This document provides an overview of LC-FTIR, a hyphenated technique that combines liquid chromatography (LC) and Fourier transform infrared spectroscopy (FTIR). LC is used to separate mixtures into individual components, which are then analyzed by FTIR to obtain structural information. There are two main interfaces used: flow cell interfaces analyze components in real-time without solvent removal, while solvent elimination interfaces remove interfering solvent before analysis. LC-FTIR has applications in trace analysis, isomer detection/distinction, and analysis of pharmaceuticals, polymers, and environmental pollutants.

1. LC-FTIR
PRESENTED BY:
GAMPALA DIVYA VANI,
M PHARMACY 1st year 2nd semester,
PHARMACEUTICAL CHEMISTRY,
UNIVERSITY COLLEGE OF TECHNOLOGY,
OSMANIA UNIVERSITY, HYDERABAD.

2. CONTENTS
• INTRODUCTION
• PRINCIPLE
• INTRUMENTATION
• APPLICATIONS
• CONCLUSION
• REFERENCE

3. LC-FTIR
• LC and FTIR can be combined together for the detection and identification of certain separated
compounds.
• HPLC works on the principle of the separation of the material according to their molecular
weight and polarity. FTIR deals with the quantitative measurement of the interaction between
IR radiation and materials and reveals molecular-vibrational transitions and provides
characteristic information on molecular structure
• The application of FTIR spectroscopy in LC is limited because the solvents used commonly in
LC are strong IR absorbers, limiting both sensitivity and the spectral information that may be
obtained.
• Therefore LC-FTIR is an hyphenated technique in which separated compounds from liquid
chromatography are interpreted for their structural determination
• The coupling of LC and FTIR has been pursued primarily to achieve specific detection and/or
identification of sample constituents.

4. • Fourier transform infrared spectroscopy is preferred over dispersive or filter methods of
infrared spectral analysis for several reasons like non-destructive, precise, speed,
sensitivity ,optical throughput, mechanically simple.

5. PRINCIPLE:
• Liquid chromatograph (LC) is a separation process used to isolate the individual components of a mixture.
This process involves mass transfer of a sample through a polar mobile phase and non- polar stationary
phase. The components eluted from LC now enters the FTIR through an interface, there are 2 types of
interfaces that are used to connect LC and FTIR they are flow cell interface and solvent elimination
interface.
• Now these components enter FTIR it comprises of beam splitter, moving mirror, and fixed mirror, the
light splits into two by beam splitter is reflected from the moving mirror and fixed , before being
recombined by beam splitter.
• As the moving mirror takes reciprocating movements, the optical path difference to fixed mirror changes,
such that the phase difference changes with the time. The beam finally passes to the detector for final
measurement.
• The detectors used are specially designed to measure the signal interferogram signal, the measured signal
is digitalized and sent to the computer where fourier transformation takes place

6. INSTRUMENTATION
• The combination of 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.

7. LC SYSTEM
• PUMP: the role of the pump is to force a liquid through the liquid chromatography at a
specific flow rate(1-2ml/min).various pumps like reciprocating pump, syringe type pump,
pneumatic pump etc.. are used to deliver constant mobile phase composition.
• MIXING UNIT: mixing unit is used to mix solvents in different proportions and pass
through the column. Mixing of solvent is done either with astatic mixer which is packed
with beads or a dynamic mixer which uses magnetic stirrer and operates under high
pressure
• INJECTOR: the injector serves to introduce the liquid sample into the flow stear the
mobile phase, an autosampler may also be used.
• Types of injectors: septum injector, Stop injector, Rheodyne injectors.

8. • COLUMN: It is the heart of the chromatography. stainless steel, glass are used for
construction for the tubing and packing material used are SILICA particle & ALUMINIA.
• In the development techniques two important coupling methodologies are:
FTIR interface and
Solvent elimination interface
1)Transmission flow cell
2)ATR flow cell
3)Specular reflection
measurement flow cell
1)LC-DRIFT interface
2)Buffer memory technique
3)Spray type interface
a- thermos spray interface
b-particle beam interface
c- electrospray interface
d-pneumatic interface
e-ultrasonic interface

9. FTIR INTERFACE
1) FLOW CELL INTERFACE:
• Flow cell offers 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 are acquired in real time.
• The analyte can be studied without any orientation or crystallization effects, oxidation 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 a online reaction
monitoring.
Merits: Demerits:
• Low cost Limited choice of eluents.
• Instrumental simplicity ex: chloroform
• Ease of operation
• Low maintenance
• Possible use of non volatile buffers.

10. Cell window material:
The choice for a specific window material is mainly determined by the properties of Lc eluent and the
spectral region that has to be monitored. A fully IR transparent material such as KBr cannot be used in
RPLC, instead water-insoluble material such as ZnSe have to be chosen.
Types of flow cells:. The spectral range of these interfaces is determined by the IR characteristics of
the applied cell-window material and by the mobile phase used for the chromatographic separation.
Three types of flow cells can be discerned for on-line LC-FTIR coupling.
• Transmission flow cell
• ATR flow cell
• Specular reflection measurements flow cell

11. TRANSMISSION FLOW CELL
• It can either consists 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.
• Path length 0.001-2mm

12. ATR FLOW CELL
• 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

13. SPECULAR REFLECTION MEASUREMNTS FLOW CELL
• 1.Cell body
• 2.IR-transparent window
• 3. flow-cell cavity
• 4.LC-Flow path
• 5.IR-beam path
• 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.
• The actual optical length is twice the thickness of the sample cavity and it can be adjusted from50um to
2mm,volume of 1-40µ.

14. 2) Solvents elimination interface:
• Solvent elimination LC-FTIR offers a number of distinct advantages when compareds with flow-cell LC-FTIR
approaches.
• 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. the separated analyte
deposited on a substrate
• After deposition , IR spectra from immobilized chromatogram are acquired

15.  Types of solvent elimination interfaces
• LC-DRIFT interface
• Buffer memory technique
• Spray type interface –a) thermos spray interface, b) particle beam interface, c) electrospray interface,
d)pneumatic nebulizer, e)ultrasonic nebulizer
LC-DRIFT INTERFACE:
• The LC effluent was dripped via a heated tube into discrete KCI-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
Buffer memory interface:
• In this technique KBr plates for transmission measurements are used. Here complete chromatogram is
immobilized and stored on a substrate, allowing offline scanning.
• For rapid evaporation of eluent micro-bore LC and low flow rate are used.
• In this interface the eluent was directed to a constantly moving substrate via a stainless steel capillary.

16. • Spray type interface:
a) Thermo spray interface- LC eluent is passed through a directly heated vaporized tube. part of
the liquid evaporates to an expanding vapour and as a result, mist of de-solvating droplets
emerge from the end of the tube.
b) Particle beam interface-for deposition of LC-separated compounds on KBr substrates . Three
components like monodisperse aerosol, de-solvation chamber, momentum separator.
c) Electrospray interface- a spray of charged droplets a produced using a high electric field. The
initial droplets are further breakdown into smaller droplets as a results of solvent evaporation
and charge density. Deposit surface-ZnSe plate.
d) Pneumatic nebulizer- high speed gas flow is used to disrupt the liquid surface and to form
small droplets which are dispersed by the gas.
e) Ultrasonic nebulizer- a spray is formed by depositing the LC effluent on a transducer that is
vibrating at ultrasonic frequencies. The vibration cause the solvent to break up into small,
de-solvating droplets which are transported by a carrier gas towards a substrate.

17. FTIR SYSTEM:
• The heart of FTIR is a Michelson interferometer. The monochromators is replaced by an
interferometer, which divide radiant beam, then recombines them in order to produce
repetitive interference signals measured as a function of optical path length difference as
the name implies, the interferometer produces interference signals, which contain IR
spectral information generated after passing through a sample.
• Consists of 3 active components –
1)A moving mirror
2)A fixed mirror
3)A beam splitter

19. WORKING:
• Radiation from the IR source is collimated and directed into the interferometer, and
impinges on the beam splitter.
• At the beam splitter half of the IR beam is transmitted to the fixed mirror and the
remaining half is reflected to the moving mirror.
• After the divided beams are reflected from the two mirrors, they are recombined at the
beam splitter.
• Due to changes in the relative position of the moving mirror, an interference pattern is
generated. The resulting beam then passes through the sample and is eventually focused
on the detector
• The detected interferogram can not be directly interpreted . It has to be decoded with a
well known mathematical technique in terms of Fourier transformation

20. APPLICATIONS OF LC-FTIR
1. In trace analysis
2. Detection and distinction of isomers.
3. Separation of secondary structures of protein such as lysosome, beta globulin.
4. Analysis of pharmaceutical products such as steroids and analgesics.
5. Analysis for polymer additives, dyes, non ionic surfactants.
6. Analysis of samples with relatively high analyte concentration ex: analysis of sugar in non-
alcoholic beverages.
7. Characterization of synthetic polymers.
8. Analysis of environmental pollutants such as polycylic aromatic hydrocarbons, pesticides
and herbicides.

21. CONCLUSION:
• From previous slides we get basic knowledge about the LC & FTIR and types of modes,
cells and instrumentation on how the LC-FTIR works on.
• LC-FTIR is an hyphenated technique in which separated compounds from liquid
chromatography are interpreted for their structural determination. Fourier transform
infrared spectroscopy is preferred over dispersive or filter methods of infrared spectral
analysis for several reasons like non-destructive, precise, speed, sensitivity ,optical
throughput, mechanically simple.

22. REFERENCES:
• P.R Griffiths, J.A.de Haseth, Fourier transform infrared spectroscopy ,wiley, new York
1986.
• Liquid chromatography Fourier transform infrared spectroscopy by Shubham Kumar
from Gitam university.
• LC-FTIR by Gangas from govt college of pharmacy Bengaluru.