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LIQUID
CHROMATOGRAPHY

FST-401
Dr. Imran Pasha
Associate Professor
National Institute of Food Science & Technology,
UAF
Liquid Chromatography
• There are several liquid chromatography techniques applied in food analysis, namely,
• Paper chromatography
• Thin layer chromatography (TLC)

Both of these techniques may be referred to as planar chromatography and column

liquid chromatography
• All of which involve a liquid mobile phase and either a solid or a liquid stationary phase
• However, the physical form of the stationary phase is quite different in each case
• Separation of the solutes is based on their physicochemical interactions with the two
phases

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Paper Chromatography
• Paper chromatography was introduced in 1944
• In paper chromatography the stationary phase and the mobile phase are both liquid
• Paper generally serves as a support for the liquid stationary phase
• The stationary phase in paper partition chromatography is usually water
• However, the support may be impregnated with a nonpolar organic solvent and developed with water or
other polar solvents or water
• The sample is spotted in one corner of a square sheet of paper
• One solvent is used to develop the paper in one direction
• The chromatogram is then dried, turned 90◦, and developed again, using a second solvent of different
polarity
• In paper and thin-layer chromatography, components of a mixture are characterized by their relative
mobility (Rf) value
Rf = Distance moved by component
Distance moved by solvent
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Thin Layer Chromatography
• Thin-layer chromatography (TLC), first described in 1938, has largely replaced paper
chromatography because it is,
• Faster
• More sensitive
• More reproducible

• TLC utilizes a thin (250 μm thick) layer of sorbent or stationary phase bound to an inert support
in a planar configuration.
• The support is often a glass plate (traditionally, 20 cm × 20 cm), but plastic sheets and aluminum
foil also are used
• Pre-coated plates, of different layer thicknesses, are commercially available in a wide variety of
sorbents, including chemically modified silicas
• Four frequently used TLC sorbents are silica gel, alumina, diatomaceous earth, and cellulose
• Both normal and reversed-phase thin-layer separations may be carried out 4
Paper and Thin Layer Chromatography

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Column Chromatography
• Column chromatography is the most useful method of separating compounds in a mixture
• Fractionation of solutes occurs as a result of differential migration through a closed tube of
stationary phase
• Analytes can be monitored while the separation is in progress
• In column liquid chromatography, the mobile phase is liquid
• The stationary phase can be either solid or liquid supported by an inert solid

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High Performance Liquid Chromatography
(HPLC)
• High-performance liquid chromatography (HPLC) developed during the 1960s as a direct branch
of classic column liquid chromatography
• Through improvements in the technology of columns and instrumental components (Pumps,
injection valves, and detectors)
• HPLC can be applied to the analysis of any compound with solubility in a liquid that can be used as
the mobile phase
• Although most frequently employed as an analytical technique, HPLC also may be used in the
preparative mode

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Components of HPLC
The main components of this system include:
• Pump
• Injector
• Column
• Detector
• Data system

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Pump
• The HPLC pump delivers the mobile phase through the system
• Typically at a flow rate of 0.4–1 ml/min, in a controlled, accurate, and precise manner
• The majority of pumps currently used in HPLC (>90%) are reciprocating, piston-type pumps
• The dual piston pump systems with ball check valves are the most efficient pumps available
• Gradient elution systems for HPLC are used to vary the mobile phase concentration during the
run, by mixing mobile phase from two or more reservoirs
• This is accomplished with low-pressure mixing, in which mobile phase components are mixed
before entering the high-pressure pump

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Injector
• The role of the injector is to place the sample into the flowing mobile phase for introduction
onto the column
• Virtually all HPLC systems use valve injectors, which separate sample introduction from the
high pressure eluent system
• With the injection valve in the LOAD position the sample is loaded into an external, fixed-
volume loop using a syringe
• Eluent, meanwhile, flows directly from the pump to the column at high pressure
• When the valve is rotated to the INJECT position, the loop becomes part of the eluent flow
stream and sample is carried onto the column
• Such injectors are generally trouble free and afford good precision

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Column
• An HPLC column is usually constructed of stainless steel tubing with terminators that allow it to be
connected between the injector and detector of the system
• Columns also are made from glass, fused silica, titanium, and polyether ether ketone (PEEK) resin
• The most commonly used analytical HPLC columns are 10, 15, or 25-cm long with an internal diameter of
4.6 or 5mm
• Short (3 cm) columns, packed with ≤3 μm particles, are gaining popularity for fast separations; for
example, in method development or process monitoring
• In recent years, the use of columns with smaller internal diameters (<0.5–2.0 mm), including wall-coated
capillary columns, has increased
• The advantages of using smaller diameter columns include:
 Decreased consumption of mobile phase
 An increased peak concentration
 Increased resolution and
 The ability to couple HPLC with mass spectrometry (MS)

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Detector
• A detector translates sample concentration changes in the HPLC column effluent into
electrical signals
• Spectrochemical, electrochemical, or other properties of solutes may be measured by a variety
of instruments, each of which has advantages and disadvantages
• The choice of which to use depends on solute type and concentration, and on detector
sensitivity, linear range, and compatibility with the solvent and elution mode to be used
• Cost also may influence detector selection
• One common feature of most HPLC detectors is the presence of a flow cell, through which the
eluent flows as it is analyzed by the detector system

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LC Detectors
Common types of LC Detectors

1. Refractive Index Detector (RI)

2. UV/Vis Absorbance Detector

3. Fluorescence Detector

4. Conductivity Detector

5. Electrochemical Detector (ECD)

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2) UV/Vis Absorbance Detector
 Measures the ability of solutes to absorb light at a particular wavelength(s) in the
ultraviolet (UV) or visible (Vis) wavelength range (Most common type of LC detector)
 Three Common types of UV/Vis Absorbance Detectors
1. Fixed wavelength detectors
2. Variable wavelength detectors
3. Photodiode array detectors

LC Detectors

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a. Fixed Wavelength Detector absorbance of only one given wavelength is monitored by
the system at all times (usually 254 nm)
1. Simplest and cheapest of the UV/Vis detectors
2. Limited in flexibility
3. Limited in types of compounds that can be monitored
b) Variable Wavelength Detector a single wavelength is monitored at any given time, but
any wavelength in a wide spectral range can be selected
1. Wavelengths vary from 190-900 nm.
2. More expensive, requires more advanced optics
3. More versatile, used for a wider range of compounds
c) Photo Diode Array Detector (DAD) operates by simultaneously monitoring absorbance
of solutes at several different wavelengths
1. Uses a series or an array of several detector cells within the instrument, With each responding to
changes in absorbance at different wavelengths
2. Entire spectrum of a compound can be taken in a minimum amount of time
3. Useful in detecting the presence of poorly resolved peaks or peak contaminants
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3) Fluorescence Detector
 A selective LC detector that measures the ability of eluting solutes to fluoresce at a
given set of excitation and emission wavelengths
Applications
I. Fluorescence used to detect any
compound that absorbs and emits
light at the chosen set of excitation
and emission wavelengths
a. High selectivity
b. Low background signal
II. It is used in detection of
a. Drugs
b. Food additives
c. Environmental pollutants
d. Any compound that can be converted
to a fluorescent derivative: alcohols,
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amines, amino acids and proteins
4) Conductivity Detector
 Used in analytical applications of ion-exchange chromatography for the detection
of ionic compounds
 Detector measures the ability of the mobile phase to conduct a current when

placed in a flow-cell between two electrodes

 Current conducted within the cell will depend on the number and types of ions

present in the mobile phase

Applications
Can be used to detect any
compound that is ionic or
weakly ionic
a. Food components
b. Industrial samples
c. Environmental samples
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 5) Electrochemical Detector
 Used to monitor any compound in the mobile phase that can undergo an oxidation or
reduction
 Electrochemical detection in liquid chromatography is sometimes referred to as

LC/EC
 Generally includes two or more electrodes which monitor the current that is

produced by the oxidation or reduction of eluting compounds at a fixed potential

 Generally electrical output is an electron flow generated by a reaction that takes

place at the surface of the electrodes

Compounds that can be detected by reduction
Aldehydes, ketones, esters and
unsaturated compounds
Compounds that can be detected by oxidation
Phenols

Mercaptans (RSH) Column flow

Aromatic amines

Dihydroxy compounds

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Advantages of HPLC over traditional low pressure column liquid chromatography:
1. Speed (many analyses can be accomplished in 30 min or less)
2. A wide variety of stationary phases
3. Improved resolution
4. Greater sensitivity (various detectors can be employed)
5. Easy sample recovery (less eluent volume to remove)

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Comparison between LC and GC

Advantages of LC compared to GC
1. LC can be applied to the separation of any compound that is soluble in a liquid phase.
2. Liquid mobile phase allows LC to be used at lower temperatures than required by GC
LC better suited than GC for separating compounds that may be thermally labile
3. Retention of solutes in LC depend on their interaction with both the mobile phase and
stationary phase
GC retention based on volatility and interaction with stationary phase
LC is more flexible in optimizing separations  change either stationary or mobile
phase
4. Most LC detectors are non-destructive
Most GC detectors are destructive
LC is better suited for preparative or process-scale separations
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Comparison between LC and GC

Disadvantage of LC compared to GC

1. LC is subject to greater peak or band-broadening

Much larger diffusion coefficients of solutes in gases vs. liquids

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