Foundations

of Biochemistry
Laboratory Skills

Practical Notes

Table of Contents 
Introduction ............................................................................................................................................. 1 
Practical classes are important! ........................................................................................................................... 1 
What you need to do for all practical classes… ............................................................................................. 2 
Important points about your practical classes .............................................................................................. 4 
Unsure what to do? ..................................................................................................................................................... 4 
How are you assessed? .............................................................................................................................................. 5 
Your responsibility ...................................................................................................................................................... 5 
Feedback .......................................................................................................................................................................... 5 
Skills are Learning Objectives ................................................................................................................................ 5 
Questions for Learning .............................................................................................................................................. 6 
Laboratory Notebook as evidence of good work practice ........................................................................ 6 
Practical 1 .................................................................................................................................................. 7 
Skill 1. Identify Health and Safety Equipment in the laboratory .......................................................... 7 
Skill 2. Identify hazards in the laboratory ....................................................................................................... 8 
Skill 3. Identify correct disposal of wastes ....................................................................................................... 9 
Skill 4. Assess Risks ................................................................................................................................................... 11 
Practical 2 ................................................................................................................................................ 12 
Skill 5. Knowledge of measurement, uncertainty and significant figures ...................................... 12 
Skill 6. Using graphs to summarise data. ...................................................................................................... 17 
Practical 3 ................................................................................................................................................ 18 
Skill 7.  Use an analytical balance to correctly weigh a dry chemical ....................................... 18 
Skill 8. Identify volumetric & non‐volumetric equipment ...................................................................... 19 
Skill 9. Correctly use appropriate pipettes to measure and dispense liquids. .............................. 20 
Practical 4 ................................................................................................................................................ 23 
Skill 10. Prepare an aqueous molar solution. .............................................................................................. 23 
Skill 11. Prepare a stock solution and a diluted solution. ...................................................................... 24 
Skill 12. Describe how to prepare a multi‐solute solution using stock solutions ........................ 24 
Practical 5 ................................................................................................................................................ 25 
Skill 13. Prepare a serial dilution ...................................................................................................................... 25 
Skill 14. Prepare a buffer solution .................................................................................................................... 25 
Skill 15. Describe how to prepare a multi‐solute buffer solution ...................................................... 26 
Practical 6 ................................................................................................................................................ 27 
Skill 16. Prepare standard solutions of an analyte and measure absorbance ............................. 27 
Skill 17. Plot a standard curve to estimate the concentration of an analyte .............................. 29 
Practical 7 ................................................................................................................................................ 30 
Skill 18. Estimate the concentration of a protein ...................................................................................... 30 
Practical 8 ................................................................................................................................................ 33 
Skill 19. Use the Henderson‐Hasselbalch Equation to calculate pH of glycine ........................... 33 
Skill 20. Experimentally titrate glycine with hydrochloric acid ......................................................... 36 
Skill 21. Plot the titration curve of glycine ................................................................................................... 38 

© Dr Steven Bottomley 2018

 Practical 9 ................................................................................................................................................ 39 
Skill 22. Assay of catalase in potato. ................................................................................................................ 39 
Skill 23. Relationship between substrate concentration and enzyme activity ............................ 42 

© Dr Steven Bottomley 2018

 Introduction

Welcome to the Foundations of Biochemistry practical class! You may be in enrolled
in the Molecular Genetics and Biotechnology, Human Biology (Preclinical),
Laboratory Medicine, or Pharmacy degree. In each case, however, you are learning
to be a biomedical scientist and a biomedical scientist often works in a laboratory.
Consequently, it is important for you to learn both the theory, and practice, of basic
laboratory skills and techniques.

The practical skills you will learn during your practical classes may be basic but
they are important skills. Basic also doesn’t necessarily mean easy and you will
have to work at practicing these skills and learning the necessary knowledge that
supports these skills. These basic skills may not be as attractive as using expensive
instruments to determine your genetics, or identifying some exotic
microorganisms, but they are necessary in all laboratory sciences. In fact, these
basic techniques are crucial to the precision and accuracy of almost all techniques
in laboratory sciences.

You will learn the knowledge of laboratory skills and techniques by carefully
studying the learning materials (e.g. the laboratory techniques study guide) and by
completing online practical reports. You learn to practice laboratory skills by
carefully studying the learning materials and practicing the skills described in these
notes during your scheduled practical classes.

Practical classes are important!

Your practical classes are important for the following reasons:

 Practice, and learn, necessary and fundamental laboratory skills in
biochemistry.
 Provide a place and time to collaborate, and learn, with your fellow students.
 Provide a place a time to discuss questions and exercises with your fellow
students.
 Provide a place and time to learn and receive guidance from experienced
laboratory supervisors.

A lot of work has gone into carefully designing these practical classes to help you
learn. However, you do have to make an effort to learn to derive the greatest
benefit.

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 What you need to do for all practical classes…

Participation, study, and professional conduct

 Attend, and participate in, all practical classes. You will put yourself at a
considerable disadvantage if you fail to attend practical classes, because you
will not be able to practice the required skills and you will miss out on useful
discussion with your team members. You will also miss out on useful
discussion, guidance, and feedback from your laboratory supervisors.
Moreover, you will lack the appropriate knowledge or skill needed for the
practical appraisal.
 Establish a ‘learning team’ with your fellow students (see later).
 Demonstrate a professional approach for all your work. That is, arrive on
time for class, attempt all skills, and think about what you are doing.
 Prepare for the practical class by carefully reading the appropriate chapter
in the laboratory study guide, the practical notes, and the risk assessment.
 Conscientiously, and carefully, perform the skills for each class as described.
 Commence the online practical report as soon as possible after it opens.
 Learn from any advice proffered by the experienced laboratory supervisors.
 Study and learn all appropriate information regarding the skill, so that you
can correctly answer theoretical or practical questions in the practical
report, in the appraisals, or when asked by the laboratory supervisor.
 Ask a laboratory supervisor to sign the record of work attempted to validate
that you attempted some, or all, of the practical skills during a practical class.
You should keep the form in your laboratory notebook at all times. The
laboratory supervisor will inspect your laboratory notebook, and possibly
ask you questions, to determine the skills you attempted during class.

Clothing

 Bring your laboratory coat and safety glasses to the laboratory if you are a
Pharmacy student. If you are a Biomedical Sciences student the laboratory
coat and safety glasses are provided.
 Wear closed in shoes to protect your feet.
 Always wear appropriate personal protective equipment (PPE) during the
practical class.

Laboratory notebook and Equipment

 You need to bring these Practical Notes, so you know what to do in a
practical class. Alternatively, you may access to the online version of these
notes.

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  Always bring your laboratory notebook, and student ID, to your practical
class. If you do not have your laboratory notebook in class, then you will not
have the appropriate evidence of your participation in the class. Your
laboratory notebook should be a bound A4 size hard cover, or soft cover,
‘exercise book’ or similar notebook. A spiral bound book or recording results
on unbound paper or on the back of practical notes is unacceptable. Please
read Chapter 1 of the laboratory study guide for more information.
 Always, write, and record, all appropriate measurements, results, notes, and
other appropriate details in your laboratory notebook. Do not use pieces of
paper, these practical notes, or any other book to record these items.
 Your laboratory notebook should be logically organised and your writing
should be legible, organised, and understandable. All tables or graphs should
also be inserted into your laboratory notebook.
 Write your laboratory notebook according to the required format as detailed
in Chapter 1 of the laboratory study guide.
 You need a pen, a pencil, a permanent marker, a ruler, and a calculator for
each practical class.

Learning Teams and Responsible Learning

 Establish a ‘learning team’ with your fellow students. A ‘learning team’ is an
informal association of up to four people and is your immediate support
system and forum for discussion. Your learning team can consist of students
who share the same laboratory bench or are located in different parts of the
same laboratory class. However, all practical work must be conducted only
on one laboratory bench (except for those practical skills where you may
have to use equipment located in other parts of the laboratory).
 Each member of your learning team has the responsibility to practice the
skills, attempt questions, and help other team members understand the
material. Helping others to learn will also help you further your own
understanding and knowledge.
 If you and your learning team cannot understand a skill, are ‘stuck’ on a
question, or can’t resolve a disagreement, then you should actively seek
guidance and feedback from your laboratory supervisors.
 Take responsibility for your learning.
 Ask questions! Ask questions for every single activity you perform in the
laboratory. Ask yourself if you are performing the skill correctly. Ask
yourself why you are performing an activity and why your think it is
important. Ask yourself how something works in a laboratory. You learn by
asking questions because your brain will try and seek an answer! The
process of seeking an answer will guide your study and help you become an
independent learner and critical thinker.

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  Seek answers! Answer questions in the online report, answer ‘Questions For
Learning’, and answer questions posed by the laboratory supervisor. By
answering questions, you will learn!

Important points about your practical classes

 All of the skills listed in this document must be performed in a practical class
according to the schedule shown in the unit outline.
 If you miss a practical class, then it is unlikely that you will have the
opportunity to practice the skills, receive feedback, demonstrate your
competence in the skills, or gain appropriate knowledge. Consequently, your
lack of knowledge and skill will put you at a disadvantage when it comes to
the practical appraisal.
 More than one skill may be performed during a practical class, but you must
ensure that you have sufficient time to complete all skills.
 Skills will become more involved as you progress through semester, but they
will be built on skills that you should have learned in class.
 You will need to work carefully, conscientiously, and diligently to ensure
that your work is of a sufficient quality.
 Do not write on separate pieces of paper or in a book that is not your
laboratory notebook.
 You may work collaboratively in your learning team to discuss the skills and
in some cases, share the workload. However, in some instances you will need
to perform the skill individually.
 Your laboratory supervisor may approach you at any time during the
practical class to offer guidance and feedback. Please accept these
interventions not as criticism but as your opportunity to learn from
experienced professionals who are willing to guide you.

Unsure what to do?

If you are unsure what to do, or do not understand what is required of you, you
should perform the following steps:

 Carefully re‐read the instructions in these practical notes.
 Carefully study the background material in the laboratory techniques
study guide.
 Ask your team members for help.
 Ask the laboratory supervisor for further clarification and guidance. Your
laboratory supervisor may, in turn, ask questions to help them help you.
Essentially, they will ask questions to assess your current knowledge,
help correct any misunderstanding, and guide you.

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 How are you assessed?

The practical appraisal is an exam, worth 35% of your total semester mark, which
will cover all aspects (theory and practice) of the skills you have learned in each
practical class. The exam will take place in the laboratory, at your usual scheduled
class, during the last week of semester. Please see the unit outline for more details.

Your responsibility

You are responsible for:

 Attending, and participating in, all practical classes and performing all
required skills within the stated timelines.
 Establishing, and participating in, your learning team.
 Completing all required practical reports within the stated timelines.
 Reading all necessary materials, practicing skills, and learning the content.
 Bringing your laboratory notebook, student ID, record of completed work,
and your practical notes, to each practical class.
 Writing appropriate experimental, or activity, details in your laboratory
book.
 Asking laboratory supervisors to check your work, for feedback and
guidance, on the required in‐class practical skills during your usual
scheduled practical class.
 Asking laboratory supervisors to sign, and validate, the skills you attempted
in each practical class in your record of work completed form
 Asking questions and seeking answers ‐ to help you learn!

Feedback

One of the benefits of the ‘Foundations of Biochemistry’ unit is that you receive
constant feedback. Feedback comes in the form of notes, lectures, practical reports,
topic quizzes, comments made during class, individual advice, discussion within
your learning team, and individual guidance. We would also be pleased to receive
your comments and suggestions at any time during semester. We prefer to receive
feedback during semester rather than at the end of semester as it is more relevant
and timely. Please also let us know if you spot any mistakes in your notes, or your
laboratory study guide, as we would like to correct them. We always aim to
improve your learning experience.

Skills are Learning Objectives

Every skill is a learning objective! If you satisfactorily complete the skill, then you
have completed the learning objective.
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 Questions for Learning

The ‘Questions for Learning’ are, as the name suggests, questions that develop
your knowledge and help you learn. They are also designed to:

1. Show you the kind of questions that you should be asking yourself about
the material.
2. Help you better understand the skills, concepts, and knowledge of
the practical syllabus (both theory and practical).
3. Help you think about related concepts and to integrate them.
4. Promote discussion within your learning team.

You should attempt all ‘Questions for Learning’ and you can do this before, during,
or after the practical classes. However, you can easily discuss these questions with
members of your learning team during the practical class. Some of the questions
may appear in the practical report, and practical tests, in a different context or
format. Consequently, it will help you learn if you write your answers to each
question in your laboratory book. The ‘answers’ to the QFL are only available from
the laboratory supervisors. The laboratory supervisors will only provide an
answer after you have written your attempted answer in your laboratory
notebook. The laboratory supervisor may also engage in further discussion with
you regarding your answers.

Laboratory Notebook as evidence of good work practice

Your laboratory notebook may be inspected by the laboratory supervisor at any
time during semester to give you advice and guidance. This may also occur when
you ask the laboratory supervisor to sign, and validate, your ‘record of work
completed’ form. You can also ask the laboratory supervisor, at any time during
practical class, for advice on your laboratory notebook. Your laboratory notebook
is not a part of the assessment and you do not need to submit your laboratory
notebook for assessment at the end of semester.

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 Practical 1

Carefully read, and study, Chapters 1 and 2 in your laboratory study guide before
commencing this practical. Establish your ‘learning team’ during this practical class.

Skill 1. Identify Health and Safety Equipment in the laboratory

Demonstrate awareness of health and safety issues in the laboratory. Identify and
locate the following items in the laboratory:

 Fire extinguisher
 Emergency shower
 Emergency shower for eyes
 Fire blanket
 First Aid cabinet or First Aid Kit
 Chemical spill kit (may be enclosed in a cabinet depending upon the lab)
 Emergency guidelines and procedures booklet or Emergency procedure A4
sized poster.
 Chemical hazard chart
 Sinks
 Biohazard and chemical waste boxes for collecting biological and chemical
waste (usually just one container in biochemistry labs)
 Small yellow plastic collection bins for small ‘sharps’ (e.g. usually pipette tips
and for small pieces of broken glass)
 Large yellow collection bins for large ‘sharps’ (e.g. usually large pieces of
glass from broken volumetric glassware)
 Fume hood for volatile chemicals
 Lab coats (if in a lab where coats are provided)
 Latex or other protective gloves
 Protective eye glasses
 Fire sprinklers
 Smoke alarms
 Telephone
 Exit signs and evacuation routes

You may draw an approximate map of the location of all the items listed above, cite
the location next to the item in the list, or do both. Please note that some items on
this list cannot be found.

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 Skill 2. Identify hazards in the laboratory

2.1 Inspect the chemicals on your bench and complete the following table:

Chemical Hazard(s)





The hazard column for each chemical in the table above should be labelled one, or
more, of the following:

 Flammable
 Toxic
 Health Hazard
 Acidic

Try and match each hazard with its appropriate hazard symbol in the study guide.

2.2 Complete the following table for potential hazards with laboratory
equipment:

Equipment Potential Hazard(s)
Mechanical air‐displacement
pipette with tip

Centrifuge (small benchtop

centrifuge or large centrifuge)

Spectrophotometer

A water bath that can have the

temperature of water adjusted

Note: some of equipment in the table may not be in your laboratory but you can still
think about the potential hazards.

NOTE: For any table in these notes you may manually draw the table in your
laboratory notebook or cut and paste the table into your notebook.

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 Skill 3. Identify correct disposal of wastes

Discover the appropriate disposal of the following wastes:

 Plastic pipette tip from a mechanical air displacement micropipette.
 Piece of bovine liver left over after an experiment.
 Paper towel after drying hands at the sink.
 An aqueous solution of 100mM sodium phosphate, pH 7.5
 Piece of paper tissue soaked with 80% alcohol after wiping the bench top.
 Piece of filter paper used for filtering a non‐toxic, non‐biological aqueous
solution at pH 8.
 Non‐toxic and non‐biological coloured water left over at the end of an
experiment.
 A dry, non‐toxic, non‐biological, powdered chemical.
 Broken glass after a volumetric cylinder had been knocked over on the
bench.

Write answers to each point above in your laboratory notebook. You may discuss
this with your team members.

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 Skill 4. Assess Risks

You should learn how to assess risks in the laboratory. Assessing risks means that
you need to do at least two things: (1) Identify the hazards and associated risks of
each activity. (2) Reduce those risks, if possible, by using appropriate procedures or
equipment. An activity is what you do in the laboratory to achieve a particular
outcome (e.g. prepare a solution).

Assess the activities, hazards, and risks for the following scenario:

A laboratory officer is asked to prepare a 1M solution of sulphuric acid. The
laboratory officer has concentrated sulphuric acid in a 2.5L glass bottle appropriately
stored in an acids cupboard the laboratory. Assume that the laboratory officer has
appropriate non‐volume, and volume‐measuring, instruments and a fume hood
available in another part of the laboratory. The acids cupboard and fume hood are
separated by about 25metres.

You may use the following table to assess the hazards and risk in the given scenario
(note you may need to expand the table). You should:

1. List all the relevant activities that need to take place in the given scenario.
Hint: there are at least two main activities in the above scenario.
2. List the hazard for each activity
3. List the risk of each hazard
4. What should be done to control, reduce, or eliminate the risk
5. Find and read an appropriate material safety data sheet (MSDS) for
sulphuric acid.

Activity Hazard Risk Risk Control*

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Practical 2

Carefully read, and study, Chapters 3, 4, and 5 in your laboratory study guide before
commencing this practical. Write all answers in your laboratory notebook.

Skill 5. Knowledge of measurement, uncertainty and significant figures

The following questions, and exercises, are designed to help you learn about
measurement, uncertainty, and significant figures. Remember to write your
answers, or points of discussion, in your laboratory notebook.

1. Each member of your team writes in their own words why measurement is
important in science. Discuss your respective answers within your team.

2. Which of the following is primary data and which is transformed or secondary
data?

 Weighing the same sample on a balance five times consecutively.
 Averaging the weights of the five consecutive measurements of the
weight of the same sample.
 Calculating the standard deviation of the five consecutive measurements
of the weight of the same sample.
 Measuring the pH of a solution

3. What do each of the following units represent? Write the full name and
property that is represented by each unit (e.g. volume, mass, etc).

 M
 mM
 nM
 nm
 mm
 cm
 kg
 g
 g
 l
 ml
 L
 kJ

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 4. A powdered chemical was weighed and found to be 1.25 g. What four pieces of
information can be determined from this weight? What cannot be determined
by this weight but may be important?

5. Read the following and determine the variable(s), the parameter(s), and
constant(s) where applicable.

 An enzyme is assayed at 37°C in a buffer by observing the conversion of
one chemical into another over 1 minute.
 Absorbance of a solute in solution is related to its concentration (c),
pathlength (l), and molar extinction coefficient () as shown in the
formula: A = cl. The pathlength is usually set to 1cm for most
measurements.
 Energy and mass are related by Einstein’s famous formula E=mc2

6. Determine the implicit uncertainty in the following measurements and
express them as: value ± implicit uncertainty

 555 nm
 0.545 M
 250 mM
 50.0 mM
 2.0034 g
 7.02 g
 37.2 m

7. Label each of the following calculations as measures of accuracy or precision.

 Tolerance
 Absolute error
 % relative error when using a reference value as a standard
 Standard Deviation (SD)
 Coefficient of variation (CV)

8. Find the following instruments in the laboratory and write the manufacturer’s
stated tolerance for each instrument:
 10 ml graduated pipette (serological or Mohr)
 10 ml volumetric (bulb) pipette
 50 ml graduated cylinder
 100 ml graduated cylinder
 500 ml or 1000 ml volumetric flask

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 9. If the tolerance of a 100 ml graduated cylinder was stated as ± 1ml does this
imply that a measurement of 50 ml, made with a 100 ml graduated cylinder,
would also have a tolerance of ± 1ml? Explain.

10. What are the estimated percentage errors of measuring 75 ml, 50 ml, and 25
ml, when each is measured with a 100ml graduated cylinder with a reported
tolerance of ± 1ml? What do you notice about the relationship between
percentage errors and the three volumes?

11. Have members of your team fill a graduated pipette and a graduated cylinder
with a volume of coloured water. Don’t worry about your pipetting technique
with the graduated pipette, because you will learn the proper pipetting in
Practical 3. The purpose of this exercise is to practice reading volumetric
instruments. The team member can choose any volume, but should not state
the volume. Each team member then reads, and records, the volume to the
appropriate number of significant figures. Discuss any discrepancies between
team members and try and resolve those discrepancies.

12. Have each member of your team take a mechanical pipette and set up any
volume. You do not need to fit a pipette tip because you are not going to
pipette any liquid. The purpose of this exercise is to practice reading
volumetric instruments. Each team member then reads, and records, the
volume on the mechanical pipettes to the appropriate number of significant
figures. Discuss any discrepancies between team members and try and
resolve those discrepancies.

13. Go to the nearest balance and turn it on. Record the units, number of decimal
places, and inherent uncertainty of the balance.

14. The following image is from a display of a spectrophotometer. Record the
units, number of decimal places, number of significant figures, and inherent
uncertainty (expressed as value ± uncertainty) of the wavelength () value
shown in the display. Repeat the exercise for the absorbance (Data) value
shown in the display.

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15. A biotechnology company manufactures a product that they state has less
than, or equal to, 0.03% impurities (0.03%). Label each of the following
batches of the product as either meeting the specification or not meeting the
specification.

A. Batch 1: 0.035%
B. Batch 2: 0.025%
C. Batch 3: 0.037%
D. Batch 4: 0.033%

16. Serum albumin is the main blood protein and an assay of its concentration in a
person’s serum is used to help diagnose liver disease, kidney disease, or the
nutritional status of a person. Medical laboratory scientists in three
laboratories each tested the same standard preparation of serum albumin
four times in the same day. The serum albumin standard was 40.0 g/L. The
results of the tests are shown in the following table.

Lab 1 Lab 2 Lab 3
(g/L) (g/L) (g/L)
39.1 40.2 40.6
39.8 40.3 39.7
39.6 39.1 41.1
39.4 39.6 39.0

A. What is the implicit uncertainty in the standard?
B. For each laboratory calculate the following:
 Mean
 Absolute error
 % relative error
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  standard deviation
 coefficient of variation
You can use your own calculator, or an online calculator, for these
calculations. Do not attempt these calculations manually. You must take
care to report all values of uncertainty with one significant figure.
C. Label each of the above calculations with appropriate units where
applicable.
D. Which laboratory is the most precise and which laboratory is the most
accurate when measuring the serum albumin standard?
E. Which laboratory (if any) seems to display a slight systematic error.
Explain.

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 Skill 6. Using graphs to summarise data.

The absorbance, at 525nm, of a series of potassium permanganate solutions was
measured. The relationship between absorbance and concentration of a solute in
solution is known as the Beer‐Lambert law and is expressed in the formula: A = cl.
This measurement of absorbance of known concentrations of a solute is a standard
curve (also known as a calibration curve). A standard curve can be used to
determine the unknown concentration of the same solute in another solution. The
table below shows the absorbance for each concentration of potassium
permanganate.



Absorbance Concentration
At 525nm (mg/L)
0.00 0
0.25 20
0.64 40
0.75 60
1.22 80
1.35 100
1.55 120

1. What is the implicit uncertainty of the absorbance values?
2. What is the implicit uncertainty of the concentration values given that
the values from 20 to 80 contain two significant figures and the values
from 100 to 120 contain three significant figures?

6.1 Manually plot the data on an appropriate graph.
 Manually plot a scatter graph of the data from the table above.
 Draw a linear line of best fit to the data on the graph.
 Have a team member check that your graph is drawn according to the
requirements stated in your laboratory study guide.
 Have a team member, or laboratory supervisor, check that your line of
best fit is appropriate.

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 Practical 3

Carefully read, and study, Chapters 6 and 7 in your laboratory study guide before
commencing this practical. Write all answers in your laboratory notebook.

Skill 7. Use an analytical balance to correctly weigh a dry chemical

Please practice the following skills. Ensure that you are wearing appropriate PPE!

7.1 Estimate the inherent uncertainty of the analytical balance from the display
when it is set to read grams. Do not have any sample on the balance pan.

7.2 Accurately weigh 1.0 g NaCl using the proper procedure (as detailed in your
Laboratory Techniques Guide).

 Record in your laboratory notebook the range of uncertainty of 1.0g.
 Record in your laboratory notebook the actual measured weight of your
1.0g sample using all the information (decimal places) given in the
balance’s display. Your actual measured weight should be within the range
of uncertainty for the 1.0g sample.

7.3 Accurately weigh 1.000 g NaCl using the proper procedure.

 Record in your laboratory notebook the range of uncertainty of 1.000g
sample
 Record in your laboratory notebook the actual measured weight of your
1.000 g sample. Remember to use all decimal places given in the
balance’s display. Your actual measured weight should be within the
range of uncertainty for the 1.000g sample.

7.4 Write in your laboratory book the procedure you used for weighing a sample
on a balance. Write in your own words. Do not simply copy from the study
guide.

Questions for Learning

1. What is meant by the term ‘accurately weigh’?
2. What did you notice about the time needed to weigh 1.0g compared with the
time (and effort) needed to weigh 1.000g? What is the essential difference in
weighing 1.0g compared with weighing 1.000g?
3. Describe at least three factors that can influence the measurement of weight.

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 Skill 8. Identify volumetric & non‐volumetric equipment

8.1 Identify the following pipettes:
 Mohr pipette
 Serological pipette
 Pasteur pipette
 Transfer pipette
 Bulb pipette (if in Pharmacy)
 Mechanical air‐displacement micropipettes

Distinguish between volumetric, and non‐volumetric, pipettes in the list (a)
above.

8.2 Identify the following equipment:

 Volumetric flask
 Beaker
 Erlenmeyer flask
 Graduated cylinder
 Microfuge tube
 3DT plastic test tube

Distinguish between volumetric, and non‐volumetric, instruments in the list
(2) above.

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 Skill 9. Correctly use appropriate pipettes to measure and dispense liquids.

9.1 Pipetting with a Mechanical Air Displacement pipette

Use a mechanical air‐displacement micropipette to practice measuring and
dispensing liquid according to the recommended procedure for:

(a) forward pipetting.

(b) reverse pipetting.

Practice using both the forward and reverse pipetting technique, for all
micropipettes available to you.

Pipetting is an important basic skill, so you should practice your pipetting
before you progress to skill 9.3. You should also ask your laboratory
supervisor to check your pipetting technique. Ensure that you are wearing
appropriate PPE whilst practicing this skill.

9.2 Pipetting with a Mohr, Serological, or Bulb pipette

Practice measuring and dispensing at least 3ml of liquid using a Mohr pipette,
a Serological pipette, or a volumetric bulb pipette (the choice is yours). Ensure
that you are wearing appropriate PPE whilst practicing this skill.

Ask your laboratory supervisor to check your pipetting skills.

9.3 Self‐assessment of your accuracy and precision with a mechanical air‐
displacement micropipette.

Preparation for Forward & Reverse pipetting self‐assessment

a) Take six empty weighing trays to your bench. Label three of the trays F1,
F2, F3 and three of the trays R1, R2, R3. The trays labelled with the letter
‘F’ are to be used for collecting volumes dispensed using the forward
pipetting technique. The trays labelled with the letter ‘R’ are to be used for
collecting the volumes dispensed using the reverse pipetting technique.
b) Take each empty, and now labelled, weighing tray to an analytical balance
and accurately weigh each tray using the proper technique.
c) Record the weight of each tray to three or four decimal places (depending
upon your balance) in your notebook.
d) Take all of the weighing trays back to your bench.

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 Forward pipetting Technique

a) Use a 1000 l micropipette, with an appropriate tip, to take up 500 l of
coloured solution (provided for you on the bench) using the forward
pipetting technique. Please note that the measurement of 500l has three
significant figures.
b) Dispense the solution into one of the weighing trays.
c) Repeat the previous two steps until you have three weighing trays each
containing 500l of solution.
d) You do not need to change the pipette tip between each use of the pipette
because you are pipetting the same solution. However, if you find that
some of the solution remains in the tip after pipetting one sample, then
you should change the tip. Note that if you use the correct technique there
should not be any solution remaining in the tip.

Reverse pipetting technique

a) Use a 1000 l micropipette, with an appropriate tip, to take up 500 l of
solution using the reverse pipetting technique.
b) Dispense the solution into one of the appropriate weighing trays.
c) Repeat the previous two steps until you have four weighing trays each
containing 500l of solution.
d) You do not need to change the pipette tip, or expel the solution, between
each use of the pipette because you are pipetting the same solution. Only if
you were pipetting different solutions, or if you are pipetting viscous
solutions, then it would be better to expel the solution and change the tip.

Measure your dispensed volumes

a) Take all weighing trays, with added solution, and accurately weigh each of
them.
b) Record the weight of each tray plus solution to three or four decimal
places (depends on your balance) in your notebook.
c) Subtract the initial (empty) weight of the tray from the weight of the same
tray with the added solution to calculate the weight of the volume that you
dispensed. Your result should also have three or four decimal places.
d) Complete the table shown below and calculate the mean, SD, and CV of the
volumes you dispensed using the forward pipetting and reverse pipetting
techniques. Remember to record the appropriate number of significant
figures in your results.
e) Convert the mean of the weights that you measured to volumes using the
density of water ~ 1.0 g/ml. Record this value in the table.

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 Record the weights of the volumes you dispensed to three or four decimal
places (depending upon your balance). Remember, all uncertainties should be
reported with only one significant figure.

Final weights of pipetted volumes (g)
500 l solution 500 l solution
using a 1000l using a 1000l
pipette pipette

forward pipetting reverse pipetting

1

2

3

Mean (g)

Mean (ml)

% error

SD

CV %

Rating

You may ‘cut and paste’ this completed table into your laboratory notebook.

Rate yourself based on your CV%!
 CV less than or equal to 1% = Excellent
 CV greater than 1% less than 5% = Good
 CV greater than 5% less than 7% = OK
 CV greater than 10% = Poor

Questions for Learning
1. List some of the necessary practical techniques to ensure reliable pipetting.

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 Practical 4

Carefully read, and study, Chapter 8 in your laboratory study guide before
commencing this practical. Write all answers in your laboratory notebook.

It is important to note that when you prepare any solution in a biochemistry or
pharmacy laboratory you should use only distilled water or similar (e.g. double
distilled water, reverse osmosis water, filtered water). Do not use tap water to
prepare solutions. You can get suitable water from the white taps at the end of your
bench (in the Biomedical Sciences laboratory). Wear gloves when handling small
magnetic stirrers and wash the stirrers before placing in your solution.

 Ensure that you record all weights, volumes, and calculations in your
laboratory notebook.
 Ensure that you are wearing appropriate PPE.
 Ensure you are using appropriate volume‐measuring instruments that
account for the uncertainty in your measurements. Remember the relative
percentage error, tolerances, and level of uncertainty!
 Record in your laboratory notebook the type of volume measuring
instrument used to prepare the solution.

Skill 10. Prepare an aqueous molar solution.

Prepare a 100 ml solution of 0.154M NaCl. This solution only contains one solute.
The molecular weight of NaCl = 58.44 g/mol

Questions for Learning

 What two basic questions do you need to ask before you prepare any
solution?
 What is the implicit uncertainty and percent relative uncertainty for the
solution you are asked to prepare? Based on this information what volume‐
measuring instrument should you use to prepare this solution?
 What should be the target range of uncertainty for your measured weight of
NaCl? Explain.
 What is meant by the term ‘quantitative transfer’?

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 Skill 11. Prepare a stock solution and a diluted solution.

11.1 Prepare a 100 ml solution of 5.0% (w/v) glucose stock solution.

11.2 Use the stock solution, prepared in 11.1, to make a 20 ml solution of 2.0%
(w/v) glucose.


Skill 12. Describe how to prepare a multi‐solute solution using stock solutions

In this skill, you do not need to prepare the solution, but you do need to describe,
with all supporting calculations, how you would prepare a total of 100ml of the
following solution using the indicated stock solutions.

50ml of 0.32 M NaCl
1.1 ml of 0.5% w/v phenylmethylsulphonyl fluoride (PMSF) in ethanol
49.9ml of 20% w/v Glucose

 Assume that you initially started with the following stock solutions: 200ml
0.32 NaCl solution, 5ml of 0.5% (w/v) PMSF, and 100 ml of 20% (w/v)
glucose solution.
 Ensure that you record all weights, volumes, and calculations in your
laboratory notebook.
 Ensure, when you explain the procedure, you state the appropriate volume‐
measuring instruments that account for the uncertainty in your
measurements.
 State the concentration of each solute in the final completed solution.

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 Practical 5

Carefully read, and study, Chapter 8 in your laboratory study guide before
commencing this practical. Write all answers in your laboratory notebook.

Skill 13. Prepare a serial dilution

Prepare three 1:4 serial dilutions of a coloured stock aqueous solution (provided
for the class). Ensure that you have no less than 4ml in each of your dilutions. You
may use an air displacement pipette to dispense all solutions.

 If you assume that the stock coloured solution contains 20mM of fructose.
Calculate the concentration of fructose in each dilution.

Skill 14. Prepare a buffer solution

Prepare 100ml of 50mM Tris buffer solution at pH 8.0, and at room temperature,
according to the procedure detailed in the laboratory study guide.

 Check the container for the molecular weight of Tris
 Use a pH meter to measure the pH. You do not need to calibrate the pH
meter. Simply remove the cap and place the electrode into the solution to
measure pH.
 If you do not know how to use the pH meter please ask the laboratory
supervisor.
 If you have a magnetic stirrer in the solution please ensure that it doesn’t hit
the pH electrode.
 Ensure you use the recommended titration procedure to prepare the buffer.
 The molecular weight of Tris is usually 121.14 g/mol. However, you need to
check the correct molecular weight of the Tris you use in class.
 Assume that 50mM has two significant figures respectively
 State the relative uncertainty and percent percentage uncertainty of the
solution.

Remember:
 Ensure that you record all weights, volumes, and calculations in your
laboratory notebook.
 Ensure that you are wearing appropriate PPE.
 Ensure you are using appropriate volume‐measuring instruments that
account for the uncertainty in your measurements.

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 Skill 15. Describe how to prepare a multi‐solute buffer solution

In this skill, you do not need to prepare the solution, but you do need to describe,
with supporting calculations, how you would prepare 500ml of the following multi‐
solute buffer solution without using stock solutions.

A buffer solution contains the following components:
20 mM Tris, pH 8.0
0.1 M NaCl
1.0 mM MgSO4

Assume two significant figures in the values for each chemical.

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 Practical 6
Carefully read, and study, Chapter 9 in your laboratory study guide before
commencing this practical. Write all answers in your laboratory notebook. In the
following exercises you will be using a spectrophotometer to measure the
absorbance of standard, and unknown, solutions of an analyte.

Skill 16. Prepare standard solutions of an analyte and measure absorbance

An analyte is a chemical in a solution (i.e. a solute) that we would like to measure.
You first prepare a stock solution of a particular analyte and then use the stock
solution to prepare diluted solutions of that analyte. These diluted solutions are
known as standards because they are used as reference values for a particular
measurement technique such as absorbance1. The absorbance of each standard is
measured and the values are used to draw a standard curve where absorbance is
plotted against concentration of the standards.

16.1 Prepare a stock solution of potassium permanganate.

Make 500 mg/L (2 sig fig) stock solution of potassium permanganate (KMnO4)
in a final volume of 100 ml. You should be accurate and precise with your
weighing and use appropriate volumetric glassware. The molecular weight of
KMnO4 is 158 g/mol, but you must check that this corresponds with the
molecular weight of the chemical (read what is printed on the container) used
in the laboratory.

16.2 Prepare six standard solutions of potassium permanganate

Use the 500 mg/L stock solution of potassium permanganate to prepare six
standard solutions. Dilute the stock, in an appropriate volume of water, to give
5.0 ml solutions containing 10, 20, 40, 80, 100, and 120 mg/L respectively.
Complete the table below. Please note that this is a stepwise (addition)
dilution not a serial dilution. You can copy and paste the table in the results, or
draw a similar table, in your laboratory book.

Volume of
stock
Volume of
water
Concentration 10mg/L 20mg/L 40mg/L 80mg/L 100mg/L 120mg/L

1 Other physical or chemical characteristics may be measured but measuring absorbance is a

common technique.

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 16.3 Measure the absorbance of each standard solution using a spectrophotometer

Transfer (using a transfer pipette or by dispensing) each of your standard
solutions into individual plastic cuvettes. Fill the cuvettes to about ¾ of its
volume with your standard solution (you may see a faint line inscribed on the
cuvette at this position). Fill one cuvette with distilled water because this will
be your ‘blank’.

Please refer to the separate instructions on how to use the spectrophotometer.
Ensure that the spectrophotometer is adjusted to a wavelength of 525nm and
that it is measuring absorbance.

The light from the spectrophotometer goes through the solution in the cuvette
from left to right (as you face the front of the machine), so you should place
the filled cuvette with its clear faces on the left and right as it sits in the holder.
Please note that the position of the cuvette given in the spectrophotometer
display is 1 to 6 from the front to the back of the cuvette holder.

Place the cuvette containing water into the first cuvette holder (position 1) of
the spectrophotometer (the one closest to the front of the machine), close the
lid, and auto zero the instrument. This is your blank sample. You may then
remove your blank sample and immediately place all of your standard
solutions in the cuvette holder.

You then need to measure, and record, the absorbance at 525nm of each of
your standard solutions. Please ensure that you record the absorbance of each
solution in your lab book.

Warning: Do NOT throw away your standard solutions until you are sure you
have reliable measurements, because you may need to repeat one or more
measurements.

16.4 Measure the absorbance of the unknown concentration of analyte.
You are given an unknown concentration of a potassium permanganate
solution. Measure its absorbance, at 525nm, of this solution and then later
determine its concentration by interpolating from your standard curve (next
skill).

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 Skill 17. Plot a standard curve to estimate the concentration of an analyte

In this skill, you are required to draw a standard curve (also known as a calibration
curve) using the absorbance values of your standard potassium permanganate
(KMnO4) solutions from the previous skill. A standard curve is a graphical plot that
relates the absorbance of each standard against their concentration. This curve is
then used to help determine the unknown concentration of an analyte in another
solution. Note that although the word ‘curve’2 is used you will actually draw a linear
line of best fit to your data.

17.1 Manually draw3 an appropriately formatted standard curve, with a linear line
of best fit to the data, on graph paper. You may later fix the graph paper into
your laboratory notebook. Remember, your line of best fit needs to account
for the zero point (0,0) but the line itself need not pass through the zero point.

17.2 Estimate the concentration of the unknown potassium permanganate solution
by interpolating from your line of best fit of your standard curve. Record your
estimate in your laboratory notebook. Your estimate should be within about ±
10% of the actual concentration, but this obviously depends upon how you
performed the assay and how well you drew your standard curve. Ask your
laboratory supervisor for the actual concentration after you have made your
estimate. Ask your laboratory supervisor for guidance and advice.

17.3 Estimate the molar absorption coefficient () of potassium permanganate at
525nm from the gradient of the curve in units of (mol L‐1)‐1 cm‐1. Be careful to
ensure you have the correct units! Compare your estimated value with the
published molar absorption coefficient of potassium permanganate.

Important: Ask your laboratory supervisor for feedback after you have completed
all the steps. You may be asked to repeat parts of the assay, or the whole assay, if
there seems to be any problems. You cannot do this assay at any other time.












2 A straight line may be thought of as a ‘curve’ with no change in its slope (gradient).
3 It is preferable that you first draw this graph manually. Later you may use excel, or other

appropriate programs, to draw these graphs.

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 Practical 7

Carefully read, and study, Chapter 10 in your laboratory study guide before
commencing this practical. Write all answers in your laboratory notebook. In these
exercises you will again be using a spectrophotometer to measure the absorbance
of standard, and unknown, protein solutions.

Skill 18. Estimate the concentration of a protein

In this skill, you will work as a team (preferably as a pair) and use your practical
knowledge gained in the previous skills to estimate the unknown concentration of a
protein. Each member of the pair should contribute and should be allocated ‘jobs’ to
do in this skill.

You are given two protein samples: labelled A and B which you will assume are
from the serum of two different human patients. You need to determine the
concentration of the protein in each of these samples by interpolation from your
standard curve of known (standard) protein solutions. You will be using the Biuret
assay to determine protein. below. Normally we would assay all standards, and the
unknown proteins, in triplicate but for this activity we only have the resources to
assay one sample of each.

Please note that these are biological samples, so you must wear appropriate
personal protective equipment.

Your estimate of the protein in each sample should be within ± 10% of the actual
protein concentration in each sample. You may ask the laboratory supervisor for
the actual concentration after you have made your estimate.

18.1 Prepare Protein Standard solutions
You are given a 100 g/L (3 significant figures) bovine serum albumin (BSA)
solution that you must dilute appropriately, with deionized or double distilled
water (dH2O), to give three other solutions: 25.0 g/L, 50.0 g/L, and 75.0 g/L in
a final volume of 1ml. You should have some of the 100 g/L stock left over
after preparing these three solutions (you need at least 300 l of the 100 g/L
stock for your assay). Consequently, you will have four standard solutions,
including the 100 g/L stock, to use for your standard curve.

18.2 Biuret assay of protein
In the following procedure, you react a chemical (called the Biuret Reagent)
with the protein in your samples (your standard samples and your two
unknown protein samples) to create a coloured compound that can then be
measured by absorbance.

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 Carefully read the following protocol before doing anything!

Make sure you know what to do and plan your experiment before you start. You
need to use a test tube that can take up to 5ml of solution (e.g. a 3DT tube). Use the
following protocol for the Biuret assay of protein:

a) Pipette 100 l of each of your standard solutions into separate tubes giving a
total of 4 tubes of standards.
b) Pipette 100 l of your unknown protein sample A into a separate tube
c) Pipette 100 l of your unknown protein sample B into separate tube
d) Pipette 100 l of deionized water into a separate tube (this is your water‐
reagent sample otherwise known as a ‘blank’).
e) You should now have a total of seven separate test tubes.
f) Add 2.4 ml deionized water to all tubes
g) Add 2.5 ml working Biuret reagent to all tubes. The Biuret reagent should be
provided in a bottle dispenser in the laboratory. Please ask your laboratory
supervisor if you don’t know how to use the dispenser.
h) Incubate all of the 5ml assay solutions in a 37°C water bath for 20 minutes
i) After the incubation you should briefly rest, and cool, the samples.
j) Set the spectrophotometer to 555nm.
k) Dispense the water‐reagent blank assay solution into a plastic cuvette. You
may use a disposable transfer pipette to dispense the solution and fill to at
least three quarters of the volume of the cuvette. Place the cuvette into the
first sample holder in the spectrophotometer. In a multiple sample holder,
the water‐reagent blank should be placed in the holder at the front of the
machine.
l) Set the absorbance of the spectrophotometer to zero. This blanks the
spectrophotometer.
m) Remove the cuvette containing the blank assay solution.
n) Dispense each standard assay solution into a cuvette, place the cuvette(s)
into the spectrophotometer, and read the absorbance of each standard.
o) Remove the cuvettes containing the standard assay solutions.
p) Dispense each unknown assay solution into a cuvette, place the cuvette(s)
into the spectrophotometer, and read the absorbance of each unknown.

Do not throw out the plastic cuvettes after you have finished measuring the
absorbance of your solutions and analysed your results. Place the filled cuvettes in a
safe place to prevent them from spilling. When appropriate dispose of the liquid
waste in the collection bottle provided for the class, wash the cuvettes at least three
times with deionized water, and return cuvettes to their storage boxes.

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 18.3 Draw a standard curve
Each member of your team should draw a standard curve using the
absorbance data of your standard solutions. If you find that any of your
standards looks ‘wrong’ (out of place on the graph), then you should repeat
the assay of those standards.

18.4 Estimate the concentration of the two protein solutions
Each member of your team should estimate the protein concentration of the
two protein samples by interpolating from their own standard curve. Your
estimate should be within about ± 10% of the actual concentration, but this
obviously depends upon how you performed the assay and how well you
drew your standard curve. Ask your laboratory supervisor for the actual
concentration after you have made your estimate.

Important: Ask your laboratory supervisor for feedback after you complete all
steps. You may be asked to repeat parts of the assay, or the whole assay, if there
seems to be any problems. You cannot do this assay at any other time.

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 Practical 8

Carefully read, and study, Chapter 11 in your laboratory study guide before
commencing this practical.

Skill 19. Use the Henderson‐Hasselbalch Equation to calculate pH of glycine

In this skill, you need to perform calculations to complete Table 8.1 below and learn
how to use the Henderson‐Hasselbalch equation to calculate the pH of a solution of
glycine when titrated with hydrochloric acid (HCl). This skill helps you prepare for
the next skill (Skill 18), which is the experimental titration of glycine. You may like
to commence, or complete, Skill 17 before attending the practical session and use the
practical session to start immediately on the experimental titration of glycine. You
may work as a team (preferably as a pair), and you may discuss this skill with your
team, but each member of the team must complete the table. An explanation of the
calculations is as follows:

Explanation of the calculations in Table 8.1

The rows labelled ‘Initial’ were completed to help you begin the calculations for
both functional groups in glycine. We assume that we first titrate only the amine
group of glycine (the top part of the table). Some of the carboxyl group would also
be titrated (remember it is an equilibrium between the three structural forms of
glycine), but we assume that this is so very small that it doesn’t affect what we are
doing in this calculation. Thus, the added acid (H+) reacts preferentially with the
basic form of glycine functional group (NH2) to give the acid form of the glycine
functional group (NH3 + ) as a product in the following equilibrium reaction:

H+ + NH2  NH3 +

When 0.5 ml of 1M HCl is added (containing 0.5mmol of H+) it will react with
0.5mmol NH2 to give 0.5mmol NH3 +.

It is then a matter of simply subtracting the amount of acid added (0.5mmol) from
the original starting amount of the glycine base (9.96mmol) and adding the same
amount to the amount to the original starting amount of the glycine acid
(0.04mmol). We get the values in step # 1 of the table by the following:

9.96 – 0.5 = 9.46 mmol of basic form of glycine (NH2)
0.04 + 0.5 = 0.54 mmol of acidic form of glycine (NH3+)

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 You then take the ratio of these numbers and calculate the log of the ratio. You can
then use the pKa (given to you in the table) and the log of the ratio to complete the
Henderson‐Hasselbalch equation:

pH = pKa + log [base]/[acid]

Thus:
pH = 9.6 + 1.2 = 10.8

The same logic we used for the amine group also applies to calculating the pH when
titrating the carboxyl group. In this case, however, the pKa = 2.3

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 Table 8.1. Calculating pH of glycine solution when titrated with acid using
the Henderson‐Hasselbalch Equation
1 2 3 4 5 6 7 8 9
Step # stepwise cumulative mmoles mmoles base/acid Log pKa2 Calculated
addition mmoles glycine glycine ratio base/acid pH
1.0M HCl H+ in base in acid
(mL) added form form
(NH2) (NH3+)

Assume that you begin to titrate the amine group first.

Initial 0 0 9.96 0.04 249 2.4 9.6 12.0
1 0.5 0.5 9.46 0.54 17.5 1.2 9.6 10.8
2 0.5 1.0 9.6
3 1.0 2.0 9.6
4 1.0 3.0 9.6
5 1.0 4.0 9.6
6 1.0 5.0 9.6
7 1.0 6.0 9.6
8 1.0 7.0 9.6
9 1.0 8.0 9.6
10 1.0 9.0 9.6
11 0.5 9.5 9.6
12 0.4 9.9 9.6
At this stage after adding 9.9ml of 1.0M HCl (sum of volumes in column 2) you will have added
a total of 9.9 mmol of H+ (shown in column 3) and you have almost finished titrating the 9.96
mmol of amine group (Initial value in column 4). In the next part of the table you assume that
you are now starting to titrate the carboxyl group.
Step # stepwise cumulative mmoles mmoles base/acid Log pKa1 Calculated
addition mmoles glycine glycine ratio base/acid pH
1.0M HCl H+ in basic in acidic
(mL) added form form
(COO ) (COOH)
‐

Initial 0 9.9 9.96 0.04 249 2.4 2.3 4.7

1 0.5 10.4 9.46 0.54 17.5 1.2 2.3 3.5
2 0.5 10.9 2.3
3 1.0 11.9 2.3
4 1.0 12.9 2.3
5 1.0 13.9 2.3
6 1.0 14.9 2.3
7 1.0 15.9 2.3
8 1.0 16.9 2.3
9 1.0 17.9 2.3
10 1.0 18.9 2.3
11 0.5 19.4 2.3
12 0.4 19.8 2.3

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 Skill 20. Experimentally titrate glycine with hydrochloric acid

In this skill, you will experimentally titrate a glycine solution with hydrochloric acid
to alter its pH. This titration is done for the following reasons:

 Determine the titration curve of glycine
 Determine the buffering regions (in terms of pH) of glycine
 Compare the experimental titration with the calculations you performed in
the previous skill
 Define the following: ionizable group, pH, pKa, pI, buffer, weak acid, weak
base, strong acid, strong base
 Understand the importance of ionisable groups in proteins.

Titration of Glycine

Titration is the process of adding acid or base to a solution. In this experiment, you
add hydrochloric acid (HCl) acid to a solution of glycine and use a pH meter to
measure the change in pH with each addition of the acid. In this way you can use
titration to determine the ionizable groups in a glycine. You need to perform the
following steps:

a) Measure 100ml (3 sig fig) of 0.10M glycine, pH 12, into an appropriate clean
beaker
b) Place the beaker on a magnetic stirrer, insert a magnetic ‘flea’, and stir the
solution evenly. If you don’t have a magnetic stirrer, then you can use a
plastic transfer pipette (turned upside down with the ‘bulb’ part in solution)
to stir the solution.
c) Once the solution is stirring evenly, suspend the pH probe in the solution
(you can use a retort stand to hold the pH probe or simply hand‐hold the pH
probe in solution). Make sure that the porous plug of the pH probe is below
the level of the solution and that the probe is not in contact with the side of
the beaker or the magnetic flea. Be careful that the magnetic flea does not
make contact with the pH probe and break it! Record the initial pH in the
table below!
d) Add the appropriate volumes of 1.0M HCl using an appropriate air
displacement micropipette4, as indicated in Table 8.2 below. If you don’t
have a magnetic stirrer you should stir the solution after every addition of
acid. Wait about 20 seconds or so to let the reading from the pH meter
stabilise after each addition of acid and then record the pH in the table.

4 Please note that if we were to titrate the glycine solution precisely then we would

use a volumetric burette to measure the volumes of acid added to the solution.

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 Table 8.2 – Experimental Titration of glycine

Step stepwise cumulative Observed Step stepwise cumulative Observed pH
addition mmoles pH addition mmoles
1.0M HCl H+ 1.0M HCl H+
(mL) added (mL) added
1 0.5 13 0.5

2 0.5 14 0.5

3 1.0 15 1.0

4 1.0 16 1.0

5 1.0 17 1.0

6 1.0 18 1.0

7 1.0 19 1.0

8 1.0 20 1.0

9 1.0 21 1.0

10 1.0 22 1.0

11 0.5 23 0.5

12 0.4 24 0.4

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 Skill 21. Plot the titration curve of glycine

In this skill, you plot the titration curve of glycine using the results of the two
previous skills: Skill 19 and Skill 20.

Use graph paper to plot both the calculated and experimental data on the same
graph. Your plot should be appropriately formatted. In this case you may ‘join the
dots’ to give continuous curves. You also need to show the following:

1. On the curve for your calculated data show:
 the two regions where glycine can act as a buffer
 the two pKa’s of glycine
 the approximate location of the two equivalence points
 the calculated pI of glycine

2. On the curve for your experimental data show:
 the two regions where glycine can act as a buffer

3. Compare and contrast the calculated and experimental curves.

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 Practical 9

Carefully read, and study, Chapter 12 in your laboratory study guide before
commencing this practical.

Skill 22. Assay of catalase in potato.

In this skill you will set up, and conduct, an assay for the enzyme catalase5.

22.1 Prepare assay buffer

Prepare 100ml of 50 mM HEPES pH 7.5 at room temperature

22.2. Prepare Potato slices
 Cut the potato in half laterally across the thickest part of the potato. The
place where you cut then gives you a large area of potato to work with.
 Cut one thin slice of about 5mm thick from the centre of the cut end of the
potato. Then cut out a 2cm square piece of potato. To do this easily, just
measure and draw a square with sides equal to 2cm on a piece of plain
paper, cut the paper around the perimeter of the square, and then use it
as a cutting template on your slice of potato. You should then have a
square slice of potato that is 2cm x 2cm x 0.5cm.
 Cut the slice into two equal pieces (1cm x 2cm).
 Weigh and record the weight of each slice
 Place the two slices of potato into a container (e.g. test tube) and cover
with 50mM HEPES, pH 7.5, buffer solution. Store on ice. One slice will be
used in the assay of catalase and the other slice will be used as a backup if
needed.

22.3 Prepare potato cubes
 Cut one slice of potato as in the previous steps (i.e. about 2cm x 2cm x
0.5cm) and cut each slice into 16 small cubes of about 5mm for each side
of the cube.
 Weigh, and record, two batches of cubes where each batch has eight
cubes. That is, you should have two measured weights each of eight
potato cubes.
 Place each of the two sets of cubed pieces of potato into separate
containers and cover with 50mM HEPES, pH 7.5, buffer solution. Store on
ice. One set of cubes will be boiled in the next step and both sets will be
used for the assay of catalase.

5 Adapted from Iwase, T., Tajima, A., Sugimoto, S., Okuda, K. I., Hironaka, I., Kamata, Y., ... & Mizunoe, Y. (2013). A
simple assay for measuring catalase activity: a visual approach. Scientific reports, 3, 3081.

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 22.4 Boil Potato cubes in water
 Remove one complete set of eight cubed pieces of potato from the buffer
by dispensing into a filter paper placed in a funnel, wash with distilled
water, blot excess water with filter paper, and place all the cubes into a
clean Erlenmeyer flask containing about 20ml of water. Boil the cubed
pieces of potato in the Erlenmeyer flask for about 5 minutes using the
Bunsen burner and stand or a heating plate (whichever is provided for
you in your practical).
 Empty the boiled cubed pieces of potato into filter paper placed in a
funnel. Careful of hot water and steam!
 Wash with cold distilled water about three times and leave to cool.

22.5 Conduct an assay of catalase

a. Label four test tubes as follows: zero, slice, cube, boiled cube
b. Pipette 0.5ml of buffer into each tube
c. Pipette 0.5ml of 1% (w/w) Triton X 100 into each tube
d. Mix each tube

To start the enzyme catalysed reaction
e. Place one potato slice (1cm x 2cm) into the appropriately labelled test
tube. Ensure that the slice is covered by solution.
f. Place one set of cubed potato (8 cubes) into the appropriately labelled
test tube. Ensure that all cubes are covered by solution.
g. Place one set of boiled cubed potato (8 cubes) into the appropriately
labelled test tube. Ensure that all cubes are covered by solution.
h. Pipette 0.5ml of 30% (w/w) hydrogen peroxide into each tube and mix
immediately. This will start the reaction and you should see foam
developing in some tubes.
i. Wait for about 15 minutes until the foam has stabilised and then
measure, and record, the height of the foam in each tube with a ruler. The
height of the foam should be measured from its interface with the
solution to its crest.
j. Use your results to complete Table 9.1.

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 Table 9.1. Sliced and Cubed Potato in hydrogen peroxide

Potato Rate Rate per Rate per

Height (mm) gram of surface area
per 15 min potato of potato
(Rate/g) (Rate/mm2)
Slice

Cubes

Boiled
cubes


Questions for Learning

1. What was the purpose of the tube labelled zero?
2. What is the probable identity of the gas released by the enzyme catalysed
decomposition of hydrogen peroxide?
3. What simple test could you use to determine the probable identity of the gas?
Hint: a glowing splint.
4. What was the purpose of the Triton X‐100 in the assay?
5. What was the purpose of hydrogen peroxide in the assay?
6. Estimate the concentration of hydrogen peroxide in the assay.
7. Why keep the cut potatoes, in buffer, and on ice before using them in the
experiment?
8. Why did you weigh your potato cubes and slices?
9. Why did you boil the potato?
10. When you measured the rate of reaction was this likely to be the initial rate?
11. Briefly explain the differences you saw in the rate of reactions between slice,
cubed, and boiled cube potato preparations.
12. Was the total volume of the solution properly controlled in the experiment?
13. What do your results show when you express the rate (i.e. height of foam per 15
min) as ‘rate per gram’ or ‘rate per surface area’?

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 Skill 23. Relationship between substrate concentration and enzyme activity

Figure 9.1 shows the experimental equipment that can be used to measure the
pressure of a gaseous product released from a chemical reaction.

Figure 9.1 The enzyme could be delivered by the syringe, through a valve, into the
Erlenmeyer flask containing one concentration of substrate in buffer. The valve is
immediately closed after the enzyme is delivered. The pressure of the gas released by the
reaction is measured by the sensor (black box in the image) over time. The Erlenmeyer
flask is in a beaker and surrounded by water. A temperature probe is also in the beaker.
Figure courtesy of: https://goo.gl/Mo5sRU

Table 9.2 shows simulated enzyme kinetic data. In an enzyme kinetic experiment
this type of data would be obtained from a series of progress curve experiments
(also known as product‐time curves if the product was measured). In this skill, we
assume that we have an experimental scenario that uses the gas pressure
equipment shown in Figure 9.1 to perform the progress curve experiments. The
equipment would measure the increase in pressure of a gaseous product, released
from the enzyme‐catalysed chemical reaction, over time. This rate of change in
pressure may then be converted to a rate with the units of M/s. We also assume
that these experiments were performed using catalase purified from potato and
hydrogen peroxide as the substrate.

23.1 Complete Table 9.2.

Complete the calculations for columns headed 1/[S] and 1/vo in Table 9.2.

23.2 Draw a Michaelis‐Menton graph.

Use the appropriate data in Table 9.2 to manually draw a Michaelis‐Menton
polt on the graph paper provided. You may ‘join the dots’ on this curve.

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 Estimate the Vmax and Km from the graph. If the catalase concentration was
5M in the experiment, what would be the estimates of kcat and the specificity
constant? Remember, to use the appropriate units for each parameter.

Table 9.2 Enzyme kinetics data
Substrate Initial Velocity (vo) 1/[S] 1/vo
Concentration [S]
(M) (M/s)
0.0 0.0
0.4 3.8
0.8 7.4
1.0 9.1
4.0 28.6
5.0 33.3
10.0 50.0
18.0 64.3
24.0 70.6
30.0 75.0


23.3 Draw a Lineweaver‐Burk graph

Use the appropriate data in Table 9.2 to manually draw a Lineweaver‐Burk
plot on the graph paper provided. Draw a line of best fit to the data points.

Estimate the Vmax and Km from the graph. If the catalase concentration was
5M in the experiment, what would be the estimates of kcat and the specificity
constant? Remember, to use the appropriate units for each parameter.


Questions for Learning

1. What would be an appropriate control experiment for the experiment
shown in Figure 9.1?
2. Consider the experiment shown in Figure 9.1, could the substrate be added
to the reaction mixture via the syringe instead of the enzyme? Explain how
this would alter the experiment and potential advantages or disadvantages.
3. What are the possible reasons for the water bath and the temperature
probe shown in Figure 9.1? Explain how this could be used to devise
another experiment on catalase activity.
4. Draw what you would reasonably expect to be the progress curve for one
concentration of substrate assuming you conducted a real experiment using
the equipment shown in Figure 9.1. What would be the minimum number of

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 progress curves, and hence experiments, you would need to perform to
acquire sufficient data to eventually plot a Michaelis‐Menton curve? Also
assume that you only need to do one set of experiments and you do not
need to perform duplicates or replicates.
5. Compare your estimates of Vmax and Km determined by a Michaelis‐
Menton plot and a Lineweaver‐Burk plot. For example, which plot would
give the ‘better’ estimates and why?
6. You estimated turnover numbers and specificity constants using both your
Michaelis‐Menton and Lineweaver‐Burk plots. What is one reason you were
asked to do this? Hint: think about the estimates of Vmax and Km for both
plots.
7. Table 9.2 shows initial velocity (vo). Describe how the initial velocity of the
product would be measured for each substrate concentration.
8. Table 9.2 shows initial velocity (vo) with the units of M/s. However, in this
experimental scenario the rate of reaction was measured as in increase in
gas pressure over time. Describe how the measurement of pressure may be
converted to estimate initial velocity with units of M/s.

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