This ppt tells in detail about an important Hyphenated technique LC-FTIR. It covers FTIR principle and working, HPLC definition, LC-FTIR interfaces and advantages, disadvantages and application of Interfaces.

1. ADVANCED SPECTRAL
ANALYSIS
LC-FTIR
GAGAN S
Govt. College of Pharmacy, Bengaluru
Dept. of Pharmaceutical Chemistry.

2. CONTENTS:
INTRODUCTION
FTIR
HPLC
LC-FTIR
FLOW CELL INTERFACE
SOLVENT ELIMINATION INTERFACE

3. INTRODUCTION:
• HPLC – High performance liquid chromatography is for separation of
the materials.
• FTIR – Fourier Transform Infrared Spectroscopy is to interpret the
structure of molecules.
• Therefore LC – FTIR is an hyphenated technique in which separated
compounds from Liquid chromatography are interpreted for their
structural determination.

4. FTIR: FOURIER TRANSFORM INFRARED SPECTROSCOPY
• Fourier transform IR spectroscopy deals with the quantitative
measurement of the interaction between IR radiation and Materials
for its structural determination.
• Principle: It is the production of path difference between the two
optical pathways which results in interference. This path difference is
directly proportional to the intensity of radiation passing into the
sample which is then converted into wave number. So in FTIR path
difference is related to wave number passing into the sample.

5. • It is a method for measuring all of the infrared frequencies simultaneously, rather
than individually as with dispersive instruments.
• For this to happen FTIR uses a very simple optical device called an
“INTERFEROMETER”.
• Working:

6. • FTIR spectrometer obtains an infrared spectra by collecting an
interferogram of a sample signal using an Interferometer and then
perform a fourier transform on the Interferogram to obtain a
spectrum.
• Fourier transform defines a relationship between signal in the time
domain and its representation in frequency domain.

7. SOURCE
.
INTERFEROMET
ER SAMPLE
DETECTOR
INTERFEROGRA
M (encoded data
of sample at each
frequency)
DECODING
BY FT
IR SPECTRA

8. PRINCIPLE ADVANTAGES OVER DISPERSIVE IR
• The multiplex or FELLGETT’S advantage: Information
from all wavelengths is collected simultaneously.
• The throughput or JACAUINOT’S advantage: In Dispersive
monochromators are used but in FTIR no Monochromators
are used so there is no restriction for amount of light to pass
through.

9. HPLC
• HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution.
• It is characterized by the use of high pressure to push a
mobile phase solution through a column of stationary phase
allowing separation of complex mixtures with high
resolution.

10. COMBINATION OF HPLC AND FTIR: LC-FTIR

11. • The combination of liquid chromatography (LC) and FTIR can be
highly useful when specific detection or identification is required.
• The application of FTIR spectroscopy in LC is, however, still rather
limited, mainly because solvents commonly used in LC are strong IR
absorbers, limiting both sensitivity and obtainable spectral
information.
• In the development of LC-FTIR techniques two important coupling
methodologies are:
 Flow cell interfaces and
 Solvent elimination interfaces

12. FLOW CELL INTERFACES
• In the flow cell approach , the eluent is lead directly
through a cell where IR spectra are recorded continuously.
• Flow cells offer a simple and straight forward means for the
on line coupling of LC and FTIR.
• The effluent of the LC is passed directly through a flow cell
and IR spectra acquired in real time.

13. MERITS
• Low cost, instrumental simplicity, ease of operation, low
maintenance and possible use of non volatile buffers.
• The analyte can be studied without any orientation or
crystallization effects, oxidative degradation or evaporation
which might occur during or after solvent elimination .
• Because flow cell detection takes place in real time, it is also
potentially useful for online reaction monitoring.

14. DEMERITS
The major drawback of flow cell LC-FTIR is the limited
choice of eluents.
Example: water obscures big parts of the mid IR region,
prohibiting a practical combination of reversed phase LC and
FTIR using a flow cell. Only some organic solvents (e.g.,
chloroform) show sufficient transparency in the IR spectrum.

15. CELL WINDOW MATERIALS
• Cell windows or crystals must be chemically resistant to the eluent
used in the chromatographic method.
• Withstand high pressure and
• It should offer sufficient transmittance to maintain a reasonable IR
energy throughput.
• E.g., calcium fluoride (CaF2), zinc selenide(ZnSe), potassium bromide
(KBr), sodium chloride(NaCl)

16. TYPES OF FLOW CELLS
• Transmission flow cell
• ATR flow cell
• Specular reflection measurements flow cell

17. TRANSMISSION FLOW CELL

18. CONT……
• It can either consist of an IR transparent cavity or of two
IR transparent windows separated by a metal or Teflon
spacer.
• The LC eluent enters and exits the cell through capillary
tubing and is sampled by the IR beam passing
perpendicularly.

19. ATR FLOW CELL
.

20. CONT….
• Consists of a cylindrically shaped ATR crystal with cone shaped ends.
• The crystal is incorporated in a flow cell with cone ends outside the cell
body.
• The effluent passes through the flow-cell cavity surrounding the crystal.
• Cassegrain optics are used to focus the IR beam on the crystal at one end
and to direct the IR radiance emerging from the other end to the
detector.

21. SPECULAR REFLECTION MEASUREMENTS
FLOW CELL

22. CONT….
• Consists of a trough shaped stainless steel cell body covered with an
IR transparent window.
• An external mirror is used to direct the IR beam towards the flow-cell
window under near- normal incidence angle, reducing the reflection
losses at the air- window interface.
• After passing the cell window, the IR beam is reflected via a mirror
surface inside the cell cavity crossing the effluent flow path twice and
directed towards the detector via a second external mirror.

23. APPLICATIONS OF FLOW CELL INTERFACE
• The analysis of sugars in non alcoholic beverages
• Since flow- cell-FTIR is a valuable tool for the rapid,
selective and quantizable determination of the chemical
composition of polymers as function of their hydrodynamic
volume.

24. SOLVENT ELIMINATION INTERFACE
• The solvent elimination approach involves an evaporation interface for the
removal of the interfering eluent and subsequent analyte deposition on to a
suitable substrate, prior to FT-IR detection.
• In this case detection is no longer affected by the IR characteristics of the
mobile phase.
• To accomplish solvent elimination the eluent is generally directed to
nebulizer, often aided with (heated) nebulizer gas.
• Almost simultaneously, the separated analytes are deposited on a substrate.
• After deposition, IR spectra from immobilized chromatogram are acquired.

25. ADVANTAGES
• In the absence of interfering eluent absorption bands permits
spectral interpretation over entire wavenumber range.
• The immobilized chromatogram is still available after the
chromatographic run has been completed.
• The signal to noise ratio (SNR) can be greatly enhanced.
• Spectra with a greater optical resolution can be obtained.
• Sensitivity can be increased.

26. DEPOSITION SUBSTRATES
• Deposition of analytes in solvent elimination in LC-FTIR is
performed on powdered substrates, mirrors or IR
transparent windows

28. TYPES OF SOLVENT ELIMINATION
INTERFACES
• LC-DRIFT interfaces
• Buffer memory technique
• Thermo-spray interface
• Pneumatic nebulization technique

29. LC-DRIFT INTERFACES
• The LC effluent was dripped via a heated tube into discrete
KCl-filled cups and residual solvent was removed under a
gentle stream of nitrogen before the acquisition of spectra.
• It is more sensitive and produced spectra of better quality
than flow cell based LC-FTIR.

30. BUFFER MEMORY TECHNIQUE
• In this technique flat KBr plates for transmission
measurements are used.
• In this technique complete chromatogram is immobilized
and stored on a substrate, allowing offline scanning.
• For rapid evaporation of eluent micro-bore LC and low
flow rates are used.
• In this interface, the eluent was directed to a constantly
moving substrate via a stainless steel capillary.

31. THERMO-SPRAY INTERFACE
• In order to increase the flow rates (i.e., > 5µl/min) in LC-
FTIR, interfaces with an enhanced evaporation capacity are
essential one such LC-MS interface type is TSP.
• TSP incorporates a heated capillary. It produces a
supersonic vapor jet when the eluent exits the capillary
thereby breaking up the eluent into a mist of fine droplets
and enhancing evaporation of the eluent.

32. PNEUMATIC NEBULIZATION TECHNIQUE
• It is a most successful solvent elimination interface.
• The nebulizers use a high speed gas flow to break up the eluent into
small, fastmoving droplets, thereby greatly enhancing the evaporating
capacity.
• The nebulizer gas is heated when (almost ) complete removal of aqueous
eluents is required. Following eluent evaporation, the analytes are
deposited on a step wise or continuously moving IR-transparent
substrate.
• Several commercially available interfaces are concentric flow nebulizer

33. APPLICATIONS
• Sample enrichment procedure, such as solid phase extraction,
will be necessary to allow analyte detection by LC-FTIR.
• Identification of compounds like environmental pollutants,
pharmaceuticals ,drug metabolites, polymer derivatives etc……
• FTIR detection can be especially useful when isomeric
compounds have to be distinguished.

35. • LC-FTIR can be particularly beneficial in the analysis of synthetic
polymers revealing the chemical composition of co-polymers

36. REFERENCE
1. Kok, S.J. (2004), Coupling of liquid chromatography and FTIR for
characterization of Polymers, UvA-DARE, University of Amsterdam
2. P.R.Griffiths, J.A.de Haseth, Fourier Transform Infrared Spectrometry, Wiley,
New York, 1986.