Spectrophotometry

Question 1

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

Answer 1

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

Question 2

Define incident light and transmitted light

Answer 2

• INCIDENT LIGHT - light before cuvette
• TRANSMITTED LIGHT - light after cuvette

Question 3

Outline the scientific method

Answer 3

• 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

Question 4

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

Answer 4

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

Question 5

Define transmission (T)

Answer 5

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

Question 6

Define absorbance (A)

Answer 6

Measure of the amount of light absorbed by a molecule

Question 7

Explain molar absorptivity or molar extinction coefficient

Answer 7

• 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

Question 8

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

Answer 8

Transmission exponentially declines and cannot be easily graphed

Question 9

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

Answer 9

• 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

Question 10

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

Answer 10

Question 11

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

Answer 11

Higher absorbance at 260nm, the higher the dsDNA concentration

Question 12

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

Answer 12

dsDNA is hypochromic to ssDNA due to less intense peak

Question 13

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

Answer 13

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

Question 14

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?

Question 15

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

Question 16

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

Question 17

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

Question 18

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

Answer 18

• 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

Question 19

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

Answer 19

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

Question 20

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

Answer 20

• 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

Question 21

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?

Answer 21

• 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

Question 22

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?

Answer 22

• 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

Question 23

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

Answer 23

• 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

Question 24

What is the difference between absorbance and optical density?

Answer 24

• 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

Question 25

Explain the method that uses Glucose Oxidase for measuring glucose

Answer 25

* 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

Question 26

Explain the method that uses Hexokinase for measuring glucose

Answer 26

* 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**

Question 27

Explain the method that uses Glucose Dehydrogenase for measuring glucose

Answer 27

* 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

Question 28

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

Answer 28

* 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

Question 29

Explain how electrochemistry can be used to detect glucose

Answer 29

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