High Performance Liquid Chromatography (HPLC)

Jeannette Comeau

CHEE 450

2 April 2004

Where are we in the process?

HPLC

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#

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.

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

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 H2O, 0.1% trifluoroacetic acid (TFA) B = HPLC grade acetonitrile, 0.08% TFA

Inlet stream containing Insulin ester

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

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

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).

How much $$?

Column from Micro-Tech:$420.00 USD

The equipment (system) will be quoted:Comes with a computer too!

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 Corporation  HPLCs, 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.

Any alternatives?

Gel Chromatography

High-Performance Ion Exchange Chromatography

QUESTIONS??