Laboratory Methods of Organic Chemistry - Sciencemadness Dot Org

HYDKOLYSIS OF CANE SUGAK 389
50 c.c. of water, a few drops of toluene are added, and the mixture is
left for fifteen hours at about 30° to allow the enzyme to be set free.
In order now to remove proteins from the thin sludge it is vigorously
stirred with 0-05 i\f-acetic acid in amount just sufficient to
change the colour of methyl red (p^ = 4:) (test with a sample), and
is then filtered as above after shaking with a little kieselguhr if
necessary. The filtrate is made neutral to litmus with dilute
ammonia and in this condition, protected by a little toluene, can be
kept unchanged for several days.
(6) The Inversion.-—Cane sugar (40 g.) is dissolved in 200 c.c. of
water in a 250 c.c. measuring flask, 25 c.c. of 10 per cent sodium
dihydrogen phosphate solution are added and the mixture is warmed
on a large water bath (or in a thermostat) at 30°. Now 10 c.c. of
the enzyme solution prepared as described under (a) are added and
the time at which the latter has run out of the pipette is noted. The
flask is filled at once to the mark with water at 30° and shaken up.
The first 25 c.c. sample is removed for polarisation and the time is
noted in the same way as was done just before. Bach sample is
run into 5 c.c. of 2iV^-sodium carbonate solution in order to stop
the action of the enzyme and at the same time to accelerate the
" mutarotation " (p. 395). After shaking it with a little animal
charcoal the sample is poured through a dry filter and the clear
solution is polarised in a 2-dm. tube. Each time the average of
three readings is taken.
The main solution in which the reaction goes on is kept at 30°.
From it samples are taken for polarisation every twenty minutes
during the first hour after the beginning of the experiment, and every
thirty minutes during the second hour. During this reaction period
that stage of the inversion which is shown by zero rotation is usually
passed. This indicates that about 75 per cent of the cane sugar
taken has been hydrolysed.
When the rotations are plotted as ordinates against the times
as abscissae, the observed points can be connected by a logarithmic
curve, which is flatter in the later stages of the reaction and indicates
the order as unimolecular. The abscissa of the point in this curve corresponding
to a rotation of 0° gives the " zero-rotation time ", which
is to some extent a measure of the activity of the enzyme solution
employed.
The course of the curve already indicates that the logarithmic law
is not strictly obeyed. By exterpolation we can obtain the point of