The purpose of this
experiment was to analyze some of the fundamental properties of a class of
macromolecule referred to as carbohydrates. Experiment 1 and 2 use two
techniques – dialysis and gel filtration – to separate carbohydrate polymers from
their monomers. Dialysis removes or separates small molecules (monomers) from
large molecules (polymers) by allowing the diffusion of small molecules through
a semi-permeable membrane while molecules that exceed 10 000 Da remain trapped.
(“Introduction to Biochemistry”, pg.12, 2018). Gel filtration – size exclusion
chromatography – uses a column loaded with hydrated beads to slow down the
passage of molecules smaller than the beads while larger molecules move faster.
Experiment 3 and 4 will test for the presence of glucose and starch (from the
dialysis and gel filtration solutions) using glucose oxidase and iodine respectively
and will determine the concentration of glucose and starch.
Results and discussion
R1. Estimate the glucose and
starch content of the two dialysis compartments at both the initial and the end
point of the dialysis. Shown an example of calculations for both.
1. Absorption, concentration (in g/L),
and mass (in mg) of glucose and starch in two dialysis compartments (internal
and external) at the beginning (t=0min) and at the end of dialysis (t=45min). A piece of semi-permeable
cellophane dialysis bag (MW cut-off: 12000-14000 Da) was filled with 10 mL of
starch (0.10 mg/mL) and glucose (2.0 mg/mL) solution. Clipped at both ends, the
bag was placed in a 200 mL beaker of water and the experiment proceeded for 45
min and 30 s. The internal and external solutions were tested for glucose and
starch using glucose oxidase-peroxidase and iodine respectively. Using a
spectrophotometer, the absorbance intensity of each solution was measured at a
wavelength of 420nm for the glucose oxidase assays and at a wavelength of 600nm
for the iodine assays. The mass and concentration of glucose and starch were
calculated using the data collected in the lab.