Unformatted text preview: RAMBLE'S SOUP CO.Ăż
TO: Product Testing Lab FROM: Vice President, New Products PROBLEM: The product development group is proposing to use a carbohydrate polymer as a
thickening agent in a new product line. The plant gums (polysaccharides) they
are considering (arabic or ghatti) are reported to contain some portion of the
following monosaccharide units: galactose, mannose, xylose, arabinose, rhamnose
and glucuronic acid. We are concerned about the breakdown of the product by
acid hydrolysis when the soup is heated which will adversely affect the
consistency. Some soups in the Lumpy Soup product line contain about two
tablespoons of vinegar per serving. We would like to know if significant
hydrolysis occurs during normal heating times. Investigate hydrolysis of the
gums under acidic conditions and at boiling. Also determine the relative amounts
of hexose and pentose sugars in the gums (o-toluidine colorimetric method).
Determine which of the monosaccharide components listed are contained in each
of the gums by thin layer chromatography following hydrolysis. REPORT: For your sample, report the hydrolysis results and conclusions, the component
residues and the percentages of pentose and hexose components. Your report is
needed in two weeks. Test for Polysaccharide Hydrolysis
Benedicts's Test - This is a test for reducing sugars. These are any carbohydrate with a potential aldehyde group
(hemiacetal) or potential ketone group (hemiketal). Carbohydrates yielding positive tests are
monosaccharides or small oligosaccharides without linked anomeric carbons. Large polysaccharides,
where the majority of anomeric carbons are involved in linkages, will test negative. Therefore,
unhydrolyzed polysaccharide will test negative while hydrolyzed will test positive.
Benedict's qualitative reagent is a solution of sodium carbonate, sodium citrate and copper
sulfate. When it is heated (5 minutes boiling water) with a reducing sugar, the Cu 2+ ion is reduced to
Cu1+ which then precipitates as a brick-red oxide, Cu2O. Red precipitate indicates a positive test.
Compare results with a 1% galactose and a 1% sucrose solution.
4) Glacial acetic acid
Concentrated hydrochloric acid
10% sodium hydroxide
Benedictâ€™s reagent Laboratory:
1. Add about one gram of the polysaccharide gum to 10 mL of cold water. Make note of the solubility.
2. Heat 200 mL of water to boiling and add the gum suspension to the hot water. Do an initial Benedictsâ€™
test by removing about 2 mL from the solution and transfering to a test tube containing 5 mL of
Benedict's reagent. (Heat tube in a boiling water bath for 5 minutes).
3. Next, add 5 ml glacial acetic acid to the boiling â€śsoupâ€ť. After 10 minutes, remove about 2 mL from the
solution and transfer to a test tube, cool and neutralize with 10% NaOH and do another Benedictâ€™s test.
4. Repeat this procedure again after 20 minutes.
5. After removing the 20 minute sample, add 5 mL of concentrated HCl to the hot solution and continue
heating. After 10 minutes with HCl, remove 2 mL, cool and neutralize with 10% NaOH, and do another
5 mL conc. HCl (12 M) 5 mL glacial acetic acid (17.5 M) 2 mL samples
every 10 min, 1 gram of gum +
10 mL water
200 mL boiling water 5 mL Benedicts's reagent o-Toluidine Colorimetric Method for Hexoses and Pentoses
Reference: Goodwin, J.F. Anal. Biochem., 1972, 48, pp 120-128. Aldohexoses react with o-toluidine in hot acetic acid to produce a
mixture of glycosylamines and the corresponding Schiff base which
have an absorbance maximum around 630 nm. Aldopentoses react and the
products show an absorbance maximum around 480 nm. Simultaneous
quantification of both components is possible by measuring the molar
absorptivities of the individual components at both wavelengths, and
then measuring the absorbances of samples at the same wavelengths.
1) Acetic acid reagent - 1.5 g thiourea and 3.5 g boric acid in one liter
of glacial acetic acid.
2) Amine reagent - add 5.0 mL of o-toluidine to 95.0 mL of the acetic acid
3) Standard monosaccharide solutions - accurately weigh 0.1 g of hexose or
pentose into 100 mL volumetric flask and dilute to mark with
distilled water. Calculate molarity.
4) Polysaccharide sample - accurately weigh 0.1 g of polysaccharide into a
test tube. Add 10 mL water and 1 mL concentrated HCl. Heat in
boiling water bath for 30 minutes. Pour solution into 100 mL
volumetric flask and dilute to the mark. Calculate concentration in
1) Prepare a blank by mixing 5.00 mL of amine reagent and 0.1 mL water.
2) Prepare a standard solution for each of the hexose and pentose
solutions. Mix 5.00 mL of amine reagent with 0.050 ml of standard
sugar and 0.050 mL of distilled water to total 0.100 mL.
3) Prepare two standard mixture samples by combining 5.00 mL of amine
reagent, 0.050 mL of the standard hexose and 0.050 mL of the standard
4) Prepare two unknown solutions by mixing 5.00 mL of amine reagent, 0.05
mL of hydrolyzed and diluted polysaccharide sample, and 0.05 mL
5) Heat all solutions in boiling water for 10 minutes - cool. Read the
absorption spectra of the individual pentose and hexose solutions
from 450 nm to 650 nm. Read the absorbances of mixtures and unknowns
at 480 nm and 630 nm relative to the blank.
Cramer's Rule for Simultaneous Equations A480 = a P,480 p + a H, 480 h
A630 = aP, 630 p + aH, 630h A = Absorbance at wavelength shown
a = molar absorptivity of pentose or
hexose at wavelength shown
p = molarity of pentose
h = molarity of hexose A 480 a H, 480
A630 a H, 630
a P, 480 aH, 480
a P, 630 a H, 630
a P, 480 A480
aP, 630 A 630
a P, 480 aH, 480
a P, 630 a H, 630 Thin Layer Chromatography of Monosaccharides
Reference: White, B.J.; Robyt, J.F. J. Chem. Ed., 1988, 65(2), pp 164-166.
1) Mobile phase - acetonitrile:water, 85:15 (v,v)
2) 1% (w,v) solutions of galactose, mannose, arabinose, xylose, rhamnose
and glucuronic acid.
3) Polysaccharide Acid Hydrolysate - Add 0.2 g of polysaccharide to 10 mL
of water in a test tube, and add 1 mL trifluoroacetic acid. Tightly
stopper and heat in boiling water bath for 30 minutes.
4) Developing Spray - methanol:sulfuric acid, 4:1 (v,v)
1) Prepare plate (Whatman K5 Silica Gel) by using a dull pencil to draw a
horizontal line 2.5 cm from the bottom of the plate. Place four
equally spaced dots on the line for four ascent lanes.
2) With a capillary tube, spot galactose and arabinose in lane 1; mannose
and xylose in lane 2; rhamnose and glucuronic acid in lane 3; and the
acid hydrolysate in lane 4. Allow to dry thoroughly.
3) Prepare chromatography chamber and allow to equilibrate. Run two
mobile phase ascents with thorough drying in between.
4) Allow to dry completely after second ascent. Lightly spray plate with
developing solution and place in oven at 110oC for 10 min.
5) Identify spots and measure migration distances.
Include as part of the report:
â€˘ polysaccharide (g/L?), acetic acid (M?), hydrochloric acid (M?)
concentrations in hydrolysis test mixture; procedure followed
â€˘ Standard solution M of pentose and hexose; polysaccharide (g/L) used
for o-toluidine method
â€˘ Sample calculations: Calculation of molar absorptivity from
absorption spectra; sample calculation using Cramer's Rule
â€˘ Table summarizing hydrolysis (Benedict's test) results
â€˘ Figures for each of the two standard solutions showing the absorption
spectrum from 450 nm to 650 nm.
â€˘ Spreadsheet Table showing Cramerâ€™s Rule calculation of the percent
hexose and percent pentose in the 2 known mixtures (compare these to
the known concentrations) and the 2 unknown solutions. Use the 4
molar absorptivities calculated from the absorption spectra, along
with the absorbances of the solutions.
â€˘ Table showing structures of all monosaccharides used for TLC
â€˘ Figure showing the results of the TLC experiment; all spots should be
identified and labeled ...
View Full Document