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0 SPECTROSCOPY
(part 2)
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.
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.
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
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)
• 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
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