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Determination of Sugar as Glucose

In this experiment you will determine the amount of sugar or carbohydrates in a soft
drink by a spectrophotometric (colorimetric) method. The method is based upon the
color which forms when sugars reduce 3,5-dinitrosalicylic acid (DNSA) to 3-amino-5-
nitrosalicylic acid, as shown in the equation.

One problem with this method is that sucrose, or common table sugar, does not undergo
this reaction with DNSA. Therefore, the sucrose and complex carbohydrates must be
broken down into simple sugars like glucose first. This can be done by boiling the
sample with hydrochloric acid. The pH is then adjusted to give a basic solution, under
which condition simple sugars are good reducing agents.

In addition, the sugars in soft drinks are at too high a concentration for this method to be
used unless they are diluted first. Most beverages will have to be diluted by at least a
factor of 10 (this is called a 10:100 dilution) before carrying out the analysis. However,
you will want to try more than one dilution if necessary in order to get acceptable
readings. These dilutions must be carried out quantitatively (in a precisely known
manner) using a volumetric flask. Section 4.3 of your Zumdahl/6e text discusses dilution
calculations. Recall that dilution involves the addition of additional solvent to a known
volume of the solution. Thus, the number of moles of solute is unchanged. If solution 1
is the original solution and solution 2 is the diluted sample, then the following relationship
holds:
M1V1 = M2V2
where M represents concentration and V represents volume of the original solution or
diluted solution. Some suggested dilutions include 1:100, 2:100, 3:100, 5:100, and
10:100, depending upon how much sugar is in your sample. You will most likely not
need to do all of these dilutions.

The interaction of light (electromagnetic radiation) with molecules is of great interest and
use to chemists. When compounds absorb light, various changes can occur in the
molecule, depending upon the particular wavelength of light used. The changes that
occur may involve movement of electrons to higher-energy orbitals, increased vibrational
motion of bonds, or increased rotation of molecules, among others. The wavelengths of
light absorbed are very characteristic of a given molecule.
Determination of Sugar as Glucose

One application of light absorption (spectroscopy) is to determine the concentration of a


molecule which absorbs light. If a beam of monochromatic (single-wavelength) light of
intensity Io enters a sample which absorbs some of that light, the beam of light emerging
from the other side of the sample will have a reduced intensity I. The transmittance of
the sample is then defined as T = I / Io, often expressed as percent transmittance rather
than a fraction. A device known as a spectrophotometer measures the transmittance by
calculating the ratio of the two intensities.

The Beer-Lambert Law relates the absorbance of light by a sample to the concentration
of the absorbing species. First, the quantity known as absorbance is defined as the
negative logarithm to the base 10 of the transmittance, A = - log T. Since most
instruments use the percent transmittance, and T = (%T / 100), then A = - log(%T) - (-
log 100) = 2 - log(%T). Now that we have defined absorbance, the Beer-Lambert Law is
stated thusly:
A = bc
where A = absorbance (a unitless number, since it is a logarithm), c = the concentration
of the absorbing species, b = the path length in centimeters of light passing through the
sample, and (epsilon) = the molar absorptivity, a characteristic property of the
absorbing species which depends upon the wavelength of light used and which has units
of 1/cm conc. Thus, the absorbance is directly proportional to the concentration of the
absorbing species. Absorbance is also directly proportional to the path length, but this
quantity is held constant in a given experiment (it is the inside diameter of the sample
tubes, which are called cuvettes).

In this experiment, you will test specifically for concentration of sugars using light
absorbance measurements. Specifically you will test for the concentration of the colored
product formed by the reaction of glucose with DNSA. Since all of the glucose will be
converted to the colored product, the Beer-Lambert law can still be used to determine
sugar concentration. The amount of color formed is time-dependent. Therefore, once
the DNSA is added to the test solution, a timer should be started and the absorbance
measurements for all samples should be made at the same time after the addition. A
time interval of 5 to 20 minutes will suffice.

Note that a measurement of absorbance can only yield the concentration of the
absorbing species if both the molar absorptivity and the path length are known. The
path length is no problem, as stated above; the cuvettes have an internal diameter of 1.1
cm. However, most times we do not know the molar absorptivity of the absorbing
species. Therefore, it is necessary to first prepare a calibration curve known as a Beer's
Law plot. To do this, a series of standards is prepared in which the concentrations of the
absorbing species are precisely known. The absorbances of these standards are then
measured in the spectrophotometer and a plot of absorbance versus concentration is
prepared. This plot can then be used to relate the measured absorbance of any other
sample containing the same absorbing species to the concentration of that species in
the solution. For precise work, one might wish to do a linear least-squares regression on
the data for the standards to obtain the equation of the line (slope and intercept).

According to Beer's law, the absorbance of a solution should be zero (100%T) if there is
none of the absorbing species present. A blank solution containing all components of
the solution except the sample is used to set the spectrophotometer to a reading of zero

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Determination of Sugar as Glucose

absorbance. Thus, if any other components of the mixture absorb light at the
wavelength used, their effect will be cancelled out.

Click on the following link for further information on use of the spectrophotometer:

http://chemscape.santafe.cc.fl.us/chemscape/catofp/measurea/concentr/spec20/spec20
b.htm

Safety

Check for flammables before lighting a bunsen burner! When boiling solutions, point test
tubes away from your face and those of any other students nearby. Use test tube
holders to handle hot tubes. Hydrochloric acid and sodium hydroxide are both
corrosive. Be sure to wear your goggles at all times. The DNSA solution is toxic; avoid
contact with the skin. If any is spilled, wash thoroughly with soap and water. Inform the
instructor.

Equipment and reagents

6 M HCl, 2.5 M NaOH, 0.050 M 3,5-dinitrosalicylic acid, 1000 mg/dL standard sucrose
solution, soft drink to test (non-diet, not dark-colored)
Spectronic 20 spectrophotometer and cuvettes, test tubes, test tube rack, test tube
clamp, (2) 400-mL beakers, Mohr pipets and bulb (5 & 10 mL), 25-mL volumetric pipet,
timer, (5) 100-mL volumetric flasks, Pasteur pipet and bulb, Parafilm, ring stand, ring,
wire gauze, burner, lighter

Procedure

First you must prepare the sucrose standards by suitable dilution of the stock solution.
Concentrations in the range 100 - 1000 mg/dL are suggested as a starting point. For
example, make a 20:100 dilution as follows. Pipet 20.00 mL of the stock solution into a
clean 100-mL volumetric flask. Add distilled water to a point about 1 cm below the
calibration mark. Use a Pasteur pipet to carefully add water until the bottom of the
meniscus is exactly on the line. Cover the flask with Parafilm and shake well to mix. In
a similar fashion, prepare 40:100, 60:100, and 80:100 dilutions. Prepare a data table
with columns for standard concentration, %T, and Absorbance.

Turn on the spectrometer (left-hand knob) and set the wavelength to 580 nm. Let the
instrument warm up for 15 minutes. With no sample in the instrument, use the left-hand
knob to set the meter exactly on 0 %T. Prepare five standards (the original stock
sucrose solution and the four dilutions) and a blank at the same time. The unknown
samples can also be prepared at the same time or at a later time.

Pipet 2.00 mL of each sucrose standard into a large test tube; also pipet 2.00 mL of
distilled water into a test tube for the blank solution. Pipet 2.00 mL of 6 M HCl into each
test tube and place in a boiling water bath for 10 minutes. Remove the test tubes and
carefully pipet 8.00 mL of 2.5 M NaOH into each, then 2.00 mL of 0.050 M 3,5-
dinitrosalicylic acid (DNSA) into each. As soon as the DNSA is added, cover the tube
with Parafilm, shake to mix the solution thoroughly, and place the tube in a boiling water

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Determination of Sugar as Glucose

bath for 5 minutes. Begin timing when the tube is placed in the hot water; each tube
must remain in the boiling water for the same amount of time. Remove each test
tube from the boiling-water bath at the proper time and quickly place it in an ice-water
bath for 10 minutes. Pour some of the blank solution into a clean, dry cuvette (fill
halfway), wipe the outside of the cuvette with a Kimwipe, and place it in the
spectrometer. Close the cover and use the right-hand knob to set the meter exactly on
100%T. Place some of each solution in a cuvette and record the %T to as many
significant figures as possible. If you use the same cuvette over and over, you must
rinse it with a little of each new solution before filling (to get rid of lingering drops of the
previous solution). It is a good idea to periodically check the 0 %T and 100 %T settings
and readjust them as necessary.

Convert the %T values to absorbance and draw a rough graph of Absorbance versus
concentration of sucrose. If the data are not linear, you will want to redo the standards.
It is important to heat each solution for the same amount of time when developing the
color. If the data look satisfactory, draw an accurate graph on good graph paper. If you
know how, you may wish to use the Excel spreadsheet to perform a linear least-squares
regression on the data to determine the slope and intercept of the line. This is your
Beer's Law calibration plot.

Prepare at least two dilutions of your beverage in the range of 1:100 to 10:100,
depending upon the amount of sugar or carbohydrate you expect. You might wish to
begin somewhere in the middle of that range and see whether the absorbance falls
within the range of your calibration plot. Treat 2.00-mL aliquots of the diluted samples in
the same manner that you did the standards; carry this determination out in triplicate for
each dilution. Be sure to use the same time interval after adding the DNSA that you
used for the standards. Record the %T for the diluted samples (in triplicate). Convert
the results to absorbance. Use your graph or the equation obtained from the regression
to determine the concentration of sucrose in your samples. If using the equation, c =
A/eb = A/slope. Taking dilution into account, calculate the concentration of sucrose in
the original beverage. In your discussion, compare your result to the label information
and carry out a detailed error discussion.

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