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: H 2 O 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  m; 5  m for proteins Stationary Phase: Hydrophobic alkyl chains (C-18) Mobile Phase: Solvent system: A = HPLC grade H 2 O, 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  m) 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  m 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.Agilent Alltech Associates, Inc.Alltech Associates, Inc. Instruments, GC, Sample Prep, Vials, Standards, TLC, HPLC, LC Accessories, IC. BodmanBodman chromatography systems and chromatography supplies. Cohesive TechnologiesCohesive Technologies systems for chromatographic solutions for pharmacokinetic analysis. LC Packings LC Packings capillary HPLCs, columns and capillary UV flow cells, etc. Michrom BioResources, Inc.Michrom BioResources, Inc. MAGIC 2002™ HPLC plus columns traps and other HPLC and LC/MS supplies and accessories. Mico-Tech Scientific Inc.Mico-Tech Scientific Inc. Sell capillary HPLCs along with a large selection of capillary HPLC columns and supplies. Rheodyne L.P.Rheodyne L.P. Best known as an HPLC injection valve manufacturer. ThermoFinniganThermoFinnigan Surveyor™ HPLC, and assorted UV detectors Waters CorporationWaters Corporation HPLCs, HPLC columns, mass spectrometers and more. Upchurch ScientificUpchurch Scientific chromatography products and specialize in HPLC fittings. Valco Instruments Co. Inc.Valco Instruments Co. Inc. Contact Info. HPLC injection valve manufacturer. Injectors, detectors, valves, fittings, syringes and tubing.Contact Info VydacVydac HPLC columns ranging from capillary to preparative.

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