Analysis of Algae Lipids by HPLC and Mass Spectroscopy
Martin Poenie, Jessica Jones, Morela Montoya, Schonna Manning
The University of Texas at Austin, Dept. of Molecular Cell and Developmental Biology
HPLC Instrumentation and Methods
• Bulleted Points
ABSTRACT
Analysis of Algae Lipids by HPLC and Mass Spectroscopy
Martin Poenie, Jessica Jones and Morela Montoya
Molecular Cell and Developmental Biology, University of Texas at
Austin, Austin, TX 78712
Algae hold promise as a sustainable source of biofuels based on their
rapid growth and reportedly high concentration of lipids. However
reporting of lipid content in the literature is based on a wide
variety of different measurement techniques that lead to confusion in
terms of what is actually being measured. We have developed a
comprehensive extraction and HPLC-based measurement
protocol that provides for quantitation of all the various lipid classes
including hydrocarbons, triglycerides, 1,3-diglyceride, 1,2 diglyceride,
fatty acids, monoglycerides and various polar lipids including
phosphatidyl ethanolamine. By splitting the column output to both an
ELS detector and a mass spectrometer one obtains both the amount of
material in particular lipid classes and a more detailed analysis of the
kinds of lipids within a particular class. For example, one can obtain
the molecular weights of different triglycerides within the triglyceride
class and from this infer the fatty acid composition. This approach
has been useful for analyzing lipid extraction protocols, lipid
production as a function of growth conditions, lipid breakdown during
algae processing, lipid recovery during algae processing and the
discovery of various hydrocarbons with chain lengths as high as C-60
as a significant component of some algae oil extracts. Our data show
that triglyceride and hydrocarbon content can vary dramatically with
time after seeding the culture and for different growth conditions
ranging from 2% triglyceride to over 30% triglyceride as a percentage
of dry weight for a particular algae grown in a bioreactor.
The origin of hydrocarbons in algae samples is not known but they are
present in algae from a variety of sources and can match or exceed the
– Subtext
Analytical Extraction Studies
Development of Better Solvents For
Oil Extraction and Analysis
Methods For Measuring Oil
Gravimetric
This involves weighing a solvent extract. The material in the extract varies with the
solvent used. Often this extract contains many different classes of molecules.
Nile Red
Nile Red is a hydrophobic dye that tends to partition into oil. It shows a
bathochromic shift in fluorescence in hydrophobic environments which helps
selectively measure hydrophobic materials. But Nile Red binds and fluoresces in
response to many cellular proteins as well as lipids . In practice, samples of algae
estimated to contain high “lipid” content by Nile Red have repeatedly shown low
content of triglycerides.
FAME Analysis
Fatty acid methyl esters are derived from a variety of cellular lipids including
mono-, di-, and triglycerides, phospholipids and glycolipids. Unless lipids are
fractionated into their various classes, one does not know where the fatty acids
come from. Furthermore, algae can contain lipids such as long chain alkanes that
will not be detected by FAME analysis.
Analysis of Commercial Extracts
A) Solvent A
C) Chloroform: Methanol
Algal Oil Can Vary in Composition
From Extraction Runs
Classes of Molecules Detected In
Neutral Lipid Extracts Hydrocarbons
Using the “Open Algae” extraction
system.
• Hydrocarbons
– Steranes (Cholestane)
– Paraffins (up to C60)
– Carotene (also a pigment)
• Glycerides
Triglyceride
– 1,3 Diglyceride
– 1,2 Diglyceride
– Monoglycerides
• Free Fatty Acids
• Pigments
– Chlorophylls (A&B)
– Phaeophytin
– Xanthophylls
E) Acetone
Reflux
B)
D)
F)
Cold
HPLC
HPLC of algae extracts has the potential for identifying and quantifying all lipid
classes. When combined with Mass Spectroscopy one obtains more detailed
molecular signatures for each class of lipid.
Pure triglyceride from algae is colorless. The
color of the oil depends amounts of pigments.
Typical pigments are carotene (orange) and
chlorophyll (green). Note that orange and
green mixed together gives brown.
.
triglyceride content
Analysis of Algae Oils Harvested From Algae Grown Under Different Conditions
Separation of Lipid Standards
Standards
G) Isopropanol
I)
7.000
6.000
Cold
Solvent A
HC
KT
TG
DG
ChL
FFA
MG
0.474
% of Dry Weight
4.000
Triglyeride Concentration-Response
Calibration For Different Gains
Of The ELS Detector
Mass Spec .of DG Peak Acquired During Run
Above
339 fragment arising
from loss of oleic acid
603 fragment arising
from loss of glycerol
OH group
Diolein
1.148
0.246
2.601
Chlorophyll
3.000
1.656
2.000
0.388
1.000
1.578
0.000
Simultaneous HPLC Detection By ELS and Mass
Spec.
TG
1.047
Free Fatty Acids
Reflux
HC
Monoglyceride
5.000
H)
CHCl3:MeOH
0.113
0.236
Diglyceride
Triglyceride
b-Carotene
Hydrocarbon
DG
DG
0.357
1.425
0.072
0.270
Do Algae Produce Paraffins?
Panels A-I (above)
HPLC Trace of lipids obtained by extraction of aliquots of algae with different solvents. Previous
studies showed that extraction of lipids from dried algae was very inefficient as shown by
retention of chlorophyll. Our strategy was to first reflux wet algae pellets in a solvent then dry the
algae completely under high vacuum to obtain a dry weight. Subsequently the sample is extracted
again. A. Solvent A is a mixture of methanol, 2-ethoxy ethanol, and dioxane, which was compared
to standard 2:1 chloroform:methanol mixture (C). The dried biomasses are then both extracted
twice with chloroform:methanol. For Solvent A, the extracts are combined and dried to a crude
lipid extract. The crude is separated into chloroform (A) and methanol (B) soluble fractions for
HPLC analysis. For the other sample, the extracts were combined then partitioned into chloroform
(C ) and aqueous phases using water (D). Both phases were dried then resuspended into
chloroform and methanol, respectively, for HPLC analysis. The data show that the material that
dissolves in methanol (B) is the same as the material that partitions into water (D). Neither the
methanol soluble (B) or water soluble (D) material dissolve in straight chlroform. E-H For
comparison, we show the effectiveness of cold (E) or refluxing (F) acetone and cold (G) or refluxing
(H) isopropanol. Acetone was primarily effective at removing pigments. Isopropanol surprisingly
removed very few lipids. I. The two most effective solvents for extracting lipids were solvent A and
chloroform: methanol. However the data show that solvent A extracts substantially more material
for most classes of lipids.
A) Algae Extracts
B) Purifed Hydrocarbon
C) MS Analysis
Algae from a wide variety of sources show the presence of material that migrates faster than
triglycerides by HPLC. Analysis of algae samples showed that some contain remarkably high levels
hydrocarbon –like material that had the same retention time as mineral oil. This fraction was
purified and shown to be pure by HPLC (B). C. The purified material was further analyzed by
Mass Spec. (courtesy of Karin Keller UT-Chem). The Mass Spec. shows a classic mixture of alkanes.