Chromatographic methods

High performance liquid Chromatography (HPLC)

Shishir Kanti Pramanik, PhD

Professor
Department of Chemistry, SUST

Dept. of Chemistry, SUST

 Chromatography
❑ It is define as, it is analytical method in which separation of active
constituent in complex mixture, and the mixture was distributed in two
phases i.e. stationary phase and mobile phase is known as
chromatography.

❑ It is technique is used for separation, purification, Identification and

extraction of compound.

❑ It is method it can consist of two phases stationary phase and mobile

phase.

❑ Stationary phase is constant phase or column packaging material.

❑ Mobile phase is moveable phase.

❑ The basic principle of chromatography is based on Adsorption and

partition chromatography.

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 Chromatography
❑ Adsorption chromatography - The affinity of molecules towards stationary
phase is known as Adsorption chromatography.

❑ Partition chromatography - The molecule can moves in two phases of liquidis

known as partition chromatography.

❑ It is important for qualitative and quantitative analysis.

TYPES OF LIQUID CHROMATOGRAPHY

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 HPLC

 HPLC is a High Performance liquid Chromatography.

 High Pressure Liquid Chromatography.
 High Priced Liquid Chromatography.
 It is column chromatography.
 It is Liquid Chromatography.
 It is modified from of gas chromatography, it is
applicable for both Volatile as well as Non volatile
compound.

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Dept. of Chemistry, SUST
 HPLC

 It can mainly divided by two types 1. Normal phase

HPLC 2. Reversed Phase HPLC.
 It is having a high resolution and separation capacity.
 It is used as qualitative as well as quantitative
analysis.
 High performance liquid chromatography (HPLC) is a
chromatographic technique used to separate a mixture of
compounds in analytical chemistry and biochemistry with
the purpose of identifying, quantifying or purifying the
individual components of the mixture.
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Dept. of Chemistry, SUST
 HPLC Technique

▪ Columns are tightly packed, and the eluent is forced through the column
under high pressure(up to 5,000 psi) by a pump.

▪ Allows 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.

▪ Allows a much better separation of the components of the mixture.

▪ Utilizes liquid mobile phase to separate the mixture

▪ Analytes 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.

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Dept. of Chemistry, SUST
Stationary phase and Mobile phase in 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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 Components of HPLC

• Pump
• Injector
• Column
• Detector
• Recorder or data
system

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Dept. of Chemistry, SUST
 HPLC Principle

▪ High Performance Liquid Chromatography [HPLC] is principle is based

on adsorption as well as partition chromatography is depending on
the nature of stationary phase, if stationary phase is solid principle is
based on adsorption chromatography and if stationary phase is liquid
principle is based on partition chromatography.

▪ It is important for determination of volatile and non volatile

compounds.

▪ It is important for determination qualitative and quantitative analysis.

▪ It is important for determination of Retention Time (the time is

required , after sample injection maximum angle peak reaches to
detector)

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Dept. of Chemistry, SUST
 HPLC Advantages

 It is simple, rapid , reproducible.

 High sensitivity.
 High performance.
 Rapid process and hence time saving.
 It is having a high resolution and separation capacity.
 Accuracy and Precision.
 Stationary phase was chemically innert.
 Wide varities of stationary phase.
 Mobile phase was chemically innert.
 Less requirement of mobile phase in developing chamber.
 Early recovery of separated component.
 Easy visualization of separated components.
 It is having Good reproducibility and repeatability.
 It is analytical technique is important for validation of product, quality control
studies of product.
 It is important for qualitative and quantitative analysis.
 It is used for both analytical and preparative purpose.
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Dept. of Chemistry, SUST
 TYPES OF HPLC SEPARATIONS

 Normal Phase: Separation of polar analytes by partitioning onto a polar,

bonded stationary phase.

 Reversed Phase: Separation of non-polar analytes by partitioning onto a non-

polar, bonded stationary phase.

 Adsorption: In Between Normal and Reversed. Separation of moderately polar

analytes using adsorption onto a pure stationary phase (e.g. alumina or silica)

 Ion Chromatography: Separation of organic and inorganic ions by their

partitioning onto ionic stationary phases bonded to a solid support.

 Size Exclusion Chromatography: Separation of large molecules based in the

paths they take through a “maze” of tunnels in the stationary phase.

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MODES OF HPLC

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DETECTORS

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Dept. of Chemistry, SUST
 WHY USE HPLC??
❖ Simultaneous analysis
❖ Good pumping system
❖ Rapid analysis
❖ High resolution
❖ High sensitivity
❖ Good repeatability
❖ Moderate analysis condition
❖ Easy to fractionate and purify
❖ Not destructive
❖ Special continuous flow detectors- can handle small flow
rates and detect very small amounts
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 Disadvantages of HPLC

▪ Costly
▪ Complexity
▪ Low sensitivity for some compounds
▪ Irreversibly adsorbed compounds not
detected
▪ Co-elution difficult to detect

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HPLC System

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Dept. of Chemistry, SUST
HPLC System

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Dept. of Chemistry, SUST
HPLC System

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Dept. of Chemistry, SUST
 SOLVENT/ MOBILE PHASE RESERVOIRS

 Glass or stainless-steel containers capable of holding up

to 1 liter mobile phase (pure organic solvents or aqueous
solutions of salts and buffers)

 Inert to a variety of aqueous and non aqueous mobile

phases.

 Stainless steel should be avoided for use with solvents

containing halide ions.

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Dept. of Chemistry, SUST
DEGASSING & FILTRATION OF MOBILE PHASE
 In many cases, aqueous solvents & some organic solvents are degassed prior to
use
 Degassing is done to prevent formation of gas bubbles in the pump or detector (
Mobile phases are degassed by stirring of the mobile phase under vacuum,
sonication or sparing with helium gas)
 The mobile phase are filtered to remove particulate matter that may clog the
system
Tubing
 Should be inert,
 have ability to withstand pressure
 able to carry sufficient volume

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Dept. of Chemistry, SUST
 HPLC PUMP

 A pump forces the mobile phase through the column at a much

greater velocity than gravity-flow columns.

 The solvents or mobile phase must be passed through a column

at high pressures at up to 6000 psi(lb/in²) or 414 bar.

 As the particle size of stationary phase is smaller (5 to 10μ) the

resistance to the flow of solvent will be high.

 That is, smaller the particle size of the stationary phase the
greater is the resistance to the flow of solvents.

 Hence high pressure is recommended.

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Dept. of Chemistry, SUST
 HPLC PUMP

REQUIREMENTS FOR PUMPS

❖ Generation of pressure of about 6000 psi.

❖ Pulse free output & all materials in the pump should be

chemically resistant to solvents.

❖ Flow rates ranging from 0.1 to 10 mL/min

❖ Pumps should be capable of taking the solvent from a single

reservoir or more than one reservoir containing different solvents
simultaneously.

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HPLC PUMP

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Dept. of Chemistry, SUST
HPLC PUMP

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Dept. of Chemistry, SUST
 HPLC PUMP

RECIPROCATING PUMPS

 This pump transmits alternative pressure to the solvent via a flexible

diaphragm ,which in turn is hydraulically pumped by a reciprocating pump.

 Solvent is pumped back and forth by a motor driven piston

 Two ball check valves which open & close which controls the flow
 The piston is in direct contact with the solvent
 Small internal volume 35-400μL
 High output pressure up to 10,000 psi
 Ready adaptability to gradient elution and constant flow rate

 Disadvantages
Produces a pulsed flow which is damped because pulses appear as baseline noise
on the chromatograph. This can be overcome by use of dual pump heads or
elliptical cams to minimize such pulsations.
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Dept. of Chemistry, SUST
HPLC PUMP

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Dept. of Chemistry, SUST
HPLC PUMP

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Dept. of Chemistry, SUST
HPLC PUMP

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Dept. of Chemistry, SUST
 HPLC PUMP

Advantages

 Have small internal volume of 35-400μL

 Higher output pressures up to 10,000 psi.
 Adaptability to gradient elution.
 Large solvent capacities & constant flow rates.
 Largely independent of column back pressure & solvent viscosity.

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Dept. of Chemistry, SUST
 COLUMN
1. Precolumn
- It contains a packing chemically identical to
that in analytical column.

- Mainly used to remove the impurities from

the solvent and thus prevents contamination
of the analytical column, it can protect
analytical column.

- It is also called as guard column or

protective column.

- it is having large particle size.

- It is having short length of 2 to 10 cm, so does

not affect separation.

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Dept. of Chemistry, SUST
 COLUMN
2. Analytical column

- The success or failure of analysis depends upon choice of column.

- Actual separation is carried out here.

- Stainless –steel tube

- size – length -25 to 100 cm

- Internal diameter – 2 to 4.6 mm

- Column is filled with small particles 5 – 10 micron. The solid support can be
silica gel, alumina.

- The separation is result of different components adhering to or diffusion into

the packing particles when the mobile phase is forced through column.

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Dept. of Chemistry, SUST
 COLUMN
2. Analytical column
- C8 and C18 columns are considered as examples of reversed phase liquid
chromatography (RP).
- The stationary phase here is seen as a thin film of non-polar liquid phase that
has been designed to be chemically similar to an inert material (Silica gel
particles).
- The non-polar layer is chemically linked to the silica particles surface by
reaction with the polar silanol groups on the stationary phase surface and so
rendering them less polar or non-polar.
- The difference between the two columns will be in the length of the carbonchain
attached to the silica surface.
- Accordingly C8 hplc columns have packing material composed of silica
particles attached to C8 carbon units.
- C18 will, of course, have packing materials coated with C18 hydrophobic
units.
- Categorically both are reversed phase but C18 columns will definitely be
more "hydrophobic rather than the C8 columns.

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Dept. of Chemistry, SUST
 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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Dept. of Chemistry, SUST
7.DERIVATISATION IN
HPLC:Inordertoincreasethedetectabilityofvariousclassesofcompounds(forwhichsensitivedetec
torsarenotavailable)derivatisationiscarriedoutinHPLC.Agoodamountofworkhasbeenperforme
donthelabellingofcompoundswithchromophoresandflurophoresfordetectionusingUVspectro
metersandfluorimetersrespectively.There are 2 important types of derivatisation. These are1.
Pre column derivatisation2. Post column derivatisation30
PRE COLUMN DERIVATISATION:
Inprecolumnderivatisationtherearenorestrictionsonthesolvents,reagents,orreactionrateschos
enandexcessofreagentscanberemovedbeforetheinjection.However,artifactformation,ifpresent
,canbecheckedbypositiveidentificationoftheelutedpeaks.Forexample,inthederivatisationofatri
ketonewithmorethanonefunctionalgroupcapableofbeingderivatisedthereisapossibilityofrange
ofderivativesbeingformedfromonesolute.Itisclearlynecessarytocheckthatthederivatisationreac
tionsarequantitativeorthesamplederivatisationsproceedinamanneranaloguestothederivatisati
onofstandards.
ExamplesofprecolumnderivatisationtoformUVchromophoresincludethetreatmentofketosteroi
dswith2,4,DNPandthebenzoylationofhydroxysteroidsortheesterificationoffattyacids.Similarly,fl
uorophoreshavebeenintroducedintoaminoacids,biogenicamines,andalkaloidsbytreatmentwit
hdansylchloride.
POST COLUMN DERIVATISATION:
Itiscarriedoutontheseparatedsolutesastheyemergefromthechromatographiccolumn.InHPLC,th
isplacesseriousrestrictiononthederivatisationreactions,becausedilutionoftheeluentpeakmustb
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Dept. of Chemistry, SUST
eminimized.Consequently,veryfastreactionsmustbeusedandthereagentsandmobilephasemust
ExamplesofpostcolumnderivatisationreactionsforusewithUVdetectorsinclude:
A.Reactionofaminoacidswithninhydrinandfluorescamine.
B.Reactionoffattyacidwithorthonitrophenol.
C.Reactionofketoneswith2,4,DNP.
D.Thermaloracidtreatmentofcarbohydrates.Anoxidationdetectorforthefluorimetricanalysis
ofcarbohydratesinbodyfluidsusingCe(III)flourescencehasalsobeenreported.

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Dept. of Chemistry, SUST