Lecture 3

Question 1

What does a spectrophotometer look like ? (give the main components) What does it do?

Answer 1

Light source, monochromator, cuvette, output / diode array

It measures transmission of light (=absorbance)

Question 2

What is a diode array detector ? A couple advantages and what does the accuracy depend on ?

Answer 2

2D pattern of diodes and prisms, used to detect organic compounds, any light transmitted through the sample is dispersed by the prism so that light of different wavelengths falls on different diodes

+: very fast, no monochromator needed

Accuracy depends on nb of pixels

Question 3

what is the difference between cuvettes and well plates regarding the optical length ?

Answer 3

it is fixed for cuvettes (length L) because horizontal measurement.
It is variable for well plates because vertical measurement -> length depends on the volume of sample and shape of bottom of plate.

Question 4

mention some applications of spectrophotometry

Answer 4

• detection of concentration of substances (OD)
• detection of impurities
• structure elucidation of organic compounds
• molecular weight determination
• characterization of proteins-

Question 5

what is the assumption taken during OD measurements ?

Answer 5

OD value = proportional to the cell number (concentration of sample)

Question 6

3 things that impact OD measurements

Answer 6

• single VS multiple scattering
• size of cells / medium they grow in
• bacterial aggregation

Question 7

what is a nephelometer ? When does it work better ?

Answer 7

It measures the scattering instead of the transmission.
Works better when more turbidity (high cell densities). It has a bigger measuring range than OD.

Question 8

What are the main components of a nephelometer

Answer 8

sample, ulbricht sphere / light trap, light detector (can have multiple ones at different positions)

Question 9

how do labeled substrates work ?

Answer 9

You add a colorful label to a substrate, and when the enzyme cuts the label -> we can measure how much of it there is with the spectrophotometer

Question 10

mention the main components of a fluorometer

Answer 10

light source
excitation monochromator
(excitation filter)
sample
emission monochromator
(emission filter)
detector

Question 11

how can filters be useful in fluorometry ?

Answer 11

for example a short pass filter (takes out high wavelengths) -> makes our peak of interest more visible and measurable

Question 12

give three examples when a fluorometer can be used

Answer 12

• GFP labeled cells
• ATP measurements (luminescence and not fluorescence, but still works)
• labeled substrates (fluorescent labels)