Total Carbohydrates Protocol Ebru Dulekgurgen UIUC'04

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TOTAL CARBOHYDRATES PROTOCOL
1. INTRODUCTION
The total saccharides moiety in a sample can be estimated by the anthron method which is a
simple colorimetric method with relative insensitivity to interferences from the other cellular
components. The first step in total carbohydrates measurement is to hydrolyze the
polysaccharides and to dehydrate the monomers (digestion with sulfuric acid addition and heat
treatment). The 5-carbon (pentoses) and 6-carbon (hexoses) sugars are converted to furfural and
hydroxymethylfurfural, respectively. When anthrone (an aromatic compound), it reacts with these
digestion products to give colored compound. The amount of total carbohydrates in the sample is
then estimated via reading the absorbance of the resulting solution against a glucose standard
curve. Please refer to Section 22.3 at “Methods for General and Molecular Bacteriology” by
Gerhardt et al. (1994) for further information and useful tips. We have this book at the Raskin Lab
(Loc: 4217).
2. MATERIALS and CHEMICALS
• COD vials : or any other type of vial durable to heating at 100oC.

• Heating-block : or a reliable water-bath that goes up to 100oC (Loc.: 4217)
• Clean Glassware
• 10 mL pipets : glass or acid-durable (disposable) pipettes
• Ice-buckets : to keep everything chilled all throughout the experiment
• Semi-micro cuvettes : Standard type plastic disposable cuvettes with 10 mm path length,
sample capacity of 1.5-3 mL (FisherBrand 14-385-942): Using these
cuvettes is optional for total carbohydrates measurements. If you
prefer to measure at the HACH spec., you can directly use the COD
vials and record the readings manually.
• Spectrophotometer
• Anthron reagent : Anthron (C14H10O), [9(10-H)-Anthracenone] Reagent, Spectrum,
AN135 (Loc: 4217)
• Glucose : D-(+)-Glucose anhydrous, Sigma G-7528 (Loc: 4217)
• Reagent-grade H2SO4
3. PREPARING the SOLUTIONS
• Prepare the 75% H2SO4 solution (preferably 1 day before the experiment, or at least 4 hours
before starting). 500 mL is good for approx 70 measurements (6 standard dilutions and 17
samples in triplicates).
• Put 100 mL of ddI water in a 500 mL volumetric flask
• Add a magnetic stirring bar, place the flask in an ice-bath, and put this on a stirring plate
• Wearing goggles, preferably acid-durable thick gloves, lab coat, and working under the fume
hood (COD station at 4130), measure 390 mL of 95-97% concentrated reagent-grade H2SO4.
• Turn on the stirrer and add the acid slowly and carefully to the volumetric flask
• Let it cool down to room temp. under the fume hood, add more ice to the ice-bath to speed up
• Adjust the volume to 500 mL with ddI water
• Prepare the anthron solution freshly, at the day of measurement:

• Weigh 0.5 g of anthron and add to a small beaker containing some absolute EtOH (i.e., 5 mL)
Total Carbohydrates Protocol

Ebru Dulekgurgen UIUC’04
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• Use some more EtOH (total of 5 mL) to rinse the anthron particle on the weighing boat into the
beaker (you can use a 1000 uL pipetman at this point)
• Put a mini stirring bar and stir to dissolve
• Add the anthron, dissolved in 10 mL EtOH, to a 250 mL volumetric flask containing the 75%
H2SO4 solution, stir to mix well and fill up to 250 mL with the 75% H2SO4 solution
4. GETTING READY
• Take your samples out of -20oC freezer to thaw
• Place the freshly prepared anthron reagent and the

75% H2SO4 solution on ice to chill
• Switch on the heating block and set the temperature to

100oC (actually for the heating-block at 4217, you
need to set the temp. to 110-115oC to get a fairly
stable 100oC reading. Check the temperature from the
digital display and also with a thermometer)
• Prepare the glucose solution and dilutions for the standard curve (prepare freshly):
• Weigh 0.05 g of glucose and add to a 500 mL volumetric flask containing ddI water
• Stir well to dissolve and adjust the volume to 500 mL with ddI water: Final concentration of the
stock is 100 mg glucose/L.
• Prepare dilutions* in 15 mL Croning tubes, following the recipe at Table 4.1.
Table 4.1 Dilutions from the glucose stock solution (100 mg/L) for the standard curve
V of stock
V ddI water Final conc.
glucose solution
10 mL 0 mL 0 mg/L
8 mL 2 mL 20 mg/L
6 mL 4 mL 40 mg/L
4 mL 6 mL 60 mg/L
2 mL 8 mL 80 mg/L
0 mL 10 mL 100 mg/L
*POINT OPEN FOR DISCUSSION: I’ve been preparing standard curves at the above range for my
measurements. I do not need to dilute the BLANK and SUPERNATANT samples from the EPS
extraction experiments, but I usually need to dilute my DOWEX-extracts (1:2 to 1:5 dilutions).
Depending on the general character of the sample and the expected total carbs values for the
supernatant and the DOWEX-extract, a higher-range calibration curve can be prepared to avoid
sample dilution.
• Dilute your samples with ddI water if necessary*.

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5. PROCEDURE
• Wear gloves, goggles, lab coat, and work very carefully
• Be careful while preparing and dispensing the 75% H2SO4 solution and the anthron solution:
Work on a bench covered with paper towels. You can easily recognize an acid spill on a paper
towel since strong acid burns the paper, turning the color to black. But during the experiment,
DO NOT use paper towels to wipe the plastic pipettes (especially the tips) or the flasks
containing the solutions (even if there are drops or spills on/around the containers). Acid
containing reagents hydrolyze the cellulose, present in paper towels, to sugars, resulting in a
high background and interfering significantly with the measurements. Though, clean the work
area thoroughly after finishing the experiment.
• Work in triplicates and on ice

• Vortex your samples/dilutions (or standards) well to mix
and transfer 1 mL to a COD tube. You can use a 1000 uL
pipetman at this step
• After transferring all of your samples/dilutions/
standards to the COD tubes, place the tube rack on ice to
chill
• Add 2 mL of already chilled 75% H2SO4 solution to the

tubes. You can use a 10 mL disposable plastic pipette to
dispense 2 mL of the acid solution to the samples.
• Cap and vortex briefly to mix (decrease the speed of the
vortex to approx. 3-4)
• Add 4 mL of already chilled anthron solution to the tubes
• Cap and vortex to mix
• Place the COD tubes on the

heating-block and boil at 100oC for 15 min
• Remove the tubes from the heating-block carefully and cool

down to room temp.
• While waiting, turn on the UV-VIS spec to warm up and stabilize (Loc.:4215)
• Before using the UV-VIS spec, please get the general training from the Lab. Manager.

4
• Transfer sufficient amount of acid-heat digested samples to

semi-micro disposable cuvettes
• Working at the Quantitative mode and @578 nm, auto-zero

the spec. with 2 semi-micro cuvettes filled with ddI water.
Then record the absorbance values for the standards
(standards mode) and the samples (unknown mode).
• Prepare the calibration curve from the abs. readings of the standards and calculate the total
carbohydrates of the samples in mg glucose/L from this curve.
6. CLEAN-UP and WASTE DISPOSAL
• When done with the spec. readings, collect all the

samples (both in the COD vials and in the semi-micro
cuvettes) and the extra anthron solution in a acid-
durable container; label
• Collect the emptied semi-micro disposable cuvettes in
a plastic bag; label
• Collect all the waste generated to a designated area to
be picked-up.
• Fill out a CWM-TRK-01 form (Request for Pickup of Chemical Waste) for the liquid and solid
wastes to be picked up and disposed.
• Rinse all the glassware and then clean with soapy water, rinse well again
• Clean the work space well
7. REFERENCES
Gerhardt, P.; Murray, R.G.E.; Wood, W.A.; Krieg, N.R. (1994) “Methods for General and
Molecular Bacteriology”, ASM, Washington DC, ISBN 1-55581-048-9, p 518.
Frolund, B.; Palmgren, R.; Keiding, K.; Nielsen, P.H. (1996). “Extraction of extracellular
polymers from activated sludge using a cation exchange resin”, Water Res 30(8), pp. 1749-1758.


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1

TOTAL CARBOHYDRATES PROTOCOL

2. MATERIALS and CHEMICALS

• COD vials : or any other type of vial durable to heating at 100oC.

3. PREPARING the SOLUTIONS

• Prepare the anthron solution freshly, at the day of measurement:

Total Carbohydrates Protocol

• Take your samples out of -20oC freezer to thaw

• Place the freshly prepared anthron reagent and the

• Switch on the heating block and set the temperature to

• Dilute your samples with ddI water if necessary*.

Total Carbohydrates Protocol

• Wear gloves, goggles, lab coat, and work very carefully

• Work in triplicates and on ice

• Add 2 mL of already chilled 75% H2SO4 solution to the

• Add 4 mL of already chilled anthron solution to the tubes

• Cap and vortex to mix

• Place the COD tubes on the

• Remove the tubes from the heating-block carefully and cool

Total Carbohydrates Protocol

• Transfer sufficient amount of acid-heat digested samples to

• Working at the Quantitative mode and @578 nm, auto-zero

6. CLEAN-UP and WASTE DISPOSAL

• When done with the spec. readings, collect all the

• Clean the work space well

Total Carbohydrates Protocol

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