2. Materials
4% phenol: 40 g phenol (reagent grade) in 1 liter distilled water, store up to 6
months at room temperature
96% sulfuric acid (reagent grade)
1 mg/ml sugar standards (reagent grade) stored in sealed tubes
Sample
10-ml test tubes
Spectrophotometer
500-µl and 2.5-ml bottle-top reagent dispensers
5 to 200-µl and 200 to 1000-µl pipets
IMPORTANT NOTE: Cellulose can interfere with the experiment; all contact with paper
dust must be avoided.
1. Wash the 10-ml test tubes with distilled water. Set the spectrophotometer at 490 nm.
Allow the lamp of the spectrophotometer to warm up and zero the instrument with a
blank solution of 500 µl 4% phenol and 2.5 ml of 96% sulfuric acid.
2a. For samples containing a single sugar unit (monosaccharides or simple polysaccha-
rides): Prepare calibration sugar standards using 1 mg/ml sugar standard solutions,
of the same sugar present in the test sample, in distilled water. Transfer aliquots to
10 different, dry 10-ml tubes in 5-µl increments ranging from 5 to 50 µl, with an
accurate pipet.
When only one sugar unit is present, only that specific sugar should be used. As an example,
maltose is a polysaccharide composed of two glucose units. To get accurate results in the
determination of maltose, use commercially available maltose or glucose.
2b. For samples containing different sugar units (monosaccharide mixtures or complex
polysaccharides): Mix stoichiometric amounts of all of the component sugar units
present in the sample and prepare a 1 mg/ml (total) solution in distilled water. Prepare
calibration sugar standards by transferring aliquots to 10 different 10-ml tubes in 5
µl increments from 5 to 50 µl with an accurate pipet.
When different sugar units are present, all the sugars units in the standards should be mixed
in the same proportion as in the sample. As an example, lactose is a disaccharide composed
of a galactose and glucose unit. To get accurate results in the determination of lactose, use
commercially available lactose or an equivalent stoichiometric mixture of galactose and
glucose .
3. Transfer a portion of the sample to be analyzed to a 10-ml test tube; note the volume
or weight of the sample taken.
At this point, all the tubes should contain between 5 to 50 ìg of sugar, and one should
contain an unknown amount of sugar to be determined.
4. To all the tubes, add 500 µl of 4% phenol followed by 2.5 ml 96% sulfuric acid.
All the glycosidic linkages are broken and the colored complex is formed in this step.
In contact with water, sulfuric acid produces some heat.
5. Transfer the solutions from the test tubes to the cuvettes and measure the A490 of the
sugar standards and unknown solutions.
If the A490 is higher than 1.0 OD unit, or higher than all the standards, dilute the sample
with distilled water.
6. To calculate the concentration of sugar present in the sample, make a graph plotting
A490 versus sugar weight (µg) of the sugar calibration standards.
The intercept of the A490 of the unknown sample with the calibration line represents the
amount (ìg) of sugar present in the sample.
Current Protocols in Food Analytical Chemistry
E1.1.2
Colorimetric
Quantification of
Carbohydrates
3. 7. Calculate the unknown sample concentration using the following equations:
where x is the mass (g) of sugar sample deduced from the graph, mol. wt. represents the
molecular weight of the monosaccharide or polysaccharide in the sample, and w is the
weight (g) of the sample (step 3).
Concentrations in ìmol/liter can be calculated by replacing w(g) of the equation by v
(liters), where v (liters) is the volume in liters (step 3).
ALTERNATE
PROTOCOL 1
DETERMINATION OF REDUCING SUGARS USING THE
SOMOGYI-NELSON METHOD
If all the sugars present in a certain medium are expected to be reducing sugars, or to
determine the content of total reducing sugar, the Somogyi-Nelson method can be
performed. This method utilizes the reducing properties of certain types of carbohydrates.
Determination of reducing sugar using Somogyi-Nelson is based on the absorbance at
500 nm of a colored complex formed between a copper-oxidized sugar and arseno-
molybdate. The amount of carbohydrate present is determined by comparison with a
calibration curve using a spectrophotometer. Under the proper conditions, the Somogyi-
Nelson method is accurate to ± 0.01 mg for D-glucose, D-galactose, and maltose. The most
consistent results are obtained when operations are carried out under inert atmosphere,
and when the measured concentrations do not exceed 1 mg/ml. If a spectrophotometer is
not available, the method can also be performed qualitatively.
Additional Materials (also see Basic Protocol)
Low-alkalinity copper reagent (see recipe)
Arsenomolybdate reagent (see recipe)
Boiling water bath
1. Wash 10-ml test tubes with distilled water. Set the spectrophotometer at 500 nm.
Allow the lamp of the spectrophotometer to warm up, and zero the instrument with
a prepared blank solution.
The blank is prepared with 1 ml of distilled water, adding low-alkalinity copper reagent
and arsenomolybdate reagent as described in step 4.
2. Prepare sugar standards using a 1 mg/ml solution of the commercially available
reducing sugar present in the sample, in distilled water. Transfer aliquots of 5 to 100
µl, or 100 to 600 µl with an accurate pipet to different 10-ml tubes.
3. Transfer a portion of the sample mixture to be analyzed to a 10-ml test tube; note the
volume or weight of the sample taken.
At this point, all test tubes should contain 5 to 100 ìg or 0.1 to 0.6 mg of sugars, with one
containing an unknown amount of sugar to be determined.
4. To all the tubes, add 1 ml low-alkalinity copper reagent. Heat the tubes in boiling
water for 10 min. Add 1 ml of arsenomolybdate reagent to tubes containing ≤0.1 mg
sugar or 2 ml arsenomolybdate reagent to tubes containing 0.1 to 0.6 mg sugar.
(g)
concentration (mol/g) =
Mol. wt (g/mol) weight (g)
x
×
(g)
percentage of sugar (% by weight) = 100
weight (g)
x
×
Current Protocols in Food Analytical Chemistry
E1.1.3
Mono- and
Oligosaccharides
4. The color should appear after the addition of 1 or 2 ml of the arsenomolybdate reagent.
All volumes should be diluted to 5 ml with distilled water and allowed to stand at least 15
min at room temperature.
5. Transfer the solutions from test tubes to cuvettes. Measure the A500 of the sugar
standards and unknown solutions.
If the A500 measured is greater than 1.0 OD unit, dilute the samples.
6. To calculate the concentration of sugar present in the sample, make a graph of A500
versus weight (mg) of sugar. Calculate the concentration from the intercept (see Basic
Protocol, step 7).
ALTERNATE
PROTOCOL 2
DETERMINATION OF AMINO SUGAR DERIVATIVES USING THE
MORGAN-ELSON METHOD
It is sometimes necessary to measure the amount of N-acetyl- or amino-sugars in a certain
medium. In theMorgan-Elsonmethod,N-acetyl-oramino-sugars areheatedinanalkaline
solution to form a chromogen, which produces a purple colored compound when reacted
with N,N-dimethyl-p-aminobenzaldehyde in an acid solution (Ehrlich reagent). The
amount of sugar present is determined by comparison with a calibration curve using a
spectrophotometer at the appropriate wavelength of 530 for amino-sugars, and either 544
or 585 nm for N-acetyl-sugars. The Morgan-Elson method is accurate to about ±10%,
under proper conditions.
Additional Materials (also see Basic Protocol)
1 mg/ml sugar standards (reagent grade): D-glucosamine, D-galactosamine, or
N-acetyl-D-glucosamine
2,4-pentadione solution (see recipe)
Ehrlich reagent solution I (see recipe)
Boiling water bath
0.8 M potassium tetraborate
Ehrlich reagent solution II (see recipe)
1. Set the spectrophotometer at 530 nm for the measurement of amino-sugars, and at
544 or 585 nm for the measurement of N-acetyl-sugars. Allow the lamp of the
spectrophotometer to warm up and zero the instrument with a prepared blank
solution.
The blank is prepared with 1 ml of distilled water, adding the reagents as described in step
4a or 4b.
2. Prepare standards using 1 mg/ml reducing sugar standards composed of solutions of
commercially available sugars in distilled water. Transfer aliquots of 5 to 20 µl or 10
to 50 µl with an accurate pipet to different 10-ml tubes.
More reproducible results are obtained with amounts of 5 to 20 ìg for N-acetyl-D-glu-
cosamine and amino-sugars, and 10 to 50 ìg for N-acetyl-D-galactosamine.
3. Transfer a portion of the sample to analyze to a 10-ml test tube; note the volume or
weight of the sample taken.
4a. For the measurement of amino-sugars: To all the test tubes, add 2 ml 2,4-pentadione
solution and heat the tubes in boiling water for 1 hr. Add 2 ml of Ehrlich reagent
solution I.
The red color produced is stable for <1 hr.
Current Protocols in Food Analytical Chemistry
E1.1.4
Colorimetric
Quantification of
Carbohydrates
5. 4b. For the measurement of N-acetyl-sugars: To all the test tubes containing sugars, add
1 ml 0.8 M potassium tetraborate solution and heat the tubes in boiling water for 10
min. Cool the tubes in ice water and add 3 ml Ehrlich reagent solution II.
The red color produced is stable for <1 hr.
5. Transfer the solution from the test tubes to the cuvettes and measure the absorbance
of the reducing sugar standards and unknown solutions.
If the absorbance of the unknowns are higher than 1.0 OD unit, or higher than all the
standards, dilute the samples.
6. To calculate the concentrations of sugar present in the sample, make a graph of the
absorbance versus weight mass (mg) of sugar. Calculate the concentration from the
intercept (see Basic Protocol 1, step 7).
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, see
APPENDIX 2A; for suppliers see SUPPLIERS APPENDIX.
Arsenomolybdate reagent
Prepare a solution of 25 g ammonium molybdate in 450 ml distilled water. Add,
with stirring, 21 ml concentrated sulfuric acid and 25 ml of distilled watercontaining
3 g disodium hydrogen arsenate heptahydrate. Continue stirring 24 hr at 37°C, then
store the solution in a 1 liter glass-stoppered brown bottle up to 6 months at room
temperature.
Instead of a brown bottle, a bottle covered with aluminum foil can be used to protect the
solution from light.
Ehrlich reagent solution I
530 mg N,N-dimethyl-p-aminobenzaldehyde
20 ml ethanol
15 ml concentrated hydrochloric acid
Store up to 1 month at 0°C
Ehrlich reagent solution II
1 g of N,N-dimethyl-p-aminobenzaldehyde
50 ml glacial acetic acid
1.5 ml 10 N hydrochloric acid
Store up to 1 month at 0°C
Low-alkalinity copper reagent
Prepare a solution of 12 g sodium potassium tartrate and 24 g anhydrous sodium
carbonate in 250 ml distilled water. Add a solution of 4 g copper sulfate pentahydrate
and 16 g sodium hydrogen carbonate in 200 ml distilled water. Separately, prepare
a solution of 180 g anhydrous sodium sulfate in 500 ml of boiling distilled water.
Combine the two solutions in a volumetric flask and dilute the final solution to 1
liter. Store up to 1 year at room temperature.
Anhydrous sodium sulfate is added to the combined alkaline copper reagent to suppress
back-oxidation by air.
2,4-Pentadione solution
Add 2 ml 2,4-pentadione to a 50 ml solution of 1 M sodium carbonate.
The solution is stable, but should preferably be prepared fresh each time.
Current Protocols in Food Analytical Chemistry
E1.1.5
Mono- and
Oligosaccharides
6. COMMENTARY
Colorimetric determination of sugars has
been recognized for a long time. The most
common method is that employing phenol and
sulfuric acid (Dubois, 1951; Koch, 1951),
which was developed particularly for the deter-
mination of lactose in milk products (Barnett,
1957). This assay is currently used because it
is simple and the material needed is readily
available. Earlier, Nelson (1944) reported a
colorimetric modification of a micro copper
titrimetric method developed by Somogyi
(1937). In the original paper by Somogyi
(1926), a component was erroneously printed
as sodium carbonate instead of sodium sulfate
(Somogyi, 1952). A major objection to the
Somogyi assay was the back-oxidation by air.
Sodium sulfate is now added to suppress back-
oxidation. Thereafter, the method first intro-
duced in 1926 was modified several times by
different groups. Consequently, the variation of
the Somogyi-Nelson method in use today has
been commonly used only since 1954 (Wager,
1954). Despite newer, similar techniques
(Cheronis, 1957), the Somogyi-Nelson method
was long used for the determination of blood
sugar level.
To measure amino-sugar derivatives from
different sources, a method developed by Mor-
gan and Elson is currently used (Elson and
Morgan, 1933; Cornforth and Firth, 1958). The
measurement of amino-sugars is occasionally
performed indirectly by condensationwithace-
tylacetone, which subsequently reacts with the
Ehrlich reagent. In this case, the method is
commonly called Elson-Morgan (Elson and
Morgan,1933).WhentheN-acetyl-sugarsreact
directly with Ehrlich reagent the method is
called Morgan-Elson (Morgan and Elson,
1934). The method in use today (Alternate
Protocol 2), includes some modifications
(Aminoff, 1952).
Most colorimetric determination methods
have not been modified since 1957 or earlier.
Therefore, colorimetric analyses described in
recent literature refer to the early protocols
(Whistler, 1962; Birch, 1985; Beauchemin,
1995). Colorimetric carbohydrate analysis for
characterization purposes is still a valuable tool
asdocumentedinrecentchemistrypublications
(Nilsson, 1997).
Critical Parameters and
Troubleshooting
Spectophotometric parameters
Disposable plastic cuvettes are more con-
venient to use in spectrophotometric detection;
however, plastic melts after extended exposure
to strong acids. In addition, for more linearity
and accuracy in the measurements, many im-
portant parameters must be considered. It is
advantageous to use cuvettes with path lengths
of (0.01 to 0.5 cm), because they use a smaller
volume of sample and often give more repro-
ducible results.
Absorbances measured at higher than 1.0
OD unit can affect the linearity of the absor-
bance versus concentration, and therefore the
samples must be diluted if the absorbance
measured is higher then 1.0 OD unit or higher
then the prepared standards. Sometimes, insol-
uble residues will also affect the experiment.
The samples must be centrifuged or allowed to
precipitate before any measurement.
Colorimetric detection
When a determination is critical, it is often
necessary to do two or more different col-
orimetric tests in parallel. A substance present
in thesamplecouldinterferewithonetestwhile
not affecting others. In most cases, interference
can be controlled. Interference can be detected
by adding a known amount of sugar to a sample
solution to be measured. The amount of sugar
will differ from what is expected if there is
interference. In addition, a very colored sample
can interfere with the results obtained. In the
case of colored samples, subtract the absor-
bance of the samples dissolved in water or
buffer alone from the value obtained after ad-
dition of the colorimetric reagents.
In the phenol-sulfuric acid assay, the major
interference known is caused by cellulose; all
contact with paper dust must therefore be
avoided (Dubois, 1979). Otherwise, there are
no reports of significantly common interfer-
ence.
In theSomogyi-Nelson assay, all substances
with reducing or oxidative properties might
cause interference. The most important factor
to consider is the back-oxidation by air. To
control air oxidation, sodium sulfate is added
during the process. In addition, citric acid has
been reported to cause interference in the esti-
mation of reducing sugars (Paleg, 1959).
Current Protocols in Food Analytical Chemistry
E1.1.6
Colorimetric
Quantification of
Carbohydrates
7. In the Morgan-Elson assay, amino acids
alone do not interfere, but could alter the result
in synergy with other type of sugars. In all
colorimetric tests, the quality and stability of
the reagents is crucial in order to obtain repro-
ducible results. To eliminate the effect of poor-
quality reagents, results should always be vali-
dated by measuring a sample of known concen-
tration.Iftheresultsdiffer,newreagentsshould
be prepared. Depending, on the assay, color
could vary with time.
Anticipated Results
The three colorimetric analyses described in
this unit should give consistent results, pro-
vided there is no major interference and the
sample concentrations do not exceed the con-
centrations of the standards. In the appropriate
range, all assays described in this unit are ac-
curatebetween2%and10%.Thespectroscopic
detection in this unit allows the measurement
of the lowest standard concentration, i.e., usu-
ally 5 µg or more of sugar.
Time Considerations
The reagents are usually prepared in large
quantity and are stable for a relatively long
period. Therefore, once the reagents are pre-
pared, 1 to 2 hr is appropriate for all manipula-
tions. The phenol-sulfuric acidassay(seeBasic
Protocol), requires an additional ∼30 min for
the preparation of the reagents. A total of ∼2 hr
is necessary from the beginning of the experi-
ments to the obtaining the results.
The Somogyi-Nelson assay (see Alternate
Protocol1)requires∼30minforthepreparation
of the low-alkalinity copper reagent and the
same amount of time for the preparation of the
arsenomolybdate reagent. An additional 24 hr
should be considered for the 37°C incubation
when preparing the arsenomolybdate reagent.
Therefore, a total of 26 hr is necessary from the
beginning of the experiments to obtaining the
results.
TheMorgan-Elsonassay(seeAlternatePro-
tocol 2) requires less than 1 hr for the prepara-
tion of all the reagents, but a one hour period is
required for the reaction of the 2,4-pentadione
solution with the amino-sugar samples. In con-
clusion, without considering the incubation
time in the preparation of the reagents, 2 to 4
hrs working time should be enough to deter-
mine a sample concentration with any of the
procedures.
In general, each method requires 1 to 2 hr
for the manipulation involving the spectrome-
ter and 1 to 2 additional hours for the manipu-
lation involving the preparation of the reagents.
Literature Cited
Aminoff, D., Morgan, W.T.J. and Watkins, W.M.
1952. The action of dilute alkali on the N-Ace-
tylhexosamines and the specific blood-group
mucoids. Biochem. J. 51:379-389.
Barnett, A.J.G. and Tawab, G.A. 1957. A rapid
method for the determination of lactose in milk
and cheese. J. Sci. Food Agr. 8:437-441.
Beauchemin, K. 1995. Optimizing structural and
nonstructural carbohydrate concentrations in
dairy cow diets/project. In Agriculture and food
industry. Agriculture Canada, Edmonton.
Birch, G.G. 1985. Analysis of food carbohydrate,
University of Reading, London.
Cheronis, N.D. and Zymaris, M.C. 1957. The mi-
crodetermination of reducing sugars in blood by
means of p-anisyl tetrazolium blue. Mikrochim.
Acta 6:769-777.
Cornforth, J.W. and Firth, M.E. 1958. Identification
of two chromogens in the Elson-Morgan deter-
mination of hexosamines. A new synthesis of
3-methylpyrrole. Structure of the “pyrolene-
phthalides”. J. Chem. Soc. 1091-1099.
Dubois, M., Gilles K., Hamilton, J.K., Rebers, P.A.,
and Smith, F. 1951. A colorimetric method for
the determination of sugars. Nature 168:167-
168.
Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers,
P.A. and Smith,F. 1979. Colorimetricmethod for
the determination of sugars and related sub-
stances. Anal. Chem. 28:350-356.
Elson, L.A. and Morgan, W.T. 1933. A colorimetric
method for the determination of glucosamine
and chrondrosamine.Biochem. J. 27:1824-1828.
Koch, R.B., and Geddes, W.F., and Smith, F. 1951.
The carbohydrates of graminae. I. The sugars of
the flour of wheat (Triticum vulgare). Cereal
Chem. 28:424-431.
Morgan, W.T. and Elson, L.A. 1934. A colorimetric
method for the determination of N-acetylglu-
cosamine and N-acetylchrondrosamine. Bio-
chem. J. 26:988-996.
Nelson, N. 1944. A photometric adaption of the
Somogyi method for the determination of glu-
cose. J. Biol. Chem.153:375-380.
Nilsson, U.J., Heerze, L.D., Liu, Y.-C., Armstrong,
G.D., Palcic, M.M., and Hindsgaul O. 1997.
Immobilization of reducing sugars as toxin bind-
ing agents. Bioconjugate Chem. 8:466-471.
Paleg, L.G. 1959. Citric acid interference in the
estimation of reducing sugars with alkaline cop-
per reagents. Anal. Chem. 31:1902-1904.
Somogyi, M. 1926. Notes on sugars determination.
J. Biol. Chem. 70:599-613.
Somogyi, M. 1937. A reagent for the copper-io-
dometric determination of very small amounts of
sugar. J. Biol. Chem. 117:771-776.
Current Protocols in Food Analytical Chemistry
E1.1.7
Mono- and
Oligosaccharides
8. Somogyi, M. 1952. Note on sugar determination. J.
Biol. Chem. 195:19-25.
Wager, H.G. 1954. An improved copper reduction
method for the micro-determination of reducing
sugars. Analyst 79:34-41.
Whistler, B.L. and Wolfrom, M.L. 1962. Methods in
Carbohydrate Chemistry, Volume I. Academic
Press, London.
Key References
Chaplin, M.F. and Kennedy, J.F. 1994. Carbohy-
drates analysis: A practical approach. Oxford
University Press, Oxford.
This document contains valuable information on the
analysis of carbohydrates. The methods are com-
pared and evaluated on the basis of their uses to
measure the content of carbohydrates in specific
matrix.
Dische, Z. 1962. Color reaction of hexosamine. In
Methods in carbohydrate chemistry, Vol. 1 (R.L.
Whistler, and M.L. Wolfrom, eds.) pp.507-514.
Academic Press, London.
This section was taken from a book treating all of
the aspects of carbohydrates, including synthesis,
properties, and analysis of carbohydrates.
Hodge, J.E. and Hofreiter, B.T.1962. Determination
of reducing sugars and carbohydrates. In Meth-
ods in carbohydrate chemistry, Vol. 1 (R.L.
Whistler, and M.L. Wolfrom, eds.) pp.380-394.
Academic Press, London.
The reference describes the analysis of reducing
sugars using the Somogyi-Nelson method. It con-
tains no experiments, but it is one of the first compi-
lations explaining the method currently in use today.
Contributed by Eric Fournier
University of Alberta
Alberta, Canada
Current Protocols in Food Analytical Chemistry
E1.1.8
Colorimetric
Quantification of
Carbohydrates
