4.

0 SPECTROSCOPY
(part 2)
BMS481
NURUL AILI ZAKARIA (Ph.D)

https://padlet.com/bms415sept2019/dnwxxk3n9srg
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 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
Outlines

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 QUANTITATION OF PROTEIN, DNA & RNA
of DNA &
Protein

Principle
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 QUANTITATION OF PROTEIN, DNA & RNA
of DNA &
Protein

Instruments
UV vis spectra and quartz cuvette
Or Nanodrop spectra

sample
Quantitation ADVANTAGES OF USING NANODROP
of DNA &
Protein
Can 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 THE 96 WELL MICROPLATE READER
assays

• 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 THE 96 WELL MICROPLATE READER
assays
Enzyme THE 96 WELL MICROPLATE READER
assays

• 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 ENZYME ASSAY
assays

• 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 BLANK/STANDARD FOR ENZYME ASSAY
assays

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 ENZYME ASSAY
assays

• 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 APPLICATIONS OF SPECTROPHOTOMETER

1. Measurement of Concentration
- Prepare samples
-Make series of standard solutions of known
concentrations
-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 APPLICATIONS OF SPECTROPHOTOMETER

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

5. Detection of Functional groups

-Absence of a band at particular wavelength regarded as an evidence for absence
of particular group
 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