMR combines liquid chromatography with nuclear magnetic resonance spectroscopy. In LC-NMR, the effluent from the LC column is introduced directly into the NMR flow probe or flow cell for analysis. This allows NMR spectra to be obtained on-line for LC fractions as they elute from the column. The key components of an LC-NMR system include the LC unit, interface, and NMR unit. The interface connects the LC and NMR and can be either direct coupling of the LC effluent to the NMR flow cell or indirect coupling using an intermediate storage loop. LC-NMR provides complementary information from the two techniques for analyzing mixtures and determining compound structures.

1. Presented By:
Rashu Raju
Sub: Advanced Spectral analysis
M. Pharm 1st year
Department of Pharmaceutical Chemistry
Krupanidhi college of pharmacy
LC-MS and LC-NMR
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2. CONTENTS
 LC-MS: Introduction, Principle, Instrumentation, Application
 LC-NMR: Introduction, Principle, Instrumentation, Application
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3. Hyphenated Techniques
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 A Hyphenated technique is a combination or coupling of two different analytical techniques with the
help of proper interface.
 Hyphenation refers to the on-line combination of a separation techniques and one or more
spectroscopic detection techniques.
 Chromatography- Produces pure or nearly pure fractions of chemical components in a mixture.
 Spectroscopy- Produces selective information for identification using standards or library spectra.
 Some of the hyphenated techniques are: GC-MS, LC-MS, LC-NMR, EC-MS, CE-MS, LC-MS-MS,
GC-MS-M,S ICP-MS, ICP-OES.

4. LIQUID CHROMATOGRAPHY- MASS
SPECTROMETRY
 LC-MS is an analytical technique that combines the physical separation capabilities of liquid
chromatography with the mass analysis capabilities of mass spectrometry.
 It has very high sensitivity and specificity.
 Mass spectrometry in LC-MS helps to determine the elemental composition and structural elucidation
of a sample.
Principle :-
 sample is separated by LC
Normal: hydrophobic or non polar stationary phase & polar mobile phase or
reverse phase mode: hydrophilic or polar stationary phase & non polar mobile phase
 sample species are sprayed into atmospheric pressure ion source (converted into ions )
 mass analyser is then used to sort ions
 mass spectrum is obtained
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5.  It is the combination of liquid chromatography and the mass spectrometry.
 In LC-MS we are removing the detector from the column of LC and fitting the column to interface of MS.
 In most of the cases the interface used in LC-MS are ionization source.
INSTRUMENTATION:
Liquid
chromatography Ionization
Mass
analyzer
Detector/
Data
collection
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6. Parts of LC-MS
LC-MS systems include
 Introducing samples devices
 Mobile Phase
 Column
 Interface for connecting such device,
 Ion source that ionizes samples,
 Electrostatic lens that efficiently introduces the generated ions,
 Mass analyzer unit that separates ions based on their mass-to-charge (m/z) ratio
 Detector unit that detects the separated ions
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7. Sample Preparation
 Sample preparation generally consists of concentrating the analyte and removing compounds that can
cause background ion or suppress ionization.
 Steps – on column concentration or preconcentration, desalting, filtration
Mobile Phase
 The mobile phase is the solvent that moves the solute through out column
 General requirements:-
low cost, UV transparency, high purity.
low viscosity, low toxicity, non flammability.
non corrosive to LC system component
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8. Column
 The use of di-functional or tri-functional silanes to create bonded groups with two or three
attachment points leading to phases with higher stability in low or higher pH and lower bleed for
LC-MS.
 Most widely used columns for LCMS are :-
fast LC column- the use of short column( 15-50mm)
micro LC column- the use of large column (20-150mm)
Bonding obtained with a: A monofunctional silane, B and C difunctional silane, D and E
trifunctional silane,
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9. Interfaces
 If the LC unit is simply connected directly to the MS unit, the liquid mobile phase would vaporize,
resulting in large amounts of gas being introduced into the MS unit.
 This would decrease the vacuum level and prevent the target ions from reaching the detector. So
interfaces are to be used.
 The commonly used ion interface sources:
1. Direct chemical ionization
2. Thermospray
3. Moving wire or belt interface
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10. Direct Chemical Ionization:
 The simplest way to introduce HPLC effluent into mass spectrometer is to split the flow. Chemical
ionization is most suitable in this technique because under CI pressure conditions, solvent rates as
high as 10 micro lit / min can be tolerated. This permits10-20 micro lit/ min (1-2%) eluate from the
HPLC to that of the ion source.
Thermospray:
The eluent from the column is vapourised and a portion of vapour is transferred to the mass
spectrometer and rest of the vapour is pumped to waste. As a result a supersonic jet vapour, containing
a mist of particles and solvent droplet is created. There vaporization takes place in presence of an
electrolyte the LC buffer, the droplets are charged. And finally they enter into the ionization chamber.
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11. Moving Wire or Belt interface:
 The moving wire or belt interface consists of an auxiliary vacuum chamber through which a continuous train
carries the column eluate, evaporates the solvent and subsequently vaporizes the solute. The residual solvent
helps to maintain vaccum in the MS. The sample is finally conducted into the ion source, where it vaporizes.
Ionization sources: -
 Electrospray ionization (ESI)
 Atmospheric pressure chemical ionization (APCI)
 Atmospheric pressure photoionization (APPI)
 Matrix-assisted laser desorption/ionization (MALDI)
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12. Electrospray ionization (ESI):
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 The method generates ions from solution of a sample by creating fine spray of
charged droplets.
 A solution of sample is pumped through a fine, charged stainless steel capillary
needle at a rate of few microlitres/minute. The needle is maintained at a high
electric field (several kilovolts) with respect to cylindrical electrode.
 The electrostatic field causes further dissociation of the analyte molecules.
 The heated drying gas causes the solvent in the droplets to evaporate. As the
droplets shrink, the charge concentration in the droplets increases.
 Eventually , the repulsive force between ions with like charges exceeds the
cohesive forces and ions are ejected (desorbed) into the gas phase.
 These ions are attracted and pass through a capillary sampling orifice into the mass
analyzer.

13. Atmospheric Pressure Chemical Ionization (APCI)
 APCI produces ions using a reagent gas generated from solvent vapour. The solvent - a mixture of
methanol, acetonitrile and water at 0.5 ml/min - is supplied to the APCI probe by a pump (either from
HPLC or LC).
 APCI vaporizes solvent and sample molecules by spraying the sample solution into a heater (heated to
about 4000C) using a gas , such as Nitrogen
 Solvent molecules are ionized by corona discharge electrode to generate stable reaction ions.
 Most commonly used ionization source in LC-MS
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14. Matrix-assisted laser desorption/ionization (MALDI):
 In this method ionization is carried out by bombarding a laser beam on the sample dissolved in a matrix
solution.
 Used for peptides, proteins, polymers, dendrimers.
 Preparation of matrix : three methods
 dried-droplet method :matrix-to-sample ratio is of about 5000:1
 thin layer method: good sensitivity, resolving power and mass accuracy, Nitrocellulose (NC) is used as a
matrix
 sandwich method :prepared followed by subsequent addition of droplets of aqouse TFA (trifluroacetic
acid) , sample and matrix
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15. Atmospheric pressure photoionization (APPI)
 Atmospheric pressure photo ionization is relatively newer technique. Here a discharge UV lamp is
placed which generates photons in a narrow range of ionization energies. It shows its ionization for
highly non polar compounds and low flow rates[<100m/min]
 The LC is vaporized using a heater at atmospheric pressure. The resulting gas is made to pass through
a beam of photons generated by a discharge lamp (UV lamp) which ionizes the gas molecules.
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16. Mass Analyser
 They deflect ions down a curved tubes in a magnetic fields based on their kinetic energy
determined by the mass, charge and velocity.
 The magnetic field is scanned to measure different ions.
 Types of mass analyzer:-
1. Quadrapole mass analyser
2. Time of flight
3. Ion trap
4. Fourier transform ion cyclotron resonance (FT-ICR)
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17. Quadrapole mass analyzer
 It consist of four parallel metal rods with different charges.
 Two opposite rods have an applied +ve potential and the other two rods have –ve potential.
 The applied voltages affect the trajectory of ions travelling down the flight path.
 Only ions of a certain mass to charge ratio pass through the quadrupole filter and all other ions are
thrown out of their original path
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18. Ion trap analyser:
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 The ion trap mass analyser operates by similar principles where it consists of circular ring electrode
Plus two end caps that form a chamber. Here AC or DC power along RF potential is applied between
the cups and the ring electrode. There the ions entering into the chamber are trapped by
electromagnetic fields and they oscillates in concentric trajectories. This process is called resonant
ejection.
 The amplitude of the applied voltage enables the analyser to trap ions of specified mass to charge
ratio within the analyzing device.

19. Time of Flight (TOF) analyzer:
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 TOF mass analyser is based on simple idea that the velocities of two ions are created by uniform
electromagnetic force applied to all the ions at same time, causing them to accelerate down a flight
tube.
 Lighter ions travels faster and strike the detector first so that the m/z ratio of ions is detected.

20. Detectors
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A. Electron multipliers:
 to convert either –ve, +ve ions into electrons and detect
 used in quadrupole and ion trap instruments.

21. Photomultiplier
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 converts the charged ions into electrons
 electrons sticks to a phosphor and emits photons
 photons are made to strike the photomultiplier

22. Application
 Pharmaceutical applications:
Rapid chromatography of benzodiazepines
Identification of bile acid metabolite
 Biochemical applications
rapid protein identification
 Clinical applications
High sensitivity detection of trimipramine and thioridazine
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23. contd
 Food applications
identification of aflatoxins in food
determination of vitamin D3 in poultry feed supplements
 Environmental applications
Detection of phenylurea herbicides
 Forensic applications
Illegal substances, toxic agents
explosives
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24. Limitation of LC-MS
 Higher operational cost
 More limited sample throughput
 Less favourable concentration sensitivity
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25. LIQUID CHROMATOGRAPHY-
NUCLEAR MAGNETIC
RESONANCE(LC-NMR)
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26. Principle of LC (HPLC).
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 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 3.Size-Exclusion HPLC
 2.Reverse phase 4.Ion-Exchange HPLC

27. Principle of NMR
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 NMR is based upon the spin of nuclei in an
external magnetic field. In absence of magnetic
field, the nuclear spins are oriented randomly.
 Once a strong magnetic field is applied, they
reorient their spins i.e., aligned with the field or
against the field. When nuclei are irradiated
with RF radiation the lower energy nuclei flip to
high state and nuclei said to be in resonance.

28. Interfaces of LC-NMR
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29. Instrumentation of LC-NMR.
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30. LC UNIT
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 Solvent Reservoir: Mobile phase contents are contained in a glass reservoir.
 Pump: A pump aspirates the mobile phase from the solvent reservoir and forces it through the system’s column
and detector.
 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.
 Columns: Columns is the heart of HPLC separation processes. Columns are usually made of polished stainless
steel. They are commonly filled a stationary phase with particle size of 3–10 µm.
 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. Signals from the detector may be collected on chart recorders or electronic integrators.

31. LC-NMR Interface:
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 DIRECT COUPLING: It include direct flow of LC effluent into NMR flow cell and
continuous recording of spectra.
 post-column splitter
 valve-switching interface i.e., BNMI (Bruker NMR-Mass Spectrometry Interface)
 INDIRECT COUPLING: Intermediate storage loop which transfer outlet of LC to NMR
flow cell at specified time interval.
 SPE unit

32. NMR UNIT
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 NMR instrumentation involves the following units.
 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.
 Types of magnets: 1.Permanent magnets 2.Electromagnets 3.Super conducting magnets

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2) The Probe Unit
 A sample probe containing coils for coupling the sample with the RF field
 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 i.e., Ho and RF
(EMR).
 The usual NMR sample cell is generally made up of the glass, which is strong and cheap. It consists 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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3) 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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4) Detector:
 The purpose of detector is to monitor the eluent coming out of the column.
 Generally two types of detectors are used in HPLC.
1. Bulk property detectors: These detectors are based on differential measurement of a property. Eg:
refractive index, conductivity and dielectric constant detectors.
2. Solute property detectors: respond to a physical property of the solute, which is not exhibited by
the pure mobile phase. Eg: UV detector, fluorescence detectors, electro-chemical and radioactivity
detectors, electron capture detector are suitable for gradient elution.

36. Advantages of LC-NMR
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 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 is impure.
 LC-NMR/MS is “THE” ultimate instrument.
 NMR data can be taken without complete separation of mixture.
 It is non-destructive technique.
 Sample can be stored for analysis by another method.

37. Disadvantages of LC-NMR
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 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.
 Stopping the pump (for NMR signal averaging) frequently may affect resolution of method
 Flow systems can clog up, and get dirty, and be hard to clean.

38. Application of LC-NMR
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 In characterization of Degradation Products. Ex application of cilazapril.
 In the separation and rapid structure elucidation of an unknown impurities
 Ex 5-amino salicylic acid.
 Structural elucidation of in vivo metabolites of isobavachalcone in rat by LC–NMR.
 Application of LC– NMR Techniques for Secondary Metabolite Identification.
 Separation and characterization of peptide libraries.
 In Combinatorial chemistry, photochemical analysis, drug discovery.

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