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METHOD OF ESTIMATION OF LACTIC ACID.
The method is a modification of that described by
F~etcher
and
Hopk1ns (J. Physiol. 35, I907, P. 247.)
Samples or 50 tG IOO gruB. of tissue are weighed out and cut up
With scissors under alcohol (Met~lated Sp1rit ~5%) and ground up until
thorough~
d1sintegrated.
Where the values of lactic acid requ1red are
belew the 'maximum for the tissue, the alcohol, mortar and 1netrumentsmust
be cooled in ,f r ee z1ng mixture and all operations done as quickly as poss­
ible.
The materia.l 1s then washed out 1nto a wide mouthed vessel and
allowed to stand tor at least 24 hours.
It is then transferred to a
500 ca. graduated cylinder, the vessel washed Gut with alcehol and the
VOltll118
made up to about 5 times as many cc , as grams of tissue were taken,
bringing the ~cohol concentration ts. about 80%.
Atter thorough1.y shak­
ing, the suspended matter is allowed to settle, the volume read off and
the liqu1d filtered into two
estimations.
yo-
roughly equal posttlons for duplioate
The filtrate 1s collected 1n 200 co. graduated cylinders
and the volumes noted (Ist
F1~tration).
Each filtrate is then evaporated to dryness on the water bath.
5 gme ,
of ammonium eui.pnat.e are stirred in during evaporation.
3 to
The
residue is rUbbed up with successive quantities Qf hot water and trans­
terred to a small tared beaker.
The volume of liquid at this stage
should be kept roughly constant between IOO and 50 cc ,
c)oc~
cQvered with a 4aPk glass and placed on the water bath.
The beaker is
If the suspended
fatty matter does not settle readl1Yt a little charcoal 1s added t and the
liquid further d1gested.
The beaker and ita contents are rapidly weighed
as
so~n
as enQugh filtrate has been collected.
p§rfectly
e~ea~ I
though 1t 1s always c&loured.
be rejected or refiltered.
The
f1ltrat~.must. b~
Cloudy filtrates must
The filtrate 1s evaporated nearly to dryness
th.t
and then transferred with ar1d of very small quantities of het water to
1\
a 100 ec , stoppered graduated cylinders.
The volume 1s brought to 10 co.
exactly' and 6 ec , or saturated Phosphoric A.c1d solut1Gn added, then
6to
The cylinder 1s v1gorousJ.;y shaken tor
6
of freshly d1stilled ether.
co.
minutes, atter the l1qu1ds have sellarated the ethereal layer 1s syphomed
otf
and
f1ltered though
a dry
filter paper 1nto
The extract zen is repeated 6 more t1mes
the same f1lter each time.
~
t
600
cc. con1cal rlask.
the ether be1ng f1ltered tbrGugh
ether of the combined extracts 1s d1stilled
off, the flask blOwn through with a1r to remove the last traces
Excess or Zinc carbonate (waShed tree or solUble matter)
added.
and
or ether.
hot wa.ter are
The whole bo11ea tor a short ttme, lett on the water bath tor
several hOurs, then f1ltered through a small tilter into a tared evaporat­
1ng d1sh.
The
flask and filter are washed several t1mea w1th bo11ing
water and the whole filtrate evaporated on the water bath. dried to
constant weight at IIOoe and weighed as Anbydrous Zinc Lactate.
The Zinc lactate on drying shoUld be crystalline
Extracts ot less than 10 mgm. are seldom cr,ystal11ne.
est1mat1~na
about IOO mgms. are needed.
and
nearly colourless
For satisfactory
coloured or noncrystalline
extracts must be regarded w1th suspicion and rejected it the Zlnc Oxide
cGntent on ign1t1on 1s found to be much below the theoret1cal.
For the f1rst and second filtrations it has been found better beth
for speed and accuracy to take an a11~ot part ~t the filtrate and not
attempt to wash the lactic ac1d quantitat1vely from the residue.
Evan
it the assumption of the equal part1tion ot lactic acid between the
residue and the f1ltrate is 1ncorrect, by us1ng s1m1lar vGlumea 1n all
cases, the error from thls source 1s neg11g1ble.
t ion about 80%
90~
or
In the f1rst f11tra­
the total can be obtained as filtrate, 1n the second
er more.
The residue trom the alcohol extractien may be used tor glycogen
estimation.
to
If the "lower carbohydrate- 1s to be estimated according
l~yerhofts
method -pflug. Arch. 182, 1920, P. 232) the alcohol
concentratlQn must be kept down to 80%_
But at th1s concentratiQn
the extract 1s much more diff1cult to handle.
With flsh muscle, espec1ally OOd, 1t 1s difficUlt to obtain a
c. ear- filtrate at the second flltratiQn. unless thls 1s done there
1s trOUble w1th the ether extract ren an<1 the zinc lactate 1s l1kely
to be contam1nated.
Combined ammon1um sulphate
and
charcoal treat­
ment usually brlngs the tatty material to a f1lterable condition.
The use of a double filter paper is often a help.
The
method of ether extraction described, though tedious, has
been found more regUlar 1n its results than contlnuous extraction
methodS (confirming the experience in Professor Hopkin'S laboratory).
Rather more than ~O% of the lactic acid 1s obtained by the met110d
descrlbed, the exact amount depending upon what i6 present 1n the
agueoue layer.
It is 1mportant to use exactly the same volumes 1n
every case and to keep the concentrations of ammonium sulphate and
phosphoric acid as constant as possible.
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D.R