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
Nonspecific (hydrophobic) interactions are at least ten times weaker than polar
small differences in component molecular structure could have a significant effect in their retention
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
Experimental Comparison of NP and RP HPLC Tocopherols Tocopherols Tocotrienols R 1 R 2 -Tocopherol ( -T) -Tocotrienol ( -3)_ Me Me -Tocopherol ( -T) -Tocotrienol ( -3) Me H -Tocopherol ( -T) -Tocotrienol ( -3) H Me -Tocopherol ( -T) -Tocotrienol ( -3) H H Tocotrienols 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 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.
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.
For cations (cation exchange) - SO 3 - (strong) , CO 2 - (weak)
For anions (anion exchange)- NR 4 + (strong) , NH 3 + (weak)
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
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 w pH range of the aqueous portion of the mobile phase by running one linear gradient with 6 different w w pH 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 w pH 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 w pH (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, pK a , UV spectra, log P, log D. Acceptable Step 5 : Verification run. Check peak purity (MS and DAD) Unacceptable Acceptable Step 4b: Screen additional columns/ mobile phases with the optimal w w pH 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).