Download Spectrophotometer 2 R

SPECTROPHOTOMETER 2
The use of absorption
spectroscopy UV/VIS
 Concentration measurements.
 Assay of chemical reactions.
 Identification of substances.
1. Concentration measurements
 Using the Beer’s law its is easy to calculate
the concentration of a given substance given
the ε and the absorbance at a given wave
length
 In addition to the use of the standard curve to
calculate the concentration.
Standard curve
x
1.0
x
0.5
x
1
4
2
3
Concentration (mg/ml)
5
There is some A vs. C where graph is linear.
Spectrometric Analysis Using Standard Curve
1.2
0.8
0.4
3
1
2
Concentration (g/l) glucose
4
Avoid very high or low absorbencies when drawing a standard
curve. The best results are obtained with 0.1 < A < 1. Plot the
Absorbance vs. Concentration to get a straight line
2. Assay of chemical reactions
 Enzyme assays are laboratory procedures
that measure the rate of enzyme reactions.
Because enzymes are not consumed by the
reactions they catalyse, enzyme assays
usually follow changes in the concentration of
either substrates or products to measure the
rate of reaction. There are many methods of
measurement. Spectrophotometric assays
observe change in the absorbance of light
between products and reactants;
Progress curve for an enzyme reaction.
3. Identification of substances
 Most substances have characteristic spectra
and can be identified thereby.
 This can be done by measuring a complete
spectrum of a certain substance.
 The absorbance will be measured at different
wavelengths.
STRUCTURAL STUDIES OF
MOLECULES USING ABSORPTION
OF LIGHT
proteins
 In a non polar environment, the λmax and the
ε of trp, tyr, phe, and his increase. Hence:
a. If these amino acids have higher λmax and ε
in a protein (dissolved in a polar solvent)
than when they are free, then they must be
“ buried “ inside the protein structure and
surrounded by non polar amino acids.
Proteins (2)
 The λmax and the ε also increases when a
charged titratable group is present. Hence ,
when the PH changes:
a. If there is no spectral change, this means
those groups are “buried” in a non polar
region of the protein.
b. If the spectral changed as a function of PH
and the pK was the same , then the amino
acid is on the surface of the protein
Nucleic acids
 For purines and pyrimidines ε decreases when
they are more “ stacked” together
 The value of ε decreases in the following
order:
Free base
base in an unstacked single
stranded polynucleotide
base in a
stacked single stranded polynucleotide
base in a double stranded poly nucleotide
Differences in the absorbance between
double and single strands of DNA
The A of DNA increases if the DNA is heated through a particular
temperature range .Hyperchromicity of DNA; Is a phenomena that
occurs when the DNA double helix is denatured by heat accompanied
by an increase of the absorbance.
Some of the important information that can be obtained is the G+ C
content .
A DNA molecule with a higher G+C content has a higher thermal
stability(since it has a 3 hydrogen bonds ) compared to a DNA with a
higher A-T content ( 2 hydrogen bonds).
Example problems 1
1. You are provided with a plot of standard
protein concentrations against their
absorbance.
a. Draw the standard curve
b. What is the concentration of the sample
shown in the figure ( A= 0.6)
c. If you have an un kown sample with an
absorbance of 1.2. How can you determine
its concntration?
Example problems 2
2. The enzyme GOT is released into the blood
stream as a result of myocardial infarction.
The enzyme is assayed in serum by
following the decrease in the absorbance of
NADH.
Calculate the concentration of GOT in the
patients serum . The cuvette had a light path
of 1 cm and the absorbance decreased at a
rate of 0.4/min ( am= 6.22 X 103 )
Example problems 3
3. A lab technician was working on two samples of
the same DNA molecule , one of those samples
was denatured , the other was native DNA
during her work she got the two samples mixed
up .How can you determine the identity of each
sample by an absorbance measurement?
Explain.
Example problems 4
4- A student was studying two different DNA
samples DNA1 and DNA2 the two DNA
molecules have the same molecular
weight , the melting temperature of DNA1
was Tm = 70°C , while DNA 2 had a Tm =
85°C . Explain the samples structure in
terms of G + C content.