LEB
12.01
Determination of the isoelectric point of an amino acid (glycine)
Related topics
Isoelectric point, acidic anions, basic cations, zwitterions,
equivalence (inflection) points, pKs value, titration, motor
piston burette
Principle and task
Amino acid molecules carry both acid and amino groups.
They can therefore form both acidic anions and basic
cations. The pH at which these two types of ions are both
present in the same concentration is called the isoelectric
point. This point is to be determined by recording the titration curve for the amino acid glycine.
Equipment (for the experimental procedure using the
Chem Unit)
Cobra3 Chem Unit
12153.00* 1
Cobra3 power supply
12151.99 1
Data cable RS232
14602.00 1
Software Cobra3 Chem Unit
14520.61* 1
pH electrode, gel-filled, BNC
46265.10* 1
Immersion probe NiCr-Ni, Teflon
13615.05* 1
Cable, Chem Unit / motor piston burette
36501.01* 1
Motor piston burette, universal type, 50 ml 36499.93 1
Magnetic stirrer mini, plastic (ABS)
47334.93 1
Magnetic stirring rod, cylindrical, l = 30 mm 46299.02 1
Volumetric pipette, 50 ml
36581.00 1
Rubber stopper, 14/18, 1 hole, d = 7 mm 39254.01 1
Safety pipettor Flip
36592.00 1
Wash bottle, 500 ml
33931.00 1
Beaker, DURAN, tall form, 250 ml
36004.00 1
Beaker, DURAN, tall form, 50 ml
Volumetric flask, 500 ml
Funnel, glass, d = 55 mm
Spoon with spatula end,
l = 150 mm, steel, wide
Balance SAS 51, 200 g / 0.01 g, RS232
Buffer solution pH 4.01, 460 ml
Buffer solution pH 10.01, 460 ml
Sodium hydroxide standard solution,
1 mol/l, 1000 ml
Hydrochloric acid standard solution,
1 mol/l, 1000 ml
Glycine, 100 g
Distilled water, 5 l
PC, Windows® 95 or higher
36001.00 3
36551.00 1
34457.00 1
33398.00
45990.93
46270.11
46272.11
1
1
1
1
48329.70 1
48454.70 1
31341.10 1
31246.81 1
Hazards and safety measures
According to its concentration, sodium hydroxide solutions cause severe burns or act as irritant to skin, eyes
and mucous membranes. Spray from them irritates the
respiratory organs. The corrosion destroys tissue and
causes severe pain. Must be kept out of the reach of children.
According to its concentration, hydrochloric acid causes
severe burns or acts as irritant. Vapours irritate respiratory organs, whereby the mucous membranes of the upper
respiratory tract are particularly affected. Concentrated
acids destroy skin and textiles.
Fig. 1: Experimental set-up using the Chem Unit
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P4120111/40
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12.01
Determination of the isoelectric point of an amino acid (glycine)
Do not inhale vapours or spray. Avoid skin contact. When
working with these chemicals wear appropriate protective
apparel, protective gloves and safety glasses.
First aid: Thoroughly rinse the affected skin or eyes
(opened wide) with plenty of water. When eyes are affected, seek immediate medical attention. After accidents or
on feeling unwell, fetch medical attention at once. When
inhaled: Fresh air, keep respiratory tract free. On respiratory distress: Transport to medical attention in a half-sitting position.
Waste disposal: Dilute the solutions with water, neutralize
them (pH 6 - 8) and wash them down the drain.
Preparatory work
Prepare the solution required for the experiment as follows:
— 0.1 molar hydrochloric acid glycine solution: Weigh
3.75 g of glycine into a 50 ml beaker. Transfer this
weight quantitatively into a 500 ml volumetric flask
(rinse the beaker several times with distilled water).
Use the volumetric pipette to add 50 ml of 1 mol/l
hydrochloric acid to this flask and make up to the
mark with distilled water.
Set-up and procedure (using the Chem Unit)
— Set the apparatus up as shown in Fig. 1
— Connect the control input of the motor-driven piston
burette to the TTL output of the Chem Unit with the
cable specifically for this
— Connect the pH electrode to the pH input and the
immersion probe to the temperature input T1 of the
Chem Unit
— Call up the Cobra3 Measure programme in Windows
and assign the Chem Unit as measuring instrument
— Set the measurement parameters as shown in Fig. 2.
In the <Displays...> menu, set both Digital display 1
and Diagram 1a (range 0-30 ml) to pH (range 1 to 13)
Fig. 2: Measurement parameters (Chem Unit)
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— Set the temperature compensation in the <Preferences / pH> menu to temperature probe T1:
pH(comp.T1)
— Calibrate the pH electrode by pouring the two buffer
solutions into separate 50 ml beakers and calling up
<Calibrate / pH>. Should the electrode which is to be
used have already been recently calibrated, however,
then a new calibration is not necessary (automatic
saving of calibration data)
— Follow the operating instructions for the motor-driven
piston burette to fill it with sodium hydroxide solution
— Set the motor-driven piston burette to <Dispense>,
<Dispensing volume = 0.2 ml> and <Rate =
100 ml/min>
— Place the 200 ml beaker, containing a magnetic stirring rod, on the magnetic stirrer
— Use the 50 ml volumetric pipette twice to pipette
100 ml of the prepared hydrochloric acid glycine solution into the beaker
— Fix the discharge tube of the motor-driven piston
burette, the pH electrode and the immersion probe to
the electrode holder of the motor-driven piston
burette, using the bored rubber stopper for the immersion probe. Adjust the electrodes and the tip of the
discharge tube so that they are completely immersed
in the solution
— Adjust the stirrer to a medium stirring speed (Take
care: Do not allow the magnetic stirring rod to hit
against the pH electrode)
— Start the titration with <Continue> and <Start measurement>. The first measured value (V = 0 ml, for the
starting point) is immediately acquired by the programme and the motor-driven piston burette dispenses the first portion of sodium hydroxide solution
— The titration is now automatically carried out, in accordance with the set parameters, until a total of 30 ml of
sodium hydroxide solution has been added. The programme then automatically stops the measurement
Fig. 3: Titration curve for hydrochloric acid glycine solution against 1 mol/l NaOH
Phywe Series of publication • Laboratory Experiments Biology • © PHYWE SYSTEME GMBH & Co. KG • D-37070 Göttingen
Determination of the isoelectric point of an amino acid (glycine)
— At the end of the measurement, save the data in menu
<File> <Save measurement as...>
Results and evaluation
Fig. 3 shows the titration curve, as it is displayed by the
programme after ending the measurements.
The prepared hydrochloric acid glycine solution contains
glycine and hydrochloric acid in a ratio of 1:1. The amino
groups of the glycine are converted by the acid to the protonized cation form:
H2N-CH2-COOH + H3O+ + Cl—> +H3N-CH2-COOH + H2O + ClOn the addition of sodium hydroxide, the pH of the solution continually increases and the concentration of the
cations continually decreases:
+H N-CH -COOH
3
2
+ Cl- + OH- + Na+
—>
+ H2O + Cl- + Na+
+H N-CH -COO3
2
At the first equivalence point of the titration, the addition
of an equimolar quantity of base has quantitatively split
the proton from the carboxyl group. The glycine is present
here in the form of zwitterions (externally neutral). The pH
at this point is called the isoelectric point (pI), as the number of amino acid cations is here equal to the number of
amino acid anions.
As the titration is continued, the increase in pH continues,
the concentration of zwitterions decreases and that of the
Fig. 4: Magnified display of the right portion of the titration curve
LEB
12.01
amino acid anions increases, until, at the second equivalence point, only amino acid anions are present in the
solution:
+H N-CH -COO- + OH- + Na+
3
2
—> H2N-CH2-COO- + H2O + Na+
In principle, therefore, the glycine cation (see above) represents a two-proton acid, and the titration curve so
exhibits two inflection points (equivalence points).
The software enables the equivalence point of the titration
to be automatically determined. Should your software not
enable this to be done, then you can have it determined
by using differentiation. For this, call up the menu
<Analysis> / <Channel modification> and select there the
options <Differentiate>, <Modify left axis> and <Add new
channel>. Select the function <Calculate> for the differentiated measurement curves to be displayed (Fig. 3). To
have the two curves displayed underneath each other,
reset the displayed pH range under <Measurement>,
<Display options> and <Left or right y-axis>. Use the
<Survey> function to have a vertical line fitted through the
peak of the differentiated curve. The intersection of this
line with the titration curve gives the equivalence point
(point of inflection). The coordinates of this equivalence
point are displayed numerically in a yellow field. Unlike the
first equivalence point in this titration, the pH increase at
the second equivalence point is too low. To determine this
second equivalence point, therefore, first enlarge the display of the second portion of the titration curve with the
zoom function (Fig. 4) and then proceed as described
above.The pKS1 value can be read from the curve as 5 ml
(half of the consumption of alkali up to the first equivalence point, see Fig. 5). The pKS2 value can be read as
15 ml (half of the difference in the consumption of alkali up
to the first and the second equivalence point).
Fig. 5: Reading of the pKS value
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12.01
Determination of the isoelectric point of an amino acid (glycine)
The respective buffering regions of the aqueous glycine
solution lie in the regions of pH around the pKS values. In
these regions, the pH changes only slightly despite the
addition of base or acid. At the equivalence points, however, a large change in the pH is caused by only small
additions of base or acid. In aqueous solution, and in
dependence on the pH, glycine forms not only cations,
but also anions and neutral zwitterions. At the isoelectric
point, the number of cations is the same as the number of
anions, the concentration of zwitterions is highest here. In
the titration described here, this point is identical with the
first equivalence point (pI = pH = 6.12).
In the example shown, the equivalence points are at V =
10.04 ml and V = 20.00 ml, and at pH values of 6.12 and
11.19. The respective pKS values were calculated to be
2.34 and 9.68.
Theoretically, the equivalence points of the amount of
hydrochloric acid glycine used should lie at V = 10.00 ml
and V = 20.00 ml, with pH values of pH = 6.01 (pH =
0.5 · pKS1 + 0.5 · pKS2) and pH = 11.32 (pH = 14 0.5 · pKb + 0.5 · log cb)
Experimental procedure using the Basic Unit
The experiment using the Chem Unit described above can
be analogously carried out using the Basic Unit. For this,
the entries in the list of materials which are marked with
an asterisk must be replaced by the materials listed
below. The set-up and procedure are then also slightly different (see below, in particular Fig. 6 and Fig. 7).
Fig. 7: Measurement parameters (Basic Unit)
The values for glycine given in the literature are:
pKS value of the carboxyl group:
2.34
pKS value of the ammonium group: 9.71
(pKb value: 4.29)
Isoelectric point:
6.03
The values found experimentally therefore comply quite
well with the theoretical values.
Fig. 6: Experimental set-up using the Basic Unit
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Phywe Series of publication • Laboratory Experiments Biology • © PHYWE SYSTEME GMBH & Co. KG • D-37070 Göttingen
Determination of the isoelectric point of an amino acid (glycine)
LEB
12.01
Changes in the equipment required for use of the Basic Unit:
Cobra3 Basic Unit
12150.00
1
Software Cobra3 pH/potential
14509.61
1
Measuring module pH/potential
12101.00
1
pH Electrode, plastic, gel-filled
18450.00
1
Temperature sensor, semiconductor type 12120.00
1
Cable, Basic Unit / motor piston burette 36501.03
1
— Call up the Cobra3 Measure programme in Windows
and assign Cobra3 pH/Potential as measuring instrument
— Set the measurement parameters as shown in Fig. 7
— Set the temperature compensation to <Automatic>
(temperature probe S2)
— Carry on from here as with the Chem Unit
Set-up and procedure using the Basic Unit
— Prepare the set-up as in Fig. 6
— Connect the control input of the motor-driven piston
burette to output S1 of the Basic Unit with the cable
specifically for this
— Connect the pH electrode to the pH measuring module and the temperature sensor to input S2 of the
Basic Unit
Note
The measurement can also be carried out without a temperature probe, but then the temperature of the solution
must be entered by hand in the menu <Preferences / pH>,
or, when using the Basic Unit, in the field <Temperature
compensation>.
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P4120111/40
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12.01
Determination of the isoelectric point of an amino acid (glycine)
Room for notes
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Phywe Series of publication • Laboratory Experiments Biology • © PHYWE SYSTEME GMBH & Co. KG • D-37070 Göttingen