Analysis of Carbohydrates and Lipids in Microalgal
Biomass with HPAE-MS and LC/MS
Linda Lopez,1 Leo Wang,1 Ting Zheng,1 Mark Tracy,1 Rod Corpuz,2 Rosanne Slingsby1
1
Dionex Corporation, Sunnyvale, CA; 2 General Atomics, San Diego, CA
INTRODUCTION
Biodiesel is rapidly becoming an attractive alternative to fossil-derived
diesel fuel, and algae are a promising feedstock. Efficient production
of biodiesel from microalgae requires analysis of all cell products,
including carbohydrates, lipids, and proteins. Profiling 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 profiling is commonly done
by GC with a flame ionization detector (FID), as specified 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 profiling 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 profiling, a complete characterization of the carbohy-
drate breakdown products in the algae is essential for efficient 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 effluent
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.
Chromeleon
Eluent Chromatography
Under N2, ΔP Data System
Signals
Water
Pump Autosampler CMD
Guard Analytical Column
To Waste
MSQ
0.5 mM LiCI Pump
25999
Figure 1. Instrument setup for on-line HPAE-MS analysis of carbohydrates.
Chromatographic separations by LC/MS analyses were performed using 220 Column: CarboPac MA1 (4 × 250 mm) with Guard
a Dionex Acclaim® RSLC C18 2.2 µm column, in 2.1 × 150 mm format. Eluent: 500 mM NaOH, isocratic
Flow Rate: 0.2 mL/min
Chromatographic separations by HPAE-MS were performed using a Dionex Temperature: 30 °C
CarboPac® MA1 (3 × 250 mm) analytical column and CarboPac MA1 Detection: Integrated Pulsed Amperometry
(4 × 50 mm) guard column at a flow rate of 200 µL/min at 30 ˚C under
nC
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 Scientific).

Sample Preparation 0

Samples of algal biomass and oil were obtained from General Atomics, 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140
Minutes
San Diego, CA. To prepare biomass samples for the HPAEC-MS analy- 26000

sis, 2 mL of lysed microalgae sample was centrifuged at 12,000 rpm for Figure 2. Separation profile of microalgae carbohydrates on the CarboPac
60 min. The supernatant was collected and centrifuged for an additional MA1 column.
30 min. The supernatant was collected and filtered through a 0.2 µm
syringe filter to remove any remaining particles. The sample was passed
through an OnGuard® RP cartridge to remove hydrophobic components. 70E6 12
Current for CMD: 400 mA
The OnGuard RP cartridge was activated first with 5 mL methanol fol- Postcolumn addition: 0.5 mM LiCI, 0.1 mL/min
lowed by 10 mL DI water using a 10 mL syringe. Next, a 400 µL sample Mode: +ESI, Full Scan 120–600 m/z
Cone voltage: 50V
of supernatant was mixed with 600 µL water and passed through the
activated cartridge using a 1 mL syringe. The cartridge was then washed counts
with 0.5 mL water and the eluted washing solution was combined with Separation conditions are show in Figure 2.

the initial eluent. The sample was then passed through another activated 2 7
RP cartridge. This step was repeated until the sample was free of color. 1 34 5 6 9 10 11 13
8
All the eluted solutions (~3 mL) were combined and concentrated down 0
to a volume of ~250 µL.
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Results Minutes
26001

Carbohydrate Analysis Figure 3. MS TC profile microalgae carbohydrates.

The separation profile 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 173.1
Peak 1
138.1 196.1
samples to confirm the carbohydrate identity of the peaks. Note the 205.1
187.1 367.1 Peak 2
similarities between the TIC and PAD profiles (Figure 3). The full scan 138.1 228.3
423.1
138.1 Peak 3
spectrum of each peak is shown in Figure 4. Because many mono- and 162.1 205.1 323.2
261.2
disaccharides have identical mass-to-charge ratios (m/z), HPAE-PAD 138.1 162.1 398.2 Peak 4
profiles of carbohydrate standards were compared with the sample 138.1 162.1
261.2
Peak 5
398.1
profile. Comparison of their retention times helped to identify the 159.1 Peak 6
peaks (Figure 5). Table 1 shows the identification results based on the 138.1 182.1
349.1 Peak 7
peak’s m/z and comparison with PAD profiles of the standards. These 138.1
data indicated that monosaccharide alditols (fucitol, arabitol, inositol), 120 180 240 300 360 420 480 540 600
monosaccharides (glucose, mannose, and others) and disaccharides 205.1
187.1
(sucrose, maltose, and others) were present in the microalgal biomass, 138.1162.1 228.1 289.3
Peak 8
sucrose being the major sugar component. 205.1
Peak 9
187.1 228.2
349.1
Peak 10
139.1 175.1 205.1 337.4
219.1
205.1 237.2 Peak 11
349.1
Peak 12
365.2
349.1 Peak 13
229.1 289.2 367.1
120 180 240 300 360 420 480 540 600
m/z 26002

Figure 4. Background-substracted ESI mass spectra of peaks shown in Figure 3.

2 Analysis of Carbohydrates and Lipids in Microagal Biomass with HPAE-MS and LC/MS
 5A Column: CarboPac MA1 (4 × 250 mm) with Guard
Lipid Analysis
Eluent: 500 mM NaOH, isocratic
Flow Rate: 0.2 mL/min
The oil samples were analyzed under conditions that give a simplified
7 Temperature: 30 °C chromatogram where triacylglycerides (TAGs) with similar Partition
6 Detection: Integrated Pulsed Amperometry
nC 5 Samples: 1. Mannitol Numbers or Effective Carbon Numbers3 tend to coelute in a single
4 2. Inositol
3
3. Xylitol peak.4 Mass spectrometry allows us to assign total carbon and total
2
1 4. D-arabitol
5. L-arabitol
unsaturation numbers to components within each peak. The notation x:y
-0.4 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.5
6. Dulcitol is used where x is the total carbon number of the acyl chains, and y is
7. Algae sample
the total number of double bonds. The typical fatty acid compositions
Column: CarboPac MA1 (4 × 250 mm) with Guard
5B Eluent: 500 mM NaOH, isocratic of lipids from various sources are well known in the literature, and with
9 Flow Rate: 0.2 mL/min
Temperature: 30 °C
few exception, they have an even number of carbon atoms.5,6 For each
8 +
7 Detection: Integrated Pulsed Amperometry x:y we enumerate the m/z value of the [M+NH4] adduct, and a theoreti-
6 Samples: 1. Galactosamine
nC 5
4
2. Arabiose cal isotope pattern. We then assign matching x:y to the components in
3 3. Xylose
4. Glucose
the chromatogram. Reference 4 shows an example of this process for
2
1 5. Mannose soybean oil. Figure 6 shows the extracted ion chromatograms and x:y
6. Fucose
14.6 20 25 30 35 40 45 50 55 60 65
7. Galactose assignments for the main components of algal oil.
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 Chromatographic Conditions Mass Spectrometric Conditions
Temperature: 30 °C
4 Column: Acclaim 120 C18 2.2 µm Interface: Electrospray (ESI)
Detection: Integrated Pulsed Amperometry
nC 3 Samples: 1. Maltose 2.1 × 150 mm Infusion: 50 µL/min of 20% NH4OAC
2 2. Lactose Column Temp.: 70 °C buffer in CH3CN (10 mM)
3. Sucrose Mobile Phase: CH3CN/IPA = 80/20 (v/v) Scan Mode: Positive full scan
1 4. Algae sample Flow Rate: 500 µL/min 100–1500 amu
Inj. Volume: 3 µL Probe Temp.: 400 °C
40 50 60 70 80 90 100 110 120 130 142 56:11 56:10 Needle Voltage: 3000 V
Minutes 26003 Cone Voltage: 50 V
52:4
Figure 5. Overlay comparisons of algae samples with carbohydrate standards with, 52:3 m/z 914.9
A: alditol standards; B: monosaccharide standards; C: disaccharide standards.
m/z 917.0
50:9
m/z 879.2
m/z 874.9
50:8
Table 1. Identification of Sample Peaks Based on 52:2
m/z 877.0
Intensity [counts]

Mass-to-Charge Ratios and Retention Times 50:7

m/z 834.9
Peak m/z Adduct Possible 48:2 m/z 836.9
Identification
48:1 m/z 838.9
1 173.1 166 + 7 (Fucitol + Li)+ 50:3
m/z 846.9
2 205.1/187.1 180 + 7, 180 + 7 + 18 (Inositol + Li)+, 48:3 50:2
m/z 848.9
(Inositol + Li + H2O)+
m/z 818.9
3 423.1 Unknown Unknown 46:1
m/z 820.9
4 261.2 Unknown Unknown m/z 822.9
5 261.2 Unknown Unknown m/z 794.9
6 159.1 152 + 7 (Monoalditol + Li)+ 0 2.5 5 7.5 10 12.5 15
(L-Arabitol) Retention Time [min]
26062
7 349.1 342 + 7 (Disaccharide + Li)+
8 205.1/187.1 180 + 7 (Monosaccharide + Figure 6. Extracted MS chromatograms of lipids in algae oil.
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)

3
 Table 2 lists the masses, peak assignments, and a partial list of Table 2. TAGs in Algal Oil
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- Retention M+NH4 Adduct Assignment Probable TAGs
time m/z
able TAGs was established according to reported fatty acid com-
position in algal oil.7 Each list is isobaric and the constituents 6.4 914.9 56:11 16:1/20:5/20:5 18:3/18:3/20:5
cannot be distinguished by a single-stage mass spectrometer.
8.1 917.0 56:10 18:2/18:3/20:5
To make a complete assignment of TAGs found in the sample
would require MS/MS. Compared to typical plant-derived oils, 3.8 872.9 52:4 16:0/18:1/18:3 16:0/18:2/18:2
there are some unusual/characteristic features to this algal oil. 4.7 874.9 52:3 16:0/18:0/18:3 16:0/18:1/18:2
First, no 54-carbon TAGs were observed in this study. Second, 11.5 877.0 52:2 16:0/18:0/18:2 16:0/18:1/18:1
the presence of highly unsaturated TAGs (unsaturations from
3.7 834.9 50:9 16:2/16:3/18:4 16:3/16:3/18:3
eicosapentaenoic acid, 20:5) was confirmed and this observa-
tion agrees with reported data.7 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
Conclusion
9.1 848.9 50:2 14:0/18:0/18:2 14:0/18:1/18:1
Using simple sample preparation procedures, the carbohy-
drates in lysed microalgal biomass and lipids in algal oil 5.7 818.9 48:3 14:0/16:0/18:3 14:0/16:1/18:2
were chromatographically separated respectively and analyzed 7.2 820.9 48:2 14:0/16:0/18:2 14:0/16:1/18:1
using on-line mass spectrometry. Peaks were identified based 9.2 822.9 48:1 14:0/16:0/18:1 14:0/16:1/18:0
on their molecular weights and retention times.
7.3 794.9 46:1 12:0/16:0/18:1 12:0/16:1/18:0

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