John E.

Benron
Gettysburg College
Geftysburg, Pennsylvania

Viscometric Determination of the

Isoeiectric Point of a Protein

This experiment was designed to present

students with an opportunity to do an experiment of a
quantitative nature in macromolecular chemistry, an
area commonly neglected in undergraduate laboratory
work or studied only by means of short qualitative experiments. The experiment can be performed in a.
3-hour laboratory period by students working in pairs
and is suitable for use by chemistry majors or premedical students in courses a t several levels. The use
and operation of viscometers and pH meters are
illustrated, and the experiment is an excellent test of
technique since the results obtained depend very much
on manipulative care and cleanliness.
Mobility, conductivity, solubility, osmotic pressure,
stability toward coagulating agents, and viscosity of
proteins are a minimum a t the isoelectric point. The
minimum in the case of viscosity is quite sharp and
easily detected by preparing protein solutions of
various pH's, determining the viscosities of these solutions, and graphically finding the pH for minimum
viscosity.
The structure of proteins, the meaning of the terms
zwitterion and isoelectric point, the principle and technique of operation of a pH meter, the measurement of
viscosity1 and viscosity units2 are discussed in advance
of the experiment, either in class or in a written introduction to the ex~eriment. The students should be

warned of the fragility of the pH electrodes, the danger

of the electrodes becoming blocked by dried protein
solutions, the inadvisability of clamping viscometers
across both arms and the absolute necessity for cleanliness. The technique of rinsing and filling viscometers
and of measuring viscosities in minimum time should be
discussed.
A viscometer of the Ostwald or Cannon-Fenskef type
with a drain time for water of around 2-3 minis suitable
for this experiment. It is convenient to keep the
viscometer in a detergent-filled graduate from period to
period. The pH meter need not be very sensitive; an
instrument graduated in 0.1 pH unit and capable of a
reproducibility of
0.02 pH unit is adequate. The
use of a constant temperature bath a t 25C is advisable, but a stirred large volume of water a t 25'C is
suitable. A 1% by weight stock gelatin solution
("Knox" unflavored will work very nicely) has been
most cornmouly used by us; it should be prepared the
day before to allow it to age to constant viscosity.

The Experiment

="

Six 0.5% solutions of gelatin of pH values approximately every 0.5 pH unit from 3.5 through 6.0 are prepared by dilution of an aged 1% gelatin stock solution.
This operation is conveniently done by pipetting 50 rnl
portions of gelatin solution into six 100-ml volumetric
flasks, diluting with water to about 20 ml short of the
marks, adding dropwise some previously prepared
dilute HCI ( p H S+ 1) until each solution has a pH near
the desired value and finally diluting to the marks.
Some drift in pH will be observed and sufficient time

J m a ~ ~ s o NB..
s . AND STRILUMANIS.
M. E.. "A Short Textbook
of Colloid he-tj," John Wiley and sons, Inc., New York,
1954,pp. 1434.

CANNON,
M. R., AND FENSKE,
M. R., Ind. Eng. Cham., Anal.
Ed., 10,297 (1938).

Presented at the eleventh annual meeting of the Pennsylvania

h c i a t i o n of College Chemistry Teachers, Diokinson College,
Carlisle, Pemylvania, March, 1962.
' DANIELS,F., ET AL., "Experimental Physical Chemistry,"
6th ed., McGraw-Hill Book Co., Inc., New York, 1962, pp. 147- 8 .

468

Journal of Chemical Education

must be allowed for the electrodes to come to equilibrium with the solutions before reading the pH. Some
difficulty with foaming may cause trouble in diluting to
the mark, but the error is slight.
The times for flow through the capillary of the viseometer a t 25C are measured for water and the six
gelatin solutions. Drying of the viscometer with
acetone, the usual procedure recommended in texts,
is not necessary so long as the viscometer is carefully
and thoroughly rinsed with the solution to be used.
Drawing the solution up through the capillary into the
storage bulb a t the top and allowing it to drain down
once of its own accord before final measurement usually
insures that the solution is a t the bath temperature.
Large bubbles in the capillary side of the viscometer
can usually be avoided by carefully forcing the solution
in and out of the capillary.

Other Applications

With suitable changes in the conditions proteins

such as albumins, fibrins, and hemoglobins may be
used. Casein is not suitable because of its tendency to
precipitate a t its isoelectric point. An interesting experiment is the determination of the changes in viscosity
of a fresh gelatin solution due to aging. Although
viscometry is perhaps the simplest method to use, other
methods such as conductance, mobility, and sensitivity
toward coagulating agents may he used to determine
the isoelectric point. The isoelectric points of purified
amino acidse may be conveniently determined by conductance or electrophoretic mobility.

Results

The relative viscosities of the six gelatin solutions are

calculated and a plot of relative viscosity versus pH
prepared. The isoelectric point is determined from
the point of intersection of the two nearly linear
branches of the plot and is compared with a literature
value.' The plot diagram presents typical results on a
0.5% gelatin solution a t 25C; the experimentally determined isoelectric point was a t pH = 4.8, the commonly given literature value being 4.7.6 Lack of
sharpness in the minimum or a large error usually indicates impurities in the protein, dirty glassware, or the
presence of fairly large amounts of salts.
'Most standard texts on colloid and surface chemistry list
the isoelectric points of the more common proteins with references
to the original literature.
ABRAMSON,
H. A,, J . Den. Physiol., 13, 169 (1930).

The author is indebted to several students for working out details of the experiment and to Professor
Charlotte I. Damerel of Wilson College for her helpful
suggestions.

' HODGMAN,
C. D., edit, "Handbook of Chemistry and Physics," 38th ed., Chemiml Rubber Publishing Co., Cleveland, Ohio,
1956, p. 1649.

Volume 40, Number 9, September 1963

469