Search for notes by fellow students, in your own course and all over the country.
Browse our notes for titles which look like what you need, you can preview any of the notes via a sample of the contents. After you're happy these are the notes you're after simply pop them into your shopping cart.
Document Preview
Extracts from the notes are below, to see the PDF you'll receive please use the links above
Unedited work on Spectrophotometry
In chemistry, spectrophotometry is the quantitative
measurement of the reflection or transmission
properties of a material as a function of wavelength
...
Spectrophotometry is a tool that hinges on the
quantitative analysis of molecules depending on
how much light is absorbed by colored compounds
...
Important features of spectrophotometers are spectral
bandwidth (the range of colors it can transmit through
the test sample), the percentage of sample-transmission,
the logarithmic range of sample-absorption, and
sometimes a percentage of reflectance measurement
...
Although many biochemicals are colored,
as in, they absorb visible light and therefore can be
measured by colorimetric procedures, even colorless
biochemicals can often be converted to colored compounds
suitable for chromogenic color-forming reactions to yield
compounds suitable for colorimetric analysis
...
Within these ranges of light, calibrations
are needed on the machine using standards that vary
in type depending on the wavelength of thephotometric
determination
...
A certain chemical reaction within a solution
may occur in a forward and reverse direction, where
reactants form products and products break down into
reactants
...
In order to
determine the respective concentrations of reactants
and products at this point, the light transmittance of
the solution can be tested using spectrophotometry
...
Invented by Arnold O
...
This would come as a solution to the previously
created spectrophotometers which were unable to
absorb the ultraviolet correctly
...
It would be found that this did not
give satisfactory results, therefore in Model B, there
was a shift from a glass to a quartz prism which allowed
for better absorbance results
...
The last
and most popular model became Model D which is better
recognized now as the DU spectrophotometer which
contained the instrument case, hydrogen lamp with
ultraviolent continuum and a better monochromator
...
In the words of Nobel chemistry
laureate Bruce Merrifieldsaid it was "probably the
most important instrument ever developed towards the
advancement of bioscience
...
Diode-assay spectrophotometers
differed from the original spectrophotometer created
by Beckman because it was the first single-beam
microprocessor-controlled spectrophotometer that
scanned multiple wavelengths at a time in seconds
...
Then
it is transmitted back by grating the photodiode array
which detects the wavelength region of the spectrum
...
Single beam scanning spectrophotometer
There are two major classes of devices: single beam and
double beam
...
A single-beam spectrophotometer measures
the relative light intensity of the beam before and
after a test sample is inserted
...
In short, the sequence of events in a scanning
spectrophotometer is as follows:
The light source is shone into a monochromator,
The light source is shone into a monochromator,
diffracted into a rainbow, and split into two beams
...
Fractions of the incident wavelengths are transmitted
through, or reflected from, the sample and the
reference
...
Electronic circuits convert the relative currents into
linear transmission percentages and/or absorbance/
concentration values
...
The transmitted light is refracted into a rainbow with the
reflection grating
...
Electronic circuits convert the relative currents into
linear transmission percentages and/or absorbance/
concentration values
Applications in biochemistry
Spectrophotometry is an important technique used in
many biochemical experiments that involve DNA, RNA,
and protein isolation, enzyme kinetics and biochemical
and protein isolation, enzyme kinetics and biochemical
analyses
...
In addition, precious sample can be saved by
utilizing a micro-volume platform where as little as 1uL
of sample is required for complete analyses
...
This coloring can be
accomplished by either a dye such as Coomasie Brilliant
Blue G-250 dye measured at 595 nm or by an enzymatic
reaction as seen between β-galactosidase and ONPG
(turns sample yellow) measured at 420 nm
...
In biochemical
experiments, a chemical and/or physical property is
chosen and the procedure that is used is specific to that
property in order to derive more information about the
sample, such as the quantity, purity, enzyme activity,
etc
...
Spectrophotometry is also a helpful
process for protein purification and can also be used
as a method to create optical assays of a compound
...
Spectrophotometric data can also be used in conjunction
with the Beer-Lambert Equation, A= -log10T=εcl=OD,
in order to determine various relationships between
transmittance and concentration, and absorbance and
concentration
...
It is possible to know
the concentrations of a two component mixture using
the absorption spectra of the standard solutions of
each component
...
Spectrophotometers have been developed and improved
over decades and have been widely used among chemists
...
It is considered to be a highly accurate
instrument that is also very sensitive and therefore
extremely precise, especially in determining color change
...
UV-visible spectrophotometry
Most spectrophotometers are used in
the UVand visible regions of the spectrum, and some
of these instruments also operate into the nearinfrared region as well
...
The concentration of a protein
can be estimated by measuring the OD at 280 nm due to
the presence of tryptophan, tyrosine and phenylalanine
...
Nucleic acid contamination can also interfere
...
Samples are usually prepared in cuvettes; depending
on the region of interest, they may be constructed
of glass, plastic (visible spectrum region of interest),
or quartz (Far UV spectrum region of interest)
...
Applications
...
Specific ultraviolet absorbance for metric of aromaticity
...
Qualitative analysis can be used and spectrophotometers
are used to record spectra of compounds by scanning
broad wavelength regions to determine the absorbance
properties (the intensity of the color) of the compound at
properties (the intensity of the color) of the compound at
each wavelength
...
Largely, spectrophotometry is best used
to help quantify the amount of purification your sample
has undergone relative to total protein concentration
...
Following this testing the sample at 420 nm for
specific interaction with ONPG and at 595 for a Bradford
Assay the amount of purification can be assessed
quantitatively
...
IR Spectrophotometry
Spectrophotometers designed for the infrared
region are quite different because of the technical
requirements of measurement in that region
...
Another complication is that quite a few materials such
as glass and plastic absorb infrared light, making it
as glass and plastic absorb infrared light, making it
incompatible as an optical medium
...
Samples for IR
spectrophotometry may be smeared between two discs
of potassium bromide or ground with potassium bromide
and pressed into a pellet
...
Spectroradiometers
Spectroradiometers, which operate almost like the visible
region spectrophotometers, are designed to measure
the spectral density of illuminants
...
Components:
The light source shines onto or through the sample
...
The detector detects how much light was reflected from
or transmitted through the sample
...
Last modified: 15:46