Determination of Total Carbohydrates in Algal Biomass: Laboratory Analytical Procedure (LAP)

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Determination of Total

Carbohydrates in Algal
Biomass
Laboratory Analytical Procedure (LAP)
Issue Date: December 2, 2013
S. Van Wychen and L. M. L. Laurens

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Office of Energy Efficiency & Renewable Energy
Operated by the Alliance for Sustainable Energy, LLC
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Technical Report
NREL/TP-5100-60957
December 2013

Contract No. DE-AC36-08GO28308


Determination of Total
Carbohydrates in Algal
Biomass
Laboratory Analytical Procedure
(LAP)
Issue Date: December 2, 2013
S. Van Wychen and L. M. L. Laurens
Prepared under Task Nos. BB07.9H10 and BB07.9G10

NREL is a national laboratory of the U.S. Department of Energy


Office of Energy Efficiency & Renewable Energy
Operated by the Alliance for Sustainable Energy, LLC
This report is available at no cost from the National Renewable Energy
Laboratory (NREL) at www.nrel.gov/publications.

National Renewable Energy Laboratory Technical Report


15013 Denver West Parkway NREL/TP-5100-60957
Golden, Colorado 80401 December 2013
303-275-3000 • www.nrel.gov
Contract No. DE-AC36-08GO28308
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DISCLAIMER

The Standard Algal Biomass Analytical Methods (Methods) are provided by the National Renewable
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1. Introduction
1.1 This Laboratory Analytical Procedure (LAP) uses a two-step sulfuric acid hydrolysis to
hydrolyze the polymeric forms of carbohydrates in algal biomass into monomeric
subunits. The monomers are then quantified by either high-performance liquid
chromatography (HPLC) or a suitable spectrophotometric method.

1.2 An optimized hydrolysis procedure is expected to yield complete hydrolysis of all


polymeric structural and storage carbohydrates into monomeric sugars. A range of
sequential and optimized inorganic acid hydrolysis conditions with respective
hydrolysis liquor collection and analysis should be carried out for algal biomass. Here,
only a two-step sulfuric acid hydrolysis and quantification of released monosaccharides
are presented, and it is noted that not all carbohydrates may be hydrolyzed in this
manner. Future updates to this procedure will involve optimization of the carbohydrate
hydrolysis procedures.

1.3 Portions of this procedure are substantially similar to ASTM E1758-01 “Standard
Method for the Determination of Carbohydrates by HPLC” and the LAP developed for
terrestrial feedstocks and reference [1].

2. Scope
2.1 This procedure was originally optimized for terrestrial biomass, but has been modified
in this LAP to apply to freeze-dried algal biomass.

3. Terminology
3.1 Oven Dry Weight (ODW) – The weight of the biomass corrected for the percent
moisture determined by drying the biomass overnight in a 60°C atmospheric pressure
or 40°C vacuum convection oven according to LAP Determination of Total Solids and
Ash in Algal Biomass [2].

3.2 Structural Carbohydrates – The hydrolysable fraction of monomeric sugars after


analytical acid hydrolysis with an inorganic acid (such as sulfuric acid), this fraction
will also include monosaccharides derived from storage polysaccharides, such as
starch. This value should be subtracted from the reporting of total structural
carbohydrates.

3.3 Storage Carbohydrates – The fraction of monomeric glucose measured after enzymatic
hydrolysis of algal biomass with an enzyme cocktail of α-amylase and
amyloglucosidase. This fraction of storage carbohydrates is referred to as starch, but
reflects any portion of α-1,4 linked glucose polymers in algal biomass.

3.4 HPLC – High performance liquid chromatography, which separates compounds based
on size exclusion and ligand exchange. Details of a comparison between
chromatography columns and conditions for HPLC can be found in reference [3].

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4. Significance and Use
4.1 This procedure was developed for use on terrestrial feedstock and has not been
optimized specifically for microalgal biomass. However, the method has been
extensively tested and shown to work for algal biomass with no apparent difficulties.

4.2 The procedure described here is used to determine the total monomeric carbohydrate
content of freeze-dried algal biomass samples after a two-step sulfuric acid hydrolysis.

4.3 For the purpose of describing this procedure, we will refer to HPLC quantification, but
care should be taken with respect to monosaccharide resolution and chromatography of
complex mixtures of algae-specific monosaccharides when choosing an HPLC system.
High-performance anion exchange chromatography (HPAEC) may be the method that
gives the best separation of major micro-algae-specific monosaccharides [3], but it is
not described here.

4.4 Alternatively, spectrophotometric quantification of carbohydrates as monosaccharides


can be achieved by complexing the free aldehyde group with MBTH (3-methyl-2-
benzothiazolinone hydrazone), which has been shown to give an almost equal response
of different aldehyde sugars in solution, and matched HPLC quantification of complex
mixtures of monosaccharides [4]. This is a novel procedure; a manuscript on the
development and validation of this procedure is in preparation and will be included as a
reference in future versions of this LAP.

4.5 This procedure is used in conjunction with other compositional analysis procedures to
determine the summative mass closure for algal biomass.

5. Interferences
5.1 Samples with ash content >10% may not be suitable for this procedure as some
components of ash may cause side reactions during hydrolysis.

5.2 Samples with moisture content >10% may not be suitable for this procedure as the
excess moisture will interfere with appropriate acid concentrations.

5.3 Samples that are moldy or wet or that have been exposed to an oxygen-rich
environment may be compromised, resulting in erroneous carbohydrate values.

5.4 This procedure is not suitable for samples containing added acid, base, or catalyst.

5.5 Carbohydrates in the hydrolysates should not be measured using a phenol-sulfuric acid
quantification procedure. This quantification is known to be susceptible to significant
interferences as well as exhibiting substantially different responses for different
monosaccharides. Quantification of total carbohydrates by phenol-sulfuric acid failed to
match HPLC-quantification of even simple mixtures of sugars.

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6. Apparatus
6.1 Analytical balance, accurate to 1 mg or 0.1 mg (e.g., Mettler Toledo XP205
DeltaRange)

6.2 Vortex mixer

6.3 Water bath, set to 30°C ± 3°C

6.4 Autoclave, suitable for autoclaving liquids, set to 121°C ± 3°C

6.5 Spectrophotometer (for spectrophotometric carbohydrate determination), set to measure


at 620 nm

6.6 HPLC system equipped with refractive index detector and the following column:
Shodex sugar SP0810 (Shodex, #F6378105) with de-ashing cartridge holder (Bio-Rad,
#125-0139) and micro-guard de-ashing cartridges (Bio-Rad, #125-0118) or equivalent
with corresponding guard column

7. Reagents and Materials Needed


7.1 Reagents

7.1.1 Sulfuric acid, 72% w/w (specific gravity 1.6338 at 20°C) (Ricca Chemical
Company R8191600-1A)

7.1.2 Calcium carbonate, ACS reagent grade (Sigma Aldrich 239216)

7.1.3 Water, 18.2 MegaOhm (MΩ) water, 0.2 μm filtered

7.1.4 HPLC method:

7.1.4.1 High purity sugar standards: D(+)glucose (Sigma Aldrich G7528),


D(+)xylose (Sigma Aldrich X3877), D(+)galactose (Sigma
Aldrich G0750), L(+)arabinose (Sigma Aldrich 10839),
D(+)mannose (Sigma Aldrich 63579).

7.1.5 MBTH spectrophotometric method:

7.1.5.1 High purity sugar standards: D(+)glucose (Sigma Aldrich G7528)

7.1.5.2 MBTH (Sigma Aldrich 129739)

7.1.5.3 NaOH, ACS reagent grade (Sigma Aldrich 221465)

7.1.5.4 Dithiothreitol, DTT (Sigma Aldrich G0632)

7.1.5.5 Ferric ammonium sulfate dodecahydrate (Fluka 09730)

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7.1.5.6 Sulfamic acid (Sigma Aldrich 242772)

7.1.5.7 Hydrochloric acid (HCl), concentrated (36.5%–38%) (J.T. Baker


9535-03)

7.2 Materials

7.2.1 10-mL glass tubes and caps, heavy walled and capable of withstanding high-
pressure in an autoclave (VWR, #89079-404). Do not use the caps that are
supplied with the vials as they may not allow the samples to vent properly.
Rubber stoppers to plug glass tubes (VWR, #15507-144), tear-away
aluminum crimp caps (VWR, #16171-851)

7.2.2 Adjustable pipet, covering 250 μL to 10 mL

7.2.3 pH paper, range 4–9

7.2.4 Cuvettes – glass, quartz, or plastic (spectrophotometric method only)

7.2.5 13 x 100-mm glass test tubes or equivalent (spectrophotometric method


only)

7.2.6 Glass marbles or caps to cover or plug the 13 x 100-mm glass test tubes
(7.2.5) (spectrophotometric method only)

7.2.7 Disposable syringes (VWR, #BD301073) and compatible 0.2-μm nylon


filters (VWR, #28144-592), if planning to filter as opposed to centrifuging
to remove solids

7.2.8 1.5-mL capacity HPLC vials (Agilent, #5182-0543) and compatible


PTFE/rubber crimp caps (Agilent, #5181-1210) (HPLC method only)

7.2.9 Manual or automatic crimpers for caps for glass tubes from 7.2.1 and for
HPLC vials from 7.2.8 (if doing the HPLC method)

8. ES&H Considerations and Hazards


8.1 Sulfuric acid is corrosive and should be handled with care.

8.2 MBTH procedure: hydrochloric acid, sodium hydroxide, and sulfamic acid are
moderate health hazards. MBTH and ammonium ferric sulfate dodecahydrate are
moderate health hazards. Dithiothreitol is a moderate health hazard with mild
flammability and reactivity. Follow all applicable chemical-handling procedures.

8.3 Use caution when handling hot pressure tubes after removal from the autoclave. The
pressurized tubes are a possible explosion hazard if not cooled properly.

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9. Sampling, Test Specimens and Test Units
9.1 The LAP Determination of Total Solids and Ash in Algal Biomass [2] should be
performed in concurrence with this LAP.

9.2 Care must be taken to ensure a representative sample is taken for analysis from an algal
biomass prepared and dried by freeze drying, spray drying, etc., ensuring moisture is
<10% and is ground/homogenized to a particle size <1mm.

10. Procedure
10.1 Prepare the sample for analysis and hydrolyze

10.1.1 Weigh out all samples in concurrence with the LAP Determination of Total
Solids and Ash in Algal Biomass [2]. The results from this LAP will be used
later to correct the biomass weight. The hydrolysis and moisture correction
samples must be weighed out on the SAME DAY. If not immediately
hydrolyzed, seal samples to prevent any changes in moisture content.

10.1.2 Weigh 25 ± 2.5 mg of freeze-dried algal biomass into a labeled and tared 10-
mL glass tube. Record the weight of the sample to the nearest 0.1 mg in a
lab notebook.

10.1.3 After all the samples have been weighed out, add 250 μL of 72% (w/w)
sulfuric acid to each tube and vortex to thoroughly mix the acid and
biomass. Vortex samples carefully to ensure all solids remain at the bottom
of the tube and immersed in the acid.

10.1.4 Place tubes in a 30°C ± 3°C water bath with a water level set to just above
the sample/acid level in the tubes. Incubate samples for 1 hour, vortexing
each tube vigorously every 5 to 10 minutes. Vortex samples carefully to
ensure all solids remain at the bottom of the tube and immersed in acid.

10.1.5 After 1 hour, remove tubes from the water bath and add 7 mL of 18.2
MegaOhm (MΩ) water to each tube. This will bring the sulfuric acid
concentration to 4% (w/w).

10.1.6 Place a rubber stopper snugly in the top of the glass tube. Place a tear-away
aluminum crimp cap on top of the rubber stopper. Crimp the caps tightly and
vortex to mix the contents thoroughly. Tubes may also be inverted to mix,
ensure that the solids remain in the liquid, and do not end up stuck to the
rubber stopper.

10.1.7 Place the tubes in a rack suitable for autoclaving. Be sure to place the rack in
a tray for secondary containment. Secondary containment should contain
any tubes and their contents if they were to break during the autoclave cycle.

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10.1.8 Autoclave the sealed samples for one hour at 121°C using the liquids (slow)
setting.

10.1.9 After completion of the autoclave cycle, open the autoclave door, but allow
tubes to cool for approximately 15 minutes before removing them from the
autoclave.

10.1.10 Once the samples are removed from the autoclave, allow the samples to cool
for 30 minutes to 1 hour or until tubes reach room temperature.

10.2 Prepare samples for HPLC or spectrophotometric analysis.

10.2.1 Hydrolyzed samples must either be neutralized the same day or the solids
must be separated from the hydrolysate and the hydrolysate (still acidic)
may be stored in a refrigerator (4°C) for up to 24 hours before being
neutralized. Neutralized and filtered/centrifuged hydrolysate may be stored
up to 24 hours in a refrigerator (4°C) before being analyzed. Watch for
precipitate formation after the sample has been refrigerated. The sample
must be vortexed thoroughly, and then re-filtered or centrifuged to remove
the precipitate before analysis by HPLC or spectrophotometer.

10.2.2 Once the samples are cooled to room temperature, vortex each sample and
take an aliquot for neutralization. Aliquots are typically about 1–3 mL; do
not attempt to neutralize large volumes of hydrolysate at once. The sample
does not need to be filtered or centrifuged to remove the solids before
aliquoting.

10.2.3 Place the aliquot in a vessel suitable for neutralizing the sample. The vessel
must be large enough to contain the sample when it off-gases and bubbles
during the neutralization process. Examples might be 25-50 mL Erlenmeyer
flasks or 50-mL plastic centrifuge tubes (preferable to remove solids and
precipitate from hydrolysate if centrifuging).

10.2.4 Neutralize the aliquot to a pH between 6 and 8 using calcium carbonate. It is


very important to neutralize the sample correctly as low and high pHs can
ruin an HPLC column or interfere with the spectrophotometric method. The
amount of calcium carbonate required to neutralize the sample will depend
on several factors, including, but not limited to: aliquot amount and sample
buffering capacity. Add calcium carbonate in small amounts and vortex
between additions. Allow the sample a short period of time to finish
bubbling before checking the pH and/or adding any additional calcium
carbonate. DO NOT add large amounts of calcium carbonate at one time,
this often leads the sample to froth and turn into a lump of precipitate. A
precipitate will form as the neutralization process progresses, this is normal.

10.2.5 For both methods of carbohydrate analysis (HPLC or spectrophotometric),


the neutralized sample must be either filtered through a 0.2-μm nylon filter

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(use the disposable syringes and compatible filters) or centrifuged to remove
ALL solids and precipitate. For the HPLC method, place the filtered or
centrifuged hydrolysate in a clean, labeled HPLC vial (1.5-mL vials) and
cap tightly with a crimp cap (caps are considered tight when they cannot be
turned by hand). For the spectrophotometric method, place the filtered or
centrifuged hydrolysate in a clean, labeled vial and cap until needed.

10.3 HPLC analysis for monomeric sugars

10.3.1 Prepare a series of calibration standards containing the compounds to be


quantified. Refer to Tables 1 and 2 for suggested concentration ranges and
sugars. Use at least a four-point calibration, for example, 0.05, 0.25, 1.25,
and 4 mg/mL with a 2.5 mg/mL calibration verification standard (CVS)
according to the dilution table below:

Table 1: Suggested Dilutions for Calibration Curve Sample Preparations.

Concentration Dilution Final


of stock concentration
(mg/mL) (stock + water) (mg/mL)
10 400 + 600 4
10 125 + 875 1.25
1 250 + 750 0.25
1 50 + 950 0.05
10 250 + 750 ~2.5 (CVS)

Table 2: Suggested Standard Concentrations for the HPLC. Choice of monosaccharides based on
SP0810 column chromatography and is not an accurate reflection of algal monosaccharides. (For
a full list of algae-specific monosaccharides and suggested calibration mixtures, see reference
[3]).

Suggested
Component concentration
range (mg/mL)
D(+)glucose 0.05-4
D(+)xylose 0.05-4
D(+)galactose 0.05-4
L(+)arabinose 0.05-4
D(+)mannose 0.05-4
CVS ~2.5

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10.3.2 Prepare an independent CVS for each calibration set. The CVS should come
from a different sugar stock solution than the standards. The concentration
of the CVS should fall somewhere in the middle of the chosen calibration
range. The CVS should be run immediately after the calibration, after every
10 samples, and at the end of the HPLC run. The CVS is used to verify the
quality and stability of the calibration curve throughout the run.

10.3.3 Analyze the calibration standards, CVS, and samples by HPLC using a
Shodex sugar SP0810 column equipped with an appropriate guard column
(keep the guard column out of the column compartment; it should be kept at
room temperature) and a refractive index detector. Be sure to flush both the
column and guard column if they are new. Flush them separately into a
waste container. DO NOT flush them into the detector or flush the guard
column into the sugar separation column. Running at the method
temperature and maximum flow rate for approximately 1 hour should be
sufficient.

• Sample volume: 50 μL
• Mobile phase: 18.2 MegaOhm (MΩ) water, 0.2 μm filtered
• Flow rate: 0.6 mL/min
• Column temperature: 85°C
• Detector temperature: 55°C
• Run time: 50 minutes

NOTE: Run time may be extended if there are late eluting compounds and/or
the addition of post-run time may be necessary to elute sugar breakdown
products from the column.

10.3.4 Quantify the monomeric sugars by Chemstation (Agilent) or equivalent LC


software. Correlation for each calibrated compound should be 0.9999 or
better.

10.4 Spectrophotometric analysis for monomeric sugars using MBTH

10.4.1 Preparation of reagents/solutions

• 0.5M NaOH solution – store at room temperature and remake once a


precipitate forms

• 3 mg/mL MBTH – store in the dark at 4°C for up to one week

• 1mg/mL DTT – store in the dark at 4°C for up to one week

• Ferric solution: 0.5% ferric ammonium sulfate dodecahydrate, 0.5%


sulfamic acid (w/v), and 0.25M HCl. Dissolve 200 mg of ferric ammonium
sulfate dodecahydrate and 200 mg of sulfamic acid into 40 mL of 0.25M

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HCl. This solution may be stored at room temperature in the dark for up to
one month.

10.4.2 Preparation of working solution, made up fresh right before adding to


samples:

• MBTH working solution: 1:1 dilution of 3 mg/mL MBTH and 1 mg/mL


DTT

10.4.3 Preparation of standards:

• Prepare separate glucose stock solutions for the standards and CVS. The
standard and CVS stock solution concentrations should be approximately
0.25 mg/mL glucose. Due to balance limits, start with a higher stock
glucose concentration and dilute to 0.25 mg/mL. ALWAYS prepare the
standards and CVS from separate stocks so that you can check the accuracy
of your calibration with the CVS. Glucose stock solutions should be stored
at 4°C for up to two weeks.

• Create a six-point calibration with the 0.25 mg/mL glucose stock solution
using the dilutions outlined in Table 3. Prepare dilutions directly into the
glass reaction vials (13 x 100 mm or equivalent).

Table 3: Suggested Standard Concentrations for the Spectrophotometric Method.

Concentration Dilution Final


of stock concentration
(mg/mL) (stock + water) (mg/mL)
0.25 0 + 500 0
0.25 20 + 480 0.010
0.25 30 + 470 0.015
0.25 50 + 450 0.025
0.25 75 + 425 0.0375
0.25 100 + 400 0.050

• Prepare the CVS at the center of the calibration range: 75 μL CVS glucose
stock solution (0.25 mg/mL) and 425 μL 18.2 MegaOhm (MΩ) water.
Prepare the CVS dilution directly into the glass reaction vials.

10.4.4 Reaction

• Preheat a digital dry block that fits 13 x 100-mm glass test tubes (or
equivalent reaction vials) to 80°C.

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• Prepare sample dilutions directly in the glass reaction vials: start with a 1:5
or 1:10 dilution of the filtered/centrifuged neutralized hydrolysate. Dilute
the sample to a total volume of 0.5 mL. If the sample is known to be high
in carbohydrates, start with a 1:10 dilution. The goal is to have the sample
absorbance fall in the middle of the calibration curve. A 1:5 dilution would
be 100 μL of filtered/centrifuged neutralized hydrolysate and 400 μL 18.2
MegaOhm (MΩ) water.

• ALWAYS run a set of standards and a CVS with each batch of samples.

• Add 500 µL 0.5M NaOH to each glass tube.

• Add 500 µL MBTH working solution (section 10.4.2) to each glass tube,
vortex carefully to mix, and cover the tubes with a glass marble or cap.
Immediately place the tubes with solutions in the preheated dry block at
80°C for 15 min. Do not exceed the incubation time. Work in smaller
sample sets (<20). Do not allow the samples and standards to sit around
after addition of the NaOH or MBTH working solution – they must be
placed on the hot block within a few minutes of adding these solutions.

• After the 15-minute incubation time, turn off the block and immediately
add 1 mL of the ferric solution while the glass tubes are still on the block
(section 10.4.1).

• Once the ferric solution has been added, remove the glass tubes from the
hot block and carefully vortex to mix.

• Allow the samples and standards to react with the ferric solution for 10–15
minutes while the samples cool to room temperature. Tubes may remain
open during this time. Allow them to cool in a hood to control fumes.

• Once the samples are at room temperature, add 2.5 mL 18.2 MegaOhm
(MΩ) water, mix by pipetting or vortexing.

• Once mixed, place an aliquot of the samples and standards into the
appropriate cuvettes and obtain an absorbance on the spectrophotometer at
620 nm. Take an absorbance within an hour after the final dilution step.
Zero the spectrophotometer on 18.2 MegaOhm (MΩ) water. DO NOT zero
the spectrophotometer on the 0 standard from the calibration standard set.

• Use the glucose calibration curve and linear regression to quantify the total
carbohydrate concentration in mg/mL. Be sure to include the 0 standard as
a point in the calibration. Remember to correct the sample carbohydrate
concentration for the amount of hydrolysate that was used in the dilution
(for a 1:5, use 100 μL) and then get a total carbohydrate content by
multiplying that concentration by the total volume from the hydrolysis –

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7.25 mL. When building a linear calibration curve, DO NOT force the
curve through the origin.

11. Calculations
11.1 Calculate the ODW of the sample, using the average total solids content as determined
by the LAP Determination of Total Solids and Ash in Algal Biomass [2]:

11.2 Calculate monomeric sugar concentrations (mg/mL) for each sample using the linear
regression coefficients for the spectrophotometric glucose determination or the HPLC
calibration.

11.3 Calculate monomeric sugar content (mg) for each sample using the following equation:

11.4 Sum the monomeric sugars to get total sugars in mg (for the HPLC method).

11.5 Calculate the amount of monomeric (structural) sugar in the sample on a percent ODW
basis:

11.6 To report or calculate the relative percent difference (RPD) between two samples, use
the following calculation

where:
X 1 and X 2 = measured values
X mean = the mean of X 1 and X 2

11.7 To report or calculate the root mean square deviation (RMS) or the standard deviation
(STDEV) of the samples, use the following calculation:

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where:
x m = the root mean square of all x values in the set
n = number of samples in set
x i = measured value from the set

12. Report Format


12.1 Report the total carbohydrate results as a percent of the ODW. Individual sugars may
also be reported as a percent of the ODW.

12.2 For replicate analyses of the same sample, report the average, standard deviation, and
%RPD.

12.3 Report the CVS recovery.

13. Precision and Bias


13.1 Precision and bias need to be determined by a round robin experiment using standard
method verification biomass. The target RPD should be less than 10%.

14. Quality Control


14.1 Reported Results – Report results with two decimal places. Report the average,
standard deviation, and %RPD.

14.2 Replicates – Run all samples in triplicate, unless prohibited by the amount of sample
available (hydrolysis only).

14.3 RPD Criterion – Determined by data quality objectives and laboratory-specific Quality
Assurance Plan.

14.4 CVS – CVSs should be independently prepared and analyzed as per the procedure.
Required agreement for calibration verification standard quantification relative to the
theoretical concentration should be within 5% RPD.

14.5 Sample Size – 25 ± 2.5 mg for each replicate.

14.6 Sample Storage – Hydrolysis liquors may be separated from the acid insoluble solids
and stored (still acidic) for up to two weeks in a refrigerator (4°C). Neutralized liquors
may be stored in a refrigerator (4°C) for up to 4 days.

14.7 Standard Storage – HPLC standards should be stored in a freezer (-20°C) and removed
when needed. Thaw and vortex standards prior to use. Filter if necessary.

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at www.nrel.gov/publications.
14.8 Standard Preparation – Standards should be prepared as described in the procedure.

14.9 Definition of a Batch – Any number of samples that are analyzed and recorded
together.

14.10Control Charts – Quality assurance/quality control material should be control charted


to verify reproducibility.

15. Appendices
15.1 List of revisions/updates

• Distribution of May 16, 2013 DRAFT version


• Revision July 8, 2013, addition of additional experimental details and dilution
descriptions
• Revision July 17, 2013, correction to dilutions in Table 2
• Revision July 26, 2013, addition of CVS recovery target precision

16. References
[1] ASTM E1758-01 “Standard Method for the Determination of Carbohydrates by
HPLC.” 2003 Annual Book of ASTM Standards, Volume 11.05. Philadelphia, PA:
American Society for Testing and Materials, International.

[2] Van Wychen, S.; Laurens, L.M.L. Determination of Total Solids and Ash in Algal
Biomass. NREL/TP-5100-60956. Golden, CO: National Renewable Energy Laboratory.
2013. http://www.nrel.gov/docs/fy14osti/60956.pdf

[3] Templeton, D.W.; Quinn, M.; Van Wychen, S.; Hyman, D.; Laurens, L.M.L.
“Separation and quantification of microalgal carbohydrates.” J. Chrom. A (1270), 2012;
pp. 225–234.

[4] Anthon, G.E.; Barrett, D.M. “Separation and quantification of microalgal


carbohydrates.” Anal. Biochem. (305), 2002; pp. 287–289.

This report is available at no cost from the


National Renewable Energy Laboratory (NREL) 13
at www.nrel.gov/publications.

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