1. New Hydrogen-Bonding Stationary Phases for Liquid Chromatography

2. Introduction Traditional liquid chromatography techniques include reversed phase (RP), normal-phase (NP), and ion-exchange (IE) based mechanism of separation. Secondary interactions such as hydrogen bonding, dipole-dipole, Pi-Pi interactions often contribute to a small extent to each of those techniques. We recently developed a new approach and corresponding stationary phase which allows conducting separation entirely based on the hydrogen bonding (HB) properties of molecules. This technique is extending the separation scope to many different molecules and it is based on presence of polar groups in the analytes. Many classes of compounds can be analyzed and separated in a simple mobile phase composition in both gradient and isocratic modes with unique selectivity. Some traditionally difficult separation methods can be easily achieved using this new technique. Some examples and key points of method development using hydrogen-bonding mechanism will be discussed.

3. 3 Types of liquid chromatography of small molecules Normal Phase Chromatography: Separation of molecules based on their difference in polar properties. Interaction with stationary phase which is more polar than mobile phase (dipol-dipol interaction). Reverse Phase Chromatography: Separation of molecules based on their difference in hydrophobic properties. Interaction with stationary phase which is less polar than mobile phase. Ion-Exchange Chromatography: Separation of ions based on their degree of interaction with the stationary phase carrying opposite charge.

4. 4 Existing column stationary phases technology Significant number of LC applications can be covered with few types of stationary phases. Reverse phase and ion-exchange mechanism are two dominant processes utilized for separation of small molecules. Reverse phase columnsIon-exchange columns Normal phase columns

5. 5 Molecule Properties Analyte Log P Log P = 4.8Log P = 2.7 Log P = 1.5Log P = 0.5 Log P = - 0.2Log P = - 1.8 Log P = - 3.2 012345- 4- 3- 2- 1 Reverse Phase Separation Column with hydrophobic surface Normal Phase Separation Column with hydrophilic surface MeCN/H 2 O (HILIC) H 2 O/MeCN H 2 O/MeOH Hexane/EtOH CO 2 / MeOH Typical mobile phases Typical conditions for different separation modes ColumnMobile phase Reverse phaseC8, C18, phenyl H 2 O/MeCN H 2 O/MeOH Normal phase Bare silica, diol, propylamino Hexane/EtOH CO 2 /MeOH HILIC (Hydrophilic Interaction Liquid Chromatography) Bare silica, diol, propylamino, zwitterionic MeCN/H 2 O

6. 6 Hydrogen bonds energy O−H :N (29 kJ/mol or 6.9 kcal/mol) O−H :O (21 kJ/mol or 5.0 kcal/mol) N−H :N (13 kJ/mol or 3.1 kcal/mol) N−H :O (8 kJ/mol or 1.9 kcal/mol) H-donor H-acceptors

7. 7 Silica gel for a hydrogen bond forming column H-donors H-acceptors

8. 8 Silica gel for a hydrogen bond forming column Advantages Available material Many versions available Stable at elevated temperature Chemically stable under acidic conditions Disadvantages Soluble in MeOH Has acidic properties Adsorbs water which affects silica gel properties Chemically unstable under basic conditions There are several different functional groups on silica surface. Their distribution is not consistent, their strength of formation of hydrogen bonding is different.

9. 9 Functional group candidates for SHARC separation

10. 10 Chiral separation S.C. Chang, G.L. Reid III, S. Chen, C.D. Chang and D.W. Armstrong. Trends in analytical chemistry, vol. 12, no. 4, 1993 p.144. Evaluation of a new polar organic high-performance liquid chromatographic mobile phase for cyclodextrin-bonded chiral stationary phases. Propranolol k’ = 4.36 Column:Cyclobond III Column size:4.6 x 250 mm Mobile phase: MeCN/MeOH/HOAc/TEA – 95/5/0.3/0.2 Flow:1.0 mL/min Detection:UV 254 nm

11. A stationary phase that provides strong hydrogen bonding (HB) donor type, acceptor type, or both types with molecules possessing a polar functional group(s). Hydrogen Bonding Stationary Phase Hydrogen Bonding LC Separation A separation technique based on the degree of hydrogen bond formation of the molecules with the polar stationary phase. It is possible in solvents such as MeCN/Hexane/CO2 with the addition of a strong polar solvent capable of competing for HB formation.

12. 12 SHARC SHARC stands for: Specific Hydrogen-bond Adsorption Resolution Chromatography

13. 13 Surface Interaction SHARC surface interacts with hydrogen bonding capable analytes in acetonitrile (weak solvent) Stationary support ANALYTE H H H H MeO H H H Stationary support ANALYTE H H H H MeCN SHARC surface interacts with hydrogen bonding capable methanol (strong solvent) which competes with analytes for stationary phase surface sites

14. 14 Mobile Phase Viscosity Viscosity profile of mixtures MeOH/water, MeCN/water, and MeCN/MeOH 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 10 2030405060708090100 MeOH/water MeCN/water MeCN/MeOH 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Viscosity, cP %

15. 15 Separation of Psychoactive Drugs min0510152025 4 1 2 3 1 2 4 3 Mobile phase: 70% MeCN, 30% MeOH with 0.25% Formic acid and 0.025% AmFm Mobile phase: 95% MeCN, 5% MeOH with 0.5% Formic acid and 0.05% AmFm 1.Pseudoephedrine 2.Norephedrine 3.Phenylephrine 4.Norphenylephrine Column: SHARC 1 Size: 3.2 x 100 mm Flow: 1.0 mL/min Detection:UV 270 nm The effect of the mobile phase composition on the separation of pseudoephedrine, norephedrine, phenylephrine, and norphenylephrine. Norephedrine NH 2 OH Pseudophedrine H N OH Phenylephrine H N OH OH Norephenylephrine NH 2 OH OH

16. 16 Separation of psychoactive drugs SHARC and HILIC technology 1 2 4 3 Column:SHARC 1 Column size: 3.2 x 100 mm Flow: 1.0 mL/min Mobile phase: 70% MeCN, 30% MeOH with 0.25% Formic acid and 0.025% AmFm Detection:UV 270 nm 1.Pseudoephedrine 2.Norephedrine 3.Phenylephrine 4.Norphenylephrine The separation mode effect on resolution of mixture of pseudoephedrine, norephedrine, phenylephrine, and norphenylephrine. Norephedrine NH 2 OH Pseudophedrine H N OH Phenylephrine H N OH OH Norephenylephrine NH 2 OH OH min0510152025 1,2 34 min 024681012 Column: Primesep N Column size: 4.6 x 150 mm Flow: 1.0 mL/min Mobile phase: 90% MeCN, 10% H 2 O with 0.25% Formic acid and 0.025% AmFm Detection:UV 270 nm HILIC SHARC

17. 17 Some Basic Drugs Column: SHARC 1 Size:3.2 x 100 mm Flow:0.5 mL/min Detection: UV 270 nm The effect of the mobile phase composition on the separation of pyrilamine, trimipramine, and pindolol. Mobile phase: 70% MeCN, 30% MeOH with 0.25% Formic acid and 0.025% AmFm Mobile phase: 70% MeCN, 30% MeOH with 0.5% Formic acid and 0.05% AmFm Mobile phase: 95% MeCN, 5% MeOH with 0.5% Formic acid and 0.05% AmFm 3 2 1 1 2 3 1 2 3 1. Pyrilamine 2. Trimipramine 3. Pindolol min 051015 Pyrilamine Trimipramine Pindolol N N N N N O HN O OH NH

18. 18 Basic Drugs in SHARC and RP The effect of the mobile phase composition on the separation of pyrilamine, trimipramine, and pindolol. 1 2 3 1. Pyrilamine 2. Trimipramine 3. Pindolol min 0510 Pyrilamine Trimipramine Pindolol N N N N N O HN O OH NH min0 12345 Column: SHARC 1 Size:3.2 x 100 mm Flow:0.5 mL/min Mobile phase: 70% MeCN, 30% MeOH with 0.5% Formic acid and 0.05% AmFm Detection: UV 270 nm 3 1 2 Column: Legacy L1 Size:3.2 x 100 mm Flow:0.5 mL/min Mobile phase: 40% MeCN, 60mM AmFm buffer pH 3.8 Detection: UV 270 nm RP SHARC

19. 19 Nucleobases The effect of mobile phase composition on the separation of nucleobases. min 051015 1 2 3 4 5 1. Thymidine 2. Uridine 3. Adenosine 4. Guanosine 5. Cytidine Column:SHARC 1 Size:3.2 x 100 mm Mobile phase: MeCN gradient from 100% to 75%, MeOH gradient from 0% to 25% in 15 min with 0.5% Formic acid with 0.05% AmFm Flow:1.0 mL/min Detection:UV 270 nm Thymidine Uridine Adenosine Cytidine Guanosine N NH 2 O N O OHOH HO NH N N O NH 2 N O OHOH HO N N N N NH 2 O OHOH HO NH O O N O O H OH HO HNNH O N O OHOH HO O O

20. 20 Nucleobases: Reverse Mode and SHARC mode The effect of mobile phase composition on the separation of nucleobases. 1. Thymidine 2. Uridine 3. Adenosine 4. Guanosine 5. Cytidine Column:SHARC 1 Size:3.2 x 100 mm Mobile phase: MeCN gradient from 100% to 75%, MeOH gradient from 0% to 25% in 15 min with 0.5% Formic acid with 0.05% AmFm Flow:1.0 mL/min Detection:UV 270 nm Thymidine Uridine Adenosine Cytidine Guanosine N NH 2 O N O OHOH HO NH N N O NH 2 N O OHOH HO N N N N NH 2 O OHOH HO NH O O N O O H OH HO HNNH O N O OHOH HO O O min 0510 1 2 3 4 5 5 2 4 1 3 Column:Legacy L1 Size:3.2x 100 mm Mobile phase: MeCN/H2O 5/95 with 0.5% Formic acid with 0.05% AmFm Flow:1.0 mL/min Detection:UV 270 nm RP SHARC

21. 21 Neutral Compounds min 024 1 2 3 4 Column:SHARC 1 Size:3.2 x 100 mm Mobile phase: MeCN/MeOH 95/5 with 0.1% Formic acid and 0.01% AmFm Flow:1.0 mL/min Detection:UV 270 nm 1. Caffeine 2. 3- Methylxanthine 3. 1- Methylxanthine 4. Xanthine The separation of caffeine, 3-methylxanthine, 1-methylxanthine, and xanthine. Caffiene 3-Methylxanthine 1-Methylxanthine Xanthine N N N N O O N N H N H N O O HN N N H N O O HN N H N H N O O

22. 22 Neutral Compounds. Xanthines. min 024 1 2 3 4 Column:SHARC 1 Size:3.2 x 100 mm Mobile phase: MeCN/MeOH 95/5 with 0.1% Formic acid and 0.01% AmFm Flow:1.0 mL/min Detection:UV 270 nm 1. Caffeine 2. 3- Methylxanthine 3. 1- Methylxanthine 4. Xanthine The separation of caffeine, 3-methylxanthine, 1-methylxanthine, and xanthine in reverse phase mode and SCHARC mode.. Caffiene 3-Methylxanthine 1-Methylxanthine Xanthine N N N N O O N N H N H N O O HN N N H N O O HN N H N H N O O min 024681012 14 4 3 1 2 Column:Legacy L1 Size:3.2 x 100 mm Mobile phase: MeCN/H2O 5/95 with 0.1% Formic acid and 0.01% AmFm Flow:1.0 mL/min Detection:UV 270 nm RP SHARC

23. 23 Neutral Compounds: Xanthines min 024 1 2 3 4 Column:SHARC 1 Size:3.2 x 100 mm Mobile phase: MeCN/MeOH 95/5with 0.1% Formic acid and 0.01% AmFm Flow:1.0 mL/min Detection:UV 270 nm 1. Caffeine 2. 3- Methylxanthine 3. 1- Methylxanthine 4. Xanthine The separation of caffeine, 3-methylxanthine, 1-methylxanthine, and xanthine in HILIC mode and SCHARC mode. Caffiene 3-Methylxanthine 1-Methylxanthine Xanthine N N N N O O N N H N H N O O HN N N H N O O HN N H N H N O O HILIC SHARC Column:SeQuant Size:4.6 x 150 mm Mobile phase: 90% MeCN with 10 mM AmFm pH 3.2 Flow:1.0 mL/min Detection:UV 270 nm min 01234 2 3

24. 24 Weak Bases 1.Pyridine 2.3-Aminopyridine 3.4-Aminopyridine 4.2-Aminopyridine The separation of aminopyridines with different column geometry. Pyridine 3-Aminopyridine 4-Aminopyridine 2-Aminopyridine Column: SHARC 1 Mobile Phase: Acetonitrile and MeOH (95/5) with 0.5% FA and 0.05% AmFm Detection:UV 270nm min 051015 Column: 4.6 x 150 mm, Particles:5  m, Flow: 1.0 mL/min Pressure:37 bar Column: 3.2 x 100 mm, Particles:3  m, Flow: 1.0 mL/min Pressure:77 bar Column: 3.2 x 100 mm, Particles:3  m, Flow: 2.0 mL/min Pressure:155 bar 1 4 2 3 1 4 2 3 1 4 2 3 N NH 2 NNH 2 N NH 2 N

25. 25 Weak Bases 1.Pyridine 2.3-Aminopyridine 3.4-Aminopyridine 4.2-Aminopyridine The separation of aminopyridines with different column geometry. Pyridine 3-Aminopyridine 4-Aminopyridine 2-Aminopyridine Column: SHARC 1 Mobile Phase:Acetonitrile and MeOH (95/5) with 0.5% FA and 0.05% AmFm min 051015 Column: 4.6 x 150 mm, Particles:5  m, Flow: 1.0 mL/min Detection: UV270 3 4 2 N NH 2 NNH 2 N NH 2 N min 02 1 1 2, 3, 4 Column: Legacy L1 Mobile Phase:Acetonitrile and H 2 O (5/95) with 0.5% FA and 0.05% AmFm RP SHARC

26. 26 Tryton-X on SHARC 1 Column Column:SHARC 1 Size:4.6 x 150 mm Flow:1.0 mL/min Mobile phase: Gradient MeCN/MeOH 100/0 to MeCN/MeOH 50/50 with 0.1% Formic acid with 0.01% AmFm in 12 min Detection:UV 270 nm The separation of components of Triton-X 100 surfactant. min 0246810 O O n

27. 27 Cytidine and Cytosine Separation The effect of mobile phase composition on the separation of cytidine and cytosine. Column:SHARC 1 Size:3.2 x 100 mm Flow:1.0 mL/min Detection:UV 270 nm 1 2 1 2 Mobile phase: MeCN/MeOH 95/5 with 0.5% Formic acid with 0.05% AmFm Mobile phase: MeCN/MeOH 50/50 with 0.5% Formic acid with 0.05% AmFm 1. Cytidine 2. Cytosine min 051015 Cytidine Cytosine N NH 2 ON O OH OH H H H H HO N H N O NH 2

28. 28 Loading Study Column:SHARC 1 Size:4.6 x 150 mm Mobile phase: MeCN /MeOH - 85/15% Flow:2.0 mL/min Pressure: 55 bar Detection:UV 250 nm Sample:0.5 mg/mL in ACN with small amount of water Injection:2, 5, 15, 50 uL N H NO O N N H min 0123456

29. 29 Benzenesulfonic acid on SHARC 1 column min 0246810 Injection: 5uL Plates: 1205 Symmetry: 5.95 Injection:1uL Plates: 7939 Symmetry: 1.84 Column:SHARC 1 Size:4.6 x 150 mm Mobile phase: MeCN/MeOH - 95/5% with 0.5% HCOOH and 0.05% AmFm Flow:1.0 mL/min Pressure: 30 bar Detection:UV 270 nm Samples:5 mg/mL in MeCN/MeOH

30. Conclusions 1.Hydrogen bond strength based separation can be a powerful LC technique 2.Different classes of compounds can be retained and resolved with a simple mixture of MeOH/MeCN/buffer as a MP 3.Low viscosity of MP allows the use of smaller particles and increased flow rate using conventional HPLC equipment 4.Unique selectivity can be obtained 5.High selectivity toward structural isomers 6.LC-MS friendly mobile phase and total organic provides high sensitivity 7.Preparative separation friendly MP allows simple solvent removal. 8.Variety of stationary phases to obtain optimum chromatography results

31. Sources www.sielc.com www.primesep.com www.hplcmethoddevelopment.com www.columnkit.com SIELC Technologies, Inc. 804 Seton Court Wheeling, IL 60090 847 229-2629 (Phone) 847 655-6079 (Fax)

32. Chicago New Year 2004-2005