HPLC works on the principle of the separation of the material according to their molecular weight and polar FTIR spectroscopy deals with the quantitative measurement of the interaction between IR radiation and materials. FTIR reveals molecular-vibrational transitions and provides characteristic information on molecular structure.

1. ADVANCED SPECTRAL ANALYSIS (MPC201T) UNIT-IV_CHROMATOGRAPHY (LC-FTIR)
Prepared by: - Subham Kumar Vishwakarma (shubhamkumarvishwakarma7@gmail.com), Guided by: - Dr. S Raja, Gitam
University Visakhapatnam (AP)
1
• 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 polar FTIR spectroscopy deals with the quantitative measurement of the interaction between
IR radiation and materials. FTIR 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.
Instrumentation: -
1. LC setup- see in previous hyphenated technique LC-MS or find
DOI: 10.13140/RG.2.2.33132.08321
2. Interface-
a. FTIR interface
b. Solvent-elimination interfaces
FTIR INTERFACE
Flow-cell interfaces: -
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 are acquired in real time. The
merits of the approach include low cost, instrumental simplicity, ease of operation, low maintenance,
and the 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

2. ADVANCED SPECTRAL ANALYSIS (MPC201T) UNIT-IV_CHROMATOGRAPHY (LC-FTIR)
Prepared by: - Subham Kumar Vishwakarma (shubhamkumarvishwakarma7@gmail.com), Guided by: - Dr. S Raja, Gitam
University Visakhapatnam (AP)
2
for on-line reaction monitoring. On the other hand, the dynamic nature of the IR measurements leaves
less time to collect spectra, limiting the signal-to-noise ratio (SNR).
Cell-window materials: - The choice for a specific window material is mainly determined by the
properties of the LC eluent and the spectral region that has to be monitored. A fully IR transparent
material such as potassium bromide (KBr), for instance, cannot be used with RPLC. Instead, more
expensive water-insoluble materials such as calcium fluoride and zinc selenide (ZnSe) have to be
chosen. The optical and physical properties of some commonly used IR-window materials are
presented in table below. In all cases, in order to obtain an identifiable IR spectrum a minimum
amount of analyte has to be present in the detection cell during the time of measurement.
Types of flow cells: -
Three types of flow cells can be discerned for on-line LC-FTIR coupling. These are based on
transmission, attenuated-total-reflection (ATR) and specular-reflection measurements, respectively.
The spectral range (i.e., detection-wavenumber 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.
1. Transmission cell: - 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 is 0.001 to 2 mm and
Detection Limits is 40-50 microgram when Chloroform is used as M.P.
2. Flow cells is based on the ATR principle: -
One type of cell consists of a cylindrically shaped ATR crystal with cone-shaped ends (Figure 3.2).
The crystal is incorporated in a flow cell with the cone ends outside the cell body. The effluent passes

3. ADVANCED SPECTRAL ANALYSIS (MPC201T) UNIT-IV_CHROMATOGRAPHY (LC-FTIR)
Prepared by: - Subham Kumar Vishwakarma (shubhamkumarvishwakarma7@gmail.com), Guided by: - Dr. S Raja, Gitam
University Visakhapatnam (AP)
3
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.
3. Flow cell is based on specular-reflection measurements: -
➢ 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 angles, 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 pathlength is twice the thickness of thee sample cavity and it can be adjusted
from 50 um to 2 mm, corresponding to cell volumes of 1 to 40μ.
Eluent absorption: -
➢ Ideally, the mobile phase used in flow-cell LC-FTIR should not exhibit serious background
absorption, because this may obscure analyte absorption bands. Unfortunately, just about all
organic solvents used in LC show intense IR spectra.
➢ In order to correct for background absorption by the eluent, background subtraction often can
be carried out quite reliably [17], provided that isocratic LC is used. FTIR allows the
acquisition of spectral data on an extremely precise wavenumber scale
Solvent-elimination interfaces: -
The eluent is removed prior to detection. Eluent is directed to a nebulizer often aided with nebulizer
gas. The Separated analytes are deposited on a substrate. IR Spectra from the immobilized
chromatogram may be obtained.
Solvent-elimination LC-FTIR offers a number of distinct advantages when compared with flow-
cell LC-FTIR approaches.
➢ Firstly, the absence of interfering eluent absorption bands permits spectral interpretation over
the entire wavenumber range, allowing full exploitation off the identification possibilities of
IR spectroscopy.
➢ Secondly, the immobilized chromatogram is still available after the chromatographic run has
been completed.
Types of Solvent Elimination Interfaces: -
1. Early LC-DRIFT
2. Buffer memory
3. Spray type interfaces

4. ADVANCED SPECTRAL ANALYSIS (MPC201T) UNIT-IV_CHROMATOGRAPHY (LC-FTIR)
Prepared by: - Subham Kumar Vishwakarma (shubhamkumarvishwakarma7@gmail.com), Guided by: - Dr. S Raja, Gitam
University Visakhapatnam (AP)
4
a) Thermo-spray interface
b) Particle beam interface
c) Electrospray interface
d) Pneumonic nebulizer
e) Ultrasonic nebulizer
1.Early LC-DRIFT Interfaces: - The LC eluent was dripped via a heated tube into discrete KCl-
filled cups and residual solvent was removed under a gentle stream of nitrogen before the aquisition
of spectra.
2.Buffer Memory Technique: - Flat KBr Plates are used for transmission measurements. A complete
chromatogram is immobilized and stored on a substrate. Micro-bore LC and low flow rates were used
for rapid evaporation of eluent. Spectra aquistion - FTIR transmission microscopy. Study of analytes
- X-ray Fluorescence Spectra.
3.Spray type Interfaces
a) Thermospray Interfaces: -
✓ LC eluent is passed through a directly heated vaporized tube.
✓ Part of the liquid evaporates to an expanding vapour and as a result, a mist of de-solvating
droplets emerge from the end of the tube.
✓ Operating Temp: 100 - 300℃
✓ Detection limits: 1mg
b) Particle beam interface :
For deposition of LC-separated compounds on KBr substrates.
Three Components:
I. A monodisperse aerosol is generated from the LC eluent by nebulization with the aid
of He.
II. Then directed into a desolvation chamber (Most liquid gets vapourized here)
III. The mixture of gas, vapour and condensed analyte molecules is accelerated towards the
“momentum separator”
IV. The separated analyte molecules of interest enter the IR-transparent substrate.

5. ADVANCED SPECTRAL ANALYSIS (MPC201T) UNIT-IV_CHROMATOGRAPHY (LC-FTIR)
Prepared by: - Subham Kumar Vishwakarma (shubhamkumarvishwakarma7@gmail.com), Guided by: - Dr. S Raja, Gitam
University Visakhapatnam (AP)
5
V. The substrate is then removed after deposition from the vaccum chamber and
transferred to the FT-IR spectrometer for
c) Electrospray interface:
A spray of charged droplets a produced using a high electric field. The initial droplets further
breakdown into smaller droplets as a result of solvent evaporation and charge density. Deposit
Surface - ZnSe Plate.
d) Pneumonic nebulizer:
High speed gas flow is used to disrupt the liquid surface and to form small droplets which are
dispersed by the gas. Deposit Surface: IR-Reflective Disc, Gas: Nitrogen Gas.
e) Ultrasonic nebulizer:
A Spray is formed by depositing the LC effluent on a transducer that is vibrating at ultrasonic
frequencies. The vibrations cause the solvent to break up into small, desolvating droplets which
are transported by a carrier gas towards a substrate. Deposition Substrate: KBr discs.
APPLICATIONS: -
• In trace analysis.
• 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.