Stationary Phase in NPC

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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
Q. What do we mean by the term bonded phases?
Covalent attachment of functional group to the
silica surface
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A variety of bonded phases (BP) can be prepared
for NPC. However, functional groups in BP are
less polar than the bare silica column
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Stationary Phases in Reversed-Phase HPLC
--Most common S.P in RPC are those in which a
functional group is attached to a silica
support. Examples are
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)
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So what happens to the SI-OH groups which cannot
be bonded?
Peak tails because of H-bonding of the basic
analyte . Therefore, end-capping is done to
cover more silanol groups with di- and tri chlor
silane reagent
Result in S.P which is more dense and and have
15-20 carbon loading
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Effect of End-Capping on Column Performance
Carbon Loading in RP-column What is meant by
carbon loading? Amount of carbon introduced On
S.P by functional group (measured at wt of the
bare silica packing)
How does it affect the retention times? Higher
the carbon loading, more non-polar the S.P and
more retention for hydrophobic compounds
Polymeric C-18 RP columns What are the advantages
of polymeric C-18 RP Columns over the
silica-based C-18 columns? Wider operating pH
range than silica based with no peak tailing
What are the disadvantages of polymeric C-18 RP
columns over the silica-based C-18
Columns? Longer retention times and less
retained, swell with organic solvents
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Effect of Functional Group on Column Selectivity
in RP-HPLC The following chromatograms
illustrates the effect of chain length of the
alkyl group on retention and selectivity
As shown to the right, longer chain length
produce packing which are more retentive.
Longer chain permits use of larger samples
because of greater carbon loading.
Sample size for C18 twice of C4 column
Adsorption
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Effect of Functional Group on Column Selectivity
in NP-HPLC
The major reason for the use of functional group
is to produce changes in selectivity rather than
retention. For example, column with
different functional group show a diffferent
separation selectivity of certain solutes in
normal phase chromatography (NPC)
Mobile phases of 2377 (A) and 3367 (B)
THFH2O Solute identities (1) dexamethansone
(2) triamcinolone acetonide (3) Hydrocortisone
acetate (4) Methyl testosterone
THF/water mixture of approximately same strength
were used. However, peak 1 and 2 is unresolved
on cyano column, but is partially resolved on
phenyl column
Peak 3 and 4 are well separated on cyano column
but unresolved on phenyl column
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Applications of Normal Phase Chromatography (NPC)
---NPC is best suited for the separation
of Solutes that are soluble in non-polar
solvents (e.g.,hexane) --Better suited for the
separation of isomers as shown by the example to
the right.
NPC is also useful to distinguish solutes based
on the number of electronegative atoms, such as
oxygen or nitrogen,
For molecules with different functional groups
--Fats and water soluble viatmins, lipids and
pesticides can also be analyzed by NPC.
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In modern HPLC, NPC is less commonly employed.
Why?
--RPC allows for greater separation selectivity
than NPC --More choice of columns, which are
stable in RPC --Require careful choice for M.P
preparation than in RPC
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Applications of Reversed Phase Chromatography
(RPC)
--RPC is the most widely used separation mode in
HPLC --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
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Applications of Reversed Phase HPLC (Contd)
Organophosphate insecticides
Soft Drink Additives
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N.P R.P
a-values are generally higher with RPC then with
NPC for Homologs/Benzologs
Isomers provided better selectivity with NPC
compared to RPC
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Comparison of NP and RP-HPLC
NPC RPC M.P relatively polar M.P
relatively non-polar S.P polar S.P
non-polar
Increasing the solvent strength (i.e., using more
non-polar solvent) decreases the k and
analysis time
Increasing the solvent strength (M.P) (i.e.,
using more polar solvent decreases the k and
analysis time)
Solvents with lower P values gives Shorter
retention and vice versa P is high ---------
weaker solvent P is low -----------stronger
solvent
Solvent with higher e values gives shorter
retention e is high ---------- stronger
solvent e is low ------------ weaker solvent
More polar the S.P greater the retention
More non-polar the S.P greater the of
polar solute retention of non-polar
solutes
Mechanism Mechanism Competition vs.
solvent interaction model Solvophobic vs.
Partitioning Competition model
Partitioning Model
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Comparison of NP and RP-HPLC (Contd)
NPC RPC
Applications Best suitable for compounds, which
are soluble in non-polar/medium
polarity Solvents Solutes with electronegative
atoms or Heteroatoms as a part of the ring
system Isomeric solutes positional
isomers optical isomers
Cover 75 of HPLC separations Best suited for
compounds that are soluble in water or partially
soluble in water Compounds with different chain
length
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Mobile phase Optimization in HPLC (RPC, NPC, IEC,
GPC) --can be performed using two different
methods (a) Isocratic Method (b) Gradient
Elution Method
(a) Isocratic Elution Method --A method which
involves a constant composition of a mixture of
two solvents during HPLC run is termed as
isocratic elution. --Ratio of two solvents is
fixed during the entire run HPLC run e.g., use of
50 acetonitrile/water during the entire run
(b) Gradient Elution Method --A method which
involves altering the composition of a mixture of
two or more solvents during HPLC run is termed as
gradient elution
For example, 20acetonitrile/80 water -------
80 acetonitrile/20 water (Start
as weaker solvent in RPHPLC) (Stronger
solvent in RP-HPLC) Regardless of the mode of
HPLC, gradient elution involves using a weak
eluting Solvent at the start of the
chromatographic run and adding increasing
proportions of a strong solvent over the course
of the separation
When gradient elution is required in
HPLC? --Commonly used when a mixture of solutes
with a wide range of k is to be Separated
(similar concept of temperature programming in GC)
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Low vs. High Pressure Gradient Formation in HPLC
Solvent passes from each reserovir Directly to a
pump and then to a Mixing manifold which it
passes through the sample valve into column. The
volume delivery of each solvent is controlled by
the speed of the respective pump, which is
determined by the speed of the power supply
Blending of two or more solvents at amospheric
pressure, require single pump. The manifold
receives and mixes solvents from each of the
programmed valves. The valves are operated
through microprocessor controller (program to
open and close the valve (connected to solvent
reservoirs) Predetermined amount of solvent
flows Into manifold


.
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Gives good resolution of all peaks in particular
for critical band pairs(1/2, 2/3) --However run
times are long, later bands are broader and
marginally detectable
As stronger M.P is used It gives shorter TR
and The later eluting peaks (e.g., 8,9) are
narrower, which can be beneficial for improved
quantitation and detection
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Isocratic Vs. Gradient Elution (Contd) None of
the isocratic conditions results in adequate
separation of this mixture Because early bands
require weaker solvent and later bands require
stronger solvents
Gradient Elution
All analytes are well separated using gradient
elution at a rate of 5/min. All bands are well
resolved and the run times are only 11 min.
Gradient elution is a better choice for this and
other samples with a wide range of Retention.
-----? Better quantitation and detection and
less peak tailing
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• Types of Gradient Solvent Profiles
• Modern HPLC allows the solvent composition to be
  varied using either
• Linear gradient porfiles (shown in curve 6 below
• Exponential gradient profiles
• Convex gradient curve (curve 2-5), (ii) Concave
  gradient curve (7-12)

Linear curve 6 A linear gradient is generated
when The rate of change of solvent B is linear
over time. Used in majority of HPLC separations
Convex curve 2-5 Approaches the final solvent
Composition more rapidly
Concave curve 7-10 Delay the onset of the final
condition composition
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Effect of Gradient Curve Profiles on the
Separation of Alkyl-Phenones
Starting gradient is too steep, a1-2, a2-3 are
smaller and a8-9 and a9-10 are even smaller
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Starting from a weaker solvent permits greater
separation selectivity between early peaks as
well as later peaks (19 min total analysis
time) slightly longer than the above curve
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Separation is further improved by using a convex
gradient which reaches the final conditions
more rapidly than linear profile 15 min total
analysis time
Use of concave gradient improve a of early peaks
much more than any linear or convex gradient but
total analysis time is longer than convex or
linear gradient
Best optimized conditions of alkylphenones
homologs-------? Convex Gradient


Curve 4
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Gel exclusion or Gel Permeation Chromatography
--Small molecules penetrate into the aqueous
spaces within the S.P beads --Large molecules
cannot penetrate the beads. They are eluted by a
volume of solvent equal to the volume of the
mobile phase Therefore, large molecules are
retained less than the small molecules
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Theory of Size Exclusion Chromatography
In SEC, the retention volume (Vr) is related to
void volume (Vo or Vm) by the following
relationship Vr Vm KVs Vr retention
volume Vm (aka. V0 void volume) Vs volume
of S.P K partition coefficient
Volume occupied by the gel beads (Vs)
Void volume (V0), 35 of the total volume
Total bed Volume (Vt)
Elution volume of the desired protein
--The volume of the solvent inside the gel
particles is also Vs
If the gel matrix occupied no volume than Vs
would be Vs Vt- V0 (since Vt Vs
V0)
However, the gel matrix does occupy some volume,
so Vt-V0Vs
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The Vs is proportional to Vt-V0 because the
liquid inside the particles occupies a constant
fraction of particles volume. We defined a
quantity Kav (read as K average)
Large size molecules --? does not penetrate the
gel (Vr V0) than Kav 0
Intermediate size molecules ? have some retention
(VrV0) than Kav range from 0-?1
Small size molecules ---? freely penetrate into
the gel (VrVt) Than Kav 1
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Determination of molecular weight using GPC can
be estimated by comparing the elution volume
with those of the standards
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Advantages of SEC Short, well-defined separation
times Narrow bands, good sensitivity No sample
loss since solutes do not interact with
stationary phase Absence of column deactivation
Disadvantages of SEC Limited number of bands
accommodated since short time scale Not
applicable to similar-sized molecules, like
isomers
What is the major drawback of calibration used to
determine Mol wt?
--Molecules with same molecular weight but
different shapes exhibit different elution
behavior. For example, large molecular wt.
molecules may be compact and fit into S.P pores
than expected.
Solution Match the structure of the unknown
with your standards
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What is the difference between Gel permeatation
and Gel Filtration Chromatography?
Types of SEC Gel Filtration Chromatography Aqueou
s solvent Hydrophilic Packings Gel Permeation
Chromatography Nonpolar Organic
Solvents Hydrophobic Packings