case of our instrument, the furnace never gets turned off to
avoid the risk of fracturing the combustion furnace tube after
cooling and bringing back up to temperature; however, this
may not be the case for other instrument manufacturers.
3. Run at least duplicates, ideally triplicates of each unknown
sample with a triplicate QC sample. A quality control
(QC) sample refers to a designated sample that is available in
large enough quantities that replicate data can be collected over
a long period of time and serves as a check to the analysis. A
running average of the quantitative data should be kept as a
control chart (see Note 6), which will make identifying and
isolating outlier batches easier. Keep batches of new samples to
a manageable size, with respect to sample preparation time, and
set up the instrument so that the reference standard (EDTA)
and the QC biomass are run both at the beginning and at the
end of a batch of samples, which will allow for the analysis and
reporting of drift within a run. Good practice is to run 5 or
more (up to 10) blanks until values are reproducible (standard
deviation <0.01) and lower than 0.375% protein and then run
4 or more EDTA standards until three consecutive values agree
within <0.15% protein of each other. Use the last three consecutive values to drift correct for the EDTA.
4. Dry samples in vials overnight in a 40
C oven under vacuum.
After drying, take a final weight to calculate the moisture-free
sample amount. Allow samples to cool in a desiccator under
vacuum before recording a final weight. If no vacuum oven is
available, samples can be stored overnight in a vacuum desiccator prior to analysis, given that a corresponding moisture measurement is available for that material. Refer to other reference
procedures for the determination of total solids and ash content in algal biomass for obtaining a moisture measurement.
Because moisture content can be variable in samples, it is
important to correct for moisture and report the nitrogen
and thus protein content on a dry weight basis.
5. The nitrogen content is calculated based on the instrument
calibration output and is expressed as a % of the dry weight of
the sample that is used for combustion. A multi-point calibration curve is recommended, and when creating the calibration
curve, at least 3 replicates of the standard (EDTA) should be
used. Depending on the instrument and software in use, a drift
factor may be used.
6. The combustion nitrogen system should be capable of measuring nitrogen in algal biomass materials containing between 0.2
and 20% nitrogen in the biomass, typically nitrogen
Protein Content Determination in Microalgae
237
avoid the risk of fracturing the combustion furnace tube after
cooling and bringing back up to temperature; however, this
may not be the case for other instrument manufacturers.
3. Run at least duplicates, ideally triplicates of each unknown
sample with a triplicate QC sample. A quality control
(QC) sample refers to a designated sample that is available in
large enough quantities that replicate data can be collected over
a long period of time and serves as a check to the analysis. A
running average of the quantitative data should be kept as a
control chart (see Note 6), which will make identifying and
isolating outlier batches easier. Keep batches of new samples to
a manageable size, with respect to sample preparation time, and
set up the instrument so that the reference standard (EDTA)
and the QC biomass are run both at the beginning and at the
end of a batch of samples, which will allow for the analysis and
reporting of drift within a run. Good practice is to run 5 or
more (up to 10) blanks until values are reproducible (standard
deviation <0.01) and lower than 0.375% protein and then run
4 or more EDTA standards until three consecutive values agree
within <0.15% protein of each other. Use the last three consecutive values to drift correct for the EDTA.
4. Dry samples in vials overnight in a 40
C oven under vacuum.
After drying, take a final weight to calculate the moisture-free
sample amount. Allow samples to cool in a desiccator under
vacuum before recording a final weight. If no vacuum oven is
available, samples can be stored overnight in a vacuum desiccator prior to analysis, given that a corresponding moisture measurement is available for that material. Refer to other reference
procedures for the determination of total solids and ash content in algal biomass for obtaining a moisture measurement.
Because moisture content can be variable in samples, it is
important to correct for moisture and report the nitrogen
and thus protein content on a dry weight basis.
5. The nitrogen content is calculated based on the instrument
calibration output and is expressed as a % of the dry weight of
the sample that is used for combustion. A multi-point calibration curve is recommended, and when creating the calibration
curve, at least 3 replicates of the standard (EDTA) should be
used. Depending on the instrument and software in use, a drift
factor may be used.
6. The combustion nitrogen system should be capable of measuring nitrogen in algal biomass materials containing between 0.2
and 20% nitrogen in the biomass, typically nitrogen
Protein Content Determination in Microalgae
237