M.S. Tswett, 1903, Warsaw
Polytechnic Institute (Separation
of the chlorophylls of green leaves
Calcium carbonate and non polar eluent
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 lu m n
T L C P a p e r
P la n a r
L I Q U I D
C H R O M A T O G R A P H Y
Adsorption Chromatography (NP, RP, IEX)
− Interactions of the analyte with the adsorbent surface
causing its slower movement compared to the eluent
− Exclusion of the analyte molecules from the
adsorbent pore volume due to their size
− No interactions with the adsorbent surface
30% 5% Silica Gel
47% 44% Silica based bonded phases
3% 1% Alumina
20% 50% Chiral Bonded Phases
Principle: Adsorption of analytes on the polar, weakly acidic surface of
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;
NP: SEPARATION PRINCIPLE
Polar (specific but nonionic) interactions of analyte with polar
adsorption sites (SiOH, -NH2, -CN, Diol) cause its retention
Different sorption affinities between analytes result in their
− More polar analytes retained longer
− Analytes with larger number of polar functional group are
− Structural isomers are often separated
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,
10% Octylsilica (C8)
5% Butylsilica (C4)
2% Cyano (CN)
Nonpolar (nonspecific) interactions of analyte with hydrophobic
adsorbent surface (-C18, C8, Phenyl, C4)
Different sorption affinities between analytes results in their
− More polar analytes retained less
− Analytes with larger hydrophobic part are retained
− structural isomers maybe more challenging in this mode
Reversed-Phase vs. Normal Phase
Nonspecific (hydrophobic) interactions are at least ten times weaker than
− small differences in component molecular structure could have a
significant effect in their retention
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
0 1 2 3 4 5 6 7 8 9 10
Separation of 2-Me-Phenol and 4-Me-Phenol in RP and NP
Tocopherols Tocotrienols R1
α-Tocopherol (α-T) α-Tocotrienol (α-3)_ Me Me
β-Tocopherol (β-T) β-Tocotrienol (β-3) Me H
-Tocopherol (γ-T) γ-Tocotrienol (γ-3) H Me
-Tocopherol (δ-T) δ-Tocotrienol (δ-3) H H
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
Principle: Reversible adsorption of ions on S.P. with oppositely charged functional
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
Cationic polymers on the other hand are known as anion exchange
resins and these resins can also be weak or strong anion
• For cations (cation exchange) - SO3
• For anions (anion exchange)- NR4
Mobile Phase: Aqueous buffer with pH and buffer strength carefully controlled.
Applications: All ionic compounds common anions, cations, sugars, amines, etc.
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 w
wpH range of the aqueous portion of the mobile phase
by running one linear gradient with 6 different w
wpH 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
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:
solubility, pKa, UV spectra, log P, log D.
Step 5: Verification run.
Check peak purity (MS and DAD)
Step 4b: Screen additional
columns/ mobile phases with the
wpH of the aqueous
phase; use column switcher
Step 4: Use Drylab or ACD (LC-Sim) to
determine the desired resolution/selectivity.
Scale flow rate/gradient time
*Choosing the buffer: Wavelength
*Choosing the Diluent: Solution
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
(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).