High performance liquid chromatography

HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY(HPLC)
By
Ujjain Chaurasia (18311127)
M.Sc. Chemistry (2nd year)
DEPARTMENT OF CHEMISTRY
DR. B.R. AMBEDKAR NATIONAL INSTITUTE OF TECHNOLOGY,
JALANDHAR
Submitted to: Prof. (Dr.) N.C. Kothiyal 4/6/2022
UJJAIN CHAURASIA/M.Sc chemistry/18311127
1

HPLC
High performance liquid chromatography
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 A form of column chromatography to separate ,
identity, and quantify the compounds.
 Developed in 1970s.
 The most widely used analytical separation technique.
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CHROMATOGRAPHY:-
 Chromatography is a technique which separates components in a mixture
due to the differing time taken for each component to travel through it by a
mobile phase.
 Basically ,all chromatographic systems consists of two phases.
 Mobile phase-liquid or gaseous and flow over or through the stationary
phase.
 Stationary phase- solid, liquid or a solid/liquid mixture which is immobilized.
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Some chromatography terms
 Analyte
 substance that is to be separated during chromatography.
 Immobilized phase
 Stationary phase which is immobilized on the support
particles or on the inner wall of column tubing.
 Mobile phase
 Phase which moves in a definite direction.(liquid/gas/fluid)
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 Consists of the sample being separated/analysed and the
solvent that moves the sample through the column.
 Effluent
 Mobile phase leaving the column.
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Different types of chromatography methods
 Paper chromatography
 Liquid chromatography
 Gas chromatography
 High performance liquid chromatography
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High performance liquid chromatography
 HPLC is an extension of conventional liquid chromatography.
 Powerful tool in analytical techniques.
 Columns are tightly packed ,and the eluent is forced through
the column under high pressure (up to 5,000 psi ) by a pump.
 Allow to use a very smaller particle size for the column packing
material which gives a much greater surface area for
interactions between the stationary phase and the molecules
flowing through it.
 Allow a much better separation of the components of the
mixture. 4/6/2022
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HPLC Technique
 Utilizes liquid mobile phase to separate the mixture.
 Analytic are first dissolved in a solvent then through the
column under high pressure of up to 400 atm.
 Mixture is resolved into its components in the column.
 The total separation time is often 5 or 10 minutes rather
than hours or even days required for some separations
by gravity flow with the larger systems 4/6/2022
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Components of HPLC
• Pump
• Injector
• Column
• Detector
• Recorder or data system
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HPLC SETUP
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Pump
 A pump forces the mobile phase through the column at a much
greater velocity than gravity-flow columns.
 The pump can be pneumatic, syringetype ,reciprocating, or
hydraulic amplifier.
 Pneumatic pumps are used for preoperative purposes.
 The most widely used pump today is the multihead pump with
two or more reciprocating pistons.
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 Pumps are designed in order to maintain a stable flow rate, avoiding pulsations
even when the composition of the mobile phase varies.
 flow range – 0.01-10 ml/min.
HPLC Pump
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Injectors
 Inject the liquid sample within range of 0.1- 100 ml of
volume under high pressure.
 Produce minimum band broadening.
 Produce possible flow disturbances.
 Volume must be small (0.1-500 uL).
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A model injector
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INJECTOR
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Columns
 Smooth-bore stainless steel or heavy walled glass tubing.
 Hundreds of packed columns differing in size and packing are available
from manufacture.
 E.g. Column packing vary in size (3 to 20 um) with the smaller particles
used mostly for analytical separations and the larger ones for
preparative separation.
 The most common material used for column packing is silica gel.
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HPLC columns
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Detector
 HPLC detectors monitor the elute as it leaves the column.
 Produce an electronic signal proportional to the
concentration of each separated component.
 Crucial in trace analysis.
 High sensitivity.
 Fast response.
 Simplifies quantitation.
 Insensitive to changes in type of solvent.
 flow rate and temp. 4/6/2022
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The most widely used detection methods
 Spectrophotometers
 Fluorometers
 Electrochemical detectors
 Mass spectrometer
 Refractive index detector
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Depending on the relative polarity of the solvent and stationary
phase, there are two variants in use in HPLC:-
1. Normal phase HPLC
 Utilize polar adsorbent surface and nonpolar eluent.
 Polar substance in the mixture sticks to polar adsorbent than
non-polar.
 Non-polar ones will pass more quickly through the column.
2. Reversed phase HPLC
 Utilize non-polar adsorbent surface and polar eluent.
 Attraction between non-polar compound in the mixture and
non-polar adsorbent. 4/6/2022
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 Polar molecules will travel through the column more quickly
because there is strong attraction between polar solvent and
polar molecules when pass through the column.
 Reversed phase HPLC is the most commonly used form of
HPLC.
 Solvents used in mobile phase
 hexane, heptane, cyclohexane, carbon tetrachloride, benzene,
toluene, diethyl ether, chloroform etc.
 Adsorbents used in stationary phase
 silica gel, alumina, celite, cellulose powder, ion-exchange,
cellulose, starch.
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Retention time
 The time taken for a particular compound to travel through the
column to the detector.
 From the time at which the sample is injected to the point at which
the display shows a maximum peak height for that compound.
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Applications
 HPLC is used for Chemistry and biochemistry research
 analyzing complex mixtures, Purifying chemical compounds
 Quality control to ensure the purity of raw materials
 Analyzing air and water pollutants,
 Monitoring materials that may jeopardize occupational safety or health
 Monitoring pesticide levels in the environment.
 To survey food and drug products,
 To identify confiscated narcotics
 To determine the amount of such chemical compounds found in new
drugs in pharmaceutics
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HPLC as compared with the classical technique
 Small diameter, reusable stainless steel columns
 Column packing with very small particles
 Control flow of mobile phase
 Precise sample introduction
 Good pumping system
 Special continuous flow detectors- can handle small flow rates and
detect very small amounts
 Rapid analysis
 High resolution
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Disadvantages of HPLC
• Cost
• Complexity
• Low sensitivity for some compounds
• Irreversibly adsorbed compounds not detected
• Co-elution difficult to detect
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Summary
 The modern day technique is greatly enhanced in terms of
selectivity, resolution, through miniaturization and the use of
very elaborate stationary phases.
 Therefore HPLC is widely used for separation of molecules in
biological, pharmaceutical, food, environmental and
industrial process.
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References:
• Harold Varley practical clinical chemistry
• http://scimedia.com
• http://www.forumsci.co.il/HPLC
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High performance liquid chromatography

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