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
Dionex Corporation, Sunnyvale, CA; 2 General Atomics, San Diego, CA
1
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 performance 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 carbohydrate 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 complex 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 separation of key saccharides in complex lysate mixtures.
eXPERIMENTAL
Instrument Setup
Carbohydrate analyses were performed on a Dionex ICS-3000 IC system, 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 water 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 adductforming cations, such as ammonium or sodium.
Lipids were detected using LC-UV/MS. Postcolumn addition of ammonium 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.
Eluent
Under N2, ΔP
Chromeleon
Chromatography
Data System
Signals
Water
Pump
Autosampler
Guard Analytical Column
CMD
To Waste
0.5 mM LiCI
MSQ
Pump
25999
Figure 1. Instrument setup for on-line HPAE-MS analysis of carbohydrates.
Chromatographic separations by LC/MS analyses were performed using
a Dionex Acclaim® RSLC C18 2.2 µm 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 flow rate of 200 µL/min at 30 ˚C under
isocratic conditions. Chromatography and MS analysis were controlled
by Chromeleon® Chromatography Data System (Dionex, version 6.8). Reference spectra were acquired using Xcalibur® (Thermo Fisher Scientific).
nC
Sample Preparation
Samples of algal biomass and oil were obtained from General Atomics,
San Diego, CA. To prepare biomass samples for the HPAEC-MS analysis, 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 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.
The OnGuard RP cartridge was activated first with 5 mL methanol followed by 10 mL DI water using a 10 mL syringe. Next, a 400 µL sample
of supernatant was mixed with 600 µL 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 µL.
0
0
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
samples to confirm the carbohydrate identity of the peaks. Note the
similarities between the TIC and PAD profiles (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
profiles of carbohydrate standards were compared with the sample
profile. Comparison of their retention times helped to identify the
peaks (Figure 5). Table 1 shows the identification results based on the
peak’s m/z and comparison with PAD profiles 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.
10
20
30
40
50
60
70 80
Minutes
90
100 110 120 130 140
26000
Figure 2. Separation profile of microalgae carbohydrates on the CarboPac
MA1 column.
70E6
Current for CMD:
Postcolumn addition:
Mode:
Cone voltage:
counts
12
400 mA
0.5 mM LiCI, 0.1 mL/min
+ESI, Full Scan 120–600 m/z
50V
Separation conditions are show in Figure 2.
2
7
1 34 5 6
8
13
10 11
9
0
0
Results
Carbohydrate Analysis
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
220
10
20
30
40
50
60
70 80
Minutes
90
100
110
120
130
140
26001
Figure 3. MS TC profile microalgae carbohydrates.
138.1
173.1
196.1
205.1
187.1
228.3
138.1
138.1
162.1
Peak 1
Peak 2
367.1
423.1
323.2
205.1
Peak 3
261.2
138.1 162.1
138.1 162.1
Peak 4
398.2
261.2
Peak 5
398.1
159.1
138.1 182.1
Peak 6
349.1
Peak 7
138.1
120
180
240
300
205.1
187.1
138.1162.1
228.1
205.1
187.1 228.2
289.3
360
420
480
Peak 9
Peak 10
337.4
Peak 11
349.1
365.2
349.1
180
600
Peak 8
349.1
139.1 175.1 205.1
219.1
205.1 237.2
120
540
229.1
289.2
240
300
Peak 12
Peak 13
367.1
360
m/z
420
480
540
600
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
7
6
nC 5
4
3
2
1
-0.4
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
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.5
7. Algae sample
5B
9
8
7
6
nC 5
4
3
2
1
14.6 20
25
30
35
40
45
50
55
60
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
65
8. Glucosamine
9. Algae sample
5C
The oil samples were analyzed under conditions that give a simplified
chromatogram where triacylglycerides (TAGs) with similar Partition
Numbers or Effective Carbon Numbers3 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 theoretical 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.
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
4
nC 3
2
1
40
Lipid Analysis
50
60
70
80
90 100 110 120 130
Minutes
Chromatographic Conditions
Column:
Acclaim 120 C18 2.2 µm
2.1 × 150 mm
Column Temp.: 70 °C
Mobile Phase: CH3CN/IPA = 80/20 (v/v)
Flow Rate:
500 µL/min
Inj. Volume: 3 µL
142
Mass Spectrometric Conditions
Interface:
Electrospray (ESI)
Infusion:
50 µL/min of 20% NH4OAC
buffer in CH3CN (10 mM)
Scan Mode:
Positive full scan
100–1500 amu
Probe Temp.: 400 °C
56:10 Needle Voltage: 3000 V
Cone Voltage: 50 V
56:11
26003
52:4
Figure 5. Overlay comparisons of algae samples with carbohydrate standards with,
A: alditol standards; B: monosaccharide standards; C: disaccharide standards.
52:3
m/z 914.9
m/z 917.0
50:9
Peak
1
2
m/z
173.1
Adduct
166 + 7
Possible
Identification
50:8
Intensity [counts]
Table 1. Identification of Sample Peaks Based on
Mass-to-Charge Ratios and Retention Times
m/z 879.2
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
12
349.1
342 + 7
(Disaccharide + Li)+
(Sucrose)
13
349.1
342 + 7
(Disaccharide + Li)+
(Maltose)
m/z 834.9
48:2
50:3
205.1/187.1 180 + 7, 180 + 7 + 18 (Inositol + Li)+,
(Inositol + Li + H2O)+
423.1
m/z 877.0
50:7
(Fucitol + Li)+
3
m/z 874.9
52:2
48:3
m/z 836.9
48:1
m/z 838.9
m/z 846.9
50:2
m/z 848.9
m/z 818.9
46:1
m/z 820.9
m/z 822.9
m/z 794.9
0
2.5
5
7.5
10
Retention Time [min]
12.5
15
26062
Figure 6. Extracted MS chromatograms of lipids in algae oil.
Unknown
3
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 probable TAGs was established according to reported fatty acid composition 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 confirmed and this observation agrees with reported data.7
Table 2. TAGs in Algal Oil
Retention M+NH4 Adduct Assignment
m/z
time
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
Conclusion
Using simple sample preparation procedures, the carbohydrates in lysed microalgal biomass and lipids in algal oil
were chromatographically separated respectively and analyzed
using on-line mass spectrometry. Peaks were identified based
on their molecular weights and retention times.
REFERENCE
1. Rocklin, R.D.; Clarke, A.P.; Weitzhandler, M. Anal. Chem. 1998, 70, 1496–1501.
2. Thayer, J.R; Rohrer, J.S.; Avdalociv, N; Gearing, R.P. Anal. Biochem. 1998, 256, 207–216.
3. Podlaha, O.; Töregård, B. Journal of High Resolution
Chromatography, 2005, 5 (10), 553–558.
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 µm Rapid Separation C18 Columns with Alternative Solvent Systems and Mass Spectrometry. 31st Biofuels Conference, San Francisco, USA, Feb 2–4, 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), 487–502.
7 Senanayake, N.; Shahidi, F. Concentration of Docosahexaenoic Acid (DHA) from Algal Oil via Urea Complexation. J. Food Lipids 2004, 7, 51–61.
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