Spectrophotometry Flashcards
How does a spectrophotometer work? (Use a labelled diagram)
- Light Source
- Collimator (Lens)
- Monochromator (Prism / Grating)
- Wavelength Selector (Slit)
- Sample Solution (Cuvette)
- Detector (Photocell)
- Digital Display
Define incident light and transmitted light
- INCIDENT LIGHT - light before cuvette
- TRANSMITTED LIGHT - light after cuvette
Outline the scientific method
- Aim to carry out each experiment by changing only one variable
- CONTROL - part of the experiment not being tested and used for comparison
- INDEPENDENT variable - what changes in the experiment
- CONTROLLED variable - what stays the same throughout the experiment
What is the Beer-Lambert Law?
(Explain each item)
- A = εcl
- A = absorbance
- ε = molar extinction coefficient
- c = sample length (cm)
- l = concentration (mol/L)
Define transmission (T)
- T = I/Io
- Transmittance is the ratio of the light that exits and enters the sample
Define absorbance (A)
Measure of the amount of light absorbed by a molecule
Explain molar absorptivity or molar extinction coefficient
- Physical constant unique to a molecule that relates the absorption of a substance to the concentration of the substance
- Higher ε relates to a higher absorbance for a given concentration of a substance
Why is transmission not typically used for the calculation of sample concentration?
Transmission exponentially declines and cannot be easily graphed
How would you actually generate a UV-Vis spectrum of a sample?
- Sample is irradiated with a range of wavelengths using a monochromator
- Absorption at that wavelength is measured
- Monochromator moves to higher or lower wavelengths incrementally and absorbance measured again
What would a typical UV-Vis spectrum of a double stranded DNA sample look like?
What is the relationship between DOUBLE stranded DNA absorbance at 260nm and DNA concentration?
Higher absorbance at 260nm, the higher the dsDNA concentration
What is the relationship between SINGLE stranded DNA absorbance at 260nm and DNA concentration?
dsDNA is hypochromic to ssDNA due to less intense peak
What is the relationship between RNA absorbance at 260nm and DNA concentration?
RNA is hypochromic to dsDNA but hyperchromic to ssDNA where concentration is equal
Assume an absorbance measurement of 1.0 was obtained for a double stranded DNA sample at OD260nm - what is the concentration of the DNA sample?
How would the presence of Phenol affect the UV-Vis spectrum of a double stranded DNA sample?
How would the presence of Guanidium Isothiocyanate affect the UV-Vis spectrum of a double stranded DNA sample?
How would the presence of EDTA affect the UV-Vis spectrum of a double stranded DNA sample?
How would the presence of DNAse 1 affect the UV-Vis spectrum of a double stranded DNA sample?
- Breaks down single and double stranded DNA
- Long dsDNA molecules are hyperchromic compared to short ssDNA
- Therefore, DNase would cause hyperchromic shift in the spectrum
Why might a scientist measure the 260/230 ratio of a DNA sample?
- Ratio of 1.8 indicates pure DNA
- Ratio values above this may indicate presence of residual phenol etc
Why might a scientist measure the 260/280 ratio of a DNA sample?
- DNA absorbs at 260nm
- RNA absorbs at 280nm
- Ratio of 2.0 indicates pure RNA
- Values above this can indicate the presence of impurities such as phenol etc
A sample of DNA was found to have a 260/230 ratio of 1.5 and a 260/280 ratio of 2.0 - what would this tell you?
- 260/230 ratio of 1.5 means that impurities are present in the sample
- Could be EDTA or Phenol
- 260/280 ratio of 2.0 means that pure RNA is present
A sample of DNA was found to have a 260/230 ratio of 2.0 and a 260/280 ratio of 1.5 - what would this tell you?
- 260/230 ratio of 2.0 indicates pure DNA as it falls within the 1.8 - 2.2 range
- 260/280 ratio of 1.5 indicates impure sample due to the presence of proteins or DNases
Assume you diluted a DNA sample 1 in 10 and then read its absorbance at 260nm. The reading obtained was 0.086. Calculate the concentration of the DNA in the sample
- If dsDNA then 0.086 x 10 x 50µg/ml = 43 µg/ml
- If ssDNA then 0.086 x 10 x 33 µg/ml = 28.38 µg/ml
What is the difference between absorbance and optical density?
- OPTICAL DENSITY (OD) is a measure of attenuation or intensity loss based on scattering of light
- ABSORBANCE (A) considers only the absorbance of light within the light component
Explain the method that uses Glucose Oxidase for measuring glucose
- β-d-glucose + O2 —> Gluconate + H202
- Glucose is oxidised by glucose oxidase in the presence of oxygen to gluconate and hydrogen peroxide
- Hydrogen peroxide is then oxidised by peroxidase in the presence of 4AAP and phenol to form the red dye quinoeimine
- Dye is then detected at 505nm spectrophotometrically
Explain the method that uses Hexokinase for measuring glucose
- Glucose converted to Glucose-6-Phosphate (G6P) by Hexokinase
- G6P then oxidised by Glucose-6-Phosphate Dehydrogenase (G6PD) to form NADH
- NADH reduces colourless probe to a coloured product with a strong absorbance at 450nm
Explain the method that uses Glucose Dehydrogenase for measuring glucose
- Glucose + NAD(P) —> Gluconolactone + NAD(P)H
- Change in concentration between NAD(P)+ and NAD(P)H can be assayed spectrophotometrically at 339nm
- Alternatively, NADH can be used to reduce tetrazolium chromogen to a coloured product
Explain the effect of ascorbic acid on the enzymatic colorimetric method for quantification of glucose
- Ascorbic acid can be oxidised and “consume” H202 which interferes with the assay
- Concentration of H202 cannot be related to glucose concentration unless effect of ascorbic acid is determined
- Causes a hypochromic shift in the spectrum of quinoneimine
Explain how electrochemistry can be used to detect glucose
- Glucose in blood sample oxidised by glucose oxidase to form gluconic acid, releasing 2 electrons and 2 protons
- Metal complex transfers electrons between electrode and glucose/gluconic acid oxidation reaction
- Electrode measures the current which is related to the number of electrons and therefore the number of glucose molecules converted to gluconic acid
