LC-FTIR #bestppt #lcftirppt

1. on
LC-FTIR
Prepared by- Kartik Tiwari
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2. CONTENTS
• Principle and Instrumentation of LC/HPLC
• Principle and Instrumentation of FTIR
• Principle and Instrumentation of LC-FTIR
• Interfaces
• Applications
• References

3.  Chromatography : physical method in which separation of
components takes place between two phases-a stationary phase
and a mobile phase
 Stationary phase : The substance on which adsorption of the
analyte (the substance to be separated during chromatography)
takes place . It can be a solid, a gel, or a solid liquid combination
 Mobile phase : solvent which carries the analyte (a liquid or a
gas)

4.  Chromatographic techniques are divided into different types based on :
 The type of chromatographic bed used
i.e. column chromatography (gas chromatography) and planar chromatography
(paper and thin layer)
 The physical state of mobile phase
i.e. gas chromatography and liquid chromatography
 The separation mechanism
i.e. ion-exchange and size exclusion
 HPLC is a type of liquid chromatography where the sample is forced through a
column that is packed with a stationary phase composed of irregularly or
spherically shaped particles, a porous monolithic layer , or a porous membrane by
a liquid (mobile phase) at high pressure.

5. PRINCILPE
 To understand the principle of HPLC , we must first look at the principle behind
liquid chromatography
 Liquid chromatography is a separation technique that involves:
 the placement (injection) of a small volume of liquid sample
 into a tube packed with porous particles (stationary phase)
 where individual components of the sample are transported along the packed tube
(column) by a liquid moved by gravity.
 The main principle of separation is adsorption .

6.  When a mixture of components are introduced into the column . various
chemical and/or physical interactions take place between the sample molecules
and the particles of the column packing .
 They travel according to their relative affinities towards the stationary
 phase. The component which has more affinity towards the
adsorbent, travels slower.
 The component which has less affinity towards the stationary phase
travels faster.
 Since no two components have the same affinity towards the stationary phase,
the components are separated

7.  HPLC is a separation technique that involves:
 the injection of a small volume of liquid sample into a tube packed with tiny particles (3 to 5 micron
(μm) in diameter called the stationaryphase)
 where individual components of the sample are moved down the packed tube (column) with a liquid
(mobile phase) forced through the column by high pressure delivered by a pump
 These components are separated from one another by the column packing that involves various
chemical and/or physicalinteractions between their molecules and the packing particles.
 These separated components are detected at the exit of this tube (column) by a flow-through device
(detector) that measures their amount.The output from the detector is called a liquid chromatogram
 In principle, LC and HPLC work the same way except the speed ,
 efficiency, sensitivity and ease of operation of HPLC is vastly superior.

8. Instrumentation

11. Switch on instrument
Check system set up
Prime the pump
Prepare the column
OPERATION
Set-up software(for system
flushing)
Software set up(for sample run)
Sample injection
Chromatograph data acquisition

13. CONTENTS
• Introduction
• Theory of FTIR
• Instrumentation of FTIR
• Advantages
• Disadvantages
• Applications

14. Introduction
• Infrared spectrum is an important record which gives sufficient information
about the functional groups of a compound.
• The region from o.8 µ to 2.5µ is called Near IR, from 2.5µ to 15µ is called Mid
IR or Ordinary IR and that from 15µ to 200µ is called Far IR.
• FTIR stands for Fourier Transform Infra Red Spectrophotometer —
the preferred method of infrared spectroscopy.
• A method for measuring all of the infrared frequencies simultaneously,
rather than individually as with dispersive instruments.

15. FTIR spectroscopy is preferred over dispersive
method of IR spectral analysis for several reasons:
 It is a non-destructivetechnique.
 It provides a precise measurement method which requires no external
calibration.
 It can increase scan speed, collecting a scan every second.
 It is mechanically simple with only one moving part.

16. Theory of FTIR
 Fourier Transform Infrared (FT-IR) spectrophotometry was developed in order
to overcome the limitations encountered with dispersive instruments.
 The main difficulty was the slow scanning process. A method for measuring all
of the infrared frequencies simultaneously, rather than individually, wasneeded.
 A solution was developed which employed a very simple optical device called
an interferometer. The interferometer produces a unique type of signal which
has all of the infrared frequencies “encoded” into it.

17.  Most interferometers employ a beam-splitter which takes the incoming infrared
beam and divides it into two optical beams.
 One beam reflects offa flat mirror which is fixed in place. The other beam
reflects offa flat mirror which is on a mechanism which allows this mirror to
move a very short distance (typically a few millimeters) away from the beam-
splitter.
 The two beams reflect off of their respective mirrors and are recombined when
they meet back at thebeam-splitter.
 Because the path that one beam travels is a fixed length and the other is
constantly changing as its mirror moves, the signal which exits the
interferometer is the result of these two beams “interfering” with eachother.

18.  The resulting signal is called an interferogram which has the unique property
that every data point (a function of the moving mirror position) which makes
up the signal has information about every infrared frequency which comes from
the source.
 Because the analyst requires a frequency spectrum (a plot of the intensity at
each individual frequency) in order to make an identification, the measured
interferogram signal cannot be interpreted directly.
 Hence Fourier transformation is performed by thecomputer which then
presents the user with the desired spectral information for analysis.

19. Spectrometer components
 Three basiccomponents of FT system are
1. Radiationsource
2. Interferometer
3. Detector

20. Radiation sources
 Incandecent lamp
 Nernstglower
 Globarsource
 High pressure mercuryarc

21. Interferometer
• The heart of the FTIR is a MichelsonInterferometer.
• The monochromator is replaced by an interferometer, which divides radiant
beam, then recombines them in order to produce repetitive interference signals
measured as a function of optical path difference as the name implies, the
interferometer produces interference signals, which contain IR spectral
information generated after passing through a sample.
• Consists of three activecomponents-
• A moving mirror
• A fixed mirror
• A beam splitter

22.  Mirrors in an FTIR are generally made of metal. The mirrors are polished on the front
surface and may be gold- coated to improve corrosionresistance.
- commercial FTIRs use a variety of flat and curved mirrors to move light within the
spectrometer, to focus the source onto the beam splitter, ant to focus light from the sample
onto thedetector.
• The beam splitter can be constructed of a materialsuch as Si or Ge deposited in a thin
coating onto an IR- transparent substance. The germanium or silicon is coated onto the
highly polished substrate by vapourdeposition.
• A common beam splitter material forthe mid-IR region is germanium and the most
common substrate for this region is KBr. Both the substrate and the coating must be
optically

23. Schematic diagram of Michelson Interferometer.

24.  Construction: The two mirrors are perpendicular to each other. The beam-
splitter is a semi-reflecting device and is often made by depositing a thin
film of Germanium onto a flat KBr substrate.
 Working: Radiation from the broadband IR source is collimated and
directed into the interferometer, and impinges on thebeam- splitter.
• At the beam-splitter half of the IR beam is transmitted to the fixed
mirrorand the remaining half is reflected to the moving mirror.
• Afterthedivided beamsare reflected from the two mirrors, they
are recombined at thebeam-splitter.
• Due to changes in the relative position of the moving mirror, an
interference pattern is generated. The resulting beamthen passes
through the sample and is eventually focused on the detector.

25. Fixed mirror Michelson
interferometer
Moving mirror
positions
sample
To
detector
IR
Source
Schematic diagram of FTIR spectrometer
ZPD
¼λ ¼λ
• NERNST GLOWER
• GLOBAR SOURCE
• HIGH PRESSURE MERCURY
ARC LAMP
• Dueterated triglycine sulphate(DTGS)
• Mercury cadmiumtelluride
(MCT)

27. FTIR spectrophotometer
 FTIR spectrophotometer obtains an infrared spectra by collecting an interferogram of
a sample signal using an interferometer and then performs a Fourier transform on
the interferogram to obtain a spectrum.
 Fourier transform defines a relationship between signal in time domain and
its representation in frequency domain.
 The sampling rate is controlled by an internal, independent reference,
a modulated monochromatic beam from a Helium-Neon Laser focused
on a separate detector.

28. Source interferometer sample detector
interferogram (encoded data of sample at each frequency)
decoding by FT IR Spectra

29. Detectors in FTIR
 Two most popular detectors for FTIRspectrometer are
• Pyroelectric Detector: Deuterated triglycine sulphate (DTGS).
• Photon-sensitive semiconducting Detectors: Mercury cadmium teluride
(MCT)

30. Advantages of FTIR
FTIR instruments have distinctadvantage over dispersive
spectrometers.
1. Simpler mechanical design.
2. Elimination of Stray light and emissioncontributions.
3. Powerful data station.
4. Majority of molecules in the universe absorbmid-infrared light, making it a highly usefultool.
5. Universal technique.
6. Sensitive, fast andeasy.
7. Relatively inexpensive and provides richinformation.
8. Sensitive to “molecules”-anything that containschemical bonds.

31. Disadvantages of FT-IR
• Cannot detect atoms or monoatomic ions - single atomic entities
contain no chemicalbonds.
• Cannot detect molecules comprised of twoidentical atoms symmetric-
such as N2 orO2.
• Aqueous solutions are very difficult to analyze - water is a strong IRabsorber.
• Complex mixtures - samples give rise to complex spectra.

32. Applications
1. Identification of an organiccompound.
2. Structuredetermination.
3. Study of chemicalreaction.
4. Study of Keto-Enol tautomerism.
5. Study of complexmolecules.
6. Detection of impurities in acompound.
7. Conformational analysis.

33. LC-FTIR
 IR spectroscopy has a high potential for the elucidation of molecular structures.
The IR spectrum of a poly-atomic molecule is based on molecular vibrations, each
specifically dependent on atomic masses, bond strengths and intra- and
intermolecular interactions.
 As a consequence, the entire IR spectrum of an organic compound provides a
unique fingerprint, which can be readily distinguished from the IR-absorption
patterns of other compounds including isomers.
 In other words, when reference spectra are available, most compounds can be
unambiguously identified on the basis of their IR spectra. Moreover, characteristic
absorption bands can be used for compound-specific detection.
.

34.  LC is a powerful and versatile separation technique, which can handle a wide range of
sample types and compound classes.
 Because of the widespread use of LC and the (growing) need for analytical procedures
that provide confirmation and/or identification of sample constituents, much effort has been
– and still is – devoted to the coupling of LC with spectrometric techniques such as mass
spectrometry (MS), IR and nuclear magnetic resonance (NMR) spectroscopy
 Today, with modern Fourier transform infrared (FTIR) instrumentation routinely available, spectra
can be recorded from nanogram, or even picogram, amounts of pure substance so that IR detection,
in principle, is suited for molecular recognition at analyte levels frequently met in LC.
 Unfortunately, because of the (spectral) characteristics of the mobile phase, the coupling of LC and
IR spectroscopy (LC/IR) is not straightforward and often requires the construction of special flow
cells or the development of rather complex interfaces.
 Therefore, compared with other LC detection modes such as ultraviolet/visible (UV/VIS) absorption
spectroscopy or MS, the use of IR detection in LC is still rather limited.
 Nevertheless, progress in interfacing techniques during the last decade has brought LC/IR to a stage of
analytical utility which suggests that LC/IR may well become a commonly available and applied
technique

38. INTERFACES
Flow-cell Approach :-
 The simplest way to couple LC and IR is to let the column effluent pass
directly through a flow cell suited for IR measurements.
 The IR absorption of the LC effluent is continuously monitored and
spectral data are collected on-the-fly and stored throughout the
chromatographic run.
 During or after the run, the spectra and/or IR chromatograms are computed
and absorption due to the mobile phase is subtracted.
 In a flow-cell design, band broadening caused by detection is easily
minimized.

39. Solvent-elimination Approach :-
 The compatibility and time-domain difficulties connected to flow-cell IR detection can be
circumvented by coupling LC and IR spectrometry via a substrate suitable for IR detection.
 In this indirect approach, the eluent is eliminated and the chromatographically separated
compounds are immobilized on the substrate prior to the collection of IR spectral data.
 The immobilization of the chromatogram is accomplished by using an interface which
evaporates the eluent and continuously deposits the column effluent onto the moving substrate.
 In this way, interference-free IR spectra of the deposited compounds can be recorded
independently from the LC conditions and the sensitivity of the FTIR spectrometer can be fully
exploited.

40. APPLICATIONS: -
Analysis of environmental pollutants such as polycyclic aromatic hydrocarbons,
pesticides and herbicides.
Analysis of Pharmaceuticals such as steroids and analgesics, and their impurities, drug
metabolites, polymer additives, dyes, non-ionic surfactants and fullerenes.
Distinction of isomers
Separation of secondary structure of proteins such as beta globulin and lysozyme.

41. References
1. Instrumental analysis. Skoog, Holler, Crouch. Pg No:488-493.
2. Fundamentals of Analytical chemistry. F James Holler,
Stanley R. Crouch. Pg No:749-750.
3. Introduction to Instrumental Analysis. Robert D.Braun. Pg No:372-373.
4. Instrumental methods of Chemical Analysis. Gurudeep
R. Chatwal, Sham K.Anand. Pg No:2.49-2.59.
5. Text book of Quantitative Chemical Analysis. Vogel’set al.,Pg No:680-681.