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Titration Curves of enzymes
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How to make the titration curve of glycine and process data regarding this. Also showing principal concepts of: pH, PKa
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Worksheet: EXPERIMENT 2.1: TITRATION CURVE OFGLYCINE
14 October 2020
05:49
SESSION ORGANISATION
All timings are approximate
10.00 a. m. Make sure you arrive in good time. Download a copy of the session
worksheet from Minerva before or as soon as you arrive. You will enter the breakout
room with your group. Check your answers to the questions with one another and
with your demonstrator.
10.30 a.m. There will be a brief introduction by the Academic Lead together with any
course-related announcements, followed by a video demonstrating the technique
and experiment.
10.45 a.m. – 12.00 p.m. Complete worksheet in your groups with help from your
demonstrator (who will be supervising 3-4 groups): remember that while you are
working as a group, each of you must complete their own worksheet, in your own
words.
PREPARATION
Before you come to the session, you should:
• Read the introduction for understanding and making buffers.
• Using the information from these sources, write a 100-150 word
summary of the background material.
• Answer the questions
• Read through the session worksheet (experiment 2.1) so you understand
what you will be doing (but don’t start to complete it)
METHOD
Watch the video/demonstration for experiment 2.1 on Minerva
(https://vimeo.com/456581720/136557b45a) and note the key points of the method
here. Record your observations as the experiment progresses.
You are supplied with an approximately 0.05 M solution of the hydrochloride of
glycine (pH 1.0), which has the structure:
H
H3N+⎯ C⎯CO2H
H
, You are also provided with accurately standardised 0.1 M potassium hydroxide
solution. Potassium hydroxide is used in the titration in preference to sodium
hydroxide since the glass of the glass electrode contains sodium ions, and the
electrode potential may be affected by the presence of sodium ions in the test
solutions at pH values higher than 9.5. Gloves and safety glasses MUST be worn
when handling 0.1 M potassium hydroxide. Never stir the solution while the pH
meter is in it: the glass electrode is extremely fragile and expensive.
1. Addition of KOH to water
• Deliver 25 mL of water into a 100 mL beaker from the labelled dispenser.
• Standardise the meter using the standard pH solution (if necessary),
wash the electrodes and measure the pH of the water.
• Remove the pH meter, and add 1.0 mL of 0.1 M KOH using a P1000
automatic pipette.
• Stir well and measure the pH.
• Remove the pH meter, and add another 1.0 mL of 0.1 M KOH using a P1000
automatic pipette. Do not wash the electrodes between additions of alkali.
• Record the pH readings in a suitable table.
1. Titration of glycine
• Deliver 25 mL of the glycine HCl solution from the labelled dispenser into
a 100 ml beaker. Wash the pH meter electrodes, and measure the pH of
the amino acid solution.
• Record your readings in a table in your lab book.
• Remove the pH meter, add 1.0 mL 0.1 M KOH, stir well, and measure
the pH.
• Remove the pH meter. Continue to make additions of 1.0 mL KOH,
stirring and measuring the pH after each addition, until the pH has risen
to about 10.0
• Finally, remove the solution, wash the pH meter and place it in standard
buffer solution. Record the pH reading. This will show whether the meter
readings have drifted during the titration.
RESULTS AND DISCUSSION
Experiment 2.1; titration of glycine
Titration with glycine
Vol KOH added (ml) pH
0 2.2
1 2.5
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