1. Methodologies in High Performance Liquid Chromatography (HPLC)
Mobile Phases
Mechanism
Stationary
Phases
Applications
Separation of
Neutral Compounds
Reversed Phase Chromatography
Normal Phase Chromatography
Gel Permeation Chromatography
Mechanism
Mobile Phase
Stationary Phase
Applications
Separation of
Ionic or Charged
Compounds
Ion-Exchange Chromatography
Ion Chromatography

2. Silica or Polymer backbone
Illustration of Mechanism of Retention of Solute Using Four Different Types of
HPLC
(a) Normal Phase Chromatography
aka. Adsorption Chromatography or
Classically as liquid solid chromatography (LSC) HO
Less-polar
M.P
Polar
S.P
(a) Adsorption
Silica backbone
- Solute is adsorbed on the surface of the
S.P ( polar interactions are dominant)
S.P = Polar (Silica gel or alumina backbone)
M.P = Non-polar (hexane mixed with polar
solvent)
(b) Reversed Phase Chromatography
aka. Partition Chromatography (LLC)
(b) Partition
More polar
M.P
Non-polar
S.P
Silica or Polymer backbone
- Solute equilibrate “in” and “out” of the liquid
S.P
-(partitioning in and out of the alkane chains)
S.P = Liquid coated on a solid support
(silica or polymer backbone)
M.P = Polar (water mixed with relatively non-polar solvent)

3. Illustration of Mechanism of Retention of Solute Using Four Different Types of HPLC (Continued)
Cl-
Br- F-
SO42-
Silica or Polymer Backbone
Li+
Na+ K+
Rb+
Cs+
Charged
S.P
M.P counter
ions
(3) Ion-Exchange Chromatography (IEC) or IC
- Solute competes with the M.P counterions
(of same charge as the solute) for the fixed
charged on the S.P)
- Separates molecules based on size,
charge and hydration
Solute
S.P = silica or polymer back bone containing
anion-exchange groups [e.g., NH+(CH3)3,] or cation exchange groups [SO3-]
bound on its surface
M.P = counterions ( inorganic salt buffers
e.g., ammonium acetate (CH3COO NH4 )

4. Silica or Polymer backbone
Illustration of Mechanism of Retention of Solute Using Four Different Types of
HPLC (Continued)
(4)Gel-Permeation Chromatography (GPC)
“Small molecules are retained longer than
large molecules”
separates molecules based on size
danalyte > dpore no trap
danalyte <dpore ----- increase tR
Porous
S.P
Gel permeation
chromatography
Silica or Polymer backbone
Small
molecules
Large
S.P = 10 um particles with uniform pore Silica: rigid- highly porous, stable at high temperature, interacts with solute
Polymer: cross-linked, different pore
size, (e.g., PSDVB)
M.P = Liquid aqueous solvent
organic solvent, mixed aqueous/organic solvent
Gel filteration: hydrophilic packing, sulfonated divinyl benzenes or polyacrylamide
used to separate polar species
Gel permeation: hydrophobic packings (PSDVB) used to separate non-polar species

5. NPC vs. RPC Normal Phase Chromatography Mobile phase (M.P)
Silica or Polymer support
Polar bonded-phase
--M.P is relatively non-polar
(hexane mixed with slightly more polar solvent like isopropanol, ethyl acetate or chloroform)
--S.P is polar.
The S.P polarity order is shown to the
right ----
Silica
NH2
Diol CN
Polar
Less Polar
Mobile phase (M.P)
Silica or Polymer support
Non-polar bonded-
phase
Reversed-Phase Chromatography
--M.P is relatively polar
(water mixed with acetonitrile)
Non-Polar
More Polar
C-18
Phenyl
C-8
C-4 CN
--S.P is non-polar
The S.P polarity order is shown to the
right

6. Relationship between polarity and elution times for
normal-phase and reversed-phase LC.
Hexane/
CHCl3
THF
Water/MeOH
Water/
NPC
*Most polar elutes last
*Increasing polarity of M.P decrease elution
time in NPC
RPC
*Most polar elutes first
*Decreasing polarity of M.P decreases
elution time in RPC

7. “Competition Model is More Generally Accepted”
MECHANISM OF SOLUTE RETENTION IN NPC
Competition Model:
--Assumes that entire S.P is covered by M.P molecules
- Retention occurs due to adsorption of process
- Competition between solute molecules and M.P molecules for the adsorption
sites on the S.P
(b) Solvent Interaction Model:
--Assumes that :
- “A bilayer of solvent molecules is formed around S.P particles, which depends
on the concentration of polar solvent in the M.P”
“Retention results from the interaction of solutes molecules with the secondary
layer of M.P molecules (no interaction with the S. P)”
“Competition Model is More Generally Accepted”

8. MECHANISM OF SOLUTE RETENTION IN RPC
Solvophobic Model
- “Retention is considered primarily due to hydrophobic interactions between
the solute and the M.P.”
--S.P is thought to behave more like a solid than a liquid and retention Is
considered primarily due to hydrophobic interaction between solute and M.P
--Because of solvophobic effects solute binds to the surface of the S.P reducing
surface area of the analyte exposed to the M.P.
(B) Partitioning Model
“Solute is fully embeded in the S.P rather than adsorbed on the surface of the S.P.
Therefore ,partitioning of solute occurs between M.P and liquid likd M.P.” “
“Partitioning model is generally accepted in RPC”

9. the strength of the solvent
Elutropic Series (e0) of Some Solvents Used in NPC
Higher the value of
e0, the greater
the strength of the solvent
which result in
faster
elution or
lower retention
of solutes
-e0 in NPC is a measure of
adsorption energy/area of solvent and
used quantitatively to define sovent
strength
Solvent strength in NPC is directly proportional to elutropic series e0 values
Which is the strongest and the weakest eluent for NPC?
Water is the strongest eluent--- fast separation (shorter retention times)
Pentane/Hexane are the weakest eluent------ long separation time
“A rule of thumb in NPC ---- An increase in e0 by 0.05 units typically decreases k’
by a factor of 3-4”

10. FA e0A = FB e0B ISOELUTROPIC MOBILE PHASES
- Mixtures of different solvents having the same overall polarity are called
isoelutropic mixtures.”  These mixtures have same eluent strength
Example:
--A 30% (v/v) mixture of chloroform (CHCl3) in hexane has approximately the
same solvent strength as 35% (v/v) mixture of toluene in hexane
-so this mixture is classified as isoelutropic mixture
Q. How can you prove this quantitatively?
--If the volume fraction (F) and the e0 values of the solvents are known we can
prove this using a relationship:
FA e0A = FB e0B
0.30 x = x 0.22 =
Similarly 69% (v/v) of ether in hexane has same solvent strength as 99%(v/v) of
methylene chloride in hexane
-- Isoelutropic M.Ps give similar total elution times, but they permit some
changes in sample selectivity

11. Why an increase in % (v/v) methyltertiary-butyl ether (MTBE) decreases the
retention time of aniline and phenol?
--Because an increase in MTBE % (v/v), increases the polarity of M.P, which in turn
increases the solvent strength-- analytes elutes faster
What care must be taken when preparing M.P of mixed solvents?
“The Solvents must be miscible”

12. Chromatogram showing the effect of Solvent strength in NPC
--Following chromatogram shows an example of changing the solvent
selectivity of a normal phase separation of phthalate esters (plastisizers)
- It can be seen that changing the solvent from methyl ethanoate to methyl
butanoate While keeping the ratio same radically changes the selectivity of
diethyl/diphenyl phthalate and this pair is better resolved with buty ethanoate
Q. Why the total analysis time increases when we use methyl butanoate instead of
Methyl ethanoate (ethy acetate)?
---Methyl butanoate (aka. butyl acetate) is a weaker solvent (e = 0.38) for NPC then
methyl ethanoate (e = 0.58), Thus weaker solvent gives longer retention of solutes

13. DPT = Diphenylphthalate
Q.2 Why the elution order of diethyl phthalate or diphenyl phthalate reversed? O C 2 H 5 M e 4 9
methylethanoate
(ethyl acetate)
methylbutanoate
(butyl acetate) d - +
DPT = Diphenylphthalate d+
Carbonyl
dipole
Induce dipole in benzene ring d
--Because electron donating groups are different in two solvents, they might
induce dipole moment in the benzene ring of phthalates differently.

14. Disadvantages of NPC
a)---Retention times tend to be too long and the chromatographic peak have a
tendency to tail
b)--Lack of selectivity--.Virtually all compounds are eluted in the same order
regardless of column selected
Changes in selectivity is only achieved by changing the M.P composition
c) Effect of Water
Perhaps the biggest disavantage of using NPC (using bare silica column or
alumina column) and to lesser extent bonded phase column in NPC is:
Water is adsorbed from the atmosphere into the M.P, which in turn when
pumped through the column is adsorbed on the adsorption sites of the S.P
What is the result of this effect on
chromatographic separations?
Solute tr and N continue to decreases
and columns becomes deactivated---
run times decreases,Rs decreases,
the longer the eluent sits around
Q. So how to avoid this problem?
--Prepare Normal phase eluent (saturated with
water) The Rx-SIL column shows less retention
variation and peak height as water is varied but low purity Zorbax takes at least 50% water to achieve some saturation.

15. Benefits of Making Saturated Eluents are:
Less variation in sample retention from run to run
Higher sample loading
Higher column efficiency and peak tailing is reduced
Reduced catalytic activity of the adsorbent

16. Polarity Index (P’)for Reversed Phase Chromatography
--Solvent strength in RPC is
inversely related to polarity index
Higher the value of P’ the weaker
the solvent strength and vice-
versa
-- Water is generally used as a
“base-solvent” and M.P strength
is increased by increasing the
volume of another solvent (organic
modifier).
--Three commonly used modifiers
are: Tetrahydrofuran (THF),
methanol (MeOH), and acetonitrile (ACN)
Which composition of mobile phase is weaker for RPC?
(a) 50/50 mixture of MeOH/H20 or (b) 50/50 mixture of THF/H20
“ A 50/50 mixture of MeOH/H20 is a weaker M.P”
A general rule of thumb in RPC  k’ of solute decreases by a factor of 2 or
more for every 10% (v/v) addition of organic modifier

17. SOLVENT STRENGTH EFFECTS IN REVERSED-PHASE CHROMATOGRAPHY
--Water is generally used as a base solvent in RPC and the separation is optimized by
Increasing the amount of water to the organic solvent.
--The following chromatograms shows the effect of solvent strength selectivity on
separation of five compounds
In RPC the primary purpose of decreasing the %(v/v) organic solvent (increases the
% (v/v) of polar solvent such as water) is to increase the Rs, a, and k’
---Resolution of compound pair A/B is poor for M.P of >50% ACN, however,
separation Of A/B improves for a decrease in % ACN upto 40% ACN
---Resolution of compound pair C/D first increases upto 50%, then decreases at 40%ACN

18. “
When the Rs
of one band pair increases and
of another band pair decreases
with a change in % organic of solvent ,
best optimization condition is the
one where
both band pair have same
at intermediate solvent strength. (i.e., 45% ACN)
Q. What kind of separation would you observe if the polarity of the M.P and the
S.P are similar?
---No separation (interaction of solute with each phase is similar)
MEASURING POLARITY OF MOBILE PHASE IN NORMAL AND REVERSED
PHASE CHROMATOGRAPHY
-In NPC the polarity of M.P is generally expressed in elutropic series (e0)
- In RC the polarity of M.P is generally expressed using polarity index (P’)

19. Only difference between
Q. What we have learned about the NPC and RPC separation optimization so far?
Easiest way to influence separation in NPC and RPC is by changing the
Solvent polarity
Changing the solvent polarity changes k’ in both NPC and RPC
k’ can be varied by changing the polarity index (P’)
Suppose we obtained a separation of analyte and we measured its k’ at a
certain volume fraction of organic solvents. Can we estimate what volume
fraction (%) v/v of organic solvent is needed if we want to increase k’ of the
analyte?
Yes, we can if we can relate k’ with P’
Relationship between capacity factor (k’) and P’ for reversed-phase
chromatography
P1’ and P2
are the initial
and final values
of solvent polarity
k2’
k1’ =
10 (P2’-P1’)/2
k1’ and k2’ are
The intial and final
values of capacity factor
of two solutes
Only difference between
the two equations
is on the exp values
Of polarities
Relationship between capacity factor (k’) and P’ for normal phase chromatography
10 (P1’-P2’)/2
k2’
k1’ =

20. Number of digits in mantissa of Logx = number of significant
In a RP-HPLC, a solute was found to have a retention time of 31.3 min while an
unretained species required 0.48 min for elution when the mobile phase was
30% (v/v) of methanol and 70% (v/v) of water.
What is capacity factor (k’) at this composition of M.P?
k’ = tR- t = t
= (Calculator answer)
= 64.2
(B) Calculate water/methanol composition that should be required to bring k’ to a
value of about 5.0
P’AB = fA P’A FB P’B
= x x 10.2 =
= 8.67
Substituting for reversed phase equation we can write:
10 (P2’-P1’)/2
k2’
k1’ =
= 10(P2’-8.67)/2
64.2
Log = -1.11
P2’ =
0.5
= P2’ -8.67/2.0
-1.11 = 0.5P2’ -4.34
Number of digits in mantissa of
Logx = number of significant
Figure in x
P2’ = 6.46

21. Substituting into equation (A) (see above) we get
= fA X (1-FA)10.2
6.46 = 5.1 fA fA
fA = = or 73% MeOH-27% H2O
5.1
Note that in RPC higher concentration of methanol means the k’ should
decreases compared to the initial conditions of 30% MeOH