HPLC Introduction. DISCOVERY. M.S. Tswett, 1903, Warsaw Polytechnic Institute ( Separation of the chlorophylls of green leaves extract ). Calcium carbonate and non polar eluent. HPLC Introduction. HPLC Retention. Adsorption Chromatography (NP, RP, IEX)
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Calcium carbonate and non polar eluent
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.
80% Octadecylsilica (ODS, C18)
10% Octylsilica (C8)
5% Butylsilica (C4)
2% Cyano (CN)
Separation of 2-Me-Phenol and 4-Me-Phenol in RP and NP
Tocopherols Tocotrienols R1 R2
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
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
Genesis silica column (250 x 4.6 mm, 4 m).
Mobile phase: Hexane-1,4-dioxane (96:4).
. J. Chromatogr. A, 881 (2000) 217-227
Food Chemistry, 76 (2002) 357 – 362.
solubility, pKa, UV spectra, log P, log D.
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 wwpH 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.
*Choosing the buffer: Wavelength considerations
*Choosing the Diluent: Solution stability/solubility
Step 3*: Gradient scouting studies (shallow/steep x 2 temps) with the
optimal wwpH (suitable buffer) of the aqueous phase.
(Autochrom Wave 3) Use crude and forced degraded samples.
Check for coeluting components
Step 4: Use Drylab or ACD (LC-Sim) to
determine the desired resolution/selectivity.
Step 4b: Screen additional
columns/ mobile phases with the
optimal wwpH of the aqueous
phase; use column switcher
Step 5: Verification run.
Check peak purity (MS and DAD)
Scale flow rate/gradient time
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).