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.
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.
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….