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

Introduction
What does HPLC mean?

Introduction to High Performance Liquid Chromatographic (HPLC) Modes

Dr. Shulamit Levin Medtechnica
Levins@medtechnica.co.il Shulal@zahav.net.il

High pressure liquid chromatography High priced liquid chromatography Hewlett-Packard liquid chromatography High performance liquid chromatography Hocus pocus liquid chromatography High patience liquid chromatography

Homepage: http://www. forumsci.co.il/HPLC

HPLC in Pharmaceutics Technique No 1

Discovery Testing (Pharmakokinetics & pharmacodynamics) Stability and Formulation

APPLICATIONS OF HPLC
Veterinary Environmental

Phase I Trial

Phase II Trial

Phase III Trial

Lead compound selection

NDA: New Drug Application

Agriculture & Food Biomedical and Clinical

Chemistry

Chemical Synthesis Scale-up

Safety Testing

Dr. Shulamit Levin, Medtechnica

Introduction
Chromatographic
B+A
Stationary Phase

Process
Mobile phase

The beginning: Gravitational Chromatography

A B Distribution: K = C s/C m B

A Elution through the Column Chromatogram

30 0

1.Fucose 2.Galactosamine 3.Glucosamine 4.Galactose 5.Glucose

6.Mannose

mV

Detector
20.00

Comparison of Performance

4 5

Control & Data Processing

00 0 . 50 0 .

Minutes

Waste
Normalized concentration

1 0.8 0.6 0.4 0.2 0 0

High Performance

10

15

20

1 0.8 0.6 0.4 0.2 0 0 5 10

Low Performance

a b cd Pump
flows 50-5000L/min)

Fraction Collector Auto Sampler

HPLC Column in Oven

15

20

Elution volume (mL)

Dr. Shulamit Levin, Medtechnica

Introduction
Quantitative Determination and Identification
AMQ

HPLC COURSE LAYOUT

Introduction & Applicability Modes of Chromatography Quantitative work and System Qualification.

Response

20.00

tR

40.00

60.00

Minutes

CHROMATOGRAPHY

SFC

PACKED COLUMN PRINCIPLE OF SEPARATION:

LIQUID

GAS

MOBILE PHASE

ADSORPTION

NORMAL PHASE

PARTITION

REVERSED PHASE SOLUTES:

PLANAR

COLUMN

STATIONARY PHASE

LIPOPHYLIC: OILS, FATS, LIPIDS

MOST OF THE BIOMEDICAL SUBSTANCES

PAPER, SILICA, ALUMINA PACKED

CAPILLARY

CONDITIONS:
ORGANIC SOLVENTS: n-HEXANE, HEPTANE, CHLOROFORM, ALCOHOLS

AQUEOUS MIXTURES WITH METHANOL, ACETONITRILE AND ADDITIVES (BUFFERS, ION -PAIRS)

Dr. Shulamit Levin, Medtechnica

Introduction
High Performance Liquid Chromatography PRINCIPLE OF SEPARATION:

REVERSED PHASE
SOLUTES:
BIO-AFFINITY CHIRALITY MOST OF THE BIOMEDICAL SUBSTANCES

ION -EXCHANGE

SIZE-EXCLUSION

SOLUTES:
INORGANIC IONS, ACIDS, BASES POLYMERS, PROTEINS, NUCLEIC ACIDS PROTEINS & ENZYMES ENANTIOMERS

CONDITIONS:

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

IONIZABLE
R4 R1 N R3 R2
AMINES - 1,2,3,4

CARBOXYLIC ACIDS

O R C OH

MOBILE PHASE
SOLVENTS: water, methanol, acetonitrile ADDITIVES: buffers, salts, ion-pairing reagents, complexants.

STATIONARY PHASE
CHEMISTRY: * BONDED HYDROCARBON: C-18, C-8, C-4, C-1 * % COVERAGE * ADSORBED SURFACTANTS * TYPE OF SILICA GEL

NH

R OH
ALCOHOLS

OH R O P OH O
PHOSPHATES

OH R P OH O
PHOSPHONATES

pores
OH R O S OH O
SULPHATES

GEOMETRY * SPHERE- IRREGULAR * PARTICLE DIAMETER * POROSITY

OH R S OH O
SULPHONATES THIOLS

R SH

silica
d

Dr. Shulamit Levin, Medtechnica

Introduction
ELUTION ORDER IN REVERSED PHASE
1 R E S P O N S E 2 3
LIPOPHYLIC

3 R E S P 0 O N S E

ISOCRATIC vs GRADIENT
9 1 2 3 4 5 VOID 9 1 2 3 4 5 67 8 10 11 6 7 8 10 11

2
CH 3

3
CH3

CH3

1
OH

2
OH

VOID 1

OH

2
OH OH

OH

0 VOID

TIME (MIN.)

TIME (MIN.)

NORMAL PHASE
ADSORPTION

NORMAL PHASE SOLUTES

O COOH OH O COOH OH OH
CH3 CH3 CH3 CH3 -CAROTENE H 3C H3 C CH3 CH3

OH

CH3 CH3

PROSTAGLANDINS

SOLUTES:
LIPOPHYLIC: OILS, FATS, LIPIDS
R1 R2 O C C O O O

PHOSPHOLIPIDS
CH2 CH H2C O O P O R3 OH
H3 C O HO
2

BILIRUBIN
H2 C OO NH NH HN HN CH3 CH2

H3 C

CONDITIONS:
ORGANIC SOLVENTS: n-HEXANE, HEPTANE, CHLOROFORM, ALCOHOLS

R 1, R2 - HYDROCARBON CHAIN OF FATTY ACID R 3 - SERINE CHOLINE CH OH ETHANOLAMINE H O HOCH INOSITOL H GLYCEROL OH H
2

CH3 O OH

HO H

O OH OH

CH2OH H

SUCROSE

Dr. Shulamit Levin, Medtechnica

Introduction
LIPOPHYLIC

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

ELUTION ORDER IN NORMAL PHASE

1 R E S P O N S E 2 3

1
CH3

2
CH 3

CH3

A PORE

1 0

2
OH

3
OH

VOID 1
OH

OH

2
OH

3
OH

silica
TIME (MIN.)

High Performance Liquid Chromatography PRINCIPLE OF SEPARATION:

Ion Exchange Chromatography

Cation Exchange vs Anion Exchange

ION -EXCHANGE

SIZE-EXCLUSION

BIO-AFFINITY

CHIRALITY

Cation Exchange SOLUTES:

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

Anion Exchange

CONDITIONS:
AQUEOUS BUFFERS, IONIC SOLUTIONS AQUEOUS BUFFERS OR ORGANIC SOLVENTS AQUEOUS BUFFERS AND SPECIAL ADDITIVES AQUEOUS OR ORGANIC SOLVENTS

Cation exchange columns have a negative charge to attract cations. Anion exchange columns have a positive charge to attract anions

Dr. Shulamit Levin, Medtechnica

Introduction
Ion Exchange Chromatography
Strong vs. Weak Exchange Materials

ION EXCHANGE
INSIDE A PORE IN THE STATIONARY PHASE
SAMPLE IONS IN

Cation exchanger

Anion exchanger

SO3STRONG

NR3+

+++ ++1. INJECTION

- -- --

COUNTER IONS OUT

COOWEAK

NH3+

+ + - -+ + -+

-- - + + -+ + +3. ELUTION

2. ADSORPTION: DISPLACEMENT OF COUNTER IONS

-- - - - -

MOBILE PHASE ADDITIVES

Strong Exchangers stay ionized as pH varies between 2 and 12. Weak exchangers can lose ionization as a function of pH.

ELUTION ORDER IN ANION EXCHANGE

1 R E S P O N S E 2 3

DENSITY OF CHARGE

ELUTION ORDER IN CATION EXCHANGE

3 FR E S P O N S E 1 2 3

DENSITY OF CHARGE

1 OAc -

2 OH -

1 K+ 1

2 Na+ 2 Zn ++

3 Li+ 3 Al+++

1 Cl 0 VOID 1 NO3 TIME (MIN.)

2 NO3 2 SO 4 --

3 NO2 3 SO 3 --

0 VOID

Ag+

TIME (MIN.)

Dr. Shulamit Levin, Medtechnica

Introduction
ELUTION ORDER IN ION EXCHANGE
ANION EXCHANGE STRONGER ACID
1 R E S P O N S E 2 3 R E S P O N S E

Analysis of Ions
Column: Waters IC-Pak Anion HC Eluent: Borate/ Gluconate Flow rate: 2.0 mL/min Injection vol.: L 100 Detection: Direct Conductivity
4 3 1 2 5 6 7 8

CATION EXCHANGE STRONGER BASE

1 2 3
1.40

uS

1. Fluoride 1 ppm 2. Carbonate -------3. Chloride 2 ppm 4. Nitrite 4 ppm 5. Bromide 4 ppm 6. Nitrate 4 ppm 7. Phosphate 6 ppm 8. Sulfate 4 ppm

VOID

VOID

0.60

TIME (MIN.)

TIME (MIN.)

0.00

10.00 Minutes

20.00

Analysis of Anions in Waste Water

Analysis of Anions
Column: Waters IC Pak A/HR Eluent: Borate / Gluconate Flow Rate: 1 mL/min Injection: 100 L
4 3

Duplicate Injections of Wastewater Diluted 1:10

Original Sample Cl NO
SO4

= 34.2 ppm
2 3 4

= 3.0 = 5.1 = 258

1 2 3 4 5 6 7 8

Fluoride = 1 ppm BiCarbonate Chloride = 2 Nitrite =4 Bromide = 4 Nitrate =4 Phosphate = 6 Sulfate =4

2.5 uS

HCO3 Cl

Column: IC Pak A/HR Eluent: Borate / Gluconate Flow: 1 mL / min Pressure: 1120 psi Conductivity: 220 uS Injection: 100 uL

NO SO

0.15 uS

ClO3? ? ? NO2

NO 3

1.7 m S

1 7 2

NO2

NO 3

0.00

5.00

Minutes

10.00

15.00

5.00

Minutes

10.00

15.00

Dr. Shulamit Levin, Medtechnica

Introduction
ENANTIOMERS: MIRROR IMAGES OF ONE ANOTHER

Circular Dichroism SPECTRA

600 400 200 0 0

a
peak II
200

d
peak II

CH3 R HOOC H H

H3C R

-200 -400 -600 -800 -400

peakI

-200

peakI

220

240

260

280

300

320

340

360

380

400

220

240

260

280

300

320

340

360

380

400

100 50 0

b
peakI

600 400 200 0 -200

e
peakI

COOH

-50 -400 -100 -150 240 260 280 300 320 340 360 380 400 1.0 200 220 240 260 280 300 320 340 360 380 400

peak II

-600 -800

peak II

c
peak II
0 0.5

peakI

100

0.0

-100 -200

peakI

-0.5

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
CH 2OH CH 2OH CH 2OCO C(CH 3)3 CH 2OCO C(CH 3)3 OH

BASIS FOR SEPARATION: CHIRAL RECOGNITION

ENANTIOMERS STATIONARY CHIRAL SELECTOR
B O C H C N H

R
O

OH

C6H13

(-)

(+)- -Pinene

HU-211
O
2

NO

CH 2OH CH 2OH CH 2OCOC(CH 3) 3 CH 2OCOC(CH 3) 3 OH

R
O O

H C N H

OH

C6H 13

(+)

(-)- -Pinene

HU-210
O
2

NO

Dr. Shulamit Levin, Medtechnica

Introduction
SEPARATION OF ENANTIOMERS OF TERPENOIDS

Chiral stationary phases:

vLigand exchange v-Donor -acceptor (Pirkle) vChiral Host-guest (cyclodextrin) vBonded macrocyclic antibiotics vImmobilized/bonded proteins vImmobilized/bonded polysaccharides

(+) (-)

cis- verbenol

cis-4-hydroxy-myrtenyl pivalate

(+)
OH

CH 2OCOC(CH ) 3 3

OH

(-)

(+)

Verbenone

4-oxo-myrtenyl pivalate
C H2 O C O C ( C H)3 3

(+) (-)

(-)

SEPARATION OF 6 ENANTIOMERIC PAIRS OF CANNABINOIDS

(+) / ( -) 1-THC (+) / ( -) CBD HU- 210 + HU 211

SIZE EXCLUSION CHROMATOGRAPHY

PRINCIPLE OF SEPARATION SEPARATION PROCESS:

(+)

(+) (-)

(-)

(-) (+)
MOBILE PHASE STATIONARY PHASE

(+)

(-)

(+)

(-)

(+) (-)

Gel Permeation mechanism

Scanning electron micrograph of an agarose gel. Magnification x 50,000. Ref. Anders S. Medin,PhD Thesis, Uppsala University 1995.

(+) / ( -) 6-THC

(+) / ( -) 7-OH 6-THC

HU- 243 + HU 251

ELUTION ORDER: LARGER ELUTE FIRST

Dr. Shulamit Levin, Medtechnica

Introduction
GPC Process
ELUTION ORDER IN SIZE EXCLUSION (GPC)
MW 1
R E S P O N S E

1
VOID

100,000 50,000 20,000

2
0

ELUTION VOLUME (mL)

Gel Filtration/Size Exclusion/Gel Permeation

THEORETICAL CURVE OF THE STERIC EXCLUSION

TOTAL EXCLUSION

Vi - V0 =K Vt - V0

Polymer distribution 1M 500 K 250 K 100 K 25 K

Narrow standards

log MW

PARTIAL PENETRATION

Vi K

0 <= K =< 1

Calibration curve

Log Mw

1M 500K 250K 100K 25K

Elution Time or Volume

TOTAL PENETRATION

V0

ELUTION VOLUME

Vt

Dr. Shulamit Levin, Medtechnica

Introduction
ELUTION CURVES OF VARIOUS STATIONARY PHASES
LINEAR (MIXED BED)

MOLECULAR WEIGHT DISTRIBUTION

log MW

PORES SIZE

106? 105? 104? 103? 500?

ELUTION VOLUME

R E S P O N S E

ELUTION VOLUME (mL)

What is GPC?
4The elution profile represents the molecular weight distribution based upon the relative content of different molecular weights

Affinity Chromatograpy
Symbolic representation of a section of an AC bead surface Target sample molecule with full affinity for the ligand Symbolic representation of a section of an AC bead surface

Mp Mw Mz Mn

Mz+1
largest elution volume (retention time) smallest

Sample molecules with no affinity for the ligand

AC relies upon a reversible highly specific binding reaction.

Dr. Shulamit Levin, Medtechnica

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

Hydrophobic Interaction Chromatography (HIC)

Slightly hydrophobic sample component.
1. Equilibration. 2. Sample application and wash.

Reasonable hydrophobic sample component

Quite hydrophobic sample component.

3. Gradient elution. Elution order:

Highly hydrophobic contaminant.

4. Regeneration

Methods Development Strategy

Seven Basic Considerations in Choosing HPLC Operating Parameters
1) Solubility- Hexane, Chloroform, Methanol, Water (buffer pH), other? 2) Molecular Weight - Would GPC be useful in either the analysis or sample prep? 3) Functional Groups - Any ionizable groups? Acidic, Basic, or Neutral? 4) Sample Matrix - What amounts are expected in matrix for either analytical or preparative isolation? 5) Levels in Matrix - What amounts are expected in matrix for either analytical or preparative isolation? 6) Detectability- Any chromophores or fluorophores? Consider Redox or derivatization. Together with point #5, an appropriate detector is chosen. 7) How Do Species Differ - An important clue to manipulate selectivity the separation, especially if compounds are similar in their in structure. Incomplete resolution Change Alpha Incomplete resolution Optimize N Incomplete resolution Validate Qualitation FAIL FAIL Validate Qualitation FAIL FAIL Evaluate and Optimize method for routine use PASS PASS Complete Resolution Complete Resolution Gather Information Make a Plan Optimize K Complete Resolution

- Step by step method development strategy -

Dr. Shulamit Levin, Medtechnica