HPLC Chromatography and NMR Spectrometry are analytical techniques. HPLC uses different principles like adsorption, ion-exchange, and size-based separation to separate mixtures. It works by pumping a mobile phase through a stationary phase column. NMR spectroscopy uses strong magnetic fields and radio waves to analyze nuclear spin properties to determine molecular structure. Both techniques have various applications in chemistry, forensics, environmental analysis, and quality control. LC-NMR coupling combines the separation power of liquid chromatography with the structural information of nuclear magnetic resonance for analyzing complex mixtures.

2. HPLC Chromatography

3.  Principle
 The fundamental principle of separation involved is Adsorption.
 The other principles involved are
 Ion-exchange: It is used to separate the mixture containing
similar charged ions.
 Ion-pair: It is the alternate to ion-exchange, used for the
components which are difficult to separate using covalent
bonded ion-exchangers.
 Gel permeation: It is separated on the basses of difference in
the size & shape of the solute molecules.
 In HPLC ,based on polarity there are two phase
Normal phase
Reversed phase
 Normal phase: Stationary phase Polar
Mobile phase Non-polar
 Reversed phase : Stationary phase Non-polar
Mobile phase Polar

4. based on Elution there are types
Isocratic separation
Gradient separation
 Isocratic separation: Mobile phase & the strength of
elution is maintained constant.
 Gradient separation: Mobile phase & the strength of
elution is varied.
Instrumentation
 Mobile phase reservoir
 columns
 Pumps
 Sample injection system
 Detectors

5. Mobile phase reservoir
 It can be an aqueous-organic mixture or buffer
solution or a mixture of organic solvents.
 The mobile phase is pumped under pressure from
one or several reservoirs and flows through the
column at a constant rate.
 Eluting power of the Mobile phase is determined by
its overall polarity, the polarity of the stationary phase
and the nature of sample component.

6. Solvent degassing system
 The constituents of the mobile phase should be
degassed and filtered before use.
 Methods are employed to remove the dissolved gases in
the mobile phase are include:
Heating and stirring,
Vacuum degassing with an aspirator and with an air-
soluble membrane,
Filtration through 0.45 filters,
Ultra sonication or combination of these methods.
 HPLC system is also provided an online degassing
system, which continuously removes the dissolved gases
from the mobile phase.

7. Characteristics of columns and column packings
The column is the heart of HPLC separation
processes.
Most column packing used for HPLC separations
make use of silica particles (SiO2 x H2O).
It consist of a network of siloxane linkages (Si-O-Si)
in a rigid three - dimensional structure containing
inter connecting pores.
Thus wide ranges of commercial products are
available with surface areas ranging from 100 and
800 m/g and particle sizes from 3 to 50 µm.

8. The useful pH range for column is 2 to 8, since
siloxane linkages are altered below pH 2 while at pH
values above 8 silica may dissolve.
Category Particle (µm ) Bonding
L1 1.5 to10 C18
L2 30 to 50 C18
L7 1.5 to10 C8
L8 3 to 10 NH2
L11 1.5 to10 Phenyl
L17 7 to 11 Cation Exchange

9. Pumps
o The most important component of HPLC in solvent
delivery system is the pump, because its performance
directly effects the retention time, reproducibility and
detector sensitivity.
o It should generate high pressure upto 6000psi
o Pumps are 2 types:
o Constant Displacement Pumps: Constant flow of mobile
phase Eg: Syringe type pumps, Reciprocating pumps
o Constant Pressure Pumps: Non-pulsating solvent flow but
has very small capacity. Eg Pneumatic pumps
o The reciprocating pump with twin or triple pistons is widely
used, as this system gives less baseline noise, good flow
rate, reproducibility etc

10. Sample injection system
 Two means for injection on the column are
 Injection into a flowing stream and
 Injection into a stop flow injection.
These techniques use a syringe or an injection valve.
Automatic injector is a microprocessor-controlled
version of a manual universal injector.
 Usually, up to 100 samples can be loaded into the auto
injector tray. The system parameters such as flow rates,
gradient, run time, volume to be injected, etc., are
chosen, stored in memory and sequentially executed on
consecutive injections.

11. Detectors
The purpose of detector is to monitor the eluent
coming out of the column. Generally two types of
detectors are used in HPLC.
a. Bulk property detectors: These detectors are
based on differential measurement of a property.
Eg: refractive index, conductivity and dielectric constant
detectors.
b. Solute property detectors: 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.

12. Working
Schematic representation of HPLC

13.  The mobile phase is pumped at high pressur into the
column.
 The sample is introduced at the top of the column by
using suitable injector system.
 As the mobile phase flows down the separation of
components occur.
 The column effluent is made to pass through a
detector which produces electrical signals
proportional to the characteristics of the solute
molecules which are amplified & recorded using a
potentiometric recorder as chromatographs.

14.  Application
 Used in inorganic chemistry for separation of anions &
cations
 Used in forensic science for the separation of phenylalkyl
amines from blood plasma,& for detection of poisons.
 Used in environmental studies.
 Used in bioassays of compounds.
 Used in controlling microbiological processes.
 Used in cosmetic industry for the assay & QC.
 Used in agrichemical industry for separation of herbicides.

15. NMR Spectrometry
Bruker 600 MHz NMR Spectrometer.

16. Principle: It 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.
NMR are 2types based on parameters that are measured
1. Single coil spectrometers (Measures
absorption)
2. Double coil spectrometers( Measures
resonant radiation)

17. It is further divided into
Absorption type: It uses a bridge circuit for
detecting radio-frequency energy absorbed through the coil
surrounding the sample.
Inductive type: It uses two coils perpendicular
to each other & absorption of energy is from the transmitter coil
resulting in the orientation of nuclei, thereby inducing voltage in
receiver coils.
Instrumentation
 Magnets
 Sample holder
 Sweep coil
 Transmitter
 Receiver
 Detector

18. Magnets
Permanent or electromagnet are used to obtain a
homogeneous magnetic field.
Permanent – field variation is impossible.
Electromagnet – field strength is varied.
As the field strength is proportional to chemical shift
it must not be less than 20,000guass.
Sample holder
 It is about 5mm in diameter & 15-20 cm in length. It
must be transparent to radio frequency radiation &
chemically inert. Glass tubes are used. 1-30mg of 2-
19% sample is used.

19. Sweep coil
 It is used to production of NMR spectra.
 It is achieved by passing direct current either through the
coli that are wound around the magnetic pole or through
a pair of Helmhotz coil located on either sides of the
sample.
 Rate of sweep is important parameter because slow rate
results in saturation effect where as fast rate results in
ringing.
Transmitter
 It generates of few MHz which irradiates the sample
molecules.
 If the energy difference between the relevant spin state
matches the radio frequency wave then the nuclei moves
to the higher spin state. The output obtained is multiplied
with the desired frequency.

20. Receiver
 Radio frequency bridge is employed under single
coil instrument.
 In double coil, the transmitter & receiver coil are
arranged perpendicular to each other & to
direction of the magnetic field.
Detector
 The NMR spectra obtained from high resolution
instrument are recorded directly via computer. To
direct the radio frequency signal exhibited due to
resonating nuclei, the receiver coil is used.

21. Procedure

22.  Transmitter generates radio waves of definite
frequency which passes through a receiver coil
surrounding the sample. The receiver coil receives the
signals & send to detector.
 The sweep is used to bring variations in the
externally applied magnetic field Bo. The frequency of Bo
is varied until it is in resonance with that of the radio
wave frequency, thereby leading to absorption of energy
which is recorded in the form of signals.
 The peak obtained in the signals represent the value
of Bo at which the frequency of the sample nuclei is in
resonance with that of the applied magnetic field Bo.
 To obtain NMR spectrum, the signals are platted
again applied magnetic field Bo

23.  Applications
 Used in qualitative & quantitative analysis.
 Used in identification testing
 Used in determining Hydrogen bonging present in metal
chelates & organic compounds.
 Determination of double bond character.
 Determination of structure.
 Identification of structural isomers.
 Distance between Cis & Trans Isomers.
 Determination of optical purity.

24. LC-NMR systems

25.  NMR detection coupled with LC offers great promise
in combining the ability to separate complex mixtures
into individual components with one of the most
structurally rich detection schemes available.
 In 1978, Watanabe reported the coupling of LC
effluent to NMR using a stopped flow approach, and
within 1 year, an on-line system had been reported.
The major advantages of on-line as opposed to off-
line NMR detection of LC are improved
chromatographic resolution, consistent response,
on-line data analysis, and rapid data acquisition.

26.  Over the past 15 years, numerous groups have reported
improved LC-NMR hyphenation methods, improved NMR
plus sequences and increased chromatographic
resolution.
 The drawbacks of continuous flow NMR include poorer
sensitivity due to the limited time available to measure
each analyze and the flow rate dependence of the NMR
line width.
 Recently radio frequency (RF) microcoils are used for
NMR spectroscopy to create 5 nL to 1- mL volume
detection cells.

27. Working

28.  The sample can be transferred during the
chromatographic separation and the NMR spectra are
then acquired either in
 on-flow mode (continuously, while the
chromatography is running) or
 in stop-flow mode (while a selected peak is parked
in the NMR probe and the chromatography is
paused).
 The Bruker LC-NMR systems are ideal for sensitive and
instable compounds, fully automated analysis, or simply
to provide a very convenient and quick path from LC
separation to NMR results.

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