Spectroscopy Flashcards
What are the features of a spectrophotometer ?
- Measures the fraction of the incident light transmitted through a solution or the amount of light absorbed
- Transmit light of narrow wavelength ranges
Different compounds absorb light at ? and what can be used to distinguish compounds ?
Different wavelengths, and as such a spectrophotometer can be used to distinguish compounds
Explain the experiment to work out the optimal NADH absorbance (the wavelength that is most absorbed by NADH) ?
- Use a UV cuvettes (a reference blank ) containing everything except NADH (or NAD) and zero spectrophotometer at each wavelength as required
- Used in case other compounds in a solution may absorb (although all other reactants are in fact colourless in this case) - Measure the absorbance of the solution containing NADH at a variety of
wavelengths - Repeat for NAD+
How do you do the Measurement of the Absorption Spectra of NADH and NAD+ ?
- You need to find a wavelength where there is a difference in absorbance between substrate and products.
- Plot a graph of absorbance vs wavelength between 240-400nm for both NADH and NAD+ and calculate difference spectrum.
What is the name of the laws of light absorption ?
Beer’s - Lambert’s law
These laws use calculations to enable ?
The concentration
of your molecule in a sample to be determined from the absorbance measurements of the spectrophotometer
What does Lambert’s Law state ?
“that the proportion of incident light absorbed by a transparent medium is independent of the light intensity”
Therefore successive layers of equal thickness will transmit ?
The same proportion of light (intensity decreases by the same proportion for equal lengths travelled)
Transmission of light decreases exponentially with ?
Increasing thickness of solution (or concentration)
What does Beer’s Law state ?
“that absorbance is proportional to the concentrations of the attenuating speciesin the sample”
Therefore absorption shows a linear increase with ?
Concentration and is thus independent of the intensity of the light (within limits!)
What does the transmittance measure ?
How much of an incident light (Io) is transmitted
What does the absorbance measure ?
How much of an incident light (Io) is absorbed
Are Transmission and Absorbance related ?
They are inversely related, more light absorbed, less transmitted.
The relationship is not ?
Linear, its logarithmic
What is LDH activity ?
A glycolytic enzyme found in all body tissues, including: the muscles, liver, heart, pancreas, kidneys, brain and blood cells
Enzyme activities are very high in tissues compared with the serum (ie 500 fold) and therefore ?
Leakage of the enzyme from even a small amount of tissue damage can increase the activity 10-fold
Elevations of the serum levels of LDH are associated ?
With a number of different disease states, for example myocardial infarctions, liver disease, anaemia, renal disease and muscular dystrophy.
Measurement of the activity in the serum is used ?
Clinically for both diagnosis of disease, and monitoring of the progression of the disease following treatment
Glycolysis produces ?
Pyruvate from Glucose
Pyruvate enters the ?
TCA cycle or fermentation depending on the oxygen state of the cell
In fermentation, pyruvate is?
Reduced to lactate to regenerate NAD+
LDH is a hydrogen transfer enzyme which ?
Catalyses the reduction of Pyruvate to L-lactate using NADH as the hydrogen donor
CH3COCOOH + NADH + H+ → CH3CHOHCOOH + NAD+
What is this reaction ?
Pyruvate to Lactate.
The reaction is reversible and the reaction equilibrium strongly favours the reduction of pyruvate to lactate
You will measure the conversion of NADH to NAD+, a decrease in ?
Light absorbance at the wavelength determined previously
