experimentation with phospholipids for hplc analysis
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Monique Honeyghan HCS Class of 2008 Professor Mark Borden Columbia University Chemical Engineering . Experimentation with Phospholipids for HPLC Analysis. Overview. Phospholipid Structures Motivation Procedure Data Conclusion References Acknowledgements. - PowerPoint PPT PresentationTRANSCRIPT
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
School Family and Friends