High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography for Research Students for separation of very polar constituents

1. HPLC
Introduction

2. DISCOVERY
M.S. Tswett, 1903, Warsaw
Polytechnic Institute (Separation of the
chlorophylls of green leaves extract)
Calcium carbonate and non polar eluent

3. HPLC IntroductionGSC GLC
GAS SFC
NP RP IEC
GPC GFC
SEC
Column
TLC Paper
Planar
LIQUID
CHROMATOGRAPHY

4. HPLC Retention
Adsorption Chromatography (NP, RP, IEX)
Interactions of the analyte with the adsorbent surface
causing its slower movement compared to the eluent
molecules
Size-Exclusion Chromatography
Exclusion of the analyte molecules from the adsorbent
pore volume due to their size
No interactions with the adsorbent surface

5. NORMAL PHASE
1980 2004
30% 5% Silica Gel
47% 44% Silica based bonded phases
•Diol
•Amino
•Cyano
3% 1% Alumina
20% 50% Chiral Bonded Phases
Principle:Adsorption of analytes on the polar, weakly acidic surface of
silica gel.
Stationary Phase.:Silica (pH 2-8), Alumina (pH 2 -12), Bonded Diol,
and NH
2, or CN
Mobile Phase:Non-polar solvents (Hexane, CHCl
3)
Applications: Non-polar and semi-polar samples; hexane soluble;
positional isomers.

6. NP: SEPARATION PRINCIPLE
Polar (specific but nonionic) interactions of analyte with polar
adsorption sites (SiOH, -NH
2, -CN, Diol) cause its retention
Different sorption affinities between analytes result in their separation
More polar analytes retained longer
Analytes with larger number of polar functional group are
retained longer
Structural isomers are often separated

7. Reversed-Phase HPLC
Principle: Partition of analytes between mobile phase and stagnant phase inside
the pore space + adsorption on the surface of bonded phase.
Stationary Phase: Hydrophobic surfaces of moieties bonded on silica (C18, C8,
C5, Phenyl, CN)
Mobile phase: Methanol or Acetonitrile and Water.
Applications: ~80% of all separations performed with RP HPLC.
80% Octadecylsilica (ODS, C18)
10% Octylsilica (C8)
5% Butylsilica (C4)
3% Phenyl
2% Cyano (CN)

8. REVERSED PHASE
SEPARATION PRINCIPLE
Nonpolar (nonspecific) interactions of analyte with hydrophobic
adsorbent surface (-C18, C8, Phenyl, C4)
Different sorption affinities between analytes results in their
separation
More polar analytes retained less
Analytes with larger hydrophobic part are retained longer
structural isomers maybe more challenging in this mode

9. Reversed-Phase vs. Normal Phase
Nonspecific (hydrophobic) interactions are at least ten times weaker than polar
small differences in component molecular structure could have a
significant effect in their retention0
5
10
15
20
25
0 1 2 3 4 5 6 7 8 9 10 0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 10 0
5
10
15
20
25
0 1 2 3 4 5 6 7 8 9 10 0
1
2
3
4
5
6
7
8
9
10
0 1 2 3 4 5 6 7 8 9 10
Separation of 2-Me-Phenol and 4-Me-Phenol in RP and NP
Reversed-Phase
MeOH/Water, Luna-C18
Normal Phase
Hexane/IPA, Luna-Si
90/10
60/40
95/5
98/2

10. R
1
CH
3
R
2
OH
O
CH
3
CH
3
CH
3
CH
3
CH
3CH
3
CH
3 TocopherolsO
CH
3
CH
3
CH
3CH
3
CH
3
OH
CH
3
R
1
R
2
Tocopherols Tocotrienols R
1
R
2
a-Tocopherol (a-T) a-Tocotrienol (a-3)_ Me Me
b-Tocopherol (b-T) b-Tocotrienol (b-3) Me H
g-Tocopherol (g-T) g-Tocotrienol (g-3) H Me
d-Tocopherol (d-T) d-Tocotrienol (d-3) H H
Tocotrienols
Genesissilicacolumn(250x4.6mm,4m).
Mobilephase:Hexane-1,4-dioxane(96:4).
.J.Chromatogr.A,881(2000)217-227
Separation of synthetic tocopherols by reversed phase
HPLC (280 nm) :1) d-tocopherol, 2) g-tocopherol,
3) b-tocopherol, 4) a-tocopherol, 5)a-tocopheryl acetate
Food Chemistry, 76 (2002) 357 –362.
Experimental Comparison of NP and RP HPLC

11. Ion Exchange
Principle: Reversible adsorption of ions on S.P. with oppositely charged functional
groups.
Anionic polymers are known as cation exchangeresins and these
resins can be strong or weak cation exchange resins which are
strongly dependent upon the anionic group that is bonded to the
polymer.
Cationic polymers on the other hand are known as anion exchange
resins and these resins can also be weak or strong anion exchange.
Stationary Phase:
For cations (cation exchange) -SO
3
-(strong)
, CO
2
-(weak)
For anions (anion exchange)-NR
4
+ (strong)
, NH
3
+ (weak)
Mobile Phase: Aqueous buffer with pH and buffer strength carefully controlled.
Applications: All ionic compounds common anions, cations, sugars, amines, etc.
Anionic polymer
Cationic polymer

12. Size Exclusion
Principle: Internal pores of stationary phase exclude analytes molecules based on their
hydrodynamic volume. Vr is correlated to M.W. by calibration curve.
Stationary Phase: Porous polymeric particles (SDVB) with pore diameters of 80, 100, 150,
300, 500 or 1000 Å.
Mobile Phase: Good solvent for polymer. Solvent must suppress all possible interactions
with the stationary phase surface.
Applications: Organic polymers, biopolymers.