5. Solvent B: 2-propanol and DCM (5:2, v/v).
6. Solvent C: 2-propanol and water (1:1, v/v) (see Note 3).
7. Nitrogen as nebulizing gas.
2.3 GC-MS System
1. 7890A gas chromatograph (Agilent Technologies Inc., Palo Alto,
CA, USA).
2. 5975C mass selective detector (Agilent Technologies).
3. Injection System: Gerstel MultiPurpose Sampler (MPS) (GERSTEL GmbH & Co. KG, M€ ulheim an der Ruhr, Germany).
4. Column for esterified samples: free fatty acid phase (FFAP)
silica capillary column (25 m  0.2 mm  0.3 μm) (Agilent
Technologies) or corresponding.
5. Column for trimethylsilylated samples: Rtx
®
-5MS column
(15 m  0.25 mm  0.25 μm) (Restek Corporation, Bellefonte, PA, USA) or DB-5 silica capillary column
(30 m  0.25 mm  0.25 μm) (Agilent Technologies) or
corresponding.
6. Helium as carrier gas in GC-MS, 99.99%.
2.4 Other
Instrumentation
1. Retsch mixer mill MM400 homogenizer (Retsch GmbH,
Haan, Germany).
2. Vortexer.
3. Centrifuge: Eppendorf 5804 R (Eppendorf Zentrifugen
GmbH, Leipzig, Germany).
4. Block heater Stuart
® SBH13OD/3 (Bibby Scientific Ltd.,
Staffordshire, UK).
5. Sample concentrator Stuart
® SBHCONC/1 (Bibby Scientific
Ltd., Staffordshire, UK).
3 Methods
The study of the widely varying algal lipid constituents requires
combinations of different analytical techniques (Fig. 1).
3.1 Sample
Preparation
3.1.1 Homogenization
and Extraction of Samples
1. Freeze-dried algal biomass samples (10 mg) are mixed with
500 μl of 15 mM saline solution and 1 ml of chloroform/
methanol (2:1, v/v) and homogenized for 2 min (20 Hz,
two steel grinding balls ø 3 mm). Samples for GC-MS analyses
are spiked with C17:0 internal standards (FFA + TAG) before
homogenization and extraction.
2. Vortex 5 min, and allow to stand for 30 min.
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Tuulikki Sepp€ anen-Laakso et al.
6. Solvent C: 2-propanol and water (1:1, v/v) (see Note 3).
7. Nitrogen as nebulizing gas.
2.3 GC-MS System
1. 7890A gas chromatograph (Agilent Technologies Inc., Palo Alto,
CA, USA).
2. 5975C mass selective detector (Agilent Technologies).
3. Injection System: Gerstel MultiPurpose Sampler (MPS) (GERSTEL GmbH & Co. KG, M€ ulheim an der Ruhr, Germany).
4. Column for esterified samples: free fatty acid phase (FFAP)
silica capillary column (25 m  0.2 mm  0.3 μm) (Agilent
Technologies) or corresponding.
5. Column for trimethylsilylated samples: Rtx
®
-5MS column
(15 m  0.25 mm  0.25 μm) (Restek Corporation, Bellefonte, PA, USA) or DB-5 silica capillary column
(30 m  0.25 mm  0.25 μm) (Agilent Technologies) or
corresponding.
6. Helium as carrier gas in GC-MS, 99.99%.
2.4 Other
Instrumentation
1. Retsch mixer mill MM400 homogenizer (Retsch GmbH,
Haan, Germany).
2. Vortexer.
3. Centrifuge: Eppendorf 5804 R (Eppendorf Zentrifugen
GmbH, Leipzig, Germany).
4. Block heater Stuart
® SBH13OD/3 (Bibby Scientific Ltd.,
Staffordshire, UK).
5. Sample concentrator Stuart
® SBHCONC/1 (Bibby Scientific
Ltd., Staffordshire, UK).
3 Methods
The study of the widely varying algal lipid constituents requires
combinations of different analytical techniques (Fig. 1).
3.1 Sample
Preparation
3.1.1 Homogenization
and Extraction of Samples
1. Freeze-dried algal biomass samples (10 mg) are mixed with
500 μl of 15 mM saline solution and 1 ml of chloroform/
methanol (2:1, v/v) and homogenized for 2 min (20 Hz,
two steel grinding balls ø 3 mm). Samples for GC-MS analyses
are spiked with C17:0 internal standards (FFA + TAG) before
homogenization and extraction.
2. Vortex 5 min, and allow to stand for 30 min.
226
Tuulikki Sepp€ anen-Laakso et al.