Introduction to High Performance Liquid

Chromatographic (HPLC) Modes

Dr. Shulamit Levin

Medtechnica
Levins@medtechnica.co.il
Shulal@zahav.net.il
Homepage: http://www.forumsci.co.il/HPLC
 High Performance Liquid Chromatography - HPLC
Introduction

What does HPLC mean?

Introduction to High Performance Liquid High pressure liquid chromatography
Chromatographic (HPLC) Modes
High priced liquid chromatography

Dr. Shulamit Levin Hewlett-Packard liquid chromatography

Medtechnica High performance liquid chromatography
Levins@medtechnica.co.il
Shulal@zahav.net.il Hocus pocus liquid chromatography
Homepage: http://www. forumsci.co.il/HPLC High patience liquid chromatography

HPLC in Pharmaceutics APPLICATIONS OF HPLC

Technique No 1
Veterinary
Environmental
Discovery Testing Stability and
(Pharmakokinetics & Formulation
pharmacodynamics)

Phase I Phase II Phase III

Trial Trial Trial

Lead NDA:
compound
selection
New Drug
Application Agriculture & Chemistry
Food
Chemical Safety
Synthesis Testing
Scale-up Biomedical and Clinical

Dr. Shulamit Levin, Medtechnica

1
 High Performance Liquid Chromatography - HPLC
Introduction

Chromatographic Process The beginning:

Mobile phase
B+A Gravitational Chromatography

Stationary
Phase

B Distribution:
K = C s/C m B

Elution through the Column

Chromatogram

Comparison of Performance
Detector
1.Fucose
2.Galactosamine
300
3.Glucosamine
4.Galactose
5.Glucose
1 6.Mannose
2

mV 3
4
6

Control & 00
.0

50
.0
Minutes
5

20.00

Data
Processing Waste 1
High Performance
0.8

Normalized concentration
0.6
0.4
0.2
0
0 5 10 15 20

1 Low Performance
0.8
0.6
Fraction 0.4
a b
cd Collector 0.2
0
Pump Auto HPLC Column
0 5 10 15 20
flows 50-5000µL/min) in Oven
Sampler Elution volume (mL)

Dr. Shulamit Levin, Medtechnica

2
 High Performance Liquid Chromatography - HPLC
Introduction

Quantitative Determination and Identification

Response AMQ HPLC COURSE LAYOUT

• Introduction & Applicability

• Modes of Chromatography
• Quantitative work and System Qualification.

20.00 40.00 60.00

tR Minutes

CHROMATOGRAPHY PACKED COLUMN

SFC
PRINCIPLE OF SEPARATION:

LIQUID GAS MOBILE

ADSORPTION
PHASE PARTITION
NORMAL PHASE
REVERSED PHASE

SOLUTES:
PLANAR COLUMN
STATIONARY
PHASE LIPOPHYLIC: MOST OF THE
OILS, FATS, LIPIDS BIOMEDICAL SUBSTANCES

PAPER, SILICA, ALUMINA CAPILLARY CONDITIONS:

PACKED
ORGANIC SOLVENTS: n-HEXANE, HEPTANE, AQUEOUS MIXTURES WITH
CHLOROFORM, ALCOHOLS METHANOL, ACETONITRILE AND
ADDITIVES (BUFFERS, ION -PAIRS)

Dr. Shulamit Levin, Medtechnica

3
 High Performance Liquid Chromatography - HPLC
Introduction

High Performance Liquid Chromatography

PRINCIPLE OF SEPARATION: REVERSED PHASE

SOLUTES:
ION -EXCHANGE SIZE-EXCLUSION BIO-AFFINITY CHIRALITY
MOST OF THE
BIOMEDICAL SUBSTANCES
SOLUTES:

INORGANIC POLYMERS, CONDITIONS:

PROTEINS &
IONS, ACIDS, PROTEINS, ENZYMES ENANTIOMERS
BASES NUCLEIC ACIDS

CONDITIONS:
AQUEOUS MIXTURES WITH METHANOL, ACETONITRILE AND
AQUEOUS AQUEOUS OR ADDITIVES (BUFFERS, ION -PAIRS)
AQUEOUS BUFFERS, AQUEOUS BUFFERS OR
IONIC SOLUTIONS BUFFERS AND ORGANIC
ORGANIC SOLVENTS SPECIAL SOLVENTS
ADDITIVES

IONIZABLE
R4
O MOBILE PHASE STATIONARY PHASE
R1 N R3 R C OH
R2 CARBOXYLIC ACIDS SOLVENTS: CHEMISTRY:
AMINES - 1,2,3,4
water, methanol, acetonitrile
* BONDED HYDROCARBON:
C-18, C-8, C-4, C-1
R OH ADDITIVES:
NH buffers, salts, ion-pairing * % COVERAGE
ALCOHOLS OH OH
reagents, complexants.
* ADSORBED SURFACTANTS
R O P OH R P OH
O O * TYPE OF SILICA GEL
PHOSPHATES PHOSPHONATES

pores
GEOMETRY
OH OH * SPHERE- IRREGULAR
R O S OH R S OH R SH silica * PARTICLE DIAMETER
O O
SULPHATES SULPHONATES
* POROSITY
THIOLS

Dr. Shulamit Levin, Medtechnica

4
 High Performance Liquid Chromatography - HPLC
Introduction
LIPOPHYLIC
ELUTION ORDER IN 1 2 3 ISOCRATIC vs GRADIENT
REVERSED PHASE 9
1
R 2 3 6 7 8 10 11
1 2 3 E 4
1
CH 3 CH3
S 5
R 2 3 P 0
O VOID
E CH3
S N
P 1 S 9
2 3
O E 1
OH OH 4 8 10
N 2 3 67 11
S 0 OH
E VOID 5
1 2 3 OH
0 VOID
OH OH

TIME (MIN.) TIME (MIN.)

NORMAL PHASE SOLUTES

NORMAL PHASE O
COOH
O
CH3 CH3 CH3
H 3C
H3 C
COOH
CH3 CH3 CH3
ADSORPTION OH OH OH OH CH3
CH3
PROSTAGLANDINS β-CAROTENE

SOLUTES:
O PHOSPHOLIPIDS BILIRUBIN
R1 C O CH2 H2 C
LIPOPHYLIC: CH3
CH2
OILS, FATS, LIPIDS
R2 C O CH OO
O H3 C
O NH HN
H2C O P O R3
CONDITIONS: OH NH HN
CH3
R 1, R2 - HYDROCARBON CHAIN OF FATTY ACID H3 C
R 3 - SERINE O O
ORGANIC SOLVENTS: n-HEXANE, CHOLINE CH OH
HEPTANE, CHLOROFORM, ALCOHOLS ETHANOLAMINE 2
HO OH
H O
INOSITOL H
HOCH 2 O H

GLYCEROL OH H
HO O CH2OH
H OH OH H
SUCROSE

Dr. Shulamit Levin, Medtechnica

5
 High Performance Liquid Chromatography - HPLC
Introduction

LIPOPHYLIC

NORMAL PHASE ELUTION ORDER IN 1 2 3

NORMAL PHASE
SOLVENTS: n-hexane, chloroform , ethanol, 2-propanol

1 1 2 3
CH3 CH 3
R 2 3
E CH3
S
P 1 2 3
A O OH OH
N
PORE S 0 OH
E VOID
1 OH 2 3
OH OH

silica
TIME (MIN.)

High Performance Liquid Chromatography

Ion Exchange Chromatography
PRINCIPLE OF SEPARATION:
Cation Exchange vs Anion Exchange

ION -EXCHANGE SIZE-EXCLUSION BIO-AFFINITY CHIRALITY

Cation Exchange Anion Exchange
SOLUTES:

INORGANIC POLYMERS, PROTEINS &

IONS, ACIDS, PROTEINS, ENZYMES ENANTIOMERS
BASES NUCLEIC ACIDS

CONDITIONS:

AQUEOUS AQUEOUS OR
AQUEOUS BUFFERS, AQUEOUS BUFFERS OR
IONIC SOLUTIONS BUFFERS AND ORGANIC
ORGANIC SOLVENTS SPECIAL SOLVENTS Cation exchange columns have a negative charge to attract cations.
ADDITIVES Anion exchange columns have a positive charge to attract anions

Dr. Shulamit Levin, Medtechnica

6
 High Performance Liquid Chromatography - HPLC
Introduction

Ion Exchange Chromatography ION EXCHANGE

INSIDE A PORE IN THE STATIONARY PHASE
Strong vs. Weak Exchange Materials
SAMPLE IONS IN
Cation exchanger Anion exchanger - -- --
SO3- NR3+ +- - COUNTER IONS OUT
+- -
+ - -- -
MOBILE PHASE
ADDITIVES
STRONG +- - - -
+- + -- -
1. INJECTION + - -- - -
COO- NH3+ + -
+ -- +
+ --
- - --
+ -
+ - - -
WEAK 2. ADSORPTION:
+ -
DISPLACEMENT OF
COUNTER IONS +-
Strong Exchangers stay ionized as pH varies between 2 and 12. 3. ELUTION
Weak exchangers can lose ionization as a function of pH.

ELUTION ORDER IN ELUTION ORDER IN

ANION EXCHANGE DENSITY OF CHARGE CATION EXCHANGE DENSITY OF CHARGE

1 1 2 3 1 1 2 3
R 2 3 OAc - OH - F- R 2 3 K+ Na+ Li+
E E
S S
P P
O
1 2 3 O 1 2 3
N N
S Cl - NO3 -
NO2 -
S Ag+ Zn ++ Al+++
E 0 E 0
VOID VOID
1 2 3

NO3 - SO 4 -- SO 3 --
TIME (MIN.) TIME (MIN.)

Dr. Shulamit Levin, Medtechnica

7
 High Performance Liquid Chromatography - HPLC
Introduction

ELUTION ORDER IN ION EXCHANGE Analysis of Ions

CATION EXCHANGE Column: Waters IC-Pak Anion HC
ANION EXCHANGE
Eluent: Borate/ Gluconate
STRONGER ACID STRONGER BASE Flow rate: 2.0 mL/min
1. Fluoride 1 ppm
1.40 Injection vol.:
100 µL 2. Carbonate --------
Detection: Direct Conductivity 3. Chloride 2 ppm
1 1 4. Nitrite 4 ppm
4
3 5. Bromide 4 ppm
R 2 3 R 2 3 6. Nitrate 4 ppm
E E
S S 2 7. Phosphate 6 ppm
1
P P 8. Sulfate 4 ppm
uS 5 6
O O
7
N N 8
S S
E 0 E 0
VOID VOID

0.60

TIME (MIN.) TIME (MIN.) 0.00 10.00 20.00

Minutes

Analysis of Anions in Waste Water

Analysis of Anions
Original Sample
Duplicate Injections of
Column: Waters IC Pak A/HR Wastewater Cl = 34.2 ppm
Eluent: Borate / Gluconate Diluted 1:10 NO 2 = 3.0
Flow Rate: 1 mL/min
1 Fluoride = 1 ppm HCO3 Column: IC Pak A/HR SO4
Injection: 100 Lµ Cl NO 3 = 5.1
2 BiCarbonate Eluent: Borate / Gluconate
4 3 Chloride = 2 Flow: 1 mL / min
Pressure: 1120 psi SO 4 = 258
3 4 Nitrite =4 Conductivity: 220 uS
5 Bromide = 4 Injection: 100 uL
6 Nitrate =4
7 Phosphate = 6
8 Sulfate =4 ClO3? NO 3
5 6

0.15 uS
?

2.5 uS
? NO2
1.7 mS

1
7 8

NO 3
NO2

0.00 5.00 Minutes 10.00 15.00

5.00 Minutes 10.00 15.00

Dr. Shulamit Levin, Medtechnica

8
 High Performance Liquid Chromatography - HPLC
Introduction

ENANTIOMERS: MIRROR IMAGES OF ONE ANOTHER

Circular Dichroism SPECTRA
600
a d
400 peak II 200
200
peak II
0
0

CH3 H3C -200

-400 peakI -200

peakI
-600

-800 -400

R
220 240 260 280 300 320 340 360 380 400 220 240 260 280 300 320 340 360 380 400

R 100 b 600
e
peakI 400
peakI
50
200

H H
0 0

-200

HOOC COOH -50

-100 peak II
-400

-600
peak II

-800
-150

240 260 280 300 320 340 360 380 400 220 240 260 280 300 320 340 360 380 400

1.0
200 c peakI f
100
peak II 0.5

0 0.0

-100
peakI -0.5

-200 peak II
-1.0

220 240 260 280 300 320 340 360 380 400 220 240 260 280 300 320 340 360 380 400

Asymmetric Synthesis BASIS FOR SEPARATION: CHIRAL RECOGNITION

ENANTIOMERS STATIONARY
CHIRAL SELECTOR
CH 2OH H
CH 2OCO C(CH 3)3
R B C R
CH 2OH CH 2OCO C(CH 3)3 OH
O N
O
C H
(-)
C6H13
O OH O

(+)-α -Pinene HU-211

O N NO 2
2

CH 2OH
CH 2OCOC(CH 3) 3
R B
H
CH 2OH CH 2OCOC(CH 3) 3 OH O C R
O N
C H
OH
(+)
O C6H 13
O

(-)-α -Pinene HU-210

O N NO 2
2

Dr. Shulamit Levin, Medtechnica

9
 High Performance Liquid Chromatography - HPLC
Introduction

SEPARATION OF ENANTIOMERS OF
TERPENOIDS
Chiral stationary phases: (+) cis- verbenol cis-4-hydroxy-myrtenyl pivalate

(+) CH 2OCOC(CH3) 3

(-)
OH OH

vLigand exchange (-)

vπ-Donor π-acceptor (Pirkle)

vChiral Host-guest (cyclodextrin)
vBonded macrocyclic antibiotics (+) Verbenone 4-oxo-myrtenyl pivalate
C H2 O C O C ( C H
3 )3

vImmobilized/bonded proteins (+) O

vImmobilized/bonded polysaccharides
O

(-) (-)

SEPARATION OF 6 ENANTIOMERIC PAIRS OF

CANNABINOIDS SIZE EXCLUSION CHROMATOGRAPHY
(+) / ( -) ∆ 1-THC
(+) / ( -) CBD HU- 210 + HU 211 PRINCIPLE OF SEPARATION SEPARATION PROCESS:
(+) (+) (-)
(-)
(+) STATIONARY
MOBILE
PHASE PHASE
(-)

(-) (+)
(+) (+) (-)
(-) Gel Permeation mechanism

Scanning electron micrograph

(+) / ( -) ∆ 6-THC
(+) / ( -) 7-OH ∆ 6-THC
of an agarose gel.
Magnification x 50,000.
Ref. Anders S. Medin,PhD
HU- 243 + HU 251 Thesis, Uppsala University ELUTION ORDER:
1995. LARGER ELUTE FIRST

Dr. Shulamit Levin, Medtechnica

10
 High Performance Liquid Chromatography - HPLC
Introduction

GPC Process ELUTION ORDER IN

SIZE EXCLUSION (GPC)
MW

1 100,000
1 2 3 VOID
R 2 50,000
E
S
P 3 20,000
O 0
N
S
E

ELUTION VOLUME (mL)

THEORETICAL CURVE OF THE STERIC

Gel Filtration/Size Exclusion/Gel Permeation EXCLUSION

TOTAL EXCLUSION
Vi - V0
=K
Polymer distribution Vt - V0
1M 500 K 250 K 100 K 25 K
log PARTIAL
PENETRATION
MW
Narrow standards Vi 0 <= K =< 1
K
Calibration
curve
1M
Log 500K
Mw 250K
100K
25K

TOTAL PENETRATION
Elution Time or Volume
V0 Vt
ELUTION VOLUME

Dr. Shulamit Levin, Medtechnica

11
 High Performance Liquid Chromatography - HPLC
Introduction

ELUTION CURVES OF VARIOUS

STATIONARY PHASES MOLECULAR WEIGHT
DISTRIBUTION
LINEAR (MIXED BED)

R
log PORES E
SIZE
MW S
106? P
O
105? N
S
104? E
103?
500?

ELUTION VOLUME ELUTION VOLUME (mL)

What is GPC? Affinity Chromatograpy

Symbolic representation of a Target sample molecule

section of an AC bead surface with full affinity for the Symbolic representation
4The elution profile represents the molecular weight distribution based upon ligand of a section of
the relative content of different molecular weights… an AC bead surface

Mp

Mw
Mn

Mz

Mz+1 Sample molecules with

no affinity for the ligand
largest smallest

elution volume (retention time)

AC relies upon a reversible highly specific binding reaction.

Dr. Shulamit Levin, Medtechnica

12
 High Performance Liquid Chromatography - HPLC
Introduction

Affinity Hydrophobic Interaction Chromatography (HIC)

Chromatograpy
Slightly Highly
1. Equilibration 2. Sample application and wash 3. Elution
Reasonable Quite
hydrophobic hydrophobic
The column is conditioned to The sample is applied under binding conditions. The target molecule is hydrophobic hydrophobic
promote adsorption of the target The target molecule binds specifically to the desorbed and eluted by
sample contaminant.
affinity ligands , while all other sample
sample sample
molecule by equilibrating it with switching to elution component.
binding buffer. components are washed through. buffer. component component.

1. Equilibration. 2. Sample 3. Gradient elution. 4.

application and Elution order: Regeneration
wash.

Methods Development Strategy

Seven Basic Considerations in Choosing HPLC Operating
Parameters
Gather Information

1) Solubility- Hexane, Chloroform, Methanol, Water (buffer pH), other? Make a Plan

2) Molecular Weight - Would GPC be useful in either the

analysis or sample prep? Optimize K’
Incomplete resolution Complete Resolution
3) Functional Groups - Any ionizable groups? Acidic, Basic, or Neutral?
4) Sample Matrix - What amounts are expected in matrix for Change Alpha
either analytical or preparative isolation? Incomplete resolution Complete Resolution
Optimize N
5) Levels in Matrix - What amounts are expected in matrix for either
analytical or preparative isolation? Incomplete resolution Complete Resolution
Validate Qualitation
6) Detectability- Any chromophores or fluorophores?
FAIL
FAIL PASS
Consider Redox or derivatization.
Together with point #5, an appropriate detector is chosen.
Validate Qualitation
7) How Do Species Differ - An important clue to manipulate selectivity in FAIL
FAIL PASS
the separation, especially if compounds are similar in their structure.
Evaluate and Optimize method for routine use

- Step by step method development strategy -

Dr. Shulamit Levin, Medtechnica

13