SlideShare a Scribd company logo
1 of 70
Download to read offline
By
Amol D Sagulale
By
Amol D Sagulale
Sr. Reseach Associae-II
Macleod Pharmaceuticals, Mumbai
Email: amol.sagulale@gmail.com
Normal phase chromatography
•It was one of the first kind of HPLC that
chemist developed .
•Also known as adsorption chromatography.
•This method uses a polar stationary phase and a
non-polar, non-aqueous mobile phase, and works
effectively for separating analytes readily soluble in
non-polar solvents.
Si
Si
Si
O
H
O
H
O
H
O
H
O
H
O
H surface of silica gel
Packing material
•The most popular packing material is silica gel.
•It is believed that silanol radicals ( -Si-OH ) on the
surface of silica gel act as the active site and the
sample is separated.
Normal Phase Chromatography : Separation mode
Stationary Phase in NPC
•Bare silica or alumina that have polar hydroxyl group
on the surface
--•silica is preferred over alumina due to its low cost,
known performance and ready availability
-
•For very basic compounds (e.g., amines) alumina is a
better choice because amines are retained longer on
silica
-A variety of bonded phases (BP) can be prepared
for NPC. However, functional groups in BP are less
polar than the bare silica column
Reversed phase HPLCReversed phase HPLC
• In the 1970s most liquid chromatography was
done on non-modified silica or alumina with a
hydrophilic surface chemistry and a stronger
affinity for polar compounds - hence it was
considered "normal".
• The introduction of alkyl chains bonded covalently
to the support surface reversed the elution order.
Now polar compounds are eluted first while non-
polar compounds are retained - hence "reversed
phase".
Reverse Phase ChromatographyReverse Phase Chromatography
• The term “Reverse Phase Chromatography”
was used because RP is a form of partition
chromatography where chemically bonded
phase is hydrophobic or non-polar (e.g.
octadecyl group), and the starting mobile
phase (e.g. water) must be more polar than
the stationary phase.
• It is the most widely used technique in HPLC.
• .
Reversed Phase ChromatographyReversed Phase Chromatography
It operates on the principle of hydrophobic
interactions which result from repulsive forces
between a relatively polar solvent, the relatively non-
polar analyte, and the non-polar stationary phase.
The components of the analyte mixture pass over
stationary-phase particles bearing pores large enough
for them to enter, where interactions with the
hydrophobic surface removes them from the flowing
mobile-phase stream.
Reversed Phase Chromatography :Separation mode
CH3 CH2COOCH3
CH3 CH2COOCH3
Silica-C18 (ODS)
Hydrophobic InteractionHydrophobic Interaction
Reverse Phase MechanismReverse Phase Mechanism
• Reversed phase chromatography is an
adsorptive process by experimental
design,which relies on a partitioning
mechanism to effect separation.
• The solute molecules partition (i.e. an
equilibrium is established) between the mobile
phase and the stationary phase.
• The distribution of the solute between the two
phases depends on the binding properties of
the medium, the hydrophobicity of the solute
and the composition of the mobile phase.
Reverse Phase ChromatographyReverse Phase Chromatography
• One common stationary phase is a silica which
has been treated with RMe2SiCl, where R is a
straight chain alkyl group such as C18H37 or
C8H17.
• With these stationary phases, retention time is
longer for molecules which are more non-polar,
while polar molecules elute more readily.
Reverse Phase ChromatographyReverse Phase Chromatography
• strong attraction between the polar solvent
and polar molecules in the mixture being
passed through the column.
• much attraction between the hydrocarbon
chains attached to the silica (the stationary
phase) and the polar molecules in the
solution.
• Polar molecules in the mixture will therefore
spend most of their time moving with the
solvent.
Reverse Phase ChromatographyReverse Phase Chromatography
• Non-polar compounds in the mixture will tend to
form attractions with the hydrocarbon groups
because of van der Waals dispersion forces.
• They will also be less soluble in the solvent
because of the need to break hydrogen bonds
as they squeeze in between the water or
methanol molecules.
• They therefore spend less time in solution in
the solvent and this will slow them down on
their way through the column.
Reverse Phase ChromatographyReverse Phase Chromatography
• The retention time can be increased by adding
more water to the mobile phase; thereby
making the affinity of the hydrophobic analyte
for the hydrophobic stationary phase stronger
relative to the now more hydrophilic mobile
phase.
• Similarly, the retention time can be decrease
by adding more organic solvent to the eluent.
Si
Si
O - Si - CH2(CH2)16CH
CH3
CH3
O - Si - CH2(CH2)16CH
CH3
CH3
O - Si - CH2(CH2)16CH3
CH3
CH3
CH3
CH3
O - Si -
CH3
Commonly used packing
materials are hydrocarbons
having 18 carbon atoms
(called the Octadecyl radical)
which are chemically bonded
to silica gel (Silica-
ODS).Since the surface of the
Silica-ODS is covered with
hydrocarbon, the polarity of
the packing material itself is
very low.
Reversed Phase Chromatography :Separation mode
Principle of reverse phase chromatographyPrinciple of reverse phase chromatography
Gradient elution
• To equilibrate the column packed with
reverse phase medium under suitable initial
mobile phase conditions of –
• pH
• Ionic strength
• Polarity ( mobile phase hydrophobicity)
• The polarity of the mobile phase is
controlled by adding organic modifiers or ion
–pairing agents.
• Polarity of initial mobile phase ( usually
reffered to as mobile phase A ) must be low
enough to dissolve the partially hydrophobic
solute
• Yet high enough to ensure binding of the
solute to the reverse phase chromatographic
matrix.
• Sample containing the solutes to be
separated is applied .
• The sample is applied to the column at a flow
rate where optimum binding will occur.
• Chromatographic bed is washed further with
mobile phase .
• Bound solutes are next desorbed from the
reverse phase medium by adjusting the
polarity of mobile phase so that the bound
molecule will sequentially desorbs and elute
from column.
• Removing the substances not previously
desorbed.
• Re-Equilibration of the chromatographic
medium from 100% mobile phase B back to
the initial mobile phase conditions.
Ion-pairing agents
• Ion-pairing agents are ionic compounds that
contain a hydrocarbon chain that imparts a
certain hydrophobicity so that the ion pair can
be retained on a reversed-phase column.
• Ion Pairing agents are added at concentrations
of 0.05 to 0.2.
• All ion-pairing agents are potentially capable of
ion-pairing with the positively charged basic
residues of peptides or proteins, thus reducing
hydrophilicity and increasing their retention time
• Hydrophobic counterions such as TFA and
HFBA in addition to ion-pairing with the
positively charged solute also increase the
affinity of the solute (peptide or protein) for the
hydrophobic stationary phase.
• While hydrophilic counterions such as following
ion-pair formation with positive charged
residues would be unlikely to interact with the
stationary phase.
Trifluoroacetic acid (TFA).
Heptafluorobutyric acid (HFBA).
Hexafluoroacetone (HFA).
Formic Acid (FA)
Phosphoric Acid.
Hydrochloric Acid.
Triethylamine Phosphate (TEAP).
Organic modifiers
• Additive that changes the character of the
mobile phase. In RP chromatography, water is
the weak solvent, and acetonitrile, the strong
solvent is added gradually to generate a
gradient.
• Acetonitrile.
• Isopropanol.
• Methanol.
• Ethanol
• Acetonitrile is the reverse phase solvent of
choice because the UV cut off for acetonitrile
is190 nm, allowing detection at lower
wavelengths.
• It is less viscous than methanol, thus causing
less fluctuations in pressure.
• Less bubble formation occurs when it is mixed
with water. It has also better selectivity for
peptides and proteins.
• Isopropanol is used either alone or in
combination with acetonitrile to elute large or
hydrophobic proteins.
Quality of Stationary phasesQuality of Stationary phases
Determined by their physical and chemical properties
Physical Properties :
Porosity
Specific surface area
Particle size
Particle shape
Pore size
Greatly determines the efficiency of
packing.
Quality of Stationary phasesQuality of Stationary phases
• Must be controlled in narrow tolerances to
enable manufacturer for reproducible packing
materials.
• Porosity : Determines the surface area &
others parameters.
• Retention
• Selectivity
Chemical propertiesChemical properties
• Result of substrare properties
&
• Applied surface bonding chemistry
• These forms the basis of retention and
selectivity.
SubtratesSubtrates
• Inorganic oxides
• Polymers
• Carbons
• Have sufficient hydrophobic properties & used
unmodified as RP stationary phases.
• Majority of presently available RPLC stationary
phases are modified substrates.
SubtratesSubtrates
• Substrates & Stationary phases must posses
physical and chemical properties to be suitable
as Stationary phase.
• Mechanical strength
• No Shrinking & swelling properties
SilicaSilica
• Silica & silica base are the ideal materials.
• Synthesized in pure form & yield a large number
of substrates.
• Well defined physical properties.
• Possess sufficient mechanical strength
• No shrinking & swelling properties.
SilicaSilica
Bonding chemistry of silica results into high
quality of RPLC-phase.
It covers a broad spectrum of different organic
ligands attached to a variety of silicas & enables
the separation of many different substances.
Eg. Neutral molecules in lower mol. Wt range.
Charge molecules by ion-pair
Silica based stationary phasesSilica based stationary phases
• Hydrogel from inorgnic silicates &
alkoxy silicates
Grinding and sieving yields irregular shape
silica substrate is obtained ( characterized as
Xerogel)
Relatively high surface area
High Porosity
Variable wall thickness
SilGel
Silica based stationary phasesSilica based stationary phases
• Consolidation of silica particles by either
• oil emulsion or Coacervation
• Results in sphere shape particles
• Lower surface areas
• Lower porosities
• Regular shape having thicker wall
• SolGel
Silica surfacesSilica surfaces
Silanol groups Siloxane bridges
Acidic reactive sites Hydrophobic unreactive
a)Single (geminal silanol) b)vicinal silanols
Silanediols
Most reactive sites
•Responsible for residual silanol activity of bonded
silicas for basic compounds.
•Silanols cause peak tailing and excessive retention
Types of silanol on surface of silica gelTypes of silanol on surface of silica gel
Pretreatment stepsPretreatment steps
• Heating
• Rehydroxylation
• Homogenization
• In order to reduce the single silanols
• To obtain as many as bonded or
associated silanols of similar avctivity.
Analytical PurposeAnalytical Purpose
• Silica usually produced of nominal 2,3,5 and 10 um
particle size
• Surface area – 100-600 m2/gm
• Particle porosity =0.6-0.7
• Surface density 8 umol/m2
• Equivalent to = ± 4.5 silanols/nm2
• For large molecules = 30 -100nm
• For unrestricted access to inner surface for smaller
molecules, the pore size is not less than 10 nm.
• Silica substrate uses alkoxysilane or chlorosilanes
to attach organic ligands through siloxysilane
linkages to the supports surface.
• To produce such covalently bonded organic
stationary phases, the reactive alkoxy or
chlorosilane reagent must contain atleast one
leaving group which is able to reacts with silanol
at substrate surface.
Reverse phsase chromatography 1
Surface hydrophobisation reactionSurface hydrophobisation reaction
• It is carried out under anhydrous condition.
• The is catalysed by base 2,6-lutidine or imidazole.
• It act as a scavenger base to neutralise acid
byproducts.
• Further step includes reflux, sonication, filtration,
rinsing and drying steps.
• Depending on the no. of leaving groups for
synthesis of RPLC phase, three groups of
organosilane reagents can be distinguished :-
• From the originally available no. of silanol groups,
at a silica substrate, approximately only 50% can
react.
• Due to the steric hindrances between ligands and
side chains.
• Silanol concentration = 8umol/m2
• Ligand concentration = 4umol/m2
• The unreacted silanol concentration is equal to
ligand concetration.
• Residual silanols may strongly influence
• Retention
• Selectivity
• For ionic and polar compounds.
• Depending on activity and actual eluent pH
silanols may influnce the chromatographic process
by
• Hydrogen bonding
• Ion exchange
• Dipole interaction
May cause severe peak tailing and leads to
irreproducible retention times.
• In order to suppress this residual silanol activity
after bonding, secondary synthesis step to end cap
or mask these group is performed.
• The endcapping is done by smallest possible silane
• EX-trimethyl (Most sensitive to hydrolyzation)
• Most common S.P in RPC are those in which a
functional group is attached to a silica support
Synthesis of ODS (octadecylsilane, C18H37Si) the
reagent used is octadecyl-chlorosilane
(C18H37Si(CH3)2Cl)
--.
--
Monofunctional S.P is prepared using the above
procedure because the reagent C18H37Si(CH3)2Cl)
used has one chloro group (only 8-12% carbon
Loading)
-
-For steric reasons it is not possible for all silanol
groups (-SiOH) groups on silica surface to react with
functional group (only ~45% are bonded)
Reverse phsase chromatography 1
Endcapping is done to
cover more silanol
groups with di- and tr-
chlorosilane reagent
Result in S.P which is more dense and
and have 15-20% carbon loading
• Chemically bonding the hydroxy groups would
generate a rugged s.p. and most importantly the
thinnest possible (monolayer) coating.
• Bonded carbon chains forms the primary s.p.
material C4 – C18 (RP).
• Derivatization of the terminal carbons allows us to
‘tailor’ s.p.’s with different polarities.
• Such derivatized s.p. are used in the reverse phase
• Isopropyl instead of methyl group or modification
by alkyl ligands carrying a polar function near the
silane group
– Octadecyl
– Octyl
– Hexyl
– Cyclohexyl
– Phenyl
– alkyl phenyl
• The most popular column is a octadecyl carbon
chain (C18) bonded silica (USP classification L1)
with 297 columns commercially available C8
bonded silica (L7 - 166 columns),
• pure silica (L3 - 88 columns),
• cyano bonded silica (L10 - 73 columns)
• phenyl bonded silica (L11 - 72 columns).
• Note that C18, C8 and phenyl are dedicated
reversed phase packings ..
• Cyano columns can be used in a reversed phase
mode depending on analyte and mobile phase
conditions.
• It should be noted at this point that not all C18
columns have identical retention properties.
RPLC phases inorganic oxidesRPLC phases inorganic oxides
• Alumina
• Titania
• Zirconia
• Higher stability 0 to13
• Interact with anlytes with ligands exchange
interaction
• These are strong secondary interaction and
usally unwanted
• The high activity of the surfaces of these oxides
• Lack of straight forward synthesis procedure
• Surface modification is done by deposition of
polymers layer on substrate.
Polymer based RPLC –SPPolymer based RPLC –SP
• Styrene divenyl benzene 0 to 14
• Methacrylate or
• Polyvinyl alcohol based phase 2 to 12
• Hydrolytical stability over wide pH range
• Have found appication in aqueous size
exclusion and ion exchange
chromatography.
Carbon RPLC-SPCarbon RPLC-SP
• High chemical stability over wide pH range
• 0 to 14
• Show ultimate hydrophobic properties.
• Sufficient hardness
• Well define pore structure
• Do not suffer from swelling and shrinking
• Porous graphitized carbon
• Black carbon
Applications of Reversed Phase Chromatography (RPC)
-RPC is the most widely used separation mode in
HPLC
-Cover ~ 75 % of HPLC separations.
Applicable to most non-polar analytes & to many
ionizable & ionic compounds.
-Best suitable for the separation of neutral solutes
that are soluble in water or relatively polar
solvents and with molecular weights less than
2000-3000
--
The following table lists a few examples of the
multitude of uses of RPC in various fields
Samples
• A) Regular Sample
a) ionic
ex. Acids,bases, organic salts
b) neutral
• B) Special Sample
very hydrophilic
or
hydrophobic compounds
Eg:- achiral isomers, chiral isomers,
enantiomers, biomolecules, inorganic ions,
synthetic polymers
Retention and selectivity in RPLC-SPRetention and selectivity in RPLC-SP
• Classical measures of retention
– capacity factors
– partition coefficients
– Van’t Hoff Plots
• Give bulk properties only - do not give
molecular view of separation process
Solvophobic TheorySolvophobic Theory
• Considers retention and selectivity mainly as
function of-
• Surface tension
• Dipole-dipole interaction
• The interaction is between polar groups of a
compound and mobile phase.
• Solvent cavities are created by the hydrophobic part
of compound.
• The assumption is the principal shortcoming.
• RPLC-phase is considered as passive part of
system.
• In many studies , it is shown that specially for
non-polar and ionic substances, this is
unrealistic.
Partitioning TheoryPartitioning Theory
• It is supported by the good correlation by
octanol-1/water partition coefficient.
• RPLC retention data not found for very polar
compounds.
• This theory insufficiently explains shape
selectivity.
Combining solvophobic and Partitioning theoryCombining solvophobic and Partitioning theory
• Solvent–stationary interphase layer is
formed.
• Depending upon the composition of eluent
and nature of the stationary phase,
enrichment by the organic modifier in that
phase takes place.
• Partition of solutes between interphase and
mobile phase is assumed to take place by
displacement of solvent molecules.
• Together with column efficiency retention,
selectivity determines the finally achievable
chromatographic peak resolution.
Reverse phsase chromatography 1
None of these theories can completely
explain all of the observed retention in
reversed phase HPLC.
Thank You….

More Related Content

What's hot (20)

Partition chromatographyfinal
Partition chromatographyfinalPartition chromatographyfinal
Partition chromatographyfinal
 
Adsorption chromatography
Adsorption chromatographyAdsorption chromatography
Adsorption chromatography
 
HPLC Principle,Instrumentation and Application
HPLC Principle,Instrumentation and ApplicationHPLC Principle,Instrumentation and Application
HPLC Principle,Instrumentation and Application
 
Ion exchange chromatography
Ion exchange chromatography Ion exchange chromatography
Ion exchange chromatography
 
High Performance Liquid chromatography (HPLC)
High Performance Liquid chromatography (HPLC)High Performance Liquid chromatography (HPLC)
High Performance Liquid chromatography (HPLC)
 
Size exclusion chromatography
Size exclusion chromatography Size exclusion chromatography
Size exclusion chromatography
 
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
 
Size exclusion chromatography
Size exclusion chromatographySize exclusion chromatography
Size exclusion chromatography
 
HPLC
HPLCHPLC
HPLC
 
Reverse phase chromatography
Reverse phase chromatographyReverse phase chromatography
Reverse phase chromatography
 
Gas chromatography
Gas chromatographyGas chromatography
Gas chromatography
 
Paper Chromatography PPT (new)
Paper Chromatography PPT (new)Paper Chromatography PPT (new)
Paper Chromatography PPT (new)
 
Hplc
HplcHplc
Hplc
 
an assignment on ion exchange chromatography
an assignment on ion exchange chromatographyan assignment on ion exchange chromatography
an assignment on ion exchange chromatography
 
HPTLC
HPTLCHPTLC
HPTLC
 
HPLC
HPLCHPLC
HPLC
 
Chromatography & Column chromatography
Chromatography & Column chromatographyChromatography & Column chromatography
Chromatography & Column chromatography
 
chromatographic techniques
chromatographic techniqueschromatographic techniques
chromatographic techniques
 
Gas chromatography
Gas chromatographyGas chromatography
Gas chromatography
 
AFFINITY CHROMATOGRAPHY
AFFINITY CHROMATOGRAPHYAFFINITY CHROMATOGRAPHY
AFFINITY CHROMATOGRAPHY
 

Viewers also liked

HPLC - High Performance Liquid Chromatography
HPLC - High Performance Liquid ChromatographyHPLC - High Performance Liquid Chromatography
HPLC - High Performance Liquid ChromatographyDivya Basuti
 
Introduction to RP-HPLC
Introduction to RP-HPLCIntroduction to RP-HPLC
Introduction to RP-HPLCKarthi Kumar
 
UPLC INTRODUCTION AND REVIEW
UPLC INTRODUCTION AND REVIEWUPLC INTRODUCTION AND REVIEW
UPLC INTRODUCTION AND REVIEWAnnette Loubriel
 
Chromatofocusing
ChromatofocusingChromatofocusing
ChromatofocusingAmlan Barai
 
The Benefits of UPLC Technology in the Analytical Laboratory
The Benefits of UPLC Technology in the Analytical LaboratoryThe Benefits of UPLC Technology in the Analytical Laboratory
The Benefits of UPLC Technology in the Analytical LaboratoryJNAlexanderIV
 
Reverse phsase chromatography 2
Reverse phsase chromatography 2Reverse phsase chromatography 2
Reverse phsase chromatography 2Amol Sagulale
 
History of chromatography
History of chromatographyHistory of chromatography
History of chromatographyHossein Hodjat
 
Fplc(fast protein liquid chromatography )
Fplc(fast protein liquid chromatography )Fplc(fast protein liquid chromatography )
Fplc(fast protein liquid chromatography )Safina Kouser
 
Cytochemical staining checked
Cytochemical staining checkedCytochemical staining checked
Cytochemical staining checkedBALRAM KRISHAN
 
Ultracentrifugation
Ultracentrifugation Ultracentrifugation
Ultracentrifugation Aswathi K S
 
Scanning tunneling microscope (STM)
 Scanning tunneling microscope (STM) Scanning tunneling microscope (STM)
Scanning tunneling microscope (STM)Balsam Ata
 
Enzymes and its applications
Enzymes and its applicationsEnzymes and its applications
Enzymes and its applicationseswar1810
 
Ion Exchange Chromatography Lecture
Ion Exchange Chromatography LectureIon Exchange Chromatography Lecture
Ion Exchange Chromatography Lectureouopened
 
Hplc presentation final
Hplc presentation    finalHplc presentation    final
Hplc presentation finalOvesh Gaikwad
 
Gel Filtration Chromatography Lecture
Gel Filtration Chromatography LectureGel Filtration Chromatography Lecture
Gel Filtration Chromatography Lectureouopened
 

Viewers also liked (20)

reversed phase chromatography
reversed phase chromatography reversed phase chromatography
reversed phase chromatography
 
HPLC - High Performance Liquid Chromatography
HPLC - High Performance Liquid ChromatographyHPLC - High Performance Liquid Chromatography
HPLC - High Performance Liquid Chromatography
 
Introduction to RP-HPLC
Introduction to RP-HPLCIntroduction to RP-HPLC
Introduction to RP-HPLC
 
UPLC INTRODUCTION AND REVIEW
UPLC INTRODUCTION AND REVIEWUPLC INTRODUCTION AND REVIEW
UPLC INTRODUCTION AND REVIEW
 
Chromatofocusing
ChromatofocusingChromatofocusing
Chromatofocusing
 
The Benefits of UPLC Technology in the Analytical Laboratory
The Benefits of UPLC Technology in the Analytical LaboratoryThe Benefits of UPLC Technology in the Analytical Laboratory
The Benefits of UPLC Technology in the Analytical Laboratory
 
Reverse phsase chromatography 2
Reverse phsase chromatography 2Reverse phsase chromatography 2
Reverse phsase chromatography 2
 
UPLC
UPLCUPLC
UPLC
 
pharmaceutical enzymes
pharmaceutical enzymespharmaceutical enzymes
pharmaceutical enzymes
 
History of chromatography
History of chromatographyHistory of chromatography
History of chromatography
 
Fplc(fast protein liquid chromatography )
Fplc(fast protein liquid chromatography )Fplc(fast protein liquid chromatography )
Fplc(fast protein liquid chromatography )
 
Cytochemical staining checked
Cytochemical staining checkedCytochemical staining checked
Cytochemical staining checked
 
Ultracentrifugation
Ultracentrifugation Ultracentrifugation
Ultracentrifugation
 
Chromatographic and High Performance Liquid Chromatography (HPLC)
Chromatographic and High Performance Liquid Chromatography (HPLC)Chromatographic and High Performance Liquid Chromatography (HPLC)
Chromatographic and High Performance Liquid Chromatography (HPLC)
 
Scanning tunneling microscope (STM)
 Scanning tunneling microscope (STM) Scanning tunneling microscope (STM)
Scanning tunneling microscope (STM)
 
Gel chromatography
Gel chromatography Gel chromatography
Gel chromatography
 
Enzymes and its applications
Enzymes and its applicationsEnzymes and its applications
Enzymes and its applications
 
Ion Exchange Chromatography Lecture
Ion Exchange Chromatography LectureIon Exchange Chromatography Lecture
Ion Exchange Chromatography Lecture
 
Hplc presentation final
Hplc presentation    finalHplc presentation    final
Hplc presentation final
 
Gel Filtration Chromatography Lecture
Gel Filtration Chromatography LectureGel Filtration Chromatography Lecture
Gel Filtration Chromatography Lecture
 

Similar to Reverse phsase chromatography 1

Stationary phases, tubings used in HPLC
Stationary phases, tubings used in HPLCStationary phases, tubings used in HPLC
Stationary phases, tubings used in HPLCVrushali Tambe
 
Introduction to Chromatography (Column chromatography)
Introduction to Chromatography (Column chromatography)Introduction to Chromatography (Column chromatography)
Introduction to Chromatography (Column chromatography)Ahmed Metwaly
 
HPLC Lecture1a.pdf
HPLC Lecture1a.pdfHPLC Lecture1a.pdf
HPLC Lecture1a.pdfMihirOza11
 
Partition chromatography & partition paper chromatography
Partition chromatography & partition paper chromatographyPartition chromatography & partition paper chromatography
Partition chromatography & partition paper chromatographyArtina Aquitania
 
HPLC in Pathology
HPLC in PathologyHPLC in Pathology
HPLC in PathologyAseem Jain
 
Chromatography, types by different approaches, HPLC
Chromatography, types by  different approaches, HPLC Chromatography, types by  different approaches, HPLC
Chromatography, types by different approaches, HPLC Muhammad Asif Shaheeen
 
Chromatographic techniques new ppt - dr. r. mallika
Chromatographic techniques   new ppt - dr. r. mallikaChromatographic techniques   new ppt - dr. r. mallika
Chromatographic techniques new ppt - dr. r. mallikamallikaswathi
 
Ion pair chromatography for pharmacy students
Ion pair chromatography for pharmacy studentsIon pair chromatography for pharmacy students
Ion pair chromatography for pharmacy studentsabhishek rai
 
Mode of Interactions.pptx
Mode of Interactions.pptxMode of Interactions.pptx
Mode of Interactions.pptxSuyogpatil86
 
instrumental cha 4
instrumental cha 4instrumental cha 4
instrumental cha 4Ibseusso
 
instrumental cha 4.pdf
instrumental cha 4.pdfinstrumental cha 4.pdf
instrumental cha 4.pdfIbsa15
 

Similar to Reverse phsase chromatography 1 (20)

Stationary phases, tubings used in HPLC
Stationary phases, tubings used in HPLCStationary phases, tubings used in HPLC
Stationary phases, tubings used in HPLC
 
Introduction to hplc
Introduction to hplcIntroduction to hplc
Introduction to hplc
 
Hplc
HplcHplc
Hplc
 
HPLC.pptx
HPLC.pptxHPLC.pptx
HPLC.pptx
 
Introduction to Chromatography (Column chromatography)
Introduction to Chromatography (Column chromatography)Introduction to Chromatography (Column chromatography)
Introduction to Chromatography (Column chromatography)
 
Chromatograph yfinal
Chromatograph yfinalChromatograph yfinal
Chromatograph yfinal
 
HPL CHROMATOGRAPHY
HPL  CHROMATOGRAPHYHPL  CHROMATOGRAPHY
HPL CHROMATOGRAPHY
 
HPLC Lecture1a.pdf
HPLC Lecture1a.pdfHPLC Lecture1a.pdf
HPLC Lecture1a.pdf
 
HPLC
HPLCHPLC
HPLC
 
Protein purification techniques
Protein purification techniquesProtein purification techniques
Protein purification techniques
 
Partition chromatography & partition paper chromatography
Partition chromatography & partition paper chromatographyPartition chromatography & partition paper chromatography
Partition chromatography & partition paper chromatography
 
HPLC in Pathology
HPLC in PathologyHPLC in Pathology
HPLC in Pathology
 
Chromatography, types by different approaches, HPLC
Chromatography, types by  different approaches, HPLC Chromatography, types by  different approaches, HPLC
Chromatography, types by different approaches, HPLC
 
Chromatographic techniques new ppt - dr. r. mallika
Chromatographic techniques   new ppt - dr. r. mallikaChromatographic techniques   new ppt - dr. r. mallika
Chromatographic techniques new ppt - dr. r. mallika
 
Ion pair chromatography for pharmacy students
Ion pair chromatography for pharmacy studentsIon pair chromatography for pharmacy students
Ion pair chromatography for pharmacy students
 
Mode of Interactions.pptx
Mode of Interactions.pptxMode of Interactions.pptx
Mode of Interactions.pptx
 
Ion exchange
Ion exchangeIon exchange
Ion exchange
 
instrumental cha 4
instrumental cha 4instrumental cha 4
instrumental cha 4
 
instrumental cha 4.pdf
instrumental cha 4.pdfinstrumental cha 4.pdf
instrumental cha 4.pdf
 
Liquid chromatography
Liquid chromatographyLiquid chromatography
Liquid chromatography
 

Recently uploaded

Different drug regularity bodies in different countries.
Different drug regularity bodies in different countries.Different drug regularity bodies in different countries.
Different drug regularity bodies in different countries.kishan singh tomar
 
ORAL HYPOGLYCAEMIC AGENTS - PART 2.pptx
ORAL HYPOGLYCAEMIC AGENTS  - PART 2.pptxORAL HYPOGLYCAEMIC AGENTS  - PART 2.pptx
ORAL HYPOGLYCAEMIC AGENTS - PART 2.pptxNIKITA BHUTE
 
blood bank management system project report
blood bank management system project reportblood bank management system project report
blood bank management system project reportNARMADAPETROLEUMGAS
 
High-Performance Thin-Layer Chromatography (HPTLC)
High-Performance Thin-Layer Chromatography (HPTLC)High-Performance Thin-Layer Chromatography (HPTLC)
High-Performance Thin-Layer Chromatography (HPTLC)kishan singh tomar
 
AORTIC DISSECTION and management of aortic dissection
AORTIC DISSECTION and management of aortic dissectionAORTIC DISSECTION and management of aortic dissection
AORTIC DISSECTION and management of aortic dissectiondrhanifmohdali
 
pA2 value, Schild plot and pD2 values- applications in pharmacology
pA2 value, Schild plot and pD2 values- applications in pharmacologypA2 value, Schild plot and pD2 values- applications in pharmacology
pA2 value, Schild plot and pD2 values- applications in pharmacologyDeepakDaniel9
 
FDMA FLAP - The first dorsal metacarpal artery (FDMA) flap is used mainly for...
FDMA FLAP - The first dorsal metacarpal artery (FDMA) flap is used mainly for...FDMA FLAP - The first dorsal metacarpal artery (FDMA) flap is used mainly for...
FDMA FLAP - The first dorsal metacarpal artery (FDMA) flap is used mainly for...Shubhanshu Gaurav
 
Trustworthiness of AI based predictions Aachen 2024
Trustworthiness of AI based predictions Aachen 2024Trustworthiness of AI based predictions Aachen 2024
Trustworthiness of AI based predictions Aachen 2024EwoutSteyerberg1
 
SGK RỐI LOẠN TOAN KIỀM ĐHYHN RẤT HAY VÀ ĐẶC SẮC.pdf
SGK RỐI LOẠN TOAN KIỀM ĐHYHN RẤT HAY VÀ ĐẶC SẮC.pdfSGK RỐI LOẠN TOAN KIỀM ĐHYHN RẤT HAY VÀ ĐẶC SẮC.pdf
SGK RỐI LOẠN TOAN KIỀM ĐHYHN RẤT HAY VÀ ĐẶC SẮC.pdfHongBiThi1
 
EXERCISE PERFORMANCE.pptx, Lung function
EXERCISE PERFORMANCE.pptx, Lung functionEXERCISE PERFORMANCE.pptx, Lung function
EXERCISE PERFORMANCE.pptx, Lung functionkrishnareddy157915
 
Clinical Research Informatics Year-in-Review 2024
Clinical Research Informatics Year-in-Review 2024Clinical Research Informatics Year-in-Review 2024
Clinical Research Informatics Year-in-Review 2024Peter Embi
 
Physiology of Smooth Muscles -Mechanics of contraction and relaxation
Physiology of Smooth Muscles -Mechanics of contraction and relaxationPhysiology of Smooth Muscles -Mechanics of contraction and relaxation
Physiology of Smooth Muscles -Mechanics of contraction and relaxationMedicoseAcademics
 
How to cure cirrhosis and chronic hepatitis naturally
How to cure cirrhosis and chronic hepatitis naturallyHow to cure cirrhosis and chronic hepatitis naturally
How to cure cirrhosis and chronic hepatitis naturallyZurück zum Ursprung
 
Neurological history taking (2024) .
Neurological  history  taking  (2024)  .Neurological  history  taking  (2024)  .
Neurological history taking (2024) .Mohamed Rizk Khodair
 
Pharmacokinetic Models by Dr. Ram D. Bawankar.ppt
Pharmacokinetic Models by Dr. Ram D.  Bawankar.pptPharmacokinetic Models by Dr. Ram D.  Bawankar.ppt
Pharmacokinetic Models by Dr. Ram D. Bawankar.pptRamDBawankar1
 
SGK NGẠT NƯỚC ĐHYHN RẤT LÀ HAY NHA .pdf
SGK NGẠT NƯỚC ĐHYHN RẤT LÀ HAY NHA    .pdfSGK NGẠT NƯỚC ĐHYHN RẤT LÀ HAY NHA    .pdf
SGK NGẠT NƯỚC ĐHYHN RẤT LÀ HAY NHA .pdfHongBiThi1
 
DNA nucleotides Blast in NCBI and Phylogeny using MEGA Xi.pptx
DNA nucleotides Blast in NCBI and Phylogeny using MEGA Xi.pptxDNA nucleotides Blast in NCBI and Phylogeny using MEGA Xi.pptx
DNA nucleotides Blast in NCBI and Phylogeny using MEGA Xi.pptxMAsifAhmad
 
BENIGN BREAST DISEASE
BENIGN BREAST DISEASE BENIGN BREAST DISEASE
BENIGN BREAST DISEASE Mamatha Lakka
 

Recently uploaded (20)

Different drug regularity bodies in different countries.
Different drug regularity bodies in different countries.Different drug regularity bodies in different countries.
Different drug regularity bodies in different countries.
 
ORAL HYPOGLYCAEMIC AGENTS - PART 2.pptx
ORAL HYPOGLYCAEMIC AGENTS  - PART 2.pptxORAL HYPOGLYCAEMIC AGENTS  - PART 2.pptx
ORAL HYPOGLYCAEMIC AGENTS - PART 2.pptx
 
blood bank management system project report
blood bank management system project reportblood bank management system project report
blood bank management system project report
 
High-Performance Thin-Layer Chromatography (HPTLC)
High-Performance Thin-Layer Chromatography (HPTLC)High-Performance Thin-Layer Chromatography (HPTLC)
High-Performance Thin-Layer Chromatography (HPTLC)
 
AORTIC DISSECTION and management of aortic dissection
AORTIC DISSECTION and management of aortic dissectionAORTIC DISSECTION and management of aortic dissection
AORTIC DISSECTION and management of aortic dissection
 
pA2 value, Schild plot and pD2 values- applications in pharmacology
pA2 value, Schild plot and pD2 values- applications in pharmacologypA2 value, Schild plot and pD2 values- applications in pharmacology
pA2 value, Schild plot and pD2 values- applications in pharmacology
 
FDMA FLAP - The first dorsal metacarpal artery (FDMA) flap is used mainly for...
FDMA FLAP - The first dorsal metacarpal artery (FDMA) flap is used mainly for...FDMA FLAP - The first dorsal metacarpal artery (FDMA) flap is used mainly for...
FDMA FLAP - The first dorsal metacarpal artery (FDMA) flap is used mainly for...
 
Rheumatoid arthritis Part 1, case based approach with application of the late...
Rheumatoid arthritis Part 1, case based approach with application of the late...Rheumatoid arthritis Part 1, case based approach with application of the late...
Rheumatoid arthritis Part 1, case based approach with application of the late...
 
Trustworthiness of AI based predictions Aachen 2024
Trustworthiness of AI based predictions Aachen 2024Trustworthiness of AI based predictions Aachen 2024
Trustworthiness of AI based predictions Aachen 2024
 
American College of physicians ACP high value care recommendations in rheumat...
American College of physicians ACP high value care recommendations in rheumat...American College of physicians ACP high value care recommendations in rheumat...
American College of physicians ACP high value care recommendations in rheumat...
 
SGK RỐI LOẠN TOAN KIỀM ĐHYHN RẤT HAY VÀ ĐẶC SẮC.pdf
SGK RỐI LOẠN TOAN KIỀM ĐHYHN RẤT HAY VÀ ĐẶC SẮC.pdfSGK RỐI LOẠN TOAN KIỀM ĐHYHN RẤT HAY VÀ ĐẶC SẮC.pdf
SGK RỐI LOẠN TOAN KIỀM ĐHYHN RẤT HAY VÀ ĐẶC SẮC.pdf
 
EXERCISE PERFORMANCE.pptx, Lung function
EXERCISE PERFORMANCE.pptx, Lung functionEXERCISE PERFORMANCE.pptx, Lung function
EXERCISE PERFORMANCE.pptx, Lung function
 
Clinical Research Informatics Year-in-Review 2024
Clinical Research Informatics Year-in-Review 2024Clinical Research Informatics Year-in-Review 2024
Clinical Research Informatics Year-in-Review 2024
 
Physiology of Smooth Muscles -Mechanics of contraction and relaxation
Physiology of Smooth Muscles -Mechanics of contraction and relaxationPhysiology of Smooth Muscles -Mechanics of contraction and relaxation
Physiology of Smooth Muscles -Mechanics of contraction and relaxation
 
How to cure cirrhosis and chronic hepatitis naturally
How to cure cirrhosis and chronic hepatitis naturallyHow to cure cirrhosis and chronic hepatitis naturally
How to cure cirrhosis and chronic hepatitis naturally
 
Neurological history taking (2024) .
Neurological  history  taking  (2024)  .Neurological  history  taking  (2024)  .
Neurological history taking (2024) .
 
Pharmacokinetic Models by Dr. Ram D. Bawankar.ppt
Pharmacokinetic Models by Dr. Ram D.  Bawankar.pptPharmacokinetic Models by Dr. Ram D.  Bawankar.ppt
Pharmacokinetic Models by Dr. Ram D. Bawankar.ppt
 
SGK NGẠT NƯỚC ĐHYHN RẤT LÀ HAY NHA .pdf
SGK NGẠT NƯỚC ĐHYHN RẤT LÀ HAY NHA    .pdfSGK NGẠT NƯỚC ĐHYHN RẤT LÀ HAY NHA    .pdf
SGK NGẠT NƯỚC ĐHYHN RẤT LÀ HAY NHA .pdf
 
DNA nucleotides Blast in NCBI and Phylogeny using MEGA Xi.pptx
DNA nucleotides Blast in NCBI and Phylogeny using MEGA Xi.pptxDNA nucleotides Blast in NCBI and Phylogeny using MEGA Xi.pptx
DNA nucleotides Blast in NCBI and Phylogeny using MEGA Xi.pptx
 
BENIGN BREAST DISEASE
BENIGN BREAST DISEASE BENIGN BREAST DISEASE
BENIGN BREAST DISEASE
 

Reverse phsase chromatography 1

  • 1. By Amol D Sagulale By Amol D Sagulale Sr. Reseach Associae-II Macleod Pharmaceuticals, Mumbai Email: amol.sagulale@gmail.com
  • 2. Normal phase chromatography •It was one of the first kind of HPLC that chemist developed . •Also known as adsorption chromatography. •This method uses a polar stationary phase and a non-polar, non-aqueous mobile phase, and works effectively for separating analytes readily soluble in non-polar solvents.
  • 3. Si Si Si O H O H O H O H O H O H surface of silica gel Packing material •The most popular packing material is silica gel. •It is believed that silanol radicals ( -Si-OH ) on the surface of silica gel act as the active site and the sample is separated. Normal Phase Chromatography : Separation mode
  • 4. Stationary Phase in NPC •Bare silica or alumina that have polar hydroxyl group on the surface --•silica is preferred over alumina due to its low cost, known performance and ready availability - •For very basic compounds (e.g., amines) alumina is a better choice because amines are retained longer on silica
  • 5. -A variety of bonded phases (BP) can be prepared for NPC. However, functional groups in BP are less polar than the bare silica column
  • 6. Reversed phase HPLCReversed phase HPLC • In the 1970s most liquid chromatography was done on non-modified silica or alumina with a hydrophilic surface chemistry and a stronger affinity for polar compounds - hence it was considered "normal". • The introduction of alkyl chains bonded covalently to the support surface reversed the elution order. Now polar compounds are eluted first while non- polar compounds are retained - hence "reversed phase".
  • 7. Reverse Phase ChromatographyReverse Phase Chromatography • The term “Reverse Phase Chromatography” was used because RP is a form of partition chromatography where chemically bonded phase is hydrophobic or non-polar (e.g. octadecyl group), and the starting mobile phase (e.g. water) must be more polar than the stationary phase. • It is the most widely used technique in HPLC. • .
  • 8. Reversed Phase ChromatographyReversed Phase Chromatography It operates on the principle of hydrophobic interactions which result from repulsive forces between a relatively polar solvent, the relatively non- polar analyte, and the non-polar stationary phase. The components of the analyte mixture pass over stationary-phase particles bearing pores large enough for them to enter, where interactions with the hydrophobic surface removes them from the flowing mobile-phase stream.
  • 9. Reversed Phase Chromatography :Separation mode CH3 CH2COOCH3 CH3 CH2COOCH3 Silica-C18 (ODS) Hydrophobic InteractionHydrophobic Interaction
  • 11. • Reversed phase chromatography is an adsorptive process by experimental design,which relies on a partitioning mechanism to effect separation. • The solute molecules partition (i.e. an equilibrium is established) between the mobile phase and the stationary phase. • The distribution of the solute between the two phases depends on the binding properties of the medium, the hydrophobicity of the solute and the composition of the mobile phase.
  • 12. Reverse Phase ChromatographyReverse Phase Chromatography • One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. • With these stationary phases, retention time is longer for molecules which are more non-polar, while polar molecules elute more readily.
  • 13. Reverse Phase ChromatographyReverse Phase Chromatography • strong attraction between the polar solvent and polar molecules in the mixture being passed through the column. • much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution. • Polar molecules in the mixture will therefore spend most of their time moving with the solvent.
  • 14. Reverse Phase ChromatographyReverse Phase Chromatography • Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces. • They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules. • They therefore spend less time in solution in the solvent and this will slow them down on their way through the column.
  • 15. Reverse Phase ChromatographyReverse Phase Chromatography • The retention time can be increased by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase. • Similarly, the retention time can be decrease by adding more organic solvent to the eluent.
  • 16. Si Si O - Si - CH2(CH2)16CH CH3 CH3 O - Si - CH2(CH2)16CH CH3 CH3 O - Si - CH2(CH2)16CH3 CH3 CH3 CH3 CH3 O - Si - CH3 Commonly used packing materials are hydrocarbons having 18 carbon atoms (called the Octadecyl radical) which are chemically bonded to silica gel (Silica- ODS).Since the surface of the Silica-ODS is covered with hydrocarbon, the polarity of the packing material itself is very low. Reversed Phase Chromatography :Separation mode
  • 17. Principle of reverse phase chromatographyPrinciple of reverse phase chromatography Gradient elution
  • 18. • To equilibrate the column packed with reverse phase medium under suitable initial mobile phase conditions of – • pH • Ionic strength • Polarity ( mobile phase hydrophobicity) • The polarity of the mobile phase is controlled by adding organic modifiers or ion –pairing agents.
  • 19. • Polarity of initial mobile phase ( usually reffered to as mobile phase A ) must be low enough to dissolve the partially hydrophobic solute • Yet high enough to ensure binding of the solute to the reverse phase chromatographic matrix.
  • 20. • Sample containing the solutes to be separated is applied . • The sample is applied to the column at a flow rate where optimum binding will occur. • Chromatographic bed is washed further with mobile phase .
  • 21. • Bound solutes are next desorbed from the reverse phase medium by adjusting the polarity of mobile phase so that the bound molecule will sequentially desorbs and elute from column. • Removing the substances not previously desorbed. • Re-Equilibration of the chromatographic medium from 100% mobile phase B back to the initial mobile phase conditions.
  • 22. Ion-pairing agents • Ion-pairing agents are ionic compounds that contain a hydrocarbon chain that imparts a certain hydrophobicity so that the ion pair can be retained on a reversed-phase column. • Ion Pairing agents are added at concentrations of 0.05 to 0.2. • All ion-pairing agents are potentially capable of ion-pairing with the positively charged basic residues of peptides or proteins, thus reducing hydrophilicity and increasing their retention time
  • 23. • Hydrophobic counterions such as TFA and HFBA in addition to ion-pairing with the positively charged solute also increase the affinity of the solute (peptide or protein) for the hydrophobic stationary phase. • While hydrophilic counterions such as following ion-pair formation with positive charged residues would be unlikely to interact with the stationary phase.
  • 24. Trifluoroacetic acid (TFA). Heptafluorobutyric acid (HFBA). Hexafluoroacetone (HFA). Formic Acid (FA) Phosphoric Acid. Hydrochloric Acid. Triethylamine Phosphate (TEAP).
  • 25. Organic modifiers • Additive that changes the character of the mobile phase. In RP chromatography, water is the weak solvent, and acetonitrile, the strong solvent is added gradually to generate a gradient. • Acetonitrile. • Isopropanol. • Methanol. • Ethanol • Acetonitrile is the reverse phase solvent of choice because the UV cut off for acetonitrile is190 nm, allowing detection at lower wavelengths.
  • 26. • It is less viscous than methanol, thus causing less fluctuations in pressure. • Less bubble formation occurs when it is mixed with water. It has also better selectivity for peptides and proteins. • Isopropanol is used either alone or in combination with acetonitrile to elute large or hydrophobic proteins.
  • 27. Quality of Stationary phasesQuality of Stationary phases Determined by their physical and chemical properties Physical Properties : Porosity Specific surface area Particle size Particle shape Pore size Greatly determines the efficiency of packing.
  • 28. Quality of Stationary phasesQuality of Stationary phases • Must be controlled in narrow tolerances to enable manufacturer for reproducible packing materials. • Porosity : Determines the surface area & others parameters. • Retention • Selectivity
  • 29. Chemical propertiesChemical properties • Result of substrare properties & • Applied surface bonding chemistry • These forms the basis of retention and selectivity.
  • 30. SubtratesSubtrates • Inorganic oxides • Polymers • Carbons • Have sufficient hydrophobic properties & used unmodified as RP stationary phases. • Majority of presently available RPLC stationary phases are modified substrates.
  • 31. SubtratesSubtrates • Substrates & Stationary phases must posses physical and chemical properties to be suitable as Stationary phase. • Mechanical strength • No Shrinking & swelling properties
  • 32. SilicaSilica • Silica & silica base are the ideal materials. • Synthesized in pure form & yield a large number of substrates. • Well defined physical properties. • Possess sufficient mechanical strength • No shrinking & swelling properties.
  • 33. SilicaSilica Bonding chemistry of silica results into high quality of RPLC-phase. It covers a broad spectrum of different organic ligands attached to a variety of silicas & enables the separation of many different substances. Eg. Neutral molecules in lower mol. Wt range. Charge molecules by ion-pair
  • 34. Silica based stationary phasesSilica based stationary phases • Hydrogel from inorgnic silicates & alkoxy silicates Grinding and sieving yields irregular shape silica substrate is obtained ( characterized as Xerogel) Relatively high surface area High Porosity Variable wall thickness SilGel
  • 35. Silica based stationary phasesSilica based stationary phases • Consolidation of silica particles by either • oil emulsion or Coacervation • Results in sphere shape particles • Lower surface areas • Lower porosities • Regular shape having thicker wall • SolGel
  • 36. Silica surfacesSilica surfaces Silanol groups Siloxane bridges Acidic reactive sites Hydrophobic unreactive a)Single (geminal silanol) b)vicinal silanols Silanediols Most reactive sites •Responsible for residual silanol activity of bonded silicas for basic compounds. •Silanols cause peak tailing and excessive retention
  • 37. Types of silanol on surface of silica gelTypes of silanol on surface of silica gel
  • 38. Pretreatment stepsPretreatment steps • Heating • Rehydroxylation • Homogenization • In order to reduce the single silanols • To obtain as many as bonded or associated silanols of similar avctivity.
  • 39. Analytical PurposeAnalytical Purpose • Silica usually produced of nominal 2,3,5 and 10 um particle size • Surface area – 100-600 m2/gm • Particle porosity =0.6-0.7 • Surface density 8 umol/m2 • Equivalent to = ± 4.5 silanols/nm2 • For large molecules = 30 -100nm • For unrestricted access to inner surface for smaller molecules, the pore size is not less than 10 nm.
  • 40. • Silica substrate uses alkoxysilane or chlorosilanes to attach organic ligands through siloxysilane linkages to the supports surface. • To produce such covalently bonded organic stationary phases, the reactive alkoxy or chlorosilane reagent must contain atleast one leaving group which is able to reacts with silanol at substrate surface.
  • 42. Surface hydrophobisation reactionSurface hydrophobisation reaction • It is carried out under anhydrous condition. • The is catalysed by base 2,6-lutidine or imidazole. • It act as a scavenger base to neutralise acid byproducts. • Further step includes reflux, sonication, filtration, rinsing and drying steps.
  • 43. • Depending on the no. of leaving groups for synthesis of RPLC phase, three groups of organosilane reagents can be distinguished :-
  • 44. • From the originally available no. of silanol groups, at a silica substrate, approximately only 50% can react. • Due to the steric hindrances between ligands and side chains. • Silanol concentration = 8umol/m2 • Ligand concentration = 4umol/m2
  • 45. • The unreacted silanol concentration is equal to ligand concetration. • Residual silanols may strongly influence • Retention • Selectivity • For ionic and polar compounds.
  • 46. • Depending on activity and actual eluent pH silanols may influnce the chromatographic process by • Hydrogen bonding • Ion exchange • Dipole interaction May cause severe peak tailing and leads to irreproducible retention times.
  • 47. • In order to suppress this residual silanol activity after bonding, secondary synthesis step to end cap or mask these group is performed. • The endcapping is done by smallest possible silane • EX-trimethyl (Most sensitive to hydrolyzation)
  • 48. • Most common S.P in RPC are those in which a functional group is attached to a silica support Synthesis of ODS (octadecylsilane, C18H37Si) the reagent used is octadecyl-chlorosilane (C18H37Si(CH3)2Cl)
  • 49. --. -- Monofunctional S.P is prepared using the above procedure because the reagent C18H37Si(CH3)2Cl) used has one chloro group (only 8-12% carbon Loading) - -For steric reasons it is not possible for all silanol groups (-SiOH) groups on silica surface to react with functional group (only ~45% are bonded)
  • 51. Endcapping is done to cover more silanol groups with di- and tr- chlorosilane reagent Result in S.P which is more dense and and have 15-20% carbon loading
  • 52. • Chemically bonding the hydroxy groups would generate a rugged s.p. and most importantly the thinnest possible (monolayer) coating. • Bonded carbon chains forms the primary s.p. material C4 – C18 (RP). • Derivatization of the terminal carbons allows us to ‘tailor’ s.p.’s with different polarities. • Such derivatized s.p. are used in the reverse phase
  • 53. • Isopropyl instead of methyl group or modification by alkyl ligands carrying a polar function near the silane group – Octadecyl – Octyl – Hexyl – Cyclohexyl – Phenyl – alkyl phenyl
  • 54. • The most popular column is a octadecyl carbon chain (C18) bonded silica (USP classification L1) with 297 columns commercially available C8 bonded silica (L7 - 166 columns), • pure silica (L3 - 88 columns), • cyano bonded silica (L10 - 73 columns) • phenyl bonded silica (L11 - 72 columns). • Note that C18, C8 and phenyl are dedicated reversed phase packings ..
  • 55. • Cyano columns can be used in a reversed phase mode depending on analyte and mobile phase conditions. • It should be noted at this point that not all C18 columns have identical retention properties.
  • 56. RPLC phases inorganic oxidesRPLC phases inorganic oxides • Alumina • Titania • Zirconia • Higher stability 0 to13 • Interact with anlytes with ligands exchange interaction • These are strong secondary interaction and usally unwanted
  • 57. • The high activity of the surfaces of these oxides • Lack of straight forward synthesis procedure • Surface modification is done by deposition of polymers layer on substrate.
  • 58. Polymer based RPLC –SPPolymer based RPLC –SP • Styrene divenyl benzene 0 to 14 • Methacrylate or • Polyvinyl alcohol based phase 2 to 12 • Hydrolytical stability over wide pH range • Have found appication in aqueous size exclusion and ion exchange chromatography.
  • 59. Carbon RPLC-SPCarbon RPLC-SP • High chemical stability over wide pH range • 0 to 14 • Show ultimate hydrophobic properties. • Sufficient hardness • Well define pore structure • Do not suffer from swelling and shrinking • Porous graphitized carbon • Black carbon
  • 60. Applications of Reversed Phase Chromatography (RPC) -RPC is the most widely used separation mode in HPLC -Cover ~ 75 % of HPLC separations. Applicable to most non-polar analytes & to many ionizable & ionic compounds. -Best suitable for the separation of neutral solutes that are soluble in water or relatively polar solvents and with molecular weights less than 2000-3000 --
  • 61. The following table lists a few examples of the multitude of uses of RPC in various fields
  • 62. Samples • A) Regular Sample a) ionic ex. Acids,bases, organic salts b) neutral • B) Special Sample very hydrophilic or hydrophobic compounds Eg:- achiral isomers, chiral isomers, enantiomers, biomolecules, inorganic ions, synthetic polymers
  • 63. Retention and selectivity in RPLC-SPRetention and selectivity in RPLC-SP • Classical measures of retention – capacity factors – partition coefficients – Van’t Hoff Plots • Give bulk properties only - do not give molecular view of separation process
  • 64. Solvophobic TheorySolvophobic Theory • Considers retention and selectivity mainly as function of- • Surface tension • Dipole-dipole interaction • The interaction is between polar groups of a compound and mobile phase. • Solvent cavities are created by the hydrophobic part of compound.
  • 65. • The assumption is the principal shortcoming. • RPLC-phase is considered as passive part of system. • In many studies , it is shown that specially for non-polar and ionic substances, this is unrealistic.
  • 66. Partitioning TheoryPartitioning Theory • It is supported by the good correlation by octanol-1/water partition coefficient. • RPLC retention data not found for very polar compounds. • This theory insufficiently explains shape selectivity.
  • 67. Combining solvophobic and Partitioning theoryCombining solvophobic and Partitioning theory • Solvent–stationary interphase layer is formed. • Depending upon the composition of eluent and nature of the stationary phase, enrichment by the organic modifier in that phase takes place.
  • 68. • Partition of solutes between interphase and mobile phase is assumed to take place by displacement of solvent molecules. • Together with column efficiency retention, selectivity determines the finally achievable chromatographic peak resolution.
  • 70. None of these theories can completely explain all of the observed retention in reversed phase HPLC. Thank You….