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
K. Wilson and J. Walker (2010) Principles and Techniques of Biochemistry and Molecular Biology. 7th ed.
Cambridge University Press.
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
INTRODUCTIONTO MOLECULAR
BIOLOGY LABORATORY

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

2. Biosafety in laboratory

3. The micropipetter

CHAPTER 1.4 Accuracy & Precision

OVERVIEW
5. Experimental error & data reproducibility

6. Units of measurement

7. Controls & standards in exp. design

WHAT IS MOLECULAR WHAT IS BIOMOLECULAR
BIOLOGY? SCIENCE?

INTRODUCTION TO
MOLECULAR BIOLOGYTECHNIQUES
 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 EQUIPMENTAND REAGENTS FOR
EFFECTIVE ASEPTICWORK

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 OFLIQUIDS

-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 ERRORAND
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 SAMEthing!

ACCURACYAND 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
INAN
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;
THANKYOU wish you were better.”
– Jim Rohn
CHAPTER 2.0
BUFFERS AND
SOLUTIONS
https://padlet.com/bms415sept2019/m0am3tq6cz64

Syllabus Content
1. Water for biological reactions and cell culture
2. pH and buffer systems
3. Commonly used buffers in molecular biology
4. Calculations in Molecular Biology
 COURSE LEARNING OUTCOME

At the end of the course, students should be able to:

1. Describe the fundamentals concepts of analytical techniques e.g.

accuracy, concentrations, molarity.

2. Illustrate the principles and applications of basic centrifugation,

chromatographic and spectroscopic analytical techniques in the
isolation and characterization of biological molecules.

3. Perform scientific experiments on biomolecules separation,

characterization and analysis.
 “Buffers often are overlooked and taken for granted by laboratory
scientists until the day comes when a bizarre artifact is observed and its
origin is traced to a bad buffer. Although mistakes in the composition of
buffers have led occasionally discoveries such as the correct number of
human chromosomes (Arduengo, 2010), using the proper buffer,
correctly prepared, can be key to success in the laboratory.”

Arduengo, P.M. (2010) Sloppy technicians and the progress of science. Promega
Connectionshttp://promega.wordpress.com/2010/03/15/sloppy-technicians
2.0 Water for biological reactions and cell
culture
Why water is important?
• Most biochemical processes essential for living organism takes
place in presence of water
• Water is the best solvent known – universal solvent
• Water organize nonpolar molecules
• Water causes hydrophobic molecules to aggregate or assume
specific shapes.
Classificatio
n for water
purity
 H+ and H3O+ is often used
interchangeably to represent the
Dissociation of water hydrated proton, commonly call
the hydronium ion.

Water is amphoteric in nature, hence it dissociates

H2O ⇋ OH- + H+
(hydroxide ion) (hydrogen ion)

As the H+ ions are formed, they bond with H2O molecules in the
solution to form H3O+

2H2O (𝓁) ⇋ OH- (aq) + H3O+ (aq) (hydronium ion)

Remember, pH of a solution is dependent on the concentration of

hydronium ions or simply put as H+
2.2 pH and buffer
system
A mixture of weak acid and its conjugate
base (or a mixture of a weak base and its
BUFFER? conjugate acid)

Solution which resist a change in pH when

small amounts of a strong acid or strong
base are added

CH3COOH CH3COONa
(weak acid) (conjugate base)
NH3(aq) + NH4Cl(aq)).
 2.2 pH and buffer Acidic
buffer
system
What buffers do?
• A buffer functions to resist changes in pH (H+ concentration)

•However, biologists often

think of buffers as doing much
more:
•Providing essential cofactors
•Providing critical salts
•Providing essential nutrients for
cells and tissues

Alkaline
buffer
 Why can’t
we used HCI
What make a buffer system to make
buffer?

• A buffer consist of a weak acid (HA) and its conjugate base (A-) or
weak base and its conjugate acid

• Weak acids and bases do not dissociate completely in water, but

instead exist in solution as an equilibrium of dissociated and
undissociated species.

• For acetic acid, we would express this equilibrium like this:

(HA) (A-)
 “A buffer solution has to remove any
H+ or OH- that can cause pH change”
How buffers
work?
 Pick buffers by choosing the nearest
pKa to the pH we want!
Buffer
Capacity
”is the amount of acid or base that can be added to a given volume
of a buffer solution before the pH changes significantly, usually by
one unit.”

 It depends on the amount of weak acid and its conjugate base

that are in the buffer mixture

1L of buffer containing 1.0 M acetic acid and 1.0 M sodium acetate has a
greater buffer capacity than a 1L buffer with 0.1 M acetic acid and 0.1 M
sodium acetate, even though both solutions have the same pH.
**The first solution has more buffer capacity because it contains more
acetic acid and acetate ion
How to calculate pH of a
buffer?
Determine by two factors:
1. The equilibrium constant (Ka) of the weak acid
2. The ratio of weak base (A-) to weak acid (HA) in the solution

1. The equilibrium constant (Ka) of the weak acid

Different weak acids have different Ka. The Ka tells us what proportion of HA
will be dissociated into H+ and A- in solution. The more H+ ions created, the
more acidic and lower the pH of the resulting solution. (pKa = -log Ka)

2. The ratio of weak base (A-) to weak acid (HA) in the solution
If a buffer has more base than acid, more OH- ions are likely to be
present and the pH will rise (vice versa). When the concentrations of
A- and HA are equal, the H+ is equal to Ka

Formalized in HHE
 useful for estimating
Henderson- the pH of a buffer

Hasselbalch
pKa can be determined using the Henderson-Hasselbalch equation.

equation
[A-] : conjugate base
[HA] : weak acid

– With the use of this equation, it is possible to calculate the

concentration of acid and conjugate base at all points of titration
curves

– The equation may be used for:

to determine the amount of acid [HA] and conjugate base [A-]
needed to make a buffer solution of a certain pH.
Solve the following problems…
1. What is the pH of a
solution consisting of
0.75M HC2H3O2 and 0.5M
NaC2H302? Ka of
HC2H3O2 is 1.8 x 10^-5
2. What is the pH of a
solution consisting of 0.15M
NH4CI and 1.5M NH3? The
Kb of NH3 is 1.8 x 10^-5
What makes a “Good’’
buffer
In 1996, Norman Good and colleagues developed criteria for buffers
for biological systems.
• A pKa between 6 and 8.
• Solubility in water.
• Exclusion by biological membranes.
• Minimal salt effects.
• Minimal effects on dissociation from changes in temperature and
concentration.
• Minimal interactions between buffer components and critical
reaction components.
• Chemical stability.
• Light absorption outside of wavelengths used for assay readout.
• Components should be easy to obtain and prepare.
Good, N.E. et al. (1966) Hydrogen ion buffers for biological research. Biochemistry. 5, 467–77.
 1. Optimal buffering at a neutral
pH
 Most biochemical reactions have an optimal pH in the range of 6–8, so
buffers for these reactions need to have pKas that support buffering at
these pH values.

 Some buffers and their pKa values (in water at 25°C):

– Acetate 4.76
– PIPES 6.76
– MOPS 7.20
Buffer is effective in a range about +/- 1 pH
– HEPES 7.48
unit of the pKa value
– Tris 8.06
– Borate 9.23
 2. Solubility in
water
• Most biochemical reactions occur in aqueous conditions, so your
buffering components should be soluble in water.

• If for some reason, you will be using a solvent other than water,
make sure you understand how that solvent affects the dissociation
of your buffer components.
4. Minimal salt
interactions
• If the system to be studied requires salts, appropriate ions can be
added. However, using an ionic buffer can adversely affect the
reaction if reaction studied is affected by salts.

• In other words, the buffer components should not interact or

affect ions involved in the biochemical reactions being explored.
• Changes in dissociation resulting from changes in concentration are
usually small, and most buffers can be made as stock solutions that
are diluted to working solutions.5. Minimal
However, effects
some ondothe
buffers dissociation
show a from
significant change in pH upon dilution.
changesin concentration
• For instance, the pH of Tris decreases approximately 0.1 pH unit per
tenfold dilution, and the pH could change dramatically if you dilute a
working solution and are at the limits of the optimal buffering range
of the Tris.
5. Minimal effects on the
dissociation from changes in
temperature
• Temperature changes can be a problem too.
• Eg: Tris exhibits a large shift in dissociation with a change in
temperature. (Tris is not one of Good’s buffers)

- If you prepare a Tris buffer at pH 7.0 at 4.0°C and perform a reaction

in that same buffer at 37°C, the pH will drop to 5.95.

- If you have a Tris buffer prepared at 20°C with a pKa of 8.3, it would
be an effective buffer for many biochemical reactions (pH 7.3–9.3),
but the same Tris buffer used at 4°C becomes a poor buffer at pH 7.3
because its pKa shifts to 8.8.
 5. Minimal effects on the
dissociation from
changes in temperature

So the take-home message: Make the buffer at the temperature you

plan to use it, and if your experiment involves a temperature shift, select
a buffer with a range that can accommodate any shift in dissociation as a
result.
6. Minimal interactions between buffer components
and critical reaction components

• If a complex forms between the buffer and a required cofactor, say a

metal cation like zinc or magnesium, your reaction might be
compromised. For example calcium precipitates as calcium phosphate
in phosphate buffers. Not only would any Ca2+-requiring reactions be
compromised, but the buffering capacity of the phosphate buffer also
is affected.

• Having excessive amounts of a chelating agent in an enzymatically

driven reaction could cause problems (e.g., a high concentration of
EDTA in a PCR amplification). Citrate is a calcium chelator, so avoid
citrate buffers in situations where calcium concentrations are critical.

• Watch buffer components that have reactive R groups. For instance

Tris has a reactive amine group. Remember buffers are not inert!
7. Chemical
stability
• The buffer should be stable and not break down under working
conditions. It should not oxidize or be affected by the system in
which it is being used. Try to avoid buffers that contain
participants in reactions (e.g., metabolites).

• Some buffers, such as MOPS, must be protected from light, but

when they are stored properly they are still extremely useful
buffers in biochemical reactions and laboratory protocols like
RNA electrophoresis.
 8. Light absorption outside of wavelength used
for assay readout

• Buffer should not absorb UV light at wavelengths that may be

used for readouts in photometric experiments.

• Should not absorb any light at wave-lengths longer than 230 nm,
since many spectrophotometric investigations are performed in
this range (determination of the concentrations of DNA, RNA and
proteins)
Preparing
buffers
• Prepare buffers at the appropriate temperature and concentration.
If you dilute a buffer or use it at a different temperature than the
one at which it was prepared, measure the pH after dilution and
equilibration to the new temperature.

• Adjust the pH of the buffer system correctly. Not all buffers are
prepared the same way. Be sure you understand how your buffer
system works and that you do not introduce any new ions into the
system during pH measurement.

• Make sure you know how to use and care for the pH meter.
Preparing
buffers
• Some buffering components may have to be heated or put in
alkaline or acidic conditions before they will dissolve.

• Some buffers cannot be autoclaved because they will degrade

upon heating (so they will need to be filter sterilized).

• When working with acids and bases be sure to wear the

appropriate protective clothing and eyewear. Do not try to
neutralize strong acids with strong bases.

• If you are using a solvent other than water, be sure you know how
that solvent affects the pKa of the buffer system.
 if they look cloudy or discolored, do not use them.
Using Such solutions may have microbial contamination or
buffers may have become chemically unstable

• Check all stored buffers before use. Swirl the bottle to check for any
contaminants that may have settled to the bottom during storage.

• Remeasure pH if you are diluting a buffer before using it in your

reaction.

• Keep detailed notes on buffer preparation so that you can replicate

your experiments (or troubleshoot confusing data). Indicate grade of
materials used, supplier, and lot no. if known. Indicate what acid or
base was used to pH the buffer and its concentration. If additional
components were added to the buffer indicate at what point pH was
measured.
3.3 Common buffer in
biology
The decision for or against a buffer is also dependent on the
method for which it is used

Buffer Application
Tris Electrophoresis
- 2-
H2PO4 /HPO4 Protein purification
-
H2CO3/HCO3 Blood buffer

Can you name a few?

pH and buffers in living
systems
• Common examples of how pH plays a very important role in our
daily lives are given below:

• Water in swimming pool is maintained by checking its pH. Acidic

or basic chemicals can be added if the water becomes too acidic
or too basic.
• Whenever we get a heartburn, more acid build up in the stomach
and causes pain. We needs to take antacid tablets (a base) to
neutralize excess acid in the stomach.
• The pH of blood is slightly basic. A fluctuation in the pH of the
blood can cause in serious harm to vital organs in the body.
• Certain diseases are diagnosed only by checking the pH of blood
and urine.
• Certain crops thrive better at certain pH range.
Using pH meter

• A pH meter basic principle is to measure the concentration of

hydrogen ion.

• A pH meter is a voltmeter which measures the voltage of an

electrode sensitive to the hydrogen ion concentration relative to
another electrode which exhibits a constant voltage.

• This is translated into pH by the instrument and reading is displayed.

higher voltages signalling acidic pH levels

and lower voltages signalling basic
Calibrating pH
meter
Why?
• To be certain of accurate and reliable measurements, you need
to perform pH meter calibration.
• Over time, aging and coating of pH electrodes can cause
changes in the measurement

This is generally done by measuring different buffer solutions with

standardized, well-defined values, and then adjusting the pH meter
based on any deviations from the buffer’s known pH value.
Calibration at three or more pH values (i.e. pH 3, 7, 10) increases
the measurement range of the device without recalibration being
required.
 3.4
Calculation in
molecular biology
Solution
Concentration
Dilution
Serial Dilutions
Metric Unit
Metric Unit
 Solution: Concentration and Calculation
 Solution: a homogenous mixture of two or more substances in
the same phase.

 Solute: substance dissolved in another substance (the solvent)

 Solvent: substance that dissolves a solute, resulting in a

solution
 Water is the “universal solvent”
 Other common lab solvents: Ethanol, Methanol, and Acetone

Solubility -the ability of one compound to

dissolve in another; maximum amount of solute
capable of being dissolved by a solvent.
 Solution: Concentration and Calculation
 Concentration: the quantity of solute present per unit volume of
the solvent

You can create:

- A “dilute”
- A “concentrated”
- Or a “saturated”
Solution: Concentration
and Calculation
Concentrations for solutions may be
expressed in multiple ways (units):
1. Molarity (unit = M; Moles/Liter)
-Concentration based on the no of mole
of solute per 1 liter of solution Ex: 1 M
solution of the alanine (mw=89.1)
 Solution: Concentration and Calculation
2. Percent by weight (unit = % w/w)
- Concentration based on the no of grams of solute per 100 g solution
Ex: A 5 % w/w alanine solution contains 5 g of alanine in a 100 g of
solution

3. Percent by volume (unit = % w/v)

- Concentration based on the no of grams of solute per 100 ml solution
Ex: A 5 % w/v alanine solution contains 5 g of alanine in a 100 ml of
solution

4. Weight per volume (unit = w/v)

- Concentration based on the no of grams, mg, μg of solute per unit
volume Ex: mg/ml, g/l, mg/100 ml
A 5 mg/ml alanine solution contains 5 mg alanine in a 1 ml of
solution
 Why would I ever need
Dilutio to make a dilution?
n
 Dilution: the process of decreasing the concentration of a stock
(original) solution by adding more solvent to the solution

The equation for dilution is M1V1=M2V2

stock solution= diluted solution

• M1= molarity of the stock solution

• M2= molarity of the diluted solution
 Type of Dilution
1. Simple Dilution: dilution of a single solution to make a
diluted solution

2. Serial Dilution: series of dilutions resulting in progressively

diluted versions of the Primary (“original” or “stock”)
solution.
Dilution
• Simple Dilution: dilution of a single solution to make a diluted
solution

Ratio/factor

• Dilution Factor:

(Volume of stock solution)

(Volume of stock solution) + (Volume of solvent)
 Serial Dilution
 Serial Dilution: series
of dilutions resulting
in progressively
diluted versions of the
stock (“original”)
solution.
 Typically made in
increments of 1000,
100, or 10
(logarithmic scale)

Dilution factor
 Solution: Concentration and Calculation
Calculate the molarity of a 5L solution containing 126g of HNO3 (63
g/mol).

=2 moles
Calculate the number of moles: 126g HNO3

• M= moles of solute
liters of solution

• M= 2mol HNO3
5L

• M= 0.4 mol/L
Molarity : Learning Check
How would you prepare 1000 mL of a 1 M solution of Tris buffer
from a 3 M stock of Tris buffer?

Calculate the mass of NaOH (MW: 40 g/mol) needed to prepare

1.0L of a 1.5M solution?

960g of NaOH is used in preparing a 1.5M solution. What volume of

solution can be made?
Molarity by dilution: Learning
check
The equation for dilution is M1V1=M2V2
stock solution= diluted solution

• M1= molarity of the stock solution

• M2= molarity of the diluted solution
• V1 = Volume of the stock solution
• V2 = Volume of the diluted solution
 Molarity by dilution: Learning check
Q: A stock solution of 1.00 M of NaCl is available. How many milliliters
are needed to make a 100.0 mL of 0.750 M?

What we know: the molarity of the stock solution which is 1.00 M, and
the two components of the diluted solution which are M2= 0.750 M and
V2= 100 mL.
Plug in the values you have into the equation to solve for the missing
value.
M1V1=M2V2
Molarity by dilution: Learning check

1) Describe how you would prepare 30 ml of 70 % ethanol

solution from 95 % ethanol stock solution?

2) You have 1 L of a 5 M solution. How would you make this into a

2.5 M solution?

3) If 8 ml of distilled water is added to 2 ml of 95 % ethanol

solution, what is the concentration of the diluted solution.
3.0
Centrifugation
https://padlet.com/bms415sept2019/dnwxxk3n9srg

i-learn portal
 CLO 02
Illustrate the principles and applications of
basic centrifugation, chromatographic and
spectroscopic analytical techniques in the
isolation and characterization of biological
molecules.

(i) The LLO(s) (ii)

Understand the fundamental of Differentiate different type of
centrifugation centrifugation techniques and
their applications

(iii)
Knows different types of centrifuges,
rotors and it cares
Outline
s

Differential &
Basic Principles Types of
Density
and Rotors and Care
Gradients centrifuges
Applications
centrifugation
Overvie
w
• Biological centrifugation is a process that uses centrifugal force to
separate and purify mixtures of biological particles in a liquid medium.

• One of widely used laboratory technique for the separation of materials in

the field of, biochemistry, molecular biology, medicine, food sciences and
industry.

• Today, centrifugation techniques represent a critical tool for modern

biochemistry and are employed in almost all invasive subcellular studies.
General steps
Bioseparations
Centrifugation is everywhere….

It is a key technique for isolating and

analysing cells, subcellular fractions,
supramolecular complexes and isolated
macromolecules such as proteins or nucleic
acids.
Centrifuge vs
Centrifugation
• A centrifuge is a device that separates particles from a solution
through use of a rotor.

• Centrifugation is a technique that uses centrifugal force to separate

and purify mixtures of biological particles in a liquid medium.
Why do we need to
centrifuge?
• Its all about gravity and mass –particles
in a heterogenous solution will, given
enough time, separate based on their
size and density.
• Smaller , less-dense particles may also
migrate down, but not always; some
particles will never settle, but remain
suspended in solution

…….But do we have the patient to wait long?????

Centrifuges force this process along much more quickly
and efficiently
Principle
of
Operation
• A centrifuge is a piece of equipment,
generally driven by an electric motor, that
puts an object in rotation around a fixed
axis, applying a force perpendicular to the
axis to separate substances of different
densities.
• Tubes in the centrifuge are tilted so
centrifugal force can pull denser substances
towards the bottom of the tube.
• Relative Centrifugal Force (RCF) measures
acceleration applied to the sample
FORCE IN A CENTRIFUGE IS
PROPORTIONAL TO TWO THINGS
• First, it depends on how fast the centrifuge spins

Speed use in centrifugation; the higher the speed the

higher the centrifugation force thus the smaller molecules
separated.

• Second, it depends on the radius of rotation

The greater the radius of
rotation, the more force that is
experienced by the molecule

Imagine this thing with longer string!!

RELATIVE CENTRIFUGAL FORCE
(RCF)
• Also called x g
• RCF = 11.17 (r) * (n/1000)2

Where r = radius in cm from centerline

n = rotor speed in RPM, revolutions/
Try
these….
1. Calculate the RCF for a centrifuge with a rotor with a spinning
radius of 6 cm that is spun at 4,000 RPM?

1. Calculate the RPM for a centrifuge with a rotor with a spinning

radius of 6 cm that needs a relative centrifugal force of 5,000 g?
Post centrifugation
sample

Supernatant – liquid at the top

Pellet – particles at the bottom

Factor affecting sedimentation
• It depends on:
• The applied relative centrifugal force (RCF)
• Size, density and radius of particles
• Density and viscosity of the suspended medium
This is how separation is achieved

www.phys.sinica.edu.tw/TIGP.../AC_Chapter%203%20Centrifugation%200321.pdf
Applications of centrifugation

• Separation of two immiscible liquids – eg: to remove cellular debris from blood to prepare
cell free plasma or serum
• To concentrate cellular elements and other components for microscopic and chemical
analysis
• To separate protein bound or antibody bound ligand from free ligand in immunological assay
• To separate and isolate subcellular organelles, macromolecules - DNA, RNA, proteins or lipids
• For determination of purity and shape of biomolecules
• To determine the relative molecular mass analysis using density gradient
Basic modes of
centrifugation
There are two (2) types:
1. Differential centrifugation
2. Density gradient centrifugation
1. Zonal
2. Isopycnic
Differential
Centrifugatio
n
• Basic concept:
Particles of different
sizes will sediment at
different rates.
• Separate particles
into two phase; a
pellet and a
supernatant

Procedure used to isolate different particles by stepwise successive

centrifugations at increasing RCF
• Rough separation of
subcellular components

• Usually performed prior

to density gradient
centrifugation

• Carried out using Fixed

Angle rotor
 2. Density
centrifugation
i) Zonal centrifugation a.k.a band or gradient centrifugation
• A density gradient is created in the tube with a suitable medium (eg. Sucrose and
glycerol) having high density at the bottom
• The sample is applied in a thin zone at the top of the centrifuge tube on a density
gradient.
• Upon centrifugal force, particles move at different rate depending on their size
and mass
2. Density
centrifugation
ii)Isopycnic or sedimentation equilibrium
centrifugation
• Where as, in isopycnic, the sample and the
medium is uniformly mixed in the tube and
the rotate in the centrifuge

The particles travel through the gradient until

they reach a point at which their density
matches with the density of surrounding
sucrose medium forming separate bands
Confused? These are they key
points….
• 1. Differential centrifugation
• Cell-free extract and supernatant are centrifuged at progressively higher speeds and longer
times

• 2. Density gradient centrifugation

• Cell free extract is centrifuged through a medium whose density gradually increases toward
the bottom of centrifuge tube

• a) Rate-zonal
• Sample applied at the top and centrifuged until most dense component approaches
bottom of centrifuge tube
• b) Isopycnic
• Sample is mixed and centrifuged until all components reach their equilibrium buoyant
density.
Types of
centrifuges
• Centrifuge are generally divided into 3 types based on their maximum
attainable speed:
• 1. Low speed ; < 10, 000 rpm
• 2. High speed; 10,000– 30, 000 rpm with temperature control
• 3. Ultracentrifuges ; up to 100, 000 -150 000 rpm with temperature control
Types of
Centrifuge
• Low –speed centrifuge
• Also called: microfuge, clinical, table top, bench top centrifuge
• Max speed < 10, 000 rpm (3000-7000 rpm)
• Do not have temperature regulatory system; operate at RT
• Fixed angle or swinging bucket can be used
• Commonly for rapid separation of coarse particles
• E.g. RBC from blood, DNA from protein, bacterial cells etc.
• The ample will be centrifuged until the particles are tightly packed into pellet at the
bottom of the tube.
• Liquid portion, supernatant is decanted
They are used to collect small amount of material that rapidly
sediment
High-speed centrifuge-
Preparative
• Max speed < 30, 000 rpm
• Often refrigerated, and requires vacuum to operate
• Fixed angle or swinging bucket can be used
• Generally used to separate macromolecules (proteins or nucleic acids)
during purification or preparative work.
• Can be used to estimate sedimentation coefficient and MW
Can be used to collect micro-organism cellular debris, larger cellular organelles
and proteins precipitated by ammonium sulphate
Ultracentrifu
ge
• The most advanced form : specialized and expensive
• Used to precisely determine sedimentation coefficient and MW of
molecules, molecular shape, protein-protein interaction
• Uses very high speed
• Uses small sample size (<1 ml)
• Uses relatively pure sample
• Built in optical system to analyze movements of molecules during
centrifugation
Can be used both for preparative & analytical
works

In this centrifuge rotors are

mounted on a rigid shaft . Shaft is
made up of aluminum or
titanium alloy of high tensile
strength to withstand the great
force generated during
centrifugation.
It is used for both preparative work
and analytical work
Instrument
Design
Type of
Rotors
• The principle component of a centrifuge is the rotor, which is the
moving part that spins at high speeds.
• Common types of rotor used:
1. Fixed Angel Rotor
2. Swinging bucket
Fixed Angle
Rotor
• It holds tubes in the exact same fixed angle all the time, usually 45
degree
• Mostly used in lab for simple pelleting applications
• Also useful for isopycnic separation
• Due to rigid design of the metal alloy material, fixed rotors can
withstand much higher gravitational forces.
• Sedimenting particles have only shorter distance to travel before pelleting
Swinging Bucket
Rotors
• Has bucket that start off in a vertical position but during acceleration
of the rotor swing out to a horizontal position
• Hence, the solution in the tube, is aligned perpendicular to the axis of
rotation
• The tube returning to its original position during decelaration of the
rotor
Longer distance of travel may allow better
separation, such as in density gradient
centrifugation. Easier to withdraw
supernatant without disturbing pellet.
When and which rotors to
use?
• Depending on the
• 1. Applications
• 2. Production
 Carefully read the manual before using
ROTORS ARE centrifuge

FRAGILE!
• Must withstand huge forces
• Regular maintenance
• Do not exceed maximum speed!
• Derate (run slower) when necessary
• Balance, balance, balance
• Proper handling and care; protect rotor from
• Scratches, Moisture, Spills and detergents
• Purchase the correct rotors for your application
• Log book- accurate record-keeping of run times and speeds
 Follow manufacturer’s directions
Proper use of
1. Place correct size of tubes in centrifuge
Rotors 2. Proper balancing and counterbalance:
• Wear safety PPE; gloves, glasses
• By mass NOT volume Ensure loads are evenly BALANCE!
• Put the tubes opposite each other in the centrifuge. If your have more
than two tubes, only the ones opposite each other have to be equal in
mass.
3. If centrifuge has variable speeds, enter RPM (do not exceed max speed!)
4. Close lid
5. Turn timer and press start
6. Remove the tubes carefully after the centrifuge has completely stopped
spinning to prevent remixing
7. Make sure you know what you are doing
 NEVER exceed the speed limit and Balance,
balance and balance….
• Result of unbalanced centrifuge
 How Did Scientists Find Cytochrome C? Preparation of Mitochondria from
mouse liver

The mouse livers were removed after sacrifice and dounce homogenized in ice-cold mitochondria
isolation buffer (MIB) containing 250 mM mannitol, 0.5 mM EGTA, 5 mM HEPES, and 0.1% (w/v)
BSA (pH 7.2) supplemented with the protease inhibitors of leupeptin (1 mg/ml), pepstatin A (1
mg/ml), antipain (50 mg/ml), and PMSF (0.1 mM). Unbroken cells and nuclei were pelleted by
centrifugation at 600g for 5 min at 4oC. The supernatants were further centrifuged at 10,000g for 10
min at 4oC to pellet the mitochondria. The mitochondria pellet was resuspended in 4 ml MIB and
loaded onto a continuous Percoll gradient consisted of 30% (v/v) Percoll (Sigma), 225 mM mannitol,
25 mM HEPES, 0.5 mM EGTA, and 0.1% (w/v) BSA (pH 7.2). The suspension/gradient was
centrifuged at 40,000g for 1 hr. The mitochondria were removed from the brownish band at 1.10
g/ml with a transfer pipette. The mitochondrial pellets were washed with MIB by centrifuging for 10
min at 6300g at 4oC. The mitochondria were then resuspended gently in mitochondria resuspension
buffer containing 400mM mannitol, 10 mM KH2PO4, and 50 mM Tris-HCl (pH 7.2) with 5 mg/ml BSA
and stored on ice for up to 4 hr.

http://www.swmed.edu/home_pages/wanglab/Wanglab-pic/protocols.htm
THANK YOU
4.0
SPECTROSCOPY
BMS481
NURUL AILI ZAKARIA (Ph.D)

https://padlet.com/bms415sept2019/dnwxxk3n9srg
i-learn portal
 CLO 02
Illustrate the principles and applications of
basic centrifugation, chromatographic and
spectroscopic analytical techniques in the
isolation and characterization of biological
molecules.

(i) The LLO(s) (ii)

Understand the fundamental of Differentiate different type of
spectroscopy spectroscopy and their
applications

(iii)
Knows different types of centrifuges,
rotors and it cares
Outline
s

Basic Principles Type of Quantitation of

and spectroscopy Enzyme assays
DNA & Protein
Applications analysis
Terminolo
gy
Spectroscopy Spectrophotometry Spectra
• The study of the light • Is a method to • Range of
from an object measure how much a electromagnetic
chemical substance energy separated by
absorb light by wavelength
measuring the intensity
of light as a beam of
light passes through
sample solution
Mars Exploration
Overvie Mission
w
• Astronomers use spectroscopy because it allows them to determine
the make up of stars….Without having to be present to take sample
• Studying an objects spectra can tell scientists the composition of an
object, its temperature, its density and its motion

• Two modern applications of spectroscopy in space…

 Mars Exploration
Mars Exploration Mission
Mission
The Mars Exploration Rovers were launched with the goal of
searching for and analyzing rock and soils on Mars. They utilized
several spectrometers to analyze samples.

Mini-TES: miniature thermal

emission spectrometer (examine
rock, soil & atmosphere)

MB: Mossbauer Spectrometer

(examine mineralogy of rocks &
soils)
APXS: Alpha Particle X-ray Spectrometer
(analyze elements in rocks & soils)
 Cassini-Hyugen’s
Mars Exploration Mission
Mission Mission: to gather information on
Titan (Saturn’s moon).

VIMS: Visual and Infrared

Mapping Spectrometer
(gather data about surface,
rings & atmosphere of Titan
and Saturn).
CIRS: Composite Infrared
Spectrometer (searches for
heat and by that gather
information on the object’s
composition.
 Basic
Principles &
Applications
Why
spectrophotometer?
• A spectrophotometer is a common tool used by scientist to
determine information about an object or substances through the
analysis of its light properties
• Every substance and element produces different light frequencies and
pattern which sort of their fingerprints.
• Using this principle, scientists can analyze unknown substances and
materials using spectrophotometers then compare the results to
known patterns to determine the composition of the test subject.
 Basic
Principles &
Applications
Why
spectrophotometer?
• Qualitatively – to determine the amount of material in a sample

• Qualitatively – to determine the chemical structure of a sample

 Basic
Principles &
Applications Why
spectrophotometer?
• Spectrophotometry is widely used for
quantitative analysis in various areas
(e.g., chemistry, physics, biology,
biochemistry, material and chemical
engineering, clinical applications,
industrial applications, etc)
• In biochemistry, for example, it is used to
• Any application that deals with chemical
substances or materials can use this determine enzyme-catalyzed reactions.
technique. • In clinical applications, it is used to examine
blood or tissues for clinical diagnosis.
• Can you name its applications?
 Basic
Principles &
Applications What is
spectrophotometer?
• A spectrophotometer is an instrument
that measures the amount of photons
(the intensity of light) absorbed after it
passes through sample solution.
• With the spectrophotometer, the amount
of a known chemical substance
(concentrations) can also be determined
by measuring the intensity of light
detected
 Basic
Principles &
Applications
• This instrument operates by passing a beam of light through a sample and
measuring the intensity of light reaching a detector.
Principle of
spectrophotometer?

It produces a desired range of wavelength of light. First a collimator (lens) transmits a straight beam of light (photons) that passes
through a monochromator (prism) to split it into several component wavelengths (spectrum). Then a wavelength selector (slit)
transmits only the desired wavelengths, as shown in Figure 1.
 Basic
Principles &
Applications

• As specific wavelengths of light pass through a sample of

Principle of
dissolved material (referred to analyte which in cuvette), the
instrument indirectly measures the amount of light absorbed by
that sample.
spectrophotometer?
• Calculated by comparing the initial intensity of light reaching the
sample (I0) with the light detected by a detector as it exits the
sample (IT).
• The ratio of the 2 readings is referred to as the Transmission (T)
of light through the sample
 Basic
Principles &
Applications
Principle of
spectrophotometer
• But, a detector in a spectrophotometer will display the Absorbance
reading (A) rather than Transmittance (T). So,
• Transmittance is related to absorption by the expression:
 Basic
Principles &
Applications
Let’s try
this…
• The intensity of the incident light on a sample is 0.5 w/m2 and the
intensity of the light entering the detector is 0.36 w/m2
a) Calculate the transmittance
b) Determine the absorbance
 Basic
Absorptio
Principles &
Applications
•With the
n amount of absorbance known
from the above equation, you can
determine the unknown concentration
of the sample by using Beer-Lambert
Law
•Beer-Lambert Law (also known as
Beer's Law) states that there is a
linear relationship between the
absorbance and the concentration of
a sample.
•For this reason, Beer's Law can only be
applied when there is a linear
 Basic
Principles &
Applications
Beer-Lambert Law
 Basic
Principles &
Applications
Let’s try this…
 Basic
Principles &
Applications
Let’s try this…
 Basic
Principles &
Applications
Let’s try this…
0.7
0.6 y = 1.0137x + 0.1378
R2 = 0.997
Is the fitting of the curve to the 0.5
equation acceptable? How can you
0.4

Abs
tell?
0.3
What is the concentration of C when 0.2
we obtain an Absorbance of 0.3321? 0.1
0
0 0.2 conc 0.4 0.6

Standard curve of an imaginary data

 Type of
spectroscopy
analysis
What are
they?
• Depending on the range of wavelength of light • Nuclear Magnetic Resonance (NMR)-
source, it can be classified into two different study protein structure and dynamic
types:
• X-ray Crystallography –study protein
• UV-visible spectrophotometer: uses light over structure
the ultraviolet range (185 - 400 nm) and visible
range (400 - 700 nm) of electromagnetic radiation• Mass-spectrometry – to characterize
spectrum. physical property of protein, accurate
measurements of protein molecular
• IR spectrophotometer: uses light over the weight and sequence analysis
infrared range (700 - 15000 nm) of
electromagnetic radiation spectrum.
Let’s look at the whole
spectrum..
 Type of
spectroscopy
analysis
UV-
VIS
Substances can absorb varying amounts of UV and/or Visible radiation at
particular wavelengths – Coloured compounds absorb energy in both UV
and visible region of the electromagnetic spectrum.
4.0
(part 2)
SPECTROSCOPYBMS481
NURUL AILI ZAKARIA (Ph.D)

https://padlet.com/bms415sept2019/dnwxxk3n9srg
i-learn portal
 CLO 02
Illustrate the principles and applications of
basic centrifugation, chromatographic and
spectroscopic analytical techniques in the
isolation and characterization of biological
molecules.

(i) The LLO(s) (ii)

Understand the fundamental of Differentiate different type of
spectroscopy spectroscopy and their
applications
Outline
s

Basic Principles Type of Quantitation of

and spectroscopy Enzyme assays
DNA & Protein
Applications analysis
Let’s look at the whole spectrum..how do
we study the different spectrum?
You need a spectrometer to produce a variety of
wavelengths because different compounds
absorb best at different wavelengths.
 Type of
spectroscopy
analysis
UV-VIS
SPECTROPHOTOMETRY
Substances can absorb varying amounts of UV and/or Visible radiation at
particular wavelengths – Coloured compounds absorb energy in both UV
and visible region of the electromagnetic spectrum.
Quantitation
of DNA &
QUANTITATION OF PROTEIN,
Protein
DNA
Principle
& RNA
Proteins in solution absorb ultraviolet light with absorbance maxima at 280
nm. Amino acids with aromatic rings are the primary reason for the
absorbance peak at 280 nm.

Nucleic acid (DNA) is at 260 nm

Why to quantify Protein and Nucleic acids?

• To check the concentration and purity of
DNA/RNA present in the solution mixture
• It is important to know the concentration
and purity of the nucleic acid for the use in
further applications like PCR, restriction
digestion etc.
Quantitation
of DNA &
QUANTITATION OF PROTEIN,
Protein
DNA
Instruments
& RNA
UV vis spectra and quartz cuvette
Or Nanodrop spectra

sample
Quantitation
of DNA &
ADVANTAGES OF USING
Protein
CanNANODROP
quantify nucleic acid from micro volumes of 0.5 µL – 1.0 µL
Measure DNA, RNA (A260) and protein (A280) concentrations and sample
purity (260/280 ratio)
Large concentration range (2 ng/µL – 15,000 ng/µL dsDNA) without
dilutions.
Enzyme
assays ENZYME
ASSAYS
 Enzyme assays are laboratory methods for measuring enzymatic
activity. They are vital for the study of enzyme kinetics and enzyme
inhibition
 The assay is the act of measuring how fast a given amount of enzyme
will convert substrate to product (the act of measuring velocity)
 Enzyme assays measure either the disappearance of substrate over
time or the formation of product over time- measure reaction rate
 An assay requires to determine the concentration of a product or
substrate at a given time after starting the reaction.
Enzyme
assays MEASURING ENZYME
ACTIVITY
 Spectrophotometer is the most common method of detection in
enzyme assays
 It used to measure the amount of light a substance’s absorbs, to
combine kinetic measurements and Beer’s law by calculating the
formation of product or reduction of substrate concentrations.
 Enzyme is colourless..is in the visible region we can actually see a
change in the color of the assay, these are called colorimetric assays
…remember BSA biuret assay?

Study the differences between

colorimeter and
spectrophotometer!
Enzyme
assays MEASURING ENZYME
ACTIVITY
UV light is often used, since the common coenzymes NADH and NADPH
absorb UV light in their reduced forms, but do not in their oxidized forms.
Enzyme
(G6PD)
Reactant Product
(G6P + NADP+) (6PG + NADPH at 340 nm)
• Something has to absorb light!
• Which component in the reaction should it be?
• We want to know if the enzyme is there, or if it is actively “doing”
something.
• The reactant can absorb light (abs with enzyme)

• The product can absorb light (abs with enzyme)

Enzyme
assays TYPES OF ENZYME
ASSAY
• 1. Continuous assay – where the assay gives a continuous reading of activity

- Manipulation necessary to detect product formation- allows continuous

observation of the change
-multiple measurements, usually of absorbance change, are made during the
reaction
-either at specific time intervals (usually every 30 or 60 seconds)
-or continuously by a continuous-recording spectrophotometer

These assays are advantageous over fixed-time methods because the linearity of the
reaction may be more adequately verified
Enzyme
assays TYPES OF ENZYME
ASSAY
• 2. Discontinuous assays – where samples are taken, the reaction stopped and then
the concentration of substrates/product determined. (at interval)

• Also called sampling assay

• The reaction is stopped (after a fixed time, usually by inactivating the enzyme
with a weak acid)
• Treating the reaction mixture to separate the product for analysis or produce a
measurable change in properties of substrates or product.
• A measurement is made of the amount of reaction that has occurred.
• Study the differences of Continuous assay over discontinuous assay
Enzyme
assays TYPES OF ENZYME
ASSAY
• 3. Coupled assays – use of one or more additional enzymes to catalyse a reaction of
one of the products to yield a compound that can be directly detected

• Additional enzyme – couple enzymes

Enzyme
assays VALIDITY OF RESULTS FOR
ENZYME ASSAY
• Reaction step should not be rate limiting
• Velocity of the reaction increases till coupling
enzyme reaches the rate of the first enzyme
• Coupling enzyme – high Km for the enzyme
and low Km for the substrate
Enzyme
assays
THE 96 WELL
MICROPLATE READER
• provide rapid and sensitive measurements
of a variety of analytes across a wide range
of concentrations
• The contents of the wells in a microplate
can be mixed automatically by shaking
before each read cycle, which makes it
possible to perform kinetic analysis of solid-
phase, enzyme-mediated reactions
Enzyme
assays
THE 96 WELL
MICROPLATE READER
Enzyme THE 96 WELL
assays
MICROPLATE READER
• Reading mode
1. Endpoint: at a single point in time
2. Kinetic : over a specified period of time.
3. Spectral scan : over a specified wavelength range.
Enzyme INSTRUMENTS FOR
assays
ENZYME ASSAY
• Cuvettes are made from glass, plastic or silica quartz.
• Make sure to choose the right cuvette for the wavelength studied !!
• It should be without impurities and scratch
Enzyme
assays BLANK/STANDARD FOR
ENZYME ASSAY Why use
blank?
•The blank contains all substances
except the analyte
• (i.e. the solvent of your solutions)

•Is used to set the absorbance to zero

• A blank = 0 or %Trans = 100%

•Analyte = substance undergoing

analysis
Enzyme FEATURES OF A GOOD
assays
ENZYME ASSAY
• Simple and specific
• Rapid (one doesn’t need to wait for hours or weeks for the results
to appear)
• Sensitive (very little sample)
• Easy to use
• Economical
Applications

1. Measurement of Concentration
- Prepare samples APPLICATIONS OF
-Make series of standard solutions of known
concentrations SPECTROPHOTOMETER
-Set spectrophotometer to the 𝜆 of max light
absorbance
-Measure the OD of the unknown
-Using the standard curve plot, find the
concentration of the unknown
Applications

2. Detection of Impurities
APPLICATIONS OF
- UV-Vis spectroscopy is one of the best methods for determination of impurities
in organic molecules
SPECTROPHOTOMETER
-Additional peaks can be observed due to impurities in the sample and it can be
compared with that of standard raw material
Applications
4. Chemical Kinetics
-Kinetics of reaction can also be studied using UV spectroscopy. The UV radiation is
APPLICATIONS OF
passed through the reaction cell and the absorbance changes can be observed

SPECTROPHOTOMETER
Applications

5. Detection of Functional groups

APPLICATIONS OF
-Absence of a band at particular wavelength regarded as an evidence for absence
of particular group
SPECTROPHOTOMETER
 BONUS….

HOW TO USE A SPECTROPHOTOMETER

Turn on the spectrophotometer
Most spectrophotometers need to warm up
before they can give an accurate reading
Turn on the machine and let it sit for at least 15
min before running any samples
Use the warm-up time to prepare your samples

Clean the cuvettes

Rinse each cuvette thoroughly with deionized water
When handling the cuvette, avoid touching the sides the
light will pass through (generally, the clear sides of the
container)
If you accidently touch these sides, wipe the cuvette
down with a kimwipe (which are formulated to prevent
scratching the glass).
Load the proper volume (~0.5 – 1 mL) of the sample
into the cuvette
As long as the laser producing the light is passing
through the liquid
If you are using a pipette to load your samples, use a
new tip for each sample to prevent cross-contamination

Prepare a control solution

Known as a blank, the control solution has only the
chemical solvent in which the solute to be analyzed is
dissolved in
e.g., if you had salt dissolved in water, your blank would
be just water. If you dye the water red, the blank must
also contain red water. The blank is the same volume as
the solution to be analyzed and kept in the same kind of
container
Wipe the outside of the cuvette
Before placing the cuvette into the spectrophotometer you want
to make sure it is as clean as possible to avoid interference from
dirt or dust particles
Using a lint free cloth, remove any water droplets or dust that
may be on the outside of the cuvette

Choose and set the wavelength of light to analyze the sample

Use a single wavelength of light (monochromatic color) to make
the testing more effective
The color of the light chosen should be one known to be
absorbed by one of the chemicals thought to be in the test solute
Set the desired wavelength according to the specifications of your
spectrophotometer
Calibrate the machine with the blank
Place the blank into the cuvette holder and shut the lid
Set the blank to ‘0’ (zero) using the adjustment knobs
When you remove the blank, the calibration will still be in place.
When measuring the rest of your samples, the absorbance from
the blank will automatically be subtracted out
Be sure to use a single blank per session so that each sample is
calibrated to the same blank. For instance, if you blank the
spectrophotometer, then analyze only some of samples and blank
it again, the remaining samples would be inaccurate. You would
need to start over.

Remove the blank and test the calibration

Measure the absorbance of your experimental sample
Remove the blank and place the experimental sample into the
machine
Slide the cuvette into the designated groove and ensure it stands
upright
Wait about 10 seconds the digital numbers stop changing
Record the values of % transmittance and/or absorbance. The
absorbance is also known as the optical density (OD)
The more light that is transmitted, the less light the sample
absorbs. Generally, you want to record the absorbance values
which will usually be given as a decimal, for example, 0.43
Repeat the reading for each individual sample at least 3 times
and average them together. This ensures a more accurate
readout
 Chapter 5 of 9

PRINCIPLE OF
CHROMATOGRAPHY

BMS481
BIOANALYTICAL CHEMISTRY
NURUL AILI ZAKARIA (PhD)
Course Learning Outcomes (CLOs)

At the end of the course, students should be able to:

1. Describe the fundamentals concepts of analytical techniques e.g. accuracy,
concentrations, molarity.
2. Illustrate the principles and applications of basic centrifugation,
chromatographic and spectroscopic analytical techniques in the isolation
and characterization of biological molecules.
3. Perform scientific experiments on biomolecules separation, characterization
and analysis.
Chapter Overview

Link to Practical
- Running a
Types & applications planar
- Column chromatography
Chromatography
Separation by -Planar
Chromatography Chromatography
- The main
components
Brief Intro -Principle
- What it is?
 Brief History

– The word Chromatography is derived from two Greek words-Chroma

means “color” and graphein to “write”
– Described by Mikhail Twsett in 1903 when separating plant pigments
using calcium carbonate
– Chromatography is a misnomer since it is no longer limited to the
separation of the colored substances.

Chromatography is a laboratory techniques for the separation of mixtures

 Link to
Practical

Introduction
Types & - Running a
applications planar
- Column chromatogra
Separation by Chromatograp phy: TLC
Chromatograp hy
hy
-Planar
- The main
Chromatograp
components
hy
Brief Intro -Principle
- What it is?

What is it for?
Chromatography is a process whereby the:
 The components of a mixture may be separated
 Purity of a sample may be determined
 The separated components may be identified

Greek: chromos = color , graphy = write

 Link to
Practical

Introduction
Types & - Running a
applications planar
- Column chromatogra
Separation by Chromatograp phy: TLC
Chromatograp hy
hy
-Planar
- The main
Chromatograp
components
hy
Brief Intro -Principle
- What it is?
How does it separates?

– A mixture can be separated using the differences in physical or chemical

properties of the individual components

 based on the states of matter of the two phases

 useful for separations of density and size
 useful chemical properties by which compounds can be separated
are: solubility, boiling point, and vapor pressure

Greek: chromos = color , graphy = write

 Link to
Practical

Terminologies
Types &
applications - Running a
planar
- Column chromatogra
Separation by Chromatograp phy:TLC
Chromatograp hy
hy
-Planar
- The main Chromatograp
components hy
Brief Intro -Principle
- What it is?

Chromatography Analyte Stationary phase Mobile phase

a physical method of • Substance which is to be • It is also called as • It is also known as
separation in which the purified or isolated adsorbent. solvent and eluent.
components to be during chromatography • May be a “solid or • May be a
separated are distributed liquid” supported on a “liquid/solvent/mixture
in h between two phases, solid (or) a gel of solvent/gas”
one of which is stationary, • Maybe ; 1) packed in a
while the other (mobile column 2) spread as a
phase) moves in a definite layer on a
direction glass/aluminium plate 3)
Distributed as a liquid
film
 Link to
Practical

Terminologies
Types & - Running a
applications planar
- Column chromatogra
Separation by Chromatograp phy: TLC
Chromatograp hy
hy
-Planar
- The main
Chromatograp
components
hy
Brief Intro -Principle
- What it is?

Eluate Elution Chromatograph Chromatogram

• A machine that • Visual output of the
Is the separated Process of removing performs chromatograph
component the components chromatography by • Different peaks or
gas or liquid patterns represent
from the column
separation different
components of the
separated mixture
 Separation by
Chromatograp
Types &
applications
- Column
Chromatograp
Link to
Practical
- Running a
planar
chromatogra
phy:TLC
The Chromatogram
hy
hy
-Planar
- The main Chromatograp
components hy
Brief Intro -Principle
- What it is?
 Introduction

 A sample that requires analysis is often a mixture of

many components in a complex matrix

 Often a standard containing either a single pure

compound or a mixture of known amounts of several
compounds will be used for comparison with the test
sample (by comparing the chromatograms).

Fig 1.
a) Chromatogram of an unknown mixture containing
five compounds as shown by five peaks
b) Chromatogram of a standard that contains caffeine
(1), aspirin (2) and salicylamide (5)
 Link to Practical
-Running a planar
Types & applications chromatography:
TLC
- Column Chromatography

Classification of Chromatography
-Planar Chromatography

Separation by * Answer questions in

i-learn
Chromatography

Brief Intro
- What it is?
- components
-Principle i. Based on physical state of mobile phase

Eg: column
Liquid-solid
adsorption TLC,
chromatography
HPLC
Liquid
Chromatography
Eg: column
Liquid-liquid partition
chromatography
Chromatography Paper partition

Gas Gas
Eg: GLC, GSC
Chromatography chromatography
 Link to Practical
- Running a planar
Types & applications chromatography:

Classification of Chromatography (
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

i-learn

Principle )
Chromatography
- components
Brief Intro
-Principle

ii. Based on physical state of stationary phase

- What it is?

Chromatography
Stationary phase: solid Stationary phase: Liquid

Adsorption Partition
chromatography chromatography

Eg: column adsorption Eg: column partition

Ion exchange, size Paper partition
exclusion, affinity, TLC,
HPLC GLC
 Link to Practical
- Running a planar
Types & applications chromatography:

Classification of Chromatography
TLC
- Column Chromatography
-Planar Chromatography

Separation by * Answer questions in

i-learn

iii. Based on polarity of stationary & mobile phase

Chromatography
- components
Brief Intro
- What it is?
-Principle

Chromatography

Normal phase Reverse phase

chromatography chromatography

Stationary phase: polar Stationary phase: nonpolar

(silica gel) (Octadecylsilane or C18)
Mobile phase: nonpolar (n- Mobile phase: polar
hexane) (Acetonitrile, water)
 Link to Practical
- Running a planar
Types & applications chromatography:
- Column Chromatography TLC
-Planar Chromatography

Separation by * Answer questions in

Separation by chromatography
i-learn
Chromatography
- components
Brief Intro
- What it is?
-Principle

– The basis of all forms of chromatography is the partition or distribution of a

compound (Kd), between two immiscible phases ; stationary phase and a mobile
phase.
– The value for Kd is a constant at a given temperature and is given by the expression:

Concentration of the compound in stationary phase

Kd =
Concentration of the compound in mobile phase

– If a mixture of compounds is applied to a chromatography column, they will

distribute between the two phases according to their Kd values
At equilibrium in a particular chromatographic system,
compound with a:
–K d above 1 has a greater concentration in the

stationary phase than in the mobile phase

–K below 1 has a greater concentration in the

d

mobile phase than in the stationary phase

The K
a particular compound.
d can also be use to describe the relative
As Kd
retention time of
increases, it
takes longer
for compounds
to separate
Requirements for column chromatography

 Column characteristics & selection

 Stationary phases
 Mobile phases
 Preparation of column
 Introduction of sample
 Development of column
 Detection of components
 Recovery of components
 Link to Practical
- Running a planar
Types & applications chromatography:
- Column Chromatography TLC
-Planar Chromatography

Separation by * Answer questions in

1. Column characteristics & selection

i-learn
Chromatography
- components
Brief Intro
- What it is?
-Principle

Materials of construction Good quality neutral glass, plastic or nylon

Adsorbent (stationary phase)/ adsorbate 30:1
(mixture) weight ratio
Column length to diameter ratio (cm) 10-15: 1 or 30-100: 1
Multi component system is present Long column
Components with similar affinities for Long column
adsorbent are present
Components with different affinities for Short column
adsorbent are present

Weak adsorbent, few compounds ????

 Link to Practical
- Running a planar
Types & applications chromatography:
- Column Chromatography TLC
-Planar Chromatography

Separation by * Answer questions in

2. Stationary phases
i-learn
Chromatography
- components
Brief Intro
- What it is?
-Principle

• Most commonly used adsorbent is silica gel (80-100 mesh or 100-200 mesh size)
• They adsorbs polar and unsaturated substances by formation of hydrogen bonds
with hydroxyl groups on the silicon atom

Type of mesh Size in microns

60/120 mesh 120-250 micron
100/200 mesh 75-150 micron
70/230 mesh *63-200 micron
230/400 mesh 37-63 micron
70/325 mesh 45-200 micron
 Link to Practical
- Running a planar
Types & applications chromatography:
- Column Chromatography TLC
-Planar Chromatography

Separation by * Answer questions in

2. Stationary phases
i-learn
Chromatography
- components
Brief Intro
- What it is?
-Principle

Table 1 Adsorbents used in column chromatography

Weak activity Medium activity Strong activity

Sucrose Calcium carbonate Activate magnesium silicate

Starch Calcium phosphate Activated alumina

Inulin Magnesium carbonate Activated charcoal
Talc Magnesium oxide Activated magnesia
Sodium carbonate Calcium oxide Activated silica
 Link to Practical

Types & applications

- Column Chromatography
- Running a planar
chromatography:
TLC
It can also be your purification
-Planar Chromatography

Separation by * Answer questions in

BUFFERS
3. Mobile phase
i-learn
Chromatography
- components
Brief Intro
- What it is?
-Principle

• Either gas or liquid which flows over/or through the stationary phase
either by use of a pumping system or applied gas pressure.

• Also referred as the eluent/solvent

• Act as a solvent to introduce the mixture into the column; as

developer to develop the zones for separation; and as an eluent to
remove the pure component out of the column

As the eluent flows through the column, the analytes separate on the basis of their
Kd and emerge individually in the eluate as it leaves the column
 Link to Practical
- Running a planar
Types & applications chromatography:
- Column Chromatography TLC
-Planar Chromatography

Separation by * Answer questions in

i-learn
Chromatography
- components
Brief Intro
- What it is?
-Principle

3. Mobile phase
 Let's get to know more….

Stationary phase aka. Mobile phase

adsorbents
What does it do? Stays put! Flows around stationary phase

What is it? (physical form) Solid or liquid Liquid or gas

How does it effect sample Retains sample by surface Moves sample along
movement? interaction

Analogous to.. An obstacle course Motivating force

Example Silica gel Ethyl acetate
 Link to Practical
- Running a planar
Types & applications chromatography:
- Column Chromatography TLC
-Planar Chromatography

Separation by * Answer questions in

Chromatography
i-learn
a) Slurry packing (wet packing method)
- components
Brief Intro
- What it is?
-Principle

The adsorbent is suspended in the mobile

phase and stirred very well to remove air
4. Preparation
bubbles. The resulted slurry is then poured in
to the column.
(packing) of
the column
At the bottom portion of the column, a piece
of glass wool or cotton/whatman filter paper
disc must be added before the slurry
application

After slurry application, the column must be

allowed to settle overnight. This is the ideal
method of column packing
https://www.youtube.com/watch?v=G4jyd8L0MWE
https://www.youtube.com/watch?v=8Ff1Xx-On-g
 Link to Practical
- Running a planar
Types & applications chromatography:
- Column Chromatography TLC
-Planar Chromatography

Separation by * Answer questions in

Chromatography
i-learn
a) Dry packing
- components
Brief Intro
- What it is?
-Principle

In this method, the dry adsorbent is

4. Preparation
poured to the column directly

(packing) of
the
Vibration is applied to getcolumn
rid of air
bubbles then the mobile phase is
passed through the adsorbent.

The demerit with this method is that

air bubbles are entrapped between
the solvent and the stationary phase.
 Link to Practical
- Running a planar
Types & applications chromatography:
- Column Chromatography TLC
-Planar Chromatography

Separation by * Answer questions in

Chromatography
i-learn
a) Dry packing
- components
Brief Intro
- What it is?
-Principle

4. Preparation
(packing) of
the column
5. Set up for the operation of a chromatography column
6. Detection and recovery of components

Each fraction is
Fractions are collected examined by TLC using
by elution analysis suitable experimental
conditions

From the column

Those fractions which
fraction, solvent is
give same Rf values in
evaporated, dried & the
TLC are added as a
material collected in
common fraction
eppendorf

After spectral analysis

(NMR, MS, XRD etc.) the
compound is identified
6. Detection and recovery of components
 Link to Practical

Applications of chromatography (in

- Running a planar
chromatography:
TLC
Types & applications
- Column
Separation by
Chromatography
- components
-Principle
Chromatography
-Planar Chromatography general)
Brief Intro * Answer questions in
- What it is?
i-learn Inorganic ions Separation of copper, cobalt, nickel etc
Organic Purification of dyes (ie sudan red, methylene blue,
malachite green)
Separation of diastereomers
Separation of tautomers and racemates
Plant constituents Separation of geometrical isomers of cis and trans
isomers of bixin and crocetin dimethyl ether using
alumina
Purification od steroids, carotenoids, chlorophils and
xanthophylls
Separation of alkaloids and glycosides
Drugs and pharmaceuticals Purification of vitamin B12, Oxytetracyclin and
oleabdomycin
Separation of amino acids, antibody, proteins,
phospholipids and triglycerides.
THANK YOU…
 Chapter 5 (part 2)

PRINCIPLE OF CHROMATOGRAPHY

BMS481
NURUL AILI
 Course Learning Outcomes (CLOs)
At the end of the course, students should be able to:
1. Describe the fundamentals concepts of analytical techniques e.g. accuracy,
concentrations, molarity.
2. Illustrate the principles and applications of basic centrifugation,
chromatographic and spectroscopic analytical techniques in the isolation and
characterization of biological molecules.
3. Perform scientific experiments on biomolecules separation, characterization
and analysis.
We have learnt about…
Recall…
The definitions related to
chromatography
How chromatography can be categorized
based on mobile, stationary and polarity
of MP and SP
The principle of Chromatography?
 Link to Practical
- Running a planar
chromatography:

Separation by
- Column
Chromatography
TLC
Types & applications
The chromatography that we are going to
focus ON..
Chromatography
- components -Planar Chromatography
-Principle
Brief Intro * Answer questions in
- What it is?

1. Planar chromatography – It may be Paper or Thin

i-learn

Layer
2. Column chromatography – It may be Gas or Liquid
Planar Chromatography : a separation technique in
which the stationary phase is present as or on a plane
Column Chromatography: a separation technique in
which the stationary bed is within a tube
 Planar vs Column

Column chromatography Planar chromatography

Differentiate between Planar chromatography and Column Chromatography !

 1. Planar chromatography
Thin layer chromatography (TLC)

TLC is a chromatographic
– Thin-layer chromatography consists of
technique
 a stationaryused to separate
phase consisting of a thin layer of
adsorbent material usually silica gel, aluminium
mixturesoxide or cellulose immobilized on a flat glass or
plastic plate
 an organic solvent (mobile phase)

Typical TLC
chromatograms
The TLC plate..

Glass plate with silica

Magnification x 500
 TLC in general

Sample is dissolved in an appropriate solvent and deposited as a

spot on the stationary phase.
The bottom edge of the plate is dipped into a solvent reservoir
(e.g. hexane or ethyl acetate), and placed in a sealed container. The
solvent moves up the plate by capillary action and meets the
sample mixture, which is dissolved and is carried up the plate by
the solvent.
Different compounds in the sample mixture travel at different rates
due to the differences in their attraction to the stationary phase
(see next!), and because of differences in solubility in the solvent
What to do if the spot is
colorless?
Visualizing the chromatogram

What if the substances you are interested in are colourless?

The separated spots are visualized by placing the place in iodine
vapour or
Placing the plate under ultraviolet light

The TLC plates contain an indicator, zinc sulfide, that

fluoresces green under 254 nm UV light. Analytes may
quench the fluorescence and exhibit as a dark spot
against a green background
 Interpretation of TLC

The Rf for a compound is

constant only in the
exact chromatographic
conditions. Always note
the conditions.
 Applications of TLC
Its wide range of uses include

Commonly employed to determine pigmentation of plants, using

extracts of leaves.
Used to detect pesticides or insecticides in foods
Analysis of the dye composition from clothing in forensic
investigations
Monitoring organic reaction
2. The Column Chromatography (CC)
Factors affecting separation in CC
 CC 1.0
SIZE EXCLUSION
CHROMATOGRAPHY
SIZE EXCLUSION
– Achieve rapid separation of molecules based on molecular size
– Mobile phase:
– Loading buffer: buffer used to bind of solutes (eg: protein) to
stationary phase or matrix
– Elution buffer: Buffer/solution used for elution of bound solute
from a column
– Stationary phase or Matrix:
– composed of cross-linked gel particles (bead form)
– The size of the pores is critical
Each gel has an ”exclusion limit”
and fractionation range
 What happened if the protein
exceed the exclusion limit of
column??
Exclusion limit
The gel acts as a molecular sieve, retaining molecules up to the exclusion limit.
E.g. Sephadex G-50 exclusion limit is 30 kDa; so molecules that exceed this limit
(larger molecules) will pass through and elution in Void volume (V0).

Fractionation range
Sephadex G-50 has a fractionation range of 1.5 to 30 kDa

What is Void
volume (V0)
??
■ Pores within beads:
 are of such sizes that are not accessible to
large molecules, but smaller molecules
can penetrate all pores
 Extent to which molecules can enter
pores depends on its shape and
Molecular Weight (MW)
 Hence separation depends on difference
in ability of various molecules to enter
■ As solvent moves through the column, 3
possible things can happen to solutes in
sample:
- can run with solvent front
- be washed out quickly
- totally adsorbed on to gel matrix
■ Very large molecules that are too large to enter
the pores move through the chromatographic bed
are the fastest to be eluted

■ Smaller molecules which enter the gel pores move

more slowly since they go in and out of the beads
as they travel down the column. It will elute later.

■ Hence, molecules are eluted in order of their

decreasing size
Chromatogram and determination of fractioned protein
Determination of fractioned protein
• Monitoring absorbance at 280 nm
• Protein assay or enzyme specific assay
Used to separate mixtures of macromolecules, particularly enzymes,
antibodies and other globular proteins
For protein purification, it is desirable to have proteins of interest to be
well separated from unwanted proteins
Choice of Buffer:
The most important when choosing a buffer for gel filtration,
make sure it is compatible with target molecule
BEWARE OF dissociating agents and detergents that can;
- induce conformational change,
- inactivate biologically active or
- dissociate proteins into subunits
Sodium phosphate buffer and Tris-HCl are used to control pH
■ Choice of column
- Theoretically, resolution increase
with column length

■ Choice of gel matrix

- 3 Common gel matrix:
 Sephadex (Pharmacia, is a
cross-linked dextrans)
 Polyacrylamide beads
(Biogels) (Bio-rad)
 Agarose (Sepharose)
1. It is commonly used in the process of desalting, that is, removing inorganic salts from
a liquid containing the biomolecules of interest

2.
Application of SEC
Other main application of size-exclusion chromatography is in the determination of
molecular weight
 5.2
ION EXCHANGE
CHROMATOGRAPHY
Introduction
Protein consist of amino acids. The amine and
carboxylic functional group make them “zwitterions”

Utilizing the principle, protein can be separates

based on their charge
Principle of Ion Exchange
Chromatography
Separation of analytes based on the attraction between
oppositely charged stationary phase and analyte
The stationary phase is called Ion exchanger
Two types of ion exchangers
Differences between anion and cation exchangers

Anion exchanger Cation exchanger

Have positively charged groups on Have negatively charged groups on
stationary phase stationary phase

Attract negatively charged ions Attract positively charged ions

It is called basic ion exchanger because of It is called acidic ion exchanger because the
the positive charges, which are formed by negative charges are due to the ionization of
the association of protons with basic groups acidic groups
Differences between anion and cation exchangers
Choice of Exchanger
Depends majorly on the stability of test
analytes
If test analytes, especially proteins, are stable
at narrow pH then an exchanger that can
operate in a narrow pH range can be selected
If a protein is stable below its isoelectric point
(net charge is positive) then cation exchanger
is used
If a protein is stable above its isoelectric point
(net charge is negative) then anion exchanger
is used
If a protein is stable over a wide range of pH values, then either
cationic or anionic exchanger can be used
Can you determine what type of IEC is this?
ANION EXCHANGER

(+) functional group act as anion

exchanger

(-) charged proteins bind to anion

exchanger by reversible electrostatic
interactions
 Eluent pH

The buffers used in IEC should have one pH unit above or below
the isoelectric point of analytes.

Anionic buffers are used for cation exchange chromatography

e.g., Tris, pyridine and Tricine

Cationic buffers are used for anionic exchange chromatography

e.g., acetic acid, HEPES and phosphate
Eluent pH
Elution
Gradient elution is common than isocratic
elution

For anion exchanger: the pH gradient decreases

and ionic strength increases during elution

For cation exchanger: the pH gradient and ionic

strength increases during elution
Procedure for IEC

Four steps:
Equilibration
Sample application and wash
Elution
Regeneration
Procedure for IEC
Procedure for IEC
Procedure for IEC
Although less common, a pH gradient
can also be used for elution
A pH gradient is chosen that
approaches the protein of interest’s
pI.
Protein will elute when the pH
gradient reaches their pI, because
they will
To elute proteins no
from longer carry
Anion exchange resin – a decreasing pH gradient is chosen
a net charge
that allows them to interact with the
Cation exchange resin – an increasing pH gradient is chosen
Procedure for IEC
 Application of ion
exchange

Water
softening/purifying
process
Removed Magnesium (Mg2+) and Calcium (Ca2+)

Separation of
carbohydrates and their
derivatives
Uronic acids separated on anion exchanger
Hexosamines separated on strong cation exchanger
Now that you have learn about IEC..

Figure out the limitations of IEC

IEC Pros IEC Contras
 5.3
Affinity chromatography
AFFINITY
CHROMATOGRAPHY

It exploits the unique

property of extremely specific
biological interactions to
achieve separation and
purification.
The material to be isolated is
capable of binding reversibly
to a specific ligand that is
attached to an insoluble
matrix.
The technique was originally developed
for the purification of enzymes, but it
has been extended to nucleotides,
nucleic acids, immunoglobulins,
membrane receptors and even to whole
cells.
Is a molecule that binds reversibly to a
What is a ligand?
specific target molecule or group of
target molecule
Selection of depended on the biological
specificity of the compound to be
purified
Absolute specificity
 Biological interactions between
ligand and target molecules is a
Interaction result of
Electrostatic or hydrophobic interactions
Van der Waal’s forces
Hydrogen bonding

between
To elute the target molecule from
target with the affinity medium, the
interaction can be reversed,
ligand either specifically using a
competitive ligand, or non-
specifically, by changing pH, ionic
strength and polarity.
Biological interactions

Some typical biological interactions, frequently used

in affinity chromatography, are listed below:
•Enzyme – substrate analogue, inhibitor, cofactor
•Antibody – antigen, virus, cell
•Nucleic acid – complementary base sequence,
histones, nucleic acid polymerase, nucleic acid
binding protein
•Hormone – receptor, carrier protein
•Metal ions – Poly (His) fusion proteins, native
proteins with histidine, cysteine and/or tryptophan
residues on their surface
HISTRAP HP HISTIDINE-TAGGED PROTEIN
 Example: purification of
staphylococcal nuclease by
affinity chromatography on
bisphosphothymidine-linked
agarose

When protein solution is

passed through such column,
only the proteins that can bind
the ligand will be retained on
the column, while the other
mixture will passes through the
column.

Then, the conditions can be

adjusted to release proteins of
interest from the ligand. Often
this can be done simply by
eluting with the soluble version
of the ligand
The goal : is to separate molecules of a particular specificity
in a mixture such as a blood serum
Eg: Antibodies in a serum sample specific for a particular
antigenic determinants
Step 1.
An immunoadsorbent is prepared. Consists: solid matrix to
which the antigen (shown in blue) has been coupled (usually
covalently). Agarose, sephadex, derivatives of cellulose, or
other polymers can be used as the matrix
Step 2.
The serum is passed over the immunoadsorbent. Antibodies
in the mixture specific for the antigen (shown in red) will bind
(noncovalently) and be retained
Antibodies of other specificities (green) and other serum
proteins (yellow) will pass through unimpeded
Step 3.
Elution.
 A reagent is passed into the column to release the
antibodies from the immunoadsorbent
 Buffers containing a high concentration of salts
and/or low pH are often used to disrupt the
noncovalent interactions between antibodies
and antigen
 A denaturing agent, such as 8 M urea, will also
break the interaction by altering the
configuration of the antigen-binding site of the
antibody molecule
 Another, gentler, approach is to elute with a
soluble form of the antigen. These compete
with the immunoadsorbent for the antigen-
binding sites of the antibodies and release the
antibodies to the fluid phase
Affinity Chromatography Chromatogram
At B : 100% binding
buffer, 0% salt

At C: Increasing % of
salt (the step-
gradient)

“red line” is the salt step- gradient

Step 4.
Dialysis.
The eluted antibodies is then
dialyzed against, for example,
buffered saline in order to
remove the reagent used for
elution

Collected protein then can

be concentrated
Has a wide number of uses and can be
Application of AC
applied to the isolation and purification
of virtually all biomolecules
heparin columns to separate cholesterol lipoproteins
lectin columns to separate carbohydrate groups
phenyl boronate columns to separate glycated haemoglobins
 Now that you have learned all types of COLUMN CHROMATOGRAPHY

Lets distinguished them..

SEC IEC AC
Basis principle
Components
Elution profile
Example
 RUNNING A TLC

****FOR PRACTICAL
P/S –PLEASE BRING YOUR OWN SAMPLES
(COLOURED PREFERABLY)
1. SAMPLE APPLICATION (SPOTTING)

1. Draw a thin line in pencil about 1 cm from bottom of

plate
2. Add hash marks perpendicular to line
3. Carefully spot small amounts of analyte on the hash
marks
4. Let spot dry completely
2. DEVELOPING THE PLATE
keep capped

1. Add 0.5 cm eluent to beaker or jar

2. Transfer plate to closed beaker using
tweezers
3. Cover beaker or jar
4. Wait until eluent reach the finishing line
(1 cm from the top)=solvent front
5. Remove the plate
6. Immediately mark the solvent front
with pencil
7. Allow plate to dry It is crucial that the level of
mobile phase (eluent) is below
the spotting line –where the
sample was spotted!!!
 3. VISUALIZING THE PLATE – UV LIGHT

1. Circle any visible spots in pencil

2. Put plate under UV light
3. Circle any spots, whether dark or colored in pencil

The TLC plates contain an indicator, zinc sulfide, that

fluoresces green under 254 nm UV light. Analytes may
quench the fluorescence and exhibit as a dark spot
against a green background
 3. carefully
1. In a fume hood, VISUALIZING
lower THE PLATE – I2
plate into iodine
2. Wait until spots turn dark
3. Remove plate and circle any spots
in pencil

The iodine will sublime from

the plate with time, so mark
spots immediately after
removing from chamber
Why use two visualizing methods?

Not all compounds can be visualized by all

method….
4. INTERPRETATION OF TLC RESULTS

The Rf for a compound is

constant only in the
exact chromatographic
conditions. Always note
the conditions.
Don’t touch the silica plate with your
bare hands TLC Tips
Draw light pencil lines
Use small spots of roughly equal size
Don’t let the solvent front reach the very
top of the plate (over develop)
Don’t allow light from UV lamp to reach
HAPPY REVISING 
H I G H P E R F O R M A NC E L I Q U I D
C H R O M AT O G R A P H Y ( H P L C)

C HA PT E R 6
BMS481
THE
LLO(S)
At the end of the course, you should be ableto
• Explain the fundamental concepts of HPLC
• Differentiate types/ mode of HPLC
• Able to identify different chromatograms of HPLC and troubleshoot.
INTRODUCTIO
N
• HPLC stands for “High- performance liquid chromatography”…sometimes incorrectly referred
to as High-pressure liquid chromatography

• HPLC is a powerful tool in analysis, it yield high performance and high speed comparedto
traditional column chromatography because of the forcibly pumped mobile phase.

• HPLC is a chromatographic technique that can separate a mixture of compounds

• Used in biochemistry and analytical chemistry to – identify,quantify and purify the individual
components of a mixture.
TERMINOLOGI
ES
• Chromatography – physical method in which separation of components takes place between
two phases:
• Stationary phase:the substance on which adsorption of the analyte (the substance to be be
separated during chromatography) takes place. It can be a solid, a gel, or a solid-liquid
combination
• Mobile phase:solvent which carries the analyte (a liquid or a gas)

HPLC is a type of liquid chromatography where the sample is

forced through a column that is packed with a stationary phase
composed of irregular or spherically shaped particles, aporous
monolithic layer, or a porous membrane by a liquid (mobile
phase) at high pressure
• Lets first look at the principle behind liquid chromatography….

Liquid chromatography is a separation technique that involves:

1. the placement (injection) of a small volume of liquid sample
2. Into a tube packed with porous particles (column = stationary phase)
3. where individual components of the sample are transported along the packed tube
(column) by a liquid.

when a mixture of components are introduced into the column,various chemical and /or physical
interactions take place between the sample molecules and the particles of the column packing
• They travel according to their relative affinities towards the stationary phase.The component
which has more affinity towards the adsorbent, travelslower

• The component which has less affinity towards the stationary phase travel faster.

• Since no two components have the same affinity towards the stationary phase,the
components are separated.
• These components are separated from one another by the column packing that involves
various chemical and/or physical interactions between their molecules and the packing
particles.

• These separated components are detected at the exit of this tube (column) by a flow-through
device (detector) that measures their amount.The output from the detector is called a liquid
chromatogram

In principle,LC and HPLC work the same way except the speed,efficiency,
sensitivity and ease of operation of HPLC is vastly superior
PRINCIPLE OF
HPL
C
• HPLC is a form of liquid chromatography used to separate compounds that are dissolved in
solution.
• Compounds are separated by injecting a small volume of liquid sample into a tube packed
(column) with tiny particles (3 to 5 micron in diameter called the stationary phase)
• Where individual components of the sample are moved down the column with a liquid (mobile
phase) forced through the column by high pressure delivered by a pump.
• The different components in the sample mixture are separated due to differences in their
adsorption/partition behavior between the mobile phase and the stationary phase

Adsorption: separation when SP is solid

Partition: separation when SP is liquid
SCHEMATIC DIAGRAM OF
HPLC
HPLC :MOBILE
PHASE
• The mobile phase in HPLC refers to the solvent being continuously applied to the column or
stationary phase

• The mobile phase acts as a carrier to the sample solution

• A sample solution is injected into the mobile phase of an assay through the injector port

• As a sample solution flows through the column with the mobile phase,the components of that
solution migrate according to the non-covalent interaction of the compound with the column
HPLC MOBILE
PHASE
• Usually two or more solvents (e.g.,water andAcetonitrile) for separation of biomolecules

• Highly purified (HPLC grade) and chemically unreactive solvents should be used

• Impurities may interfere in detection system specifically absorbance below 200 nm

 HPLC MOBILE
PHASE
• Degassing of solvents
– Should be done before HPLC experiment for all
solvents specially for eluents with aqueous ethanol
and methanol.
– Reason: High pressure results in bubbling of gasor
air present in solvents.These bubbles damages
stationary phase

• Degassing methods
– Warming
– Vigorous stirring with magnetic stirrer
– Vacuuming
– Ultrasonification
– Bubbling helium gas through eluent reservoir
HPLC MOBILE
PHASE
Selection of Mobile phase

Depends on type of separation and components in the sample. Usually the polar andnon-polar
solvents are used in certain ratios.

Should not interfere with detection system

TECHNIQUE FOR HPLC
I. TECHNIQUE FOR USING MOBILE
PHASE
Isocratic elutionVS gradient elution
1. Isocratic elution

A separation in which the mobile phase composition remains constant throughout the procedure
is termed isocratic elution
In isocratic elution,peak width increases with retention time linearly with the number of
theoretical plates.This leads to the disadvantage that late eluting peaks get very flat and broad

Best used for separation of simple mixtures

The system and column are equilibrated all the time and does not suffer from fast chemical changes
Often used in quality control applications that support and are in close proximity to a manufacturing
process
TECHNIQUE FOR HPLC
I. TECHNIQUE FOR USING MOBILE
PHASE

2. Gradient elution
A separation in which the mobile phase composition is gradually changing in the ratio of polarto
non-polar compounds during the sample run.

Gradient elution decreases the retention of the later-eluting components so that they elute
faster, giving narrower peaks.This also improves the peak shape and the peakheight

Best for analysis of complex samples

Often used in method development for unknown mixtures
Linear gradients are most popular
ISOCRATIC VS.
GRADIENT
TECHNIQUE FOR
HPLC
II. BASED ON MODE OF
Normal phase VS Reverse phasechromatography
•SEPARATION

Normal phase Reversed phase

Stationary phase Polar (Alumina or silica gel) Non-polar (C18)
Mobile phase Non-polar (organic solvents) Polar (aqueous/organic)
e.g. hexane e.g.water, methanol,
acetonitrile
Sample movement Least polar/non polar fastest Most polar fastest
Separation based on Different polarities Different hydrocarbon
(functionality) content

Reversed phase is more commonly used as

https://www.youtube.com/watch?v=MLoitPJQH3g drugs are usually polar (hydrophilic)
NOW CAN YOU GIVE ANSWER TO THE QUESTION
BELOW?

• 1. Distinguish between Normal Phase HPLC and Reversed Phase HPLC

• 2. State which form is more commonly used. Explain why.
• 3.Does a hydrophobic (Non polar) analyte has affinity to a polar stationary phase?
HPLC : STATIONARY
PHASE
• Rigid solid particles rather than soft gel as used in open column chromatography

• Spherical particles with uniform size to reduce space for diffusion

• Mainly three types of particles

– Microporous particles
– Pellicular particles
– Bonded phase particles
HPLC : STATIONARY
PHASE
HPLC : STATIONARY
PHASE
HPLC : STATIONARY
PHASE
HPLC: COMMON STATIONARY PHAS E CHOICES
HPLC
COLUMNS
• “the heart of chromatography”. The column’s stationary
phase separates the sample components of interest using
various physical and chemical parameters

• It is usually made of stainless steel to withstand high

pressure caused by the pump to move the mobile phase
through the column packing other material include PEEK
and glass

• The small particles inside the column are called the

“packing” what cause the high back pressure at normal flow
rates

• Column packing is usually silica gel because of its particle

shape, surface properties, and pore structure give us a good
separation
• HPLC is sometimes (incorrectly) called High Pressure
Liquid Chromatography

• Solutes diffuse too slowly in liquids for open tubular

columns to be practical
• Smaller packing particles lead to a better resolution
• But small particles also require high liquid pressure
because they resist flow

Analytical columns are expensive and easily

degraded by dust & particles
HPLC: GUARD
COLUMN
• Is a short, expandable columnpacked
with the same material as the
analytical column.

• Mainly used to protect the main

column from impurities.
 H P L C COLUMNS
C O NV E NT I O NA L HP LC COLUM N
H P L C COLUMNS
C A P I L L A RY COLUMN S
HPLC
PUMPS
Important requirement:Flow of mobile phase needs to be very stable (i.e.pulse free)

• The role of pump is to force a liquid (mobile phase) through the liquid chromatography at a
specific flow rate, expressed in milliliters per min(mL/min)
• Normal flow rates in HPLC are in the range 1 to 2 mL/min range
• Typical pumps can reach pressure in the range of 6000-9000 psi (400 to 600 bar)
• During chromatography, a pump can deliver a constant mobile phase composition(isocratic) or
an increasing mobile phase (gradient)
TYPES OF HPLC
PUMPS
Most commonly used are constant pressure pump and
constant volume pumps

Constant pressure pumps:

-Maintain constant pressure irrespective of stationary

phase resistance to mobile phase flow
-if the resistance increases in stationary phase, the flow
rate decreases automatically to maintain constant pressure
TYPES OF HPLC
PUMPS
• Constant Volumepumps:

• Maintains constant flow rate through stationary phase.

• Increase the pressure if there is increase in stationary phase resistance.
• Increasing pressure is within the pre-set limits of the pump.If the pressure required more than
preset limits,the pumps are inactivated automatically (safety-cut off mechanism)
• More popular in HPLC
Reciprocating piston pumps:

• Consist of a small motor driven piston which moves

rapidly back and forth in a hydraulic chamber that may vary
from 35-400 uL in volume.
• Advantages:
– Pumping motion produces a pulsed flow that must be
subsequently damped
– Small internal volume and high output pressure (up to 10,
000 psi)
– Largely independent of column back pressure and solvent
viscosity
– Employed in most modern commercial HPLC
 HPLC:
SAMPLE
APPLICATION
Most common is “loop injector”
Loop injector is operated
manually
HPLC :
DETECTORS
• The detector can detect the individual molecules that elute from the column and convert the
data into an electrical signal
• A detector serves to measure the amount of those molecules
• The detector provides an output to a recorded or computer that results in the liquid
chromatogram
• Detector is selected based on the analyte or the sample under detection
HPLC :
DETECTORS
COMMONLY USED DETECTOR IN
HPL
C
Ultraviolet (UV)
• This type of detector responds to substances that absorb light
• The UV detector is mainly to separate and identify the principle active components of a
mixture
• UV detectors are the most versatile, having the best sensitivity andlinearity
• UV detector cannot be used for testing substances that are low in chromophores (colourless
or virtually colorless) as they cannot absorb light at low range
• They are cost-effective and popular and are widely used in industry
Fluorescence
• A specific detector that senses only substances that emit light.This detector is popular for
trace analysis in environmental science.
• As it is very sensitive,its response is only linear over relatively limited concentration range.As
there are not many elements that fluoresce, samples must be synthesized to make them
detectable.

Mass spectrometry
• The mass spectrometry detector coupled with HPLC is called HPLC-MS. It is the most
powerful detector, widely used in pharmaceutical laboratories and research and development.
• The principle benefit of HPLC-MS is that it is capable of analyzing and providing molecular
identity of a wide range of components.
Refractive Index (RI) detection
• The RI detector uses a monochromator and is one of the least sensitive LC detectors
• This detector is extremely useful for detecting those compounds that are non-ionic,do not
absorb UV light and do not fluoresce
• E.g.sugar,alcohol,fatty acid and polymers
A L G O R I T H M F O R H P L C D E T E C TO R S E L E C T I O N
DATAPROCESSING UNIT
(COMPUTER)
• Frequently called the data system- the computer not only controls all the modules of the
HPLC instrument but it takes signal from the detector and uses it to determine the time of
elution (retention time) of the sample components (qualitative analysis) and the amount of
sample (quantitative analysis).

• The concentration of each detected components is calculated from the area or height of the
corresponding peak and reported.
ANALYSIS OF
HPLC
1. Qualitative analysis
Analysis of a substance in order to ascertain the nature of its chemical constituents
We can separate individual components but cannot assess the quantity in this analysis

2. Quantitative analysis
Determining the amounts and proportions of its chemical constituents
Quantity of the impurity and individual components can be assessed
HPLC
CHROMATOGRAM

How to interpret the HPLC data?

These peak can be interpreted in various ways…….

Negative peaks occur if mobile phase
absorbance is larger than sample absorbance
Peak doubling / tailing occurs due to the co-
elution of interfering compound, column
over load, channeling in column.

• Base line spikes occur due to the air bubbles in the

mobile phase and/or detector, column deterioration.
IRREGULAR PEAK
SHAPES
TROUBLESHOOTING
IDENTIF Y ING PROBLEM USING A CHROMATOGRAM
HPLC
PARAMETER
Retention time (RT)
• Time elapsed between sample introduction and maximum of response, it is the characteristic
time it takes for a particular analyte to pass through the system
HPLC
PARAMETER
• Solvent composition (mixture of Solvents A andB)

• magnitude or size of the peak (area-under-the-curve)

APPLICATIONS OF
HPLC
• HPLC is one of the most widely applied analytical separation techniques.

– Clinical: quantification of drugs in bodyfluids

– Environmental: identification of chemicals in drinkingwater
– Forensic: analysis of textile dyes
– Industrial: stability of compounds in foodproducts
– Pharmaceutical:quality control and shelf-life of a synthetic drug product
– Research:separation and isolation of components from natural samples from animals and
plants
ADVANTAGES OF
HPLC
• Fast and efficient separation (high resolution power)
• Continuous monitoring of the column effluent
• It can be applied to the separation and analysis of very complex mixtures
• Accurate quantitative measurements
• Repetitive and reproducible analysis using the same column
• Adsorption,partition,ion exchange and exclusion column separations are excellently made.
• HPLC is more versatile than GLC in some respects, because it has the advantage of not being
restricted to volatile and thermally stable solute and the choice of mobile and stationary phase
is much wider in HPLC
 LIMITATIONS
1. Cost of generation of high pressure
2. High pressure imposes some constraints on instrumentation
AND
3. Relatively low flow rates. Hence time taking
PROBABLE
SOLUTIONS
Probable solutions:
FOR HPLC
1. Decrease the pressure and increase the flow rate for comparable resolution of analytes by
HPLC – Perfusion chromatography
2. Increase the instrumentation that can withstand high pressure by considering cost
economics for better resolution than HPLC- UPLC
4.HPLC is especially useful in the
LIMITATIONS AND
separation and analysis of low
PROBABLE
SOLUTIONSBut
molecular weight biomolecules.
FOR HPLC
it does not usually for purification of
complex biopolymers (e.g. proteins)
in active form.
HPLC SUPPLEMENT
(FPLC)
• Fast Protein Liquid Chromatography (FPLC)

• Useful for biopolymer (e.g.,proteins,DNA) separation

• Major differences between HPLC and FPLC is low pressure in FPLC (approx. 1-2 MPa)
• Reciprocating pumps with two pistons are used to avoid pulses
• Columns are made up of glass or plastic because of low pressure
• Stationary phase usually compatible with aqueous buffers and salts.
SAMPLE
PREPARATION
SAMPLE
PREPARATION
 Compatibility Simplify
to further complex
analysis samples

Remove Concentrate or WHY PERFORM

interferences dilute the SAMPLE
from the matrix sample
PREPARATION?

Speed of System
analysis robustness
1. COMPATIBILITY TO FURTHER
ANALYSIS
2. SIMPLIFY COMPLEX
SAMPLES
3. REDUCE INTERFERENCES FROM
MATRIX
SAMPLE PREPARATION…SO MONY OPTIONS?

With such a big range of sample preparation products on the market, how do I choose
which is right for my analysis?
Further references:
PRINCIPLE, METHODOLOGY AND APPLICATION OF
GAS CHROMATOGRAPHY (GC)

CHAPTER 7
BMS481
At the end of this course, students
Lesson
should be able to Learning Outcome
(LLO)
1. Understand the principle of GC
2. Know the application and limitation of
GC
3. Compare between GC and LC
Uses of Gas Chromatography
Outline
GC components and Types of Columns
Factors Affecting Chromatographic
Separation
Data Analysis
 Which Industries Use
Chromatography?

Chemical/Petrochemical
Clinical/Forensic
Consumer Products
Environmental
Food
Pharmaceutical
Simple
Why Gas
Cheap (can be automated)
Chromatography?
Short analysis times
High accuracy
Qualitative and Quantitative analysis
Applicable in % to ppb level
 Should be thermally stable

Compounds Should be un-reactive and non-

Amenable to absorptive to chromatographic
system
GC????
Should be volatile Presence of
polar groups
at temperature reduces
below 350-400 C volatility
“ A physical method of separating
IUPAC Definition of
sample components from a mixture by
Chromatography
selective adsorption or partitioning of
the analyte between two phases:

A “mobile phase and a stationary phase”

 • 1. GLC – Gas Liquid Chromatography
• Stationary phase: immobilized liquids
Types of GC and their
(siloxanes, polyethylene glycols..)
• Principle: Partition

phases
• 2. GSC – Gas Solid Chromatography
• Stationary phase : Solids (alumina, silica,
polymers, carbon…)
• Principle : Adsorption

*Mobile phases
Gases (N2, He, H2, Ar)…GC
The principle of GC involves separation
GC Principle
of volatile components of the sample
based on their distribution and partition
co-efficient between two phases.
This is ratio of solubility of substance in
between gaseous mobile phase and
stationary liquid phase.
How gas chromatography works

GC consists of a carrier gas flow, an injector port, a column, a

temperature regulated oven and a detector.

The sample is injected into the heated injection port (to be

volatilised) and is carried by the mobile gas into the column.
The column is heated to provide sufficient vapor pressure to
elute the sample. Inside the column, the components get
separated by the differential partition in between the mobile
phase gas and stationary phase liquid. The separated
components flow through a heated detector for observation.
Components of a Gas Chromatography
GC components
Types of GC columns and types
of columns
Types of GC capillary columns
Components of a Gas
Chromatograph
Mobile phases (carrier gas)
N2, He and H2 are typical carrier gases
 He:
- Most common and compatible with most detectors
- Better resolution (smaller plate heights)
- Solutes diffuse rapidly  smaller mass transfer term
 N2 :
- Lower detection limit for a flame ionization detector
- Lower resolution and solute diffusion rates
 H2:
- Inexpensive
- Fastest separations (optimum velocity is 2x higher
than He)
- Can not be used with mass spectrometers might Flow rate increases N2 < He < H2
explosive mixtures with air
- Better resolution (smaller plate heights)
- Solutes diffuse rapidly  smaller mass transfer term
Injection port
• The sample to be analyzed is loaded at the injection port via
a hypodermic syringe.
• The injection port is heated (350oC )in order to volatilize the
sample
• Once in the gas phase, the sample is carried to mixing
chamber for complete vaporization mixing before entering
column
• GC is very sensitive instrument. Typically samples of one
micro liter or less are injected on the column
• These volumes can be further reduced by using what is
called a split injection system in which a controlled fraction
of the injected sample is carried away by a gas stream
before entering the column.
Types of GC columns
Commonly used in GC
Open tubular (Capillary)
Higher resolution, shorter analysis time,
columns
and greater sensitivity
Low sample capacity
Capillary column materials

Metal Tubing- MXT

Stainless steel
Almost as inert as fused silica but useable up to approx.
450 °C

Fused Silica
Synthethic, amorphous glass
Excellent inertness and useable to approx. 380 °C (400
°C)
Types of Capillary columns

WCOT
Partition chromatography
Typical phases: Siloxanes and Polyethylene glycols
0.10 to 0.53mm internal diameters
PLOT
Adsorption chromatography
Gas, light hydrocarbons/solvent analysis
Adsorbents: porous polymers, alumina (<1 um particle
diameter)
0.25 to 0.53mm internal diameter
 The critical parameters for GC column:
Column • Dimensions : internal diameter, column length, film thickness

selection • Conditions: temperature, flow rate

• Composition: stationary phase composition, carrier gas

parameters Given a sample, you will need first to choose what

stationary phase will work best
• First pick the type of column, then think about dimension
• Conditions can be optimized for given column dimension

Choice of stationary phase is very important

• It determines what kind of sample you can run

• Critical for packed columns, but less so for open tube column
because of its high efficiency
Factors impacting the separation
Carrier gas: type & linear velocity
Temperature Non-column factors
affecting
Injection bandwidth separation
Column factors: diameter, length
Column temperature

Increase in
temperature
gradient during
separation, increase
the solute vapor
Stationary phase factor:

Basic rule in GC stationary phase…

“choose a stationary phase that “looks

like” the components you want to
separate..”
The detector
Qualitative and Quantitative Analysis
 Mass Spectrometer and Fourier Transform Infrared Spectrometers can identify
compounds as part of a GC system
- Compare spectrum with library of spectra using a computer

 Compare retention times between reference sample and unknown

- Use multiple columns with different stationary phases
- Co-elute the known and unknown and measure changes in peak area

 The area of a peak is proportional to the quantity of that compound

Peak area increases proportional

Peak Area

to concentration of standard if
unknown/standard have the
identical retention time  same
compound

Concentration of Standard
Thermal conductivity
detector Type of detectors
Two columns with conducting wire in between
No sample – the thermal conduction is constant (temperature of
both gases is the same)

Injected sample - The effluent gas carries the sample components

into the detector column. Since effluent gas is mixed with sample
components there results in the difference in thermal conductivity
from prior one recording.

This difference in conductivity is specific for the component

analyzed. This is recorded for further comparison and identification
of the components and their quantity.
Flame ionisation detector
Sample components from effluent are ionized by
subjecting to flame in a chamber
These ions raise upwards and are attracted towards
anode or catode based on the charge on them
When the impinge on the electrodes, the current is
passed which is recorded.
The strength and intensity of current depending on
the sample and is specific
Useful for organic compunds, atmospheric and
aqueous environmental samples
Sample is destroyed
 GC Analysis
*the elution

Order of elution (retention

time) is mainly determined by
volatility
The volatility also depends on
polarity of the compound and
between the stationary phase

Polar compound = least volatile = most retained

Polar compound = polar stationary phase = most retained
The number of components in a
sample is determined by the number
of peaks
The amount of a given component in a
sample is determined by the area
under the peaks
The identity of components can be
determined by the given retention
times – Must be couple with another
instrument (e.g. MS)
THANK YOU

TEST 2
IS WAITING….
8.0 POLYMERASE CHAIN

REACTION (PCR)
BMS481
LESSON LEARNING
OUTCOMES (LLO)
1. Describe the fundamentals concepts of PCR

2. Illustrate the principles and applications of PCR techniques in

biology.
 The purpose of a PCR is to synthesize a huge
number of copies of a gene.

WHAT IS
PCR?
• An in vitro process that detects, identifies, and copies (amplifies) a
specific piece of DNA in a biological sample.
• Discovered by Dr. Kary Mullis in 1983.
• A technique that has revolutionized modern molecular biology.
 "Beginning with a single molecule of genetic material DNA, PCR can
generate 100 billion similar molecules in an afternoon. The reaction
is easy to execute. It requires no more than a test tube, a few simple
reagents and a source of heat. The DNA sample can be pure, or it can
be a minute part of an extremely complex mixture of biological
materials. The DNA may come from a hospital tissue specimen, from
a single human hair, from a drop of dried blood at the scene of a
crime, from the tissues of a mummified brain or from a 40,000-year-
old wooly mammoth frozen in a glacier.“

-Kary Mullis, Scientific American

PCR REACTION
COMPONENTS
• Template DNA to be amplified
• Pair of DNA primers
• Thermostable DNA polymerase (Taq polymerase)
• Deoxynucleoside triphosphates (dNTPs)
• Buffers and MgCl2
• 100 mM Tris-HCl, pH 8.3
• 500 mM KCl
• 15 mM MgCl2
• 0.1% gelatin
• Thermal cycler
PCR REACTION
COMPONENTS
1. Template DNA
• A sequence of DNA that is to be copied. Also called target DNA.
• Can amplify (copy) a piece of DNA ~50 to ~4000 base pairs long
(maybe more, depending on ingredients).
• DNA must be isolated from an organism before it can be copied
(remember Cell lysis, Protein denaturation, DNA precipitation)
 What are the
criteria for
PCR REACTION “good” primers??

COMPONENTS
2. A Pair of DNA primers
• In the cell (in vivo), primers are short RNA strands that serve as a
starting point for DNA replication
• In a PCR reaction (in vitro), Primers are short synthetic strands of
single stranded DNA that exactly match the beginning and the end of
the DNA fragment to be amplified.
PCR REACTION
Brock & Freeze,1969

COMPONENTS
3. Taq DNA polymerase or Pfu polymerase

Called Taq polymerase

DNA polymerase must be
because it is isolated from
Thermostable (Heat–stable)
the bacteria Thermus
because of high
aquaticus (they live in hot
temperatures used in PCR
springs)
PCR REACTION
COMPONENTS
3. Taq DNA polymerase or Pfu polymerase
 The Taq Polymerase builds a new DNA
strand in the 5’ to 3’ direction.
 The newly-polymerized molecule is
complementary to the template strand
and identical to the template's partner
strand.

DNA polymerase
PCR REACTION
COMPONENTS
4. dNTPs
 dNTPs (deoxynucleosides) are the building blocks in the PCR
Reaction.
 They are the monomers that DNA polymerase uses to form
DNA…..the A’s, T’s, C’s and G’s that will build the new strand of DNA.

G T
A A
C
A T G C C
G
PCR REACTION
COMPONENTS
5. Buffer
 To work properly, Taq needs mg++
 The concentration of magnesium ions needs to be optimized with
each target and primer combination.
 Buffer also maintains pH

Too little magnesium could equal little or no PCR product, too

much could mean unwanted product….a fine line.
HOW DOES PCR Each step happens at a
different temperature
WORK?
“ A THREE-STEP
PROCESS”
• STEP 1 : DENATURATION
• STEP 2 : ANNEALING
1 cycle
• STEP 3: EXTENSION
 Normally
at 94ºC
HOW DOES PCR
WORK?
• STEP 1 : DENATURATION

• Heat over 90ºC breaks the hydrogen bonds of DNA and separates
double-stranded molecule into two single strands
 HOW DOES PCR
WORK?
• STEP 2: ANNEALING – The temperature is reduced to 50-65 C ∘ to allow the
primers to bind to target DNA by forming hydrogen bonds with ends of target
sequence.

(Annealing temperature
depends on primer length
and G-C content)
 What happened if a standard
DNA polymerase is used in
PCR, instead of the Taq DNA
HOW DOES PCR ⁇
polymerase

WORK?
• STEP 3: EXTENSION – Taq DNA polymerase adds nucleotides to the 3’ end of
each primer
Taq ‘s ideal
• Temperature is increased ≈ 72ºC temperature
PUT AN EXAMPLE OF PCR
SCHEME..TAKE IT FROM
PHD WORKS
 INSTRUMENT - PCR tube


THERMOCYCLER
Thermal cyclers have metal heat blocks with holes where PCR
reaction tubes can be inserted. The thermalcycler then raises and
lowers the temperature of the block at each step (denaturation ~94 C,
annealing ~55 Cͦ and extension 72 C)
PCR – A CHAIN
REACTION
• At the end of the first PCR cycle, there are now two
new DNA strands identical to the original target
• Multiple Cycles (30-40)
• Exponential Growth
• # of Copies =2n
(Where n is the number of cycles)
EXPONENTIAL AMPLIFICATION OF
THE TARGET DNA SEQUENCE
PCR can generate 100 billion
similar molecules in an afternoon
(2 hours)
Read on advantages and
disadvantages of PCR !
BEWARE! PCR IS SPECIFIC AND
SENSITIVE!!
• Other DNA can contaminate the PCR reaction

• Sources:
 The person who is setting up the reaction
 The tubes
 The enzymes, buffers or water used in the reaction
HOW TO KNOW YOU ARE DOING
IT RIGHT??
• Do a negative control (no DNA) to validate that the PCR product is
amplified from the intended DNA, not some other source of DNA.

• A positive control using DNA with good primers validates that the
reaction conditions and thermal cycler work properly.

• Post PCR analysis

PCR
ANALYSIS
At the end of a PCR reaction, there is a A LOT more of your target
DNA than before the reaction started…billions of copies!

Now the sample is large enough to be seen on a gel and analyzed.

ANALYSIS OF PCR RESULTS
USING GEL Control – No DNA
ELECTROPHORESIS
A DNA Ladder of
known size is run
along with the
samples. This
allows analysis of the
size of the piece of
DNA amplifed by
200 bp
PCR.

100 bp

Target band
 What PCR is for?

PCR PRODUCT – AN END AND

A MEANS
An end
 PCR can be the final step in analyzing or answering a question. An
example is food samples. PCR can identify a piece of DNA that
indicates genetic modification, or contamination by bacteria.

A means
 PCR can amplify a gene for further study, or for gene cloning or
sequencing
 APPLICATION OF PCR
 DNA sequencing
 DNA profiling (fingerprinting)
A POWERFUL AND
 Making recombinant DNA for GMOs VERSATILE TOOL
 Detecting foreign organisms in food Salmonella, E. coli
 Detecting the cause of an infection or disease, HIV
Lyme Disease, Strep throat, STDs, etc., etc.
Agriculture Molecular biology
Archaeology Botany
Medicine Cell biology
Forensics
TROUBLESHOOTIN
G PCR
• PCR product/ band or interest is there but appear to faint on agarose gel.
How to improve the intensity of the band?
 9.0
Gel electrophoresis
 At the end of the lecture, students should be able to:

• Explain the importance of gel electrophoresis

• Differentiate the types of gel electrophoresis
• Understand the principle of gel electrophoresis
 Greek word phoresis, ‘being carried’,

Overview
in other words being carried by an
electrical field

• Arne Tiselius reported the moving boundary

method (MBE) of electrophoresis of proteins-
paving the way for much improved methods of
electrophoretic analysis (i.e. IEF and SDS-PAGE).

• Since 1980s many of these techniques have

evolved to become powerful bioanalytical tools,
that open new opportunities for researchers to
understand fundamental aspects of genomics,
proteomics and disease.
 Electrophoresis?

• Electrophoresis is a method whereby charged molecules in solution, chiefly

proteins and nucleic acids, migrate in response to an electrical field.
• Their rate of migration through the electrical field, depends on??👇
• As an analytical tool, electrophoresis is simple, rapid and highly sensitive -
one of the most widely used tools in biochemistry and
molecular biology.
- provides a powerful method for separating and analyzing proteins and
nucleic acids.
What effects rate of migration/electrophoretic separation?

• Mass and shape - larger molecules move more slowly than smaller ones

• Net charge - The net charge is determined by the number of positive and
negative charges in the molecule. Charges are conferred on proteins by
amino acid side chains as well as by groups arising from post translational
modifications such as deamidation, acylation or phosphorylation

• buffer viscosity - affected by the composition and ionic strength (salt

content) of the electrophoresis buffer (Somma & Querci, 2006).
 What effects rate of migration/electrophoretic separation?

• porosity of the matrix - The lower the concentration (%) of the gel,
the faster the DNA fragments migrate (less porous/large pores).

• Voltage/current - The higher the voltage/current, the faster the

DNA migrates. It is highly recommended not exceed 5-8 V/cm and 75
mA for standard size gels or 100 mA for minigels.

Be careful, higher voltage also means high heat. What will happened?
 Instrumentation
• Power supply
• Electrophoresis Chamber
with buffer reservoir
• Electrodes in buffer
reservoir
• Mini gel 8 cm X 8 cm
• Electrophoresis is usually done with gels formed in tubes, slabs,
or on a flat bed.
• In many electrophoresis units, the gel is mounted between two
buffer chambers containing separate electrodes, so that the
only electrical connection between the two chambers is through
the gel.
In most electrophoresis units, the
gel is mounted between two buffer
chambers containing separate
electrodes so that the only
electrical connection between the
two chambers is through the gel.
Tube Gel Unit
Slab Gel Unit
Components
Slab
of gel unit
Flat Bed Unit
 Gel matrix (type of gel)

• There are different kinds of gels and buffers that can be

used. The two main types of gels are agarose and
polyacrylamide
• Polyacrylamide forms a tighter matrix and is used to
separate most proteins and small oligonucleotides.
• Agarose forms a looser, more open matrix that enables
separation of macromolecules such as nucleic acids, large
proteins and protein complexes
 Types of gel
selected on the basis of its composition and porosity, which must be fit for purpose.
Agarose gel

• This is a polymer composed of a repeating disaccharide unit called agarobiose

which consists of galactose and 3,6-anhydrogalactose
• its neutral charge, low chemical complexity and relatively large pore size
make it useful for size-separation of large molecules such as DNA
• The percentage of agarose used depends on the size of fragments to be
resolved - (typically 0.3–2.0%)

Higher % gel with smaller pores to

separate smaller molecules
• In general, if the aim is to separate large DNA fragments, use a
low concentration (%) of gel
• If to separate small DNA fragments, use a high (%) of agarose
gel

*Mostly used at 0.8/1.0 %

Pro and cons using Agarose gel electrophoresis

You can prepare the gel beforehand and

keep it in closed tight container with
buffer at RT
 Step 1
Agarose is added to buffer and
heated to 95ºC (to melt it). After
cooling, it is poured into a pre-
prepared gel cast

Gel comb

Step 2
a comb is inserted immediately to
create wells for loading the samples

Step 3
Leave the gel to solidify

Step 4
the comb is removed slowly (see set
of wells that have been created)
Preparation and running a standard agarose gel

1. An electrophoresis
chamber and power
supply
2. Gel casting trays
3. Sample comb
4. Electrophoresis
buffers

6. Loading buffer , which has glycerol and tracking dye (bromophenol blue)
7. Staining using ethidium bromide
8. Visualization under UV lamp
9. Sample of DNA to be separated
10. DNA ladder – comprising DNA fragments of known size representing
molecular weight markers.
 Sample preparation

•Mix the samples of DNA with

the 6X sample loading buffer
(w/ tracking dye).
•This allows the samples to be
seen when loading onto the gel,
and increases the density of the
samples, causing them to sink
into the gel wells.

6X Loading Buffer: 
• SYBR Gold Dye (for color)
• Sample Buffer (SB)
Running an agarose gel
Polyacrylamide gel electrophoresis (PAGE)
• are chemically cross-linked gels formed by the polymerization of
acrylamide with a cross-linking agent, usually N,N’-
methylenebisacrylamide. The reaction is a free radical polymerization,
usually carried out with ammonium persulfate as the initiator and
N,N,N’,N’-tetramethylethylendiamine (TEMED) as the catalyst .
Pro and cons using PAGE gel electrophoresis
• Requirement PAGE The simplicity?
• PAGE Vs. Agarose Electrophoresis
Acrylamide/Bis acrylamide
• Ammonium persulfate (APS)
• Tetramethyl ethylenediamine (TEMED)
• Buffer/Buffers
• Agarose
• Agarose powder
• Buffer
 • Native?
P
• Denaturing? A
G
E
c
o
Photo by Irène Mangin, Christophe Lévêque, Fabien Magne, Antonia Suau, Philippe Pochart / CC BY 2.5
n
d
 PAGE condition
• Separating condition
• Native PAGE
• Native gels are run in non-denaturing conditions, so that the analytes
native structure is maintained
• Separation is based upon charge, size, and shape of macromolecules
• Useful for separation of purification of mixture of proteins
• This was the original mode of separation
• Denaturing PAGE Denaturing aka SDS-PAGE
• Separation based on molecular weight
• Common method to determine MW of proteins
• Useful for checking purity of protein sample
 SDS-PAGE gel electrophoresis

• In SDS separations, migration is determined not by intrinsic

electrical charge of polypeptides but by molecular weight

• Sodium dodecylsulfate (SDS) is an anionic detergent which denatures

secondary and non–disulfide–linked tertiary structures by wrapping
around the polypeptide backbone. In so doing, SDS confers a net
negative charge to the polypeptide in proportion to its length

• When treated with SDS and a reducing agent, the polypeptides

become rods of negative charges with equal “charge densities" or
charge per unit length.
SDS-PAGE gel
electrophoresi
s
 SDS-PAGE system
• Continuous vs.Discontinuous

👉
👉
 Continuous and Discontinuous
• A continuous system has only a single separating gel and uses
the same buffer in the tanks and the gel Buffer Systems
• In a discontinuous system a nonrestrictive large pore gel, called
a stacking gel, is layered on top of a separating gel
• The resolution obtainable in a discontinuous system is much
greater than that obtainable in a continuous one. However, the
continuous system is a little easier to set up
Continuous &
Discontinuous
Buffer
Systems
 Sample
preparation for
SDS-PAGE
• Add SDS, 2-
mercapthoethanol
• Heat
 R
• Samples to be run are loaded
in wells at the top of the gel,
u
in conjunction with tracking
dye. An electrical voltage is
n
applied between the upper
and lower reservoirs, causing
the samples to migrate down
through the gel n
i
n
Gel Analysis
Different methods can be
used to make the sample in
the gel visible to the human
eye
 Stain DNA
• Ethidium Bromide
• Fluorescent
• Toxic-handle with
care
 Stain Protein

Coomasive Blue Staining Silver Staining

 Comparison/dissimilarities
between PAGE and agarose gel
electrophoresis???
PAGE Agarose
Run in Vertical orientation Horizontal orientation
Polymerisation of acrylamide-bis- Agarose as gel medium
acrylamide to form gel
Gel staining : using colorimetric dye Gel staining: using only fluorescence
(Coomasive blue) of fluorescence using dye (e.g. ethidium bromide or Sybr
silver staining Red)

Discontinuous gel; has stacking and Continuous gel; has only one agarose
resolving gel concentration through out the whole gel

Has denaturing (with SDS) or non- Non denaturing gel, only agarose as
denaturing condition (native page) polymer
 • The most commonly used for duplex
DNA are TAE (Tris-acetate- EDTA)
and TBE (Tris-borate-EDTA)
• DNA fragments will migrate at
somewhat different rates in these
two buffers due to differences in
Buffers ionic strength
• Buffers not only establish a pH, but
provide ions to support conductivity

Can I use water instead?

 • Determination of Protein MW by SDS-
PAGE

Applications • Determination of Protein purity

• For sequencing
Determination of MW
 Gel electrophoresis
• Techniques
• 1. Native gel electrophoresis : analyte separated according to differences in
apparent mobility (charge)
• 2. SDS-PAGE : analyte separated according tosize
• 3. Isoelectric Focussing (IEF)
• 4. Two dimensional gel electrophoresis (2D)
 Isoelectric Point
• There is a pH at which there is no net charge on a protein; this
is the isoelectric point (pI).
• Above its isoelectric point, a protein has a net negative charge
and migrates toward the anode in an electrical field.
• Below its isoelectric point, the protein is positive and migrates
toward the cathode.
 Isoelectric Focusing(IEF)
• Isoelectric focusing is a method in which proteins are
separated in a pH gradient according to their isoelectric points
(pI)
• Focusing occurs in two stages; first, the pH gradient is formed
• In the second stage, the proteins begin their migrations toward
the anode if their net charge is negative, or toward the cathode
if their net charge is positive
• When a protein reaches its isoelectric point (pI) in the pH
gradient, it carries a net charge of zero and will stop migrating
 2D electrophoresis
• Two-dimensional gel electrophoresis is widely used to separate
complex mixtures of proteins into many more components than
is possible in conventional one-dimensional electrophoresis
• Each dimension separates proteins according to different
properties
THANK YOU..