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.

1 2 3 4 5

1.0

0.5

Concentration (mg/ml)

A a

t 2

80

nm

There is some A vs. C where graph is linear.

x

x

x

Standard curve

Spectrometric Analysis Using Standard Curve

1 2 3 4

0.4

0.8

1.2

A a

t 5

40

nm

Concentration (g/l) glucose

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.

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 curveb. 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.