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BC3009: Biophysical & Biochemical Methods > Spectrophotometry > Flashcards

Spectrophotometry Flashcards

1
Q

How does a spectrophotometer work? (Use a labelled diagram)

A
  • Light Source
  • Collimator (Lens)
  • Monochromator (Prism / Grating)
  • Wavelength Selector (Slit)
  • Sample Solution (Cuvette)
  • Detector (Photocell)
  • Digital Display
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2
Q

Define incident light and transmitted light

A
  • INCIDENT LIGHT - light before cuvette
  • TRANSMITTED LIGHT - light after cuvette
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3
Q

Outline the scientific method

A
  • 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
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4
Q

What is the Beer-Lambert Law?
(Explain each item)

A
  • A = εcl
  • A = absorbance
  • ε = molar extinction coefficient
  • c = sample length (cm)
  • l = concentration (mol/L)
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5
Q

Define transmission (T)

A
  • T = I/Io
  • Transmittance is the ratio of the light that exits and enters the sample
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6
Q

Define absorbance (A)

A

Measure of the amount of light absorbed by a molecule

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7
Q

Explain molar absorptivity or molar extinction coefficient

A
  • 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
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8
Q

Why is transmission not typically used for the calculation of sample concentration?

A

Transmission exponentially declines and cannot be easily graphed

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9
Q

How would you actually generate a UV-Vis spectrum of a sample?

A
  • 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
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10
Q

What would a typical UV-Vis spectrum of a double stranded DNA sample look like?

A
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11
Q

What is the relationship between DOUBLE stranded DNA absorbance at 260nm and DNA concentration?

A

Higher absorbance at 260nm, the higher the dsDNA concentration

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12
Q

What is the relationship between SINGLE stranded DNA absorbance at 260nm and DNA concentration?

A

dsDNA is hypochromic to ssDNA due to less intense peak

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13
Q

What is the relationship between RNA absorbance at 260nm and DNA concentration?

A

RNA is hypochromic to dsDNA but hyperchromic to ssDNA where concentration is equal

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14
Q

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?

A
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15
Q

How would the presence of Phenol affect the UV-Vis spectrum of a double stranded DNA sample?

A
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16
Q

How would the presence of Guanidium Isothiocyanate affect the UV-Vis spectrum of a double stranded DNA sample?

A
17
Q

How would the presence of EDTA affect the UV-Vis spectrum of a double stranded DNA sample?

A
18
Q

How would the presence of DNAse 1 affect the UV-Vis spectrum of a double stranded DNA sample?

A
  • 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
19
Q

Why might a scientist measure the 260/230 ratio of a DNA sample?

A
  • Ratio of 1.8 indicates pure DNA
  • Ratio values above this may indicate presence of residual phenol etc
20
Q

Why might a scientist measure the 260/280 ratio of a DNA sample?

A
  • 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
21
Q

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?

A
  • 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
22
Q

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?

A
  • 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
23
Q

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

A
  • 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
24
Q

What is the difference between absorbance and optical density?

A
  • 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
25
Q

Explain the method that uses Glucose Oxidase for measuring glucose

A
  • 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
26
Q

Explain the method that uses Hexokinase for measuring glucose

A
  • 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
27
Q

Explain the method that uses Glucose Dehydrogenase for measuring glucose

A
  • 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
28
Q

Explain the effect of ascorbic acid on the enzymatic colorimetric method for quantification of glucose

A
  • 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
29
Q

Explain how electrochemistry can be used to detect glucose

A
  1. Glucose in blood sample oxidised by glucose oxidase to form gluconic acid, releasing 2 electrons and 2 protons
  2. Metal complex transfers electrons between electrode and glucose/gluconic acid oxidation reaction
  3. Electrode measures the current which is related to the number of electrons and therefore the number of glucose molecules converted to gluconic acid

BC3009: Biophysical & Biochemical Methods (12 decks)

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