PRESENTATION

ON

LIQUID CHROMATOGRAPHY
 POINTS TO BE DISCUSSED:-
 Introduction
 Principle
 Instrument
 Modes of liquid chromatography
 Qualitative & Quantitative Analysis
 Preparative liquid chromatography
 Application
 Sample
 Limitation
 Conclusion
 References
 INTRODUCTION
 Chromatography means “COLOUR WRITING”
 Chromatography basically involves the
separation due to differences in the equilibrium
distribution of sample components.
 Stationary phase
 Mobile phase

 In liquid chromatography, a sample mixture is

passed through a column.
 The column may be coated or not.
 Fastest growing technique in analytical
chemistry.
 Invented by Russian botanist M.S TSWETT

Principle
A sample mixture is passed through a column packed with solid particles
which may or may not be coated with another liquid.
 With the proper solvents, packing conditions, some components in the
sample will travel the column more slowly than others resulting in the
desired separation.
 Instrument
 Pumping system:High-pressure pumping systems outputs of up to
34.5 MPa (5000 psi) and pulse less, reproducible flow deliveries of 0.1 to 10
mL/min.
 High performance column:-Columns are made from 3- to 25-cm
(1.2- to 10-in.) lengths of stainless steel tubing with inner diameters of 1 to
20 mm (0.04 to 0.8 in.). Columns are packed with small (3 to 50 μm) particles
 Injector:-Injector valves are designed to accommodate sample
introduction from microliter or millilitres syringes.
 Detector:-
 An ideal LC detector would
 (1) Have universal response to all compounds
 (2) Have no response to the mobile phase
 (3) Have good sensitivity
 (4) Have response independent of column parameters
 (5) Provide structural information
 (6) Have linear response to sample concentration
Essential components of a liquid chromatograph
 Modes of liquid chromatography

 Liquid-solid chromatograph (LSC)

 Liquid-liquid chromatography (LLC)
 Bonded-Phase Chromatography (BPC)
 Normal-phase chromatography (NPC)
 Reversed-phase chromatography (RPC)
 Ion-exchange chromatography (IEC)
 Ion-pair chromatography (IPC)
 Size-exclusion chromatography (SEC)
 Qualitative & Quantitative Analysis

 Qualitative analysis refers to the

identification of the components in a sample
mixture.
 Quantitative analysis of the components in a
sample is performed by comparison of the
height or area of the component peak with
those of standards.
 Preparative liquid chromatography
 Preparative liquid chromatographs are
especially designed for these large-scale
separations. They have large mobile phase
reservoirs, large flow rate capabilities, large
capacity columns, and automated fraction
collection systems.
 Application

 Analysis of Parabens in Baby Lotion

 The baby lotion sample was analyzed by comparison
of its chromatogram with that of mixtures of paraben
standards.
 The standards were prepared by dissolving weighed
amounts of methyl-, ethyl-, propyl-, and butylparaben
in 95/5 ethanol/water. Concentrations of the
standards were 18 to 202 ng/μL.
 Baby lotion extract was obtained by emulsifying. 932
mg of baby lotion in 5 mL of tetrahydrofuran.
 Next, 15 mL of 95/5 ethanol/water was added and
the solution was acidified with two drops of 10%
sulfuric acid.
 Finally, the solution was filtered through 2-μm filters
to remove particulates which could clog the
chromatographic flow system.
Other Applications

 · Organically bound halides and sulfur following

combustion and adsorption techniques
 · Determination of anions on contaminated surfaces
 · Plating bath solution analysis
 Sample
 Form: Solids or aqueous solutions
 · Size: Minimum of 1 to 5 mg for solids;
minimum of 1 mL for solutions;
 · Materials: Inorganic and organic materials,
geological samples, glasses, ceramics,
explosives alloys,
 · Preparation: Aqueous solutions can be
analyzed as received or after dilution; analysis
of solids must follow a sample preparation and
dissolution procedure.
 Limitation

 Detection limits below the part per million level

for many ions; part per billion under ideal
conditions
 · Cations: Limited to alkali and alkaline earths,
ammonia, and low molecular weight amines if
suppressed conductivity detection is used
 · Must be ionic in solution
 · Must be water soluble
 · Limited work has been done in organic
solvents
 Conclusion
 The analytical results provided by the combination of
liquid chromatography and mass spectrometry were
invaluable in identifying the products of chemical
reactions.
 This approach is applicable to any mixture of
compounds if the objective is to unambiguously
identify individual components in the mixture.
 Nuclear magnetic resonance and infrared
spectroscopy are also frequently used for structural
identification of compounds isolated by liquid
 Chromatography. LC is one of the fastest growing
techniques in analytical chemistry. It enables the
user to perform rapid, efficient separations of
complex mixtures of organic, inorganic,
pharmaceutical, and biochemical compounds.
 References

 • R.J. Hamilton and P.A. Sewell, Introduction to

High-Performance Liquid Chromatography, Chapman
and Hall, 1978
 • E.L. Johnson and R. Stevenson, Basic Liquid
Chromatography, 2nd ed., Varian, 1978
 • J.J. Kirkland, Ed., Modern Practice of Liquid
Chromatography, Wiley-Interscience, 1971
 • C.F. Simpson, Ed., Practical High-Performance
Liquid Chromatography, Heyden, 1976
 • L.R. Snyder and J.J. Kirkland, Introduction to
Modern Liquid Chromatography, 2nd ed., Wiley-
Interscience, 1979
THANK U