The document discusses liquid chromatography-nuclear magnetic resonance (LC-NMR), a hyphenated technique that combines liquid chromatography with NMR spectroscopy. It was proposed in 1980 as a way to couple different analytical techniques. LC-NMR allows for separation and quantification of components simultaneously, providing fast, accurate analysis with high sample throughput. The main components of an LC-NMR system are the HPLC system, NMR detection, and a coupling between the two. It operates in continuous flow, stop flow, or loop/cartridge storage modes. LC-NMR has applications in drug discovery, phytochemical analysis, and secondary metabolite identification.

1. sandeep bindra
M.PHARM,1ST YEAR

2. INTRODUCTION
 The term “Hyphenation” was proposed by Hirschfeld in 1980.
 The hyphenated technique is the combination or the coupling of the different analytical
techniques.
 Mainly chromatographic techniques are combined with spectroscopic techniques.
Liquid
Chromatography
Nuclear
Magnatic
Resonance
Liquid
chromatography-
Nuclear
Magnetic
Resonance
Separates mixture of
chemicals so each
can be identified
individually
Identifies
(detects)chemicals
based on their
molecular structure
at the atomic level
Achemical analysis
technique combining two
instrument to provide
powerful separation and
identification

3.  Separation and quantification of components at the same time.
 Fast and accurate analysis.
 A higher degree of automation.
 Higher sample throughput.
 Better reproducibility.
 Reduction of contamination due to its closed system.
Advantage of the Hyphenated technique

4. Instrumentation
 LC-NMR technique comprises three major components-
1. HPLC system
2. NMR detection
3. Couple of LC-NMR.
1. HPLC System:
 HPLC system consists of (1) solvent, (2) gradient valve, (3) high-pressure pump, (4) sample injection
loop, (5) analytical column, (6) detector, and (7) software.
 The major portion of HPLC instrumentation is software based.
 The operational qualification (OQ) unit can check for the chromatographic condition-
A. Column temperature within the limits, B. Adequate solvent, C. Presence of the vial in the
autosampler.
 The reservoir preserves the purity of the solvents by minimizing the evaporation of the volatile components
and preventing the entry of contaminants.
 The majority of HPLC systems use reciprocating pumps as they have high-pressure tolerance strength and
provide pulse-free flow.
 The injection of HPLC delivers the liquid sample with high pressure.

5.  Columns are connected in series to enhance the separation efficiency of
200,000 theoretical plates.
 The HPLC detector reads the column effluent and gives a recordable signal.
DIFFERENT TYPES OF HPLC DETECTOR

6. SCHEMATIC DIAGRAM OF HPLC SYSTEM

7. 2. NMR DETECTION
 There are three major components- I. NMR Magnet
II. Console
III.Computer
 The NMR magnet produces a static magnetic field that generates the magnetization in the sample.
 Homogeneous magnetic field has to be maintained throughout the entire experiment process.
 The magnet is equipped with shim coils that are adjusted to maintain the homogeneity of the
magnetic field.
 The NMR probe is an important part of the NMR spectrometer. The sample is placed in NMR probe
cell for irradiation that receives the pulse from the amplifier.
 Receiver collects the NMR signals from the probe.
3. Coupling of LC-NMR
 The coupling of liquid chromatography with nuclear magnetic resonance can be done by indirect and direct coupling.
 Indirect coupling consists of SPE unit and storage loop intermediate that convey the outlet of LC to the flow cell and
run on a recording of spectra.
 Direct coupling involves a valve-switching interface and post-column splitter to direct the flow of LC effluent to the
NMR flow cell and run on a recording of spectra.

8. SCHEMATIC DIAGRAM OF NMR

9. LC-NMR INSTRUMENTATION
The main component of an LC-NMR is the isolation zone (column) interface zone
and the detection zone i.e., probe for recording NMR spectra.

10. LC-NMR PRINCIPLE MODE OF OPERATIONS
LC-NMR experiments can be performed mainly in three modes. these are-
 Continuous flow. (Measurement under dynamic conditions)
 Direct stop flow.
 Loop/cartridge storage. (Measurement under static condition)

11. 1. Online-flow mode
 Online-flow mode, the outlet of the LC detector is connected directly to the NMR probe and the compound
peaks are eluting, the spectra are continuously acquired.
 Continuous flow is the easiest setup.
 It does not involve any synchronization between the separation and the detection system so it maintains a good
separation resolution.
 Disadvantage- I) It has a poor sensitivity because the eluted peaks get a short duration of exposure time in the
detection cell.
II)The chemical shift of the sample as well as the solvent depends on the solvent properties
which may cause shifting of the position of NMR peaks.
2. Stop-flow mode
 The NMR probe is directly connected to the outlet of the LC detector at the interface.
 It has a better sensitivity as compared to the online-flow mode with better signal-to-noise ratio.
 With the help of this mode we can detect of only selected peakes of our interest with the help of
the valve found in the interface.

12. The comparison of stop-flow and online-flow modes of LC-1H-NMR spectra

13. 3. Loop/Cartridge storage mode
 The loop storage mode of acquisition acts as interposer between the online and offline HPLC-NMR.
 The outlet of the LC detector must be connected directly to sample storage loop or cartridges.
 Cross-contamination can be avoided and diffusion-mediated peak broadening can be sorted out by storing and
sealing the desired band in the capillary loop.
 The major disadvantage of this method is that the soluble compounds in capillary loops on long-term storage get
degraded.

14. SAMPLE PREPARATION
• Sample is dissolved and then diluted to the desirable concentration with deuterated solvent.
• sample volume, size of NMR tube, sample composition, and excitation pulse angle, may produce NMR signals
of different sizes.
• The chemical shift of proton or carbon can differ with the sample concentration due to the aggregation
phenomena.
• There are many sample preparation processes that can be run with online liquid chromatography.These are-
a. Online Filtration
b. Online Trace Enrichment-This method is reported for the preconcentration of the drugs in saliva, serum, water
samples, and plasma.
c. Online Dilution-Highly concentrated organic solutions are diluted to reduced concentration, so the particles
settle on the column very efficiently. This method is generally utilized in the biological samples.
d. Online Membrane and Dialysis Techniques-The isolation of analyte can be done via diffusion process using a
semipermeable membrane to separate the two liquids

15. SOLVENT SUPPRESSION
 In LC-NMR, solvents and solvent suppression have to be considered very carefully during the selection of the mobile
phase.
 Some solvents due to their molecular structure produce one or more signals in the NMR spectrum.
 The peaks of these solvents overlap with the spectrum of the analyte.
 HPLC grade solvents are protonated and are easily detected by the 1H NMR causing a serious problem. Deuterated
solvents are used to avoid this problem.
 Solvent suppression can be done by using different techniques-
i. WATERGATE (WATER Suppression by Gradient-Tailored Excitation) :
 In this technique, dephasing gradient pulse, rephrasing gradient pulse, and 180° inversion pulse are used to cover up
the solvent signals.
 This technique is used for the 13C satellites suppression concurrently with on-flow experiment.
 This technique can be used for both the on-flow and stop-flow experiments.
ii. Transmitter Presaturation (PRESAT) :
 In this technique, the low-power pulse is used to suppress the signal.
 PRESAT is applicable only for stop-flow experiments.

16. iii. WET (Water Suppression Enhanced through T1 Effect) :
 WET can be employed for multiple solvents suppression by using shifted laminar pulses.
 WET is specifically used for the on-flow NMR.
 WET technique uses these pulses to suppress the various undesirable solvent frequencies in
multiple protonated resonance in LC-NMR.
iv. BPPSTE (Bipolar Gradient-Pulsed Stimulated Echo) :
 This solvent suppression technique was evolved for diffusion-ordered spectroscopy (DOSY)
experimentation.
 If the diffusion coefficients of the solvents and the desired analyte are different, this approach is
used to remove the solvent by altering the holding pattern of the analyte.
 NMR spectroscopy extends various other solvent suppression methods such as relaxation-based
sequence, binomial sequences, jump return, symmetrically shifted pulses, and other advanced
techniques.

17. APPLICATION
1. Drug Discovery 2. In the phytochemical analysis 3. Secondary metabolite identification

18. Reference
1. LC-NMR: AN OVERVIEW OF A HYPHENATED TECHNIQUE. DOI: 10.1002/0471238961.koe00037
2. LC-NMR for Natural Products Analysis: A Journey from an Academic Curiosity to a Robust Analytical Tool.
(doi:10.3390/sci1010031)