EXPERIMENTATION WITH PHOSPHOLIPIDS FOR HPLC ANALYSISMonique Honeyghan

HCS Class of 2008Professor Mark Borden Columbia University Chemical Engineering

Overview Phospholipid Structures Motivation Procedure Data Conclusion References Acknowledgements

Phospholipid Structures

Monomer SUV Bilayer MLV

Motivation Prepare a protocol for a lab assignment designated for

Columbia undergrad seniors majoring in Chemical Engineering

Create a solution of phospholipids and lysophospholipids

Run the lipid solution through the HPLC to identify and quantify both types of lipids for analysis

Future component of research involving phospholipids

Provide further understanding of lipids in relation to enzymatic digestion

Procedure Preparation of lipids solution

DSPC 1,2-Distearoyl-sn-Glycero-3-phosphocholine C44H88NO8P

○ stored in chloroform○ Evaporation using nitrogen gas○ Vacuum overnight ○ 10mg/ml Concentration vs. 15 mg/ml Concentration

PBS – Pure Buffer Solution

Procedure Sonication : sonic energy breaks up large

vesicles into SUVs. Bath vs. Probe

Clear, yet translucent (hazy blue)Heating Block above 60o C

Procedure Extrusion – mechanical energy in the form of force to push

larger vesicles though small pores in order to help form uniformed vesicles. 11 passes 0.2 micron polycarbonate membrane Heat block above 60o C * Bath sonicate stock sample to be extruded * Heat extruded samples Syringe filter (0.2 micron) vs. (0.45 micron) vs. no filtering Variable: hot bath vs. quenching with ice bath

Procedure Testing on DLS machine

Dynamic Light Scanner Size of liposomes Size distribution by volume

Procedure Enzyme Reaction

Phospholipase A2 from bovine pancreas○ 100 µL to 1mL lipid solution○ 45 minutes on rotator*

Reactant: liposome (DSPC)

Products: lysophospholipid + fatty acid

MLV

Phospholipase A2

SUV

Procedure Extraction (Method by Folch et al)

Solvents - Chloroform: methanol in a 2:1 ratio○ 0.6 mL per 1 ml reacted lipid solution

Agitation to homogenize (3 minutes) Centrifugation (2000 rpm x 3 minutes)

○ Separates phases distinctly• Upper: methanol, water, enzyme• Lower: chloroform, PC and Lyso PC

Remove upper phase Add 0.4 ml Filtered H2O Agitation to homogenize (1 minute) Centrifugation (2000 rpm x 3 minutes) Filtered using a 0.2 μm pore syringe filter Test upper phase on DLS* Test lipid phase (lower) on HPLC for efficiency of the

Phospholipase A2 enzyme

Procedure HPLC - High Performance Liquid Chromatography –

identify and quantify the phospholipids and lysophospholipids via an isocratic elution that is run through a silica columnBuffer Components: Hexane: 2-Propanol: 25 mM Potassium Acetate:

Acetonitrile: Glacial Acetic Acid 442: 490: 62: 25: 0.6 by volume1. Run on buffer to establish a baseline2. Run on PC and LysoPC to get readings (Control)3. Inject samples of prepared lipid solution to see if

the results match the standards

Procedure (HPLC Diagram)

Procedure (HPLC)

Detector

Injection site PUMPS

Column Chamber

r

Buffer

Procedure (HPLC)

Injection site

Column

Pump A

Pump B

Data Graph showing size distribution by volume of 10 mg/mL

DSPC

Data Graph showing size distribution by volume of 10 mg/mL

DSPC

Data Graph showing size distribution by volume of 15 mg/mL

DSPC

Data Graph showing size distribution by volume in 10 mg/mL

enzyme extract (upper phase)

Data Graph showing size distribution by volume of 10 mg/mL

enzyme extract

Data Graph showing size distribution by volume of 10 mg/mL

enzyme extract

Results Probe sonication is faster, but there is a risk to

contaminating the sample with particles from the metal probe.

Hot bath is more efficient than quenching with cold water, however, if samples need to be cooled, it is better to do it rapidly.

Virtually no difference between syringe filtering and not doing so

Making 10 mg/ml samples is easier and more efficient than making 15 mg/ml samples.

HPLC has no final results. Progress with the reacted liposome samples shown

by 2 peaks, which might correspond to the PC and Lyso-PC standards

Conclusion Able to produce unilamellar vesicles

averaging around 100 nm in diameter Developed protocol so the undergrads can

reproduce the experiment Safety concerns established

Working under a fume hood with chloroform and the HPLC buffer components

Wear nitrile gloves when working with chloroform Wear heat resistant gloves when using Avanti Mini

extruder on heat block

References “Interfacial Enzyme Kinetcs at the Phospholipid/Water

Interface: Practical Considerations “by Raymond A. Deems. www.ideallibrary.com

“Preparation of liposomes” and extrusion technique by Avanti Polar Lipids Inc. www. avantilipids.com

Phospholipase A2 at the Bilayer Interface” by Fausto ramirez and Mahendra Kumar Jain

“Separation and Quantitation of Phospholipids and Lysophospholipids by High Performance Lipid Chromatography” by Edward Lesnefsky, Maria Stroll, Paul Minker and Charles Hoppel

“HPLC – High Performance Liquid Chromatography” and HPLC diagram by Resource Library of www.waters.com

“Folch (et al) method of lipid extraction “ www.cyberlipid.org Cuvette image

www.krackeler.com/products/1092-Cuvettes/1071... Image of DLS machine (Malvern Zetasizer) www.azon.com Image of bath sonicator by all-spec.com Multilamellar vessicle image by encapsula.com

Acknowledgements Professor Borden Cherry Chen Melissa Moy Borden Lab group Dr. Sat Bhattacharya Harlem Children Society Dr. Zarou Bronx Health Sciences High

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