BMS481

BIOANALYTICAL
CHEMISTRY
Nurul Aili Binti Zakaria
(Ph.D)
 Lets get to know each other…

https://padlet.com/bms415sept2019/m0am3tq6cz64
 DATE & TIME VENUE
LECTURE
Nurul Aili Binti Zakaria Monday : DK Gamma
1200 to 1250
Wednesday : DK Alfa
0800 to 0850
BMS481
PRACTICAL CLASS
COURSE Tuesday :
0800 to 0950 M610
Wednesday :
1510 to 1700
* Be punctual, Be polite
 At the end of the course, students should be able to:

1. Describe the fundamentals concepts of analytical

techniques e.g. accuracy, concentrations, molarity.
COURSE
LESSON 2. Illustrate the principles and applications of basic
OUTCOME centrifugation, chromatographic and spectroscopic
analytical techniques in the isolation and
(CLO) characterization of biological molecules.

3. Perform scientific experiments on biomolecules

separation, characterization and analysis.
Continuous Assessments 50%
• Test 1 10%
• Test 2 10%
• Practical Test 20%
• Practical Report 10%
Assessments
Final Assessments 50%
• Final Examination (120 min) 50%
Recommended Text / References
Recommended text
1. K. Wilson and J. Walker (2010) Principles and Techniques of Biochemistry and Molecular Biology. 7th ed.
Cambridge University Press.
2. S.R. Mikkelsen and E. Corton (2016) Bioanalytical Chemistry. 2nd ed. Wiley & Sons, New Jersey.

✓References
1. J. W. Robinson, E.M.S. Frame and G. M. Frame II (2014) Undergraduate Instrumental Analysis (7th
Ed.) CRC Press Taylor & Francis
2. R.K. DeLong and Zhou Q.Q. (2015) Introductory Experiments on Biomolecules and their
Interactions. Elsevier Academic Press.
3. F.H. Stephenson (2016) Calculation for Molecular Biology and Biotechnology. 3rd Ed. Elsevier
Academic Press
4. A. Manz, P.S. Dittrich, N. Pamme, D. Iossifidis (2015) Bioanalytical Chemistry. 2nd ed. Imperial
College Press.
5. R. Katoch (2013) Analytical Techniques in Biochemistry and Molecular Biology. Springer.
 1) LESSON PLAN/padlet
2) NOTES /i-LEARN/padlet
3) DATE LAB STARTS (10/9/19)
4) STUDENT LEARNING TIME (SLT) – For 1 hour lecture student should spend 2 hours
study
5) RECOMMENDED TEXT
6) LAB COAT – COMPULSORY IN PRACTICAL CLASS
7) ATTENDANCE SHOULD MORE THAN 80% (MC/LETTER) – ATTENDANCE IS
COMPULSORY AND BE PUNCTUAL
PAY ATTENTION 8) DATE FOR THE TEST

TO THESE 9) ENTRANCE SURVEY (COMPULSORY) –EARLY OF THE SEMESTER (i-LEARN)

INFORMATIONS: (Week 1 to 2/ 3)
10) EXIT SURVEY (COMPULSORY) –END OF THE SEMESTER (i-LEARN)
(Typically Week 13 – 14)
11) SUFO ONLINE (COMPULSORY) –END OF THE SEMESTER (i-LEARN)
(Week 10 until one week after exam result is announced)
12) PAST YEAR QUESTIONS (LIBRARY WEBSITE & i-LEARN – EQPS)
13) CLASS REPRESENTATIVE – Submit name and contact number @padlet
 Rules and regulation:

Be punctual.

Lesson plan
Attendance above 80%: Please provide MC or
official letter if you unable to attend a lecture.

Be respectful, be polite.
 CHAPTER 1
INTRODUCTION TO MOLECULAR
BIOLOGY LABORATORY

BMS481
Nurul Aili Zakaria (Ph.D)
 1.1 Aseptic techniques

1.2 Biosafety in laboratory

1.3 The micropipetter

CHAPTER 1.4 Accuracy & Precision

OVERVIEW
1.5 Experimental error & data reproducibility

1.6 Units of measurement

1.7 Controls & standards in exp. design

WHAT IS MOLECULAR WHAT IS BIOMOLECULAR
BIOLOGY? SCIENCE?

INTRODUCTION TO
MOLECULAR BIOLOGY TECHNIQUES
 MOLECULAR BIOLOGY : field of biology that studies the
composition, structure and interactions of cellular
molecules such as nucleic acids and proteins that carry
out the biological processes essential for the cells
MOLECULAR functions and maintenance (Nature publishing website).
BIOLOGY
Overlaps with other areas of biology and chemistry,
VS. particularly genetics and biochemistry.

BIOMOLECULAR
SCIENCES
BIOMOLECULAR SCIENCES : is a discipline within
biology which focuses on cellular processes at
molecular level.
  Sterile : free from living organism
 Aseptic : Absence of pathogenic
microorganism and other contaminant

1.1 ASEPTIC TECHNIQUES

ASEPTIC  Aseptic technique : procedure to prevent
microbial and particulate contamination
TECHNIQUES
 Two types of agents used to remove and
prevent contamination are :
1. Chemical disinfectant
2. Heat
ASEPTIC TECHNIQUES – USING HEAT
 The use of a flame source is to establish an aseptic working environment.

 The heat from the flame causes air convection, generating a cone of hot air
above and around the burner (reduce any airborne contaminants away from
the vicinity of the burner) thus creating a "sterile field in which to conduct
aseptic work.

 The common heat source in the lab is the Bunsen burner.

 Advantages as flame source: - Bunsen burner flame to heat things very
quickly, ideal choice for sterilizing inoculating loops, warming glass bottle
necks, or igniting alcohol on culture spreaders.
ASEPTIC TECHNIQUES – USING CHEMICAL DISINFECTANT

 General chemical disinfectant is 70% (v/v) ethanol

 Other disinfectant also can be use – example industrial methylated

spirits (IMS; denatured alcohols)

 Simple, flammable disinfectants used for cleaning items and surfaces from
microorganisms and stains. They are also used for dipping culture spreaders

 Liquid for disinfection such as 70% (v/v) ethanol is placed in spray or squirt plastic
bottles – For cleaning of surfaces, the liquid is dispense and wipe with tissues.
LAMINAR FLOW  A laminar flow unit is a sophisticated appliance that can further
help prevent contamination of reagents and biological cultures.
CABINET/HOOD
 Used correctly, it provides the work space with clean, ultrafiltered
air.

 It also keeps room air from entering the work area and both
suspends and removes airborne contaminants introduced into the
work area by personnel.
 1. Wearing lab coat.

PRE-ASEPTIC
2. Wearing gloves or disinfect hands with chemical
WORKS disinfectant before engaging in aseptic work.
 1. Cleaning and disinfecting lab surfaces and apparatus prior to use.
2. Limiting the duration that cultures or reagents are uncapped exposed
to the air.
3. Keeping petri dishes closed whenever possible.
4. Effectively sterilizing inoculating loops and other equipment that
STEPS IN comes into contact with cultures or media.
ASEPTIC 5. Avoiding breathing on cultures or sterile instruments.
TECHNIQUES:
STERILIZING EQUIPMENT AND REAGENTS FOR
EFFECTIVE ASEPTIC WORK

1. Filter sterilization
2. Oven sterilization/dry heat
3. Autoclaving – using autoclave

Comparison between these approaches? READ!

 1.2 BIOSAFETY IN LABORATORY

UNIVERSAL PRECAUTIONS – To protect health professionals

-Include hand hygiene, gloves, gown, masks, eye protection, face shields,
safe injection practices, protective clothing, special site decontamination

-Require that all equipment or contaminated items are handled to prevent

transmission of infectious agents
 1.2 BIOSAFETY IN LABORATORY cont.

STANDARD PRACTICES
 Frequent handwashing
 door that can be kept closed when working;
 limits on access to the lab space when working;
 no smoking, eating, drinking, storage of food in laboratory;
 care to minimize splashes and actions that may create aerosols (tiny droplets);
 decontamination of laboratory waste;
 use of mechanical pipettes only (no mouth pipetting);
 “sharps” precautions, including special containers for disposing of needles and other
sharp objects;
 Maintenance of insect/rodent control program;
 use of personal protective equipment (lab coats, latex gloves, eye protection or face
shields)
 Do not dispose waste such as microorganism, animal/human tissues, chemicals/solvents
into the sink/drain.
 QUANTITATIVE TRANSFER OF LIQUIDS

-Pipettes are used for transferring volume of liquid solution - microvolumes

1.3
The
micropipettor
MICROPIPETTOR
  Set 1: Set the Volume

Operating the
micropipette
Operating the micropipette
How to read the volume
Operating the
micropipette
STEP 2:
Attaching the
disposable tip
 STEP 4: Immerse tip in sample

STEP 5:Draw up the sample

-To aspirate the sample into the
tip, allow the pushbutton to
return slowly and smoothly to the
fully extended UP POSITION.
Operating the NEVER LET THE PLUNGER SNAP
UP
micropipette

work by displacing air from

the pipette shaft, allowing
the liquid to be drawn into
STEP 6:Pause
the resulting vacuum. -Wait a few seconds to ensure that the full
STEP 3: volume of sample is drawn into the plastic
Depress the tip. WAIT LONGER FOR LARGER VOLUMES.
plunger to the WAIT LONGER FOR MORE VISCOUS
first stop (“SYRUP-LIKE”) SUBSTANCES
  STEP 7: Withdraw the tip
Remove the tip from the sample. No liquid should remain on the
OUTSIDE of the tip. Wipe away any droplets on the outside of the
tip with a lint-free tissue (KIMWIPES), but only wipe droplets from
the side of the tip. NEVER TOUCH THE TIP OPENING or you may
absorb part of your sample.
Operating the
micropipette
  STEP 8: Dispense the sample

Operating the
micropipette
 STEP 10: Release the plunger

STEP 11: Discard the tip –

Operating the depress the ejector button
micropipette
STEP 9: Withdraw the pipette

A fresh tip should be used

for each sample to prevent
sample carryover
1.4 & 1.5
ACCURACY & PRECISION
EXPERIMENTAL ERROR AND
REPRODUCIBILITY
 Accuracy and Precision are
NOT THE SAME thing!

 No physical quantity can be measured with perfect certainty

 Limits of measuring device
 Experimental error is the difference between a measurement
Experimental and the true value or between two measured values

Error  Measured by accuracy and precision

 Accuracy and Precision are NOT THE SAME thing!

ACCURACY AND PRECISION IN AN EXPERIMENT

 Accuracy refers to results that are close to the true (or accepted) value. In
science we rarely know what the true value is so this can be quite a difficult
concept to master.

 Precision refers to the closeness of two or more measurements to each

other.
- Obviously, if you are recording results to more decimal places then they could
be more precise than recording to one e.g. 12.15 and 12.16 are more precise
results than 12.
  Precision has to do with the concept of random errors and
ACCURACY & the precision of an average can always be improved by
increasing the sample size.
PRECISION
IN AN
EXPERIMENT  Accuracy is associated with the concept of bias or systematic
errors. It is caused by the procedure of taking the
measurement or the device itself.
 If a given substance was weighed five times,
and a mass of 2.70 g was obtained each time,
this shows accuracy or precision?

Example:
What can you conclude of the results if the
true mass in the above example was 3.20 g?
The difference between a measured value (in example given is 2.70 g)
and the true value (in example given is 3.20 g) is known as the
“experimental error/measurement error”.

 Accuracy is not a quantity and therefore cannot be given a numerical value. It

is allowable for a measurement to be described as being ‘ more accurate’
when its method and/or instruments clearly reduce measurement error, such
as using a triggered electronic timer system compared to a hand-operated
stopwatch.

 Accuracy may not be quantified; ‘experimental error’ is the quantity used to

evaluate how close a measured value is to the true value.

Accuracy is high, error Is low

Accuracy is low, error is high
 Lets figure these out…Combinations of accuracy and precision

• High accuracy, low precision • High accuracy, high precision

• High accuracy, high
precision
• Low accuracy, low
precision
• Low accuracy, high
precision
• High accuracy, low
precision

• low accuracy, high precision • low accuracy, low precision

 Can you think of an
analogy/situation to
describe accuracy and
precision?
RESEARCH DATA/RESULTS

 Experimental data and results must be more than one-off findings

(repetition of experiments) and should be repeatable and reproducible
to draw reasonable conclusions.

 Repeatability – the closeness of agreement between independent

results obtained with the same method on identical test material, under
the same conditions (same operator, same apparatus and/or same
laboratory).
 Reproducibility – the closeness of agreement between
independent results obtained with the same method on
identical test material, but under different conditions
(different operators, different apparatus and/or different
laboratories).

 Identical to reliability – when multiple no of scientist do the

same experiments and get the same results, that results is said
to be reliable.

 Experiments that use subjective human judgement (s) or that

involve small sample sizes or insufficient trials may also yield
results that may not be repeatable and/or reproducible.
RESEARCH
DATA/RESULTS
 Validity:

 A measurement is ‘valid’ if it measures what it claims

to be measuring. Both experimental design and the
implementation should be considered when evaluating
validity.

 Data are said to be ‘valid’ if the measurements that

have been made are affected by a single independent
variable only. They are not valid if the investigation is
flawed and control variables have been allowed to
change or there is observer bias. RESEARCH
DATA/RESULTS
Steps in experimentation (Little and Hills 1978):
Define the problem
Determine the objectives
Select the treatments
Select the experimental material
Select the experimental design
Select the experimental unit and number of replications
Ensure proper randomization and layout
Ensure proper means of data collection
Outline the statistical analysis before doing the experiment
Conduct the experiment
Analyze the data and interpret the results
Prepare complete and readable reports
Quantitative
vs. qualitative Quantitative data is measurable, Qualitative data can be
numerical information collected observed but not measured.
data in an experiment.
Examples: length, height, Example: color, smell, taste,
volume, mass appearance
 Prediction of what study will find

 Hypothesis – a proposed, scientifically testable explanation

for an observed phenomenon.

Hypotheses
 Bad
must be  If a small biobird is dropped, then it will twirl differently.
measurable
and compared  Good:
to a control  If a small biobird is dropped, then it will twirl 3x as many
times as a control bird.
 If a large biobird is dropped, then it will twirl half as
many times as a control bird.
 Must use a controlled experiment.
 Allows researcher to test the effects of a single
factor, or experimental variable.

Test the
hypothesis
  A controlled experiment uses two set-ups to ensure any
differences are due to the experimental variable:

Test the  Control setup – kept constant

 Experimental setup - contains the variable
hypothesis  Independent variable – ‘ I control’ – plot on X-
axis
 Dependent variable – ‘ Data collected’ – plot on
Y-axis
  Dependent : Relies upon something else to occur
 Independent : Manipulated to influence dependent
variable
Other ways of viewing it….

Defining
Independent Variable Dependent Variable
Variable Manipulator Result
Cause Effect
Influencer Outcome
  Example 1

 Hypothesis: “Changing the thermostat setting up or down

will cause the room temperature to change in the same way”

Example
  Hypothesis: “How does the amount of water affect how tall
a flower grows?”

 What is the…
 Independent variable?
The amount of water we give to the flowers
Let’s try this!
 Dependent variable?

How much the flower grow

 Control?
Type of flower, soil and light
  Key element of experimental and quasi (pseudo) – experimental
designs
 Subjects (one group or several) on which no variable (e.g.,
treatment) is applied
- Hold a variable or condition constant
Control
 Establishes baseline to compare changes in treatment
groups
 Attempts to reduce the effect of confounding independent
variables
  A standard is something established as a rule or basis of
comparison in measuring or judging capacity.
(Webster’s New World Dictionary)

Standard  What are characteristics of a measurements standard?

 They must have globally availability
 They must be accessible and ‘usable’
 They must be stable
 They must not change over time or location
  Application of a standard in biological science : example

1. A calibration curve in protein assay is prepared using

a standard protein solution. Standard protein
solution has known concentration and the calibration
Standard curve is plotted as absorbance vs protein
concentration.

1. DNA molecular weight standard in agarose gel

electrophoresis.
 “Don’t wish it were easier;
THANK YOU wish you were better.”
– Jim Rohn