Linda Lopez,

1
Leo Wang,
1
Ting Zheng,
1
Mark Tracy,
1
Rod Corpuz,
2
Rosanne Slingsby
1
1
Dionex Corporation, Sunnyvale, CA;
2
General Atomics, San Diego, CA
Linda Lopez,
1
Leo Wang,
1
Ting Zheng,
1
Mark Tracy,
1
Rod Corpuz,
2
Rosanne Slingsby
1
1
Dionex Corporation, Sunnyvale, CA;
2
General Atomics, San Diego, CA
Analysis of Carbohydrates and Lipids in Microalgal
Biomass with HPAE-MS and LC/MS
Analysis of Carbohydrates and Lipids in Microalgal
Biomass with HPAE-MS and LC/MS
INTRODUCTION
Biodiesel is rapidly becoming an attractive alternative to fossil-derived
diesel fuel, and algae are a promising feedstock. Efcient production
of biodiesel from microalgae requires analysis of all cell products,
including carbohydrates, lipids, and proteins. Proling of lipids in
biomass feed stocks is critical for the production of quality biodiesel.
Determination of carbon chain length and the degree of saturation is
key to ensuring high quality biodiesel that delivers optimum perfor-
mance during engine combustion. Lipid proling is commonly done
by GC with a ame ionization detector (FID), as specied in the ASTM
D6584 and EN 14105 methods; however, this methodology has several
limitations. High-boiling triglycerides often require special inlet types
to preclude discrimination which can cause thermal degradation and
interfere with the summative mass closure calculations typically done to
determine the total composition of biomass. Recently, HPLC analysis at
ambient temperatures with MS detection has emerged as the preferred
method for biomass lipid proling because it is well-suited to analysis
of nonvolatile components and can handle very complex or dirty sample
matrices without clogging of the LC/MS interface.
In addition to lipid proling, a complete characterization of the carbohy-
drate breakdown products in the algae is essential for efcient nutrient
recycle to determine which sugars are best absorbed by the algae.
Carbohydrate and lipid analyses of microalgal biomass are often com-
plex and require a high-resolution, high-sensitivity technique capable
of separating and quantifying trace-level components in the presence
of multiple interfering compounds. The use of high pH anion-exchange
chromatography mass spectrometry (HPAE-MS) permits baseline sepa-
ration of key saccharides in complex lysate mixtures.
EXPERIMENTAL
Instrument Setup
Carbohydrate analyses were performed on a Dionex ICS-3000 IC sys-
tem, which includes an ICS-3000 DP gradient pump, AS autosampler,
and ICS-3000 DC column compartment with electrochemical cell. Lipid
analyses were performed on a Dionex UltiMate

3000 RS system with

HPG-3400 RS pump, WPS-3000 RS sampler, TCC-3000 RS column
thermostat, DAD-3000 RS diode-array detector, and SofTA model 400
evaporative light scattering detector (ELSD).
Carbohydrates were detected using pulsed amperometry with a gold
electrode and Ag/AgCl reference electrode using the standard quadruple
waveform developed at Dionex.
1
Mass spectrometric analyses were run
on an MSQ Plus

single quadrupole mass spectrometer. The efuent

of the HPAEC column was passed through a CMD

desalter
2
(Dionex,
P/N 059090), then to the MSQ Plus mass spectrometer. Pressurized wa-
ter was pumped through the CMD as regenerating reagent. Postcolumn
addition of 0.5 mM lithium chloride was mixed into the eluent at 0.1
mL/min using a mixing tee and AXP-MS pump (Dionex, P/N 060684).
Detailed instrument setup is shown in Figure 1. The addition of lithium
allowed mono- and disaccharides to form lithium adducts. The use of
lithium allows detection of the sugars in the lithium form as opposed to
mixed forms which may be caused by contamination of weaker adduct-
forming cations, such as ammonium or sodium.
Lipids were detected using LC-UV/MS. Postcolumn addition of ammo-
nium acetate was applied to facilitate the formation of lipid-ammonium
or lipid-acetate adducts. The MSQ Plus was operated in the positive full
scan mode over a scan range of m/z 100 to 1500 with a cone voltage
fragmentor setting of 50 V.

Signals
Under N
2
, P
Chromeleon
Chromatography
Data System
CMD
MSQ
Pump
To Waste
Analytical Column Guard
Pump
0.5 mM LiCI
Eluent
Water
Autosampler
25999
Figure 1. Instrument setup for on-line HPAE-MS analysis of carbohydrates.
2 Analysis of Carbohydrates and Lipids in Microagal Biomass with HPAE-MS and LC/MS
Figure 2. Separation prole of microalgae carbohydrates on the CarboPac
MA1 column.
Chromatographic separations by LC/MS analyses were performed using
a Dionex Acclaim

RSLC C18 2.2 m column, in 2.1 150 mm format.

Chromatographic separations by HPAE-MS were performed using a Dionex
CarboPac

MA1 (3 250 mm) analytical column and CarboPac MA1

(4 50 mm) guard column at a ow rate of 200 L/min at 30 C under
isocratic conditions. Chromatography and MS analysis were controlled
by Chromeleon

Chromatography Data System (Dionex, version 6.8). Ref-

erence spectra were acquired using Xcalibur

(Thermo Fisher Scientic).

Sample Preparation
Samples of algal biomass and oil were obtained from General Atomics,
San Diego, CA. To prepare biomass samples for the HPAEC-MS analy-
sis, 2 mL of lysed microalgae sample was centrifuged at 12,000 rpm for
60 min. The supernatant was collected and centrifuged for an additional
30 min. The supernatant was collected and ltered through a 0.2 m
syringe lter to remove any remaining particles. The sample was passed
through an OnGuard

RP cartridge to remove hydrophobic components.

The OnGuard RP cartridge was activated rst with 5 mL methanol fol-
lowed by 10 mL DI water using a 10 mL syringe. Next, a 400 L sample
of supernatant was mixed with 600 L water and passed through the
activated cartridge using a 1 mL syringe. The cartridge was then washed
with 0.5 mL water and the eluted washing solution was combined with
the initial eluent. The sample was then passed through another activated
RP cartridge. This step was repeated until the sample was free of color.
All the eluted solutions (~3 mL) were combined and concentrated down
to a volume of ~250 L.
RESULTS
Carbohydrate Analysis
The separation prole of carbohydrates in microalgae samples on the
CarboPac MA1 is shown in Figure 2. More than a dozen peaks were
observed. On-line mass spectrometry analysis was used on the same
samples to conrm the carbohydrate identity of the peaks. Note the
similarities between the TIC and PAD proles (Figure 3). The full scan
spectrum of each peak is shown in Figure 4. Because many mono- and
disaccharides have identical mass-to-charge ratios (m/z), HPAE-PAD
proles of carbohydrate standards were compared with the sample
prole. Comparison of their retention times helped to identify the
peaks (Figure 5). Table 1 shows the identication results based on the
peaks m/z and comparison with PAD proles of the standards. These
data indicated that monosaccharide alditols (fucitol, arabitol, inositol),
monosaccharides (glucose, mannose, and others) and disaccharides
(sucrose, maltose, and others) were present in the microalgal biomass,
sucrose being the major sugar component.
26000
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Minutes
220
nC
0
Column: CarboPac MA1 (4 250 mm) with Guard
Eluent: 500 mM NaOH, isocratic
Flow Rate: 0.2 mL/min
Temperature: 30 C
Detection: Integrated Pulsed Amperometry
26001
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Minutes
70E6
counts
0
Current for CMD: 400 mA
Postcolumn addition: 0.5 mM LiCI, 0.1 mL/min
Mode: +ESI, Full Scan 120600 m/z
Cone voltage: 50V
Separation conditions are show in Figure 2.
1
2
3
5
4
6
7
8
9
10
11
13
12
Figure 3. MS TC prole microalgae carbohydrates.
120 180 240 300 360 420 480 540 600
120 180 240 300 360 420 480 540 600
Peak 4
Peak 3
Peak 2
Peak 1
Peak 7
Peak 6
Peak 5
261.2
138.1
162.1 398.2
423.1
138.1
323.2
205.1
162.1
205.1
187.1 367.1
138.1 228.3
173.1
138.1 196.1
349.1
138.1
159.1
138.1
182.1
261.2
138.1
162.1
398.1
m/z
Peak 13
Peak 12
Peak 11
Peak 10
Peak 9
Peak 8
349.1
367.1 229.1 289.2
349.1
365.2
219.1
237.2 205.1
349.1
175.1
139.1
205.1 337.4
205.1
187.1
228.2
205.1
187.1
138.1
162.1 228.1 289.3
26002
Figure 4. Background-substracted ESI mass spectra of peaks shown in Figure 3.
3
Lipid Analysis
The oil samples were analyzed under conditions that give a simplied
chromatogram where triacylglycerides (TAGs) with similar Partition
Numbers or Effective Carbon Numbers
3
tend to coelute in a single
peak.
4
Mass spectrometry allows us to assign total carbon and total
unsaturation numbers to components within each peak. The notation x:y
is used where x is the total carbon number of the acyl chains, and y is
the total number of double bonds. The typical fatty acid compositions
of lipids from various sources are well known in the literature, and with
few exception, they have an even number of carbon atoms.
5,6
For each
x:y we enumerate the m/z value of the [M+NH4]
+
adduct, and a theoreti-
cal isotope pattern. We then assign matching x:y to the components in
the chromatogram. Reference 4 shows an example of this process for
soybean oil. Figure 6 shows the extracted ion chromatograms and x:y
assignments for the main components of algal oil.
Figure 5. Overlay comparisons of algae samples with carbohydrate standards with,
A: alditol standards; B: monosaccharide standards; C: disaccharide standards.
5A
Column: CarboPac MA1 (4 250 mm) with Guard
Eluent: 500 mM NaOH, isocratic
Flow Rate: 0.2 mL/min
Temperature: 30 C
Detection: Integrated Pulsed Amperometry
Samples: 1. Mannitol
2. Inositol
3. Xylitol
4. D-arabitol
5. L-arabitol
6. Dulcitol
7. Algae sample
-0.4 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.5
14.6 20 25 30 35 40 45 50 55 60 65
7
6
5
4
3
2
1
9
8
7
6
5
4
3
2
1
5B
Column: CarboPac MA1 (4 250 mm) with Guard
Eluent: 500 mM NaOH, isocratic
Flow Rate: 0.2 mL/min
Temperature: 30 C
Detection: Integrated Pulsed Amperometry
Samples: 1. Galactosamine
2. Arabiose
3. Xylose
4. Glucose
5. Mannose
6. Fucose
7. Galactose
8. Glucosamine
9. Algae sample
5C
Column: CarboPac MA1 (4 250 mm) with Guard
Eluent: 500 mM NaOH, isocratic
Flow Rate: 0.2 mL/min
Temperature: 30 C
Detection: Integrated Pulsed Amperometry
Samples: 1. Maltose
2. Lactose
3. Sucrose
4. Algae sample

40 50 60 70 80 90
Minutes
100 110 120 130 142
4
3
2
1
nC
nC
nC
26003
Table 1. Identication of Sample Peaks Based on
Mass-to-Charge Ratios and Retention Times
Peak m/z Adduct Possible
Identication
1 173.1 166 + 7 (Fucitol + Li)
+
2 205.1/187.1 180 + 7, 180 + 7 + 18 (Inositol + Li)
+
,
(Inositol + Li + H2O)
+
3 423.1 Unknown Unknown
4 261.2 Unknown Unknown
5 261.2 Unknown Unknown
6 159.1 152 + 7 (Monoalditol + Li)
+

(L-Arabitol)
7 349.1 342 + 7 (Disaccharide + Li)
+
8 205.1/187.1 180 + 7 (Monosaccharide +
Li)
+
(Mannose)
9 201.1/187.1 180 + 7 (Monosaccharide +
Li)
+
(Glucose)
10 349.1 342 + 7 (Disaccharide + Li)
+
11 219.1 Unknown Unknown
12 349.1 342 + 7 (Disaccharide + Li)
+

(Sucrose)
13 349.1 342 + 7 (Disaccharide + Li)
+

(Maltose)
26062
0 2.5 5 7.5 10 12.5 15
I
n
t
e
n
s
i
t
y

[
c
o
u
n
t
s
]

Retention Time [min]
Chromatographic Conditions
Column: Acclaim 120 C18 2.2 m
2.1 150 mm
Column Temp.: 70 C
Mobile Phase: CH
3
CN/IPA = 80/20 (v/v)
Flow Rate: 500 L/min
Inj. Volume: 3 L
Mass Spectrometric Conditions
Interface: Electrospray (ESI)
Infusion: 50 L/min of 20% NH
4
OAC
buffer in CH
3
CN (10 mM)
Scan Mode: Positive full scan
1001500 amu
Probe Temp.: 400 C
Needle Voltage: 3000 V
Cone Voltage: 50 V
56:11 56:10
52:4
52:3
52:2

50:9

50:8

50:7
50:3

50:2 48:3
48:2

48:1

46:1

m/z 914.9
m/z 917.0
m/z 879.2
m/z 874.9
m/z 877.0
m/z 834.9
m/z 836.9
m/z 838.9
m/z 846.9
m/z 848.9
m/z 818.9
m/z 820.9
m/z 822.9
m/z 794.9
Figure 6. Extracted MS chromatograms of lipids in algae oil.
Passion. Power. Productivity.
LPN 2168-01 06/09
2009 Dionex Corporation
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Table 2 lists the masses, peak assignments, and a partial list of
probable TAGs in algal oil. The possible combinations of known
fatty acids to make any given x:y can be large; the list of prob-
able TAGs was established according to reported fatty acid com-
position in algal oil.
7
Each list is isobaric and the constituents
cannot be distinguished by a single-stage mass spectrometer.
To make a complete assignment of TAGs found in the sample
would require MS/MS. Compared to typical plant-derived oils,
there are some unusual/characteristic features to this algal oil.
First, no 54-carbon TAGs were observed in this study. Second,
the presence of highly unsaturated TAGs (unsaturations from
eicosapentaenoic acid, 20:5) was conrmed and this observa-
tion agrees with reported data.
7
CONCLUSION
Using simple sample preparation procedures, the carbohy-
drates in lysed microalgal biomass and lipids in algal oil
were chromatographically separated respectively and analyzed
using on-line mass spectrometry. Peaks were identied based
on their molecular weights and retention times.
REFERENCE
1. Rocklin, R.D.; Clarke, A.P.; Weitzhandler, M. Anal. Chem.
1998, 70, 14961501.
2. Thayer, J.R; Rohrer, J.S.; Avdalociv, N; Gearing, R.P. Anal. Biochem.
1998, 256, 207216.
3. Podlaha, O.; Tregrd, B. Journal of High Resolution
Chromatography, 2005, 5 (10), 553558.
4. Tracy, M. L.; Wang, L.; Liu, X.; Lopez, L.; Analysis for Triglycerides,
Diglycerides, and Monoglycerides by High Performance Liquid
Chromatography (HPLC) Using 2.2 m Rapid Separation C18
Columns with Alternative Solvent Systems and Mass Spectrometry.
31st Biofuels Conference, San Francisco, USA, Feb 24, 2009.
LPN 2266. Dionex Corporation, Sunnyvale, CA.
5. Gunstone, F. D. The Chemistry of Oils and Fats, Blackwell Publishing:
Oxford, UK, 2004; Page 4, Table 1.1.
6. Bajpai, D; Tyagi, V.K. J. Oleo. Sci. 2006, 55 (10), 487502.
7 Senanayake, N.; Shahidi, F. Concentration of Docosahexaenoic Acid
(DHA) from Algal Oil via Urea Complexation. J. Food Lipids 2004, 7,
5161.
Table 2. TAGs in Algal Oil
Retention
time
M+NH4 Adduct
m/z
Assignment Probable TAGs
6.4 914.9 56:11 16:1/20:5/20:5 18:3/18:3/20:5
8.1 917.0 56:10 18:2/18:3/20:5
3.8 872.9 52:4 16:0/18:1/18:3 16:0/18:2/18:2
4.7 874.9 52:3 16:0/18:0/18:3 16:0/18:1/18:2
11.5 877.0 52:2 16:0/18:0/18:2 16:0/18:1/18:1
3.7 834.9 50:9 16:2/16:3/18:4 16:3/16:3/18:3
3.6 836.9 50:8 14:0/16:3/20:5 16:2/16:3/18:3
4.5 838.9 50:7 14:0/16:2/20:5 16:1/16:3/18:3
7.4 846.9 50:3 14:0/18:0/18:3 14:0/18:1/18:2
9.1 848.9 50:2 14:0/18:0/18:2 14:0/18:1/18:1
5.7 818.9 48:3 14:0/16:0/18:3 14:0/16:1/18:2
7.2 820.9 48:2 14:0/16:0/18:2 14:0/16:1/18:1
9.2 822.9 48:1 14:0/16:0/18:1 14:0/16:1/18:0
7.3 794.9 46:1 12:0/16:0/18:1 12:0/16:1/18:0