1. High Performance Liquid Chromatography (HPLC) Jeannette Comeau CHEE 450 2 April 2004

2. Where are we in the process? HPLC

3. What is RP-HPLC? • Reverse Phase - High Performance Liquid Chromatography • High sensitivity and resolving capabilities • Mobile phase: • H2O miscible organic • Stationary phase: • Hydrophobic adsorbent • Purification technique: • Removing: • Contaminated proteins • Denatured insulin • Denatured insulin precursor • Atmospheric temperature, very high pressure Upchurch Scientific: http://www.upchurch.com/TechInfo/hplcIntro.asp#

4. What happens? • Inlet stream containing insulin ester. • Insulin ester binds to resin in aqueous mobile phase. • Insulin ester is eluted from column in organic mobile phase. • Acid is used to improve chromatographic peak shape, and to provide a source of electrons.

5. What are we looking at? • Our Process • Inlet flow: • 328.16 kg insulin ester • 36.45 kg denatured • ~825 L aqueous solution • Outlet flow: • 311.75 kg insulin ester

6. What’s inside the machine? • Particles: • Silica Particles, 3-50 mm; 5 mm for proteins • Stationary Phase: • Hydrophobic alkyl chains (C-18) • Mobile Phase: • Solvent system: • A = HPLC grade H2O, 0.1% trifluoroacetic acid (TFA) • B = HPLC grade acetonitrile, 0.08% TFA • Inlet stream containing Insulin ester

7. How big is an RP-HPLC? • Column Specs: • Bonded phase (C18) • Column dimension (large) • Particle size (5 mm) • Pore size (300 Å) • Flow from reactor = ~572.9 mL/min • Length necessary = 10504 m/daily throughput

8. The Possibilities • From Vydac: • 10mmID x 250mmL column • Particle size = 10-15 mm • Pore size = 300 angstrom TP silica • Practical benefits of the polymeric phase: • very long column lifetime • no measurable phase leaching • From Micro-tech Scientific: • Protein-M-25-C18-W-1000 • 5cm x 1mm i.d. glass lined Stainless Steel with 10-32 end unions packed with 5μ C18, 300Å particles

9. The Possibilities • Kroeff et al. • 57 x 30 cm column size • Internal volume = 40 L • Gradient = 15 to 28% (acetonitrile) • Loading = 15 g/L • Yield = 83% • But we want 95% yield • Need two columns to operate in parallel and 24 cycles per day (1 every hour).

10. How much $$? • Column from Micro-Tech: • $420.00 USD • The equipment (system) will be quoted: • Comes with a computer too!

11. Where could it come from? • Agilent 1100™ standard and capillary HPLC's. • Alltech Associates, Inc.Instruments, GC, Sample Prep, Vials, Standards, TLC, HPLC, LC Accessories, IC. • Bodman chromatography systems and chromatography supplies. • Cohesive Technologies systems for chromatographic solutions for pharmacokinetic analysis. • LC Packings capillary HPLCs, columns and capillary UV flow cells, etc. • Michrom BioResources, Inc.  MAGIC 2002™ HPLC plus columns traps and other HPLC and LC/MS supplies and accessories. • Mico-Tech Scientific Inc.  Sell capillary HPLCs along with a large selection of capillary HPLC columns and supplies. • Rheodyne L.P. Best known as an HPLC injection valve manufacturer. • ThermoFinnigan Surveyor™ HPLC, and assorted UV detectors • Waters CorporationHPLCs, HPLC columns, mass spectrometers and more.  • Upchurch Scientific chromatography products and specialize in HPLC fittings.  • Valco Instruments Co. Inc.Contact Info.  HPLC injection valve manufacturer.  Injectors, detectors, valves, fittings, syringes and tubing. • Vydac  HPLC columns ranging from capillary to preparative.

12. Any alternatives? • Gel Chromatography • High-Performance Ion Exchange Chromatography • QUESTIONS??