HPLCIntroduction

DISCOVERY

M.S. Tswett, 1903, Warsaw Polytechnic Inst itute (Separation of the chlorophylls of green leaves extract)

Calcium carbonate and non polar eluent

HPLC Introduction

G S C G L C

G A S S F C

N P R P I E C

G P C G F C

S E C

C o l u m n

T L C P a p e r

P l a n a r

L I Q U I D

C H R O M A T O G R A P H Y

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

NORMAL PHASE

1980 2004

30% 5% Si l ica Gel

47% 44% Si l ica 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 NH2, or CN

Mobile Phase: Non-polar solvents (Hexane, CHCl3)

Applications: Non-polar and semi-polar samples; hexane soluble; positional isomers.

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

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% Octadecylsi l ica (ODS, C18)

10% Octylsi l ica (C8)

5% Butylsi l ica (C4)

3% Phenyl

2% Cyano (CN)

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

Reversed-Phase vs. Normal Phase

Nonspecif ic (hydrophobic) interactions are at least ten times weaker than polar

− small differences in component molecular structure could have a signif icant effect in their retention

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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

R1

CH3

R2

OH

OCH3

CH3

CH3CH3CH3CH3 CH3

Tocopherols

O CH3

CH3CH3CH3

CH3

OH

CH3

R1

R2

Tocopherols Tocotrienols R1 R2

α-Tocopherol (α-T) α-Tocotrienol (α-3)_ Me Meβ-Tocopherol (β-T) β-Tocotrienol (β-3) Me Hγ-Tocopherol (γ-T) γ-Tocotrienol (γ-3) H Meδ-Tocopherol (δ-T) δ-Tocotrienol (δ-3) H H

Tocotrienols

Genesis silica column (250 x 4.6 mm, 4 µm). Mobile phase: 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

Ion Exchange

Principle: Reversible adsorption of ions on S.P. with oppositely charged functional groups.

Anionic polymers are known as cation exchange resins 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- ( s t r o n g ), CO 2

- ( w e ak )

• For anions (anion exchange)- NR 4+ ( s t r on g ), NH 3

+ ( w eak )

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

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.

Step 2: Determine the optimal wwpH range of the aqueous portion of the mobile phase

by running one linear gradient with 6 different wwpH mobile phases (2 – 10.8) using an

automated pH screening approach. (AutoChrom Wave 1)Step 2a Multiple columns can be screened with a pH value within the optimal w

wpH range using same linear gradient. Select column that gives the best selectivity, check for co-eluting components. (AutoChrom Wave 2). Do Stopped Flow study with best column/pH.

Step 3*: Gradient scouting studies (shallow/steep x 2 temps) with the optimal w

wpH (suitable buffer) of the aqueous phase. (Autochrom Wave 3) Use crude and forced degraded samples.Check for coeluting components

Step 1: Analyze the molecule: Physicochemical properties:

solubility, pKa, UV spectra, log P, log D.

α Acceptable

Step 5: Verification run.Check peak purity (MS and DAD)

α Unacceptable

α Acceptable

Step 4b: Screen additional columns/ mobile phases with the optimal w

wpH of the aqueous phase; use column switcher

Step 4: Use Drylab or ACD (LC-Sim) to determine the desired resolution/selectivity.

Finished

Optimize Speed:Scale flow rate/gradient time

Not spectrally homogeneous

Spectrally homogeneous

*Choosing the buffer: Wavelength considerations*Choosing the Diluent: Solution stability/solubility Considerations-

Published Jan. 2, 2007

PART I HPLC THEORY AND PRACTICE. 1 Introduction (Yuri Kazakevich and Rosario LoBrutto).

2 HPLC Theory (Yuri Kazakevich). 3 Stationary Phases (Yuri Kazakevich and Rosario LoBrutto).

4 Reversed-Phase HPLC (Rosario LoBrutto and Yuri Kazakevich). 5 Normal-Phase HPLC (Yong Liu and Anant Vailaya).

6 Size-Exclusion Chromatography (Yuri Kazakevich and Rosario LoBrutto). 7 LC/MS: Theory, Instrumentation, and Applications to Small Molecules

(Guodong Chen, Li-Kang Zhang, and Birendra N. Pramanik). 8 Method Development (Rosario LoBrutto). .

9 Method Validation (Rosario LoBrutto and Tarun Patel). . 10 Computer-Assisted HPLC and Knowledge Management (Yuri Kazakevich, Michael McBrien, and Rosario LoBrutto). PART II HPLC IN THE PHARMACEUTICAL INDUSTRY.

11 The Expanding Role of HPLC in Drug Discovery (Daniel B. Kassel). 12 Role of HPLC in Preformulation (Irina Kazakevich).

13 The Role of Liquid Chromatography–Mass Spectrometry in Pharmacokinetics and Drug Metabolism

(Ray Bakhtiar, Tapan K. Majumdar, and Francis L. S. Tse). 14 Role of HPLC in Process Development (Richard Thompson and Rosario LoBrutto). .

15 Role of HPLC During Formulation Development (Tarun S. Patel and Rosario LoBrutto).

16 The Role of HPLC in Technical Transfer and Manufacturing (Joseph Etse). PART III HYPHENATED TECHNIQUES AND SPECIALIZED HPLC SEPARATIONS.

17 Development of Fast HPLC Methods (Anton D. Jerkovich and Richard V. Vivilecchia). 18 Temperature as a Variable in Pharmaceutical Applications (Roger M. Smith).

19 LC/MS Analysis of Proteins and Peptides in Drug Discovery (Guodong Chen, Yan-Hui Liu, and Birendra N. Pramanik).

20 LC-NMR Overview and Pharmaceutical Applications (Maria Victoria Silva Elipe). 21 Trends in Preparative HPLC (Ernst Kuesters).

22 Chiral Separations (Nelu Grinberg, Thomas Burakowski, and Apryll M. Stalcup).

•LoBrutto,R.*, Kazakevich, Y.V.* “Chaotropic effects in RP-HPLC” (Invited Review) for volume 44 of “The Advances in Chromatography” series, editors, Professor Eli Grushka and Nelu Grinberg (September 2005).•LoBrutto, R. Normal Phase Stationary Phases (Encyclopedia chapter), Cazes, J., Editor, "Encyclopedia of Chromatography", New York, Marcel Dekker, 553-556 (2001).•Grinberg, N., LoBrutto, R. Efficiency in Chromatography, (Encyclopedia chapter), Cazes, J., Editor, "Encyclopedia of Chromatography", New York, Marcel Dekker, 274-276 (2001).•LoBrutto, R., Kazakevich, Y.V. Retention of Ionizable Components in Reversed Phase HPLC (Book Chapter), Practical Problem Solving in HPLC, Wiley-VCH, 122-158 (2000).•Jerkovich, A.D*, LoBrutto,R., and Vivilecchia, R.V., The Use of Acquity UPLC™ in Pharmaceutical Development, published in LC-GC,( 2005 )•Chan, F, Yeung, L.S, LoBrutto,R*, Kazakevich,Y*. Characterization of phenyl-type HPLC adsorbents • Journal of Chromatography A, 1069, Issue 2,April 2005, 217-224.•Kazakevich, Y*., LoBrutto,R* and Vivilecchia, R. Reversed-Phase HPLC Behavior of Chaotropic Counteranions, Journal of Chromatography, 1064, 9-18, (2005)•Pan, L, LoBrutto, R.*, Kazakevich, Y.*, and Thompson, R. Influence of inorganic mobile phase additives on the retention, efficiency, and peak symmetry of protonated basic compounds in reversed phase liquid chromatography, Journal of Chromatography A, 1049, 63 -73 (2004).•Jones, A., LoBrutto, R*., Kazakevich, Y.V.* Effect of the counter-anion type and concentration on the HPLC retention of beta-blockers, Journal of Chromatography A, 964, 179-187 (2002).•Rustamov, I., Farcas, T., Ahmed, F., Chan, F., LoBrutto, R., McNair, H.M,. Kazakevich, Y.V. Geometry of Chemically Modified Silica, Journal of Chromatography A, 913, 49 – 63 (2001).•Kazakevich, Y.V., LoBrutto, R., Chan, F., Patel, T. Interpretation of the excess adsorption isotherms of organic eluent components on the surface of reversed phase adsorbents: Effect on the analyte retention, Journal of Chromatography A, 913, 75-87 (2001).•LoBrutto, R., Jones, A., Kazakevich, Y.V. Effect of counter-anion concentration on HPLC retention of protonated basic analytes, Journal of Chromatography A, 913, 189 – 196 (2001).•LoBrutto, R., Jones, A., Kazakevich, Y.V., McNair, H.M. Effect of the Eluent pH and Acidic Modifiers on the HPLC Retention of Basic Analytes, Journal of Chromatography A, 913, 173-187 (2001).

HPLC References