Spectrophotometry

Question 1

What is spectrophotometry?

Answer 1

An analytical method that involves how light interacts with the molecules or atoms in a sample of matter.

Question 2

What is the UV range in spectrophotometry?

Answer 2

185-400 nm

Question 3

What is the visible range in spectrophotometry?

Answer 3

400-800 nm

Question 4

What type of molecules absorb ultraviolet and/or visible light?

Answer 4

Many organic molecules

Question 5

What is a chromophore?

Answer 5

Responsible for light absorption, containing multiple bonds.

Question 6

What is an auxochrome?

Answer 6

Modifies light absorption, e.g. hydroxyl (-OH), amino (-NH2), aldehyde (-CHO) group.

Question 7

What does an absorbance spectrum show?

Answer 7

A number of absorbance bands corresponding to the bonding structure and functional groups present within the molecule.

Question 8

What does spectrophotometry measure?

Answer 8

Light transmittance (T).

Question 9

What is the formula for transmittance (T)?

Answer 9

T = It / Io

Question 10

What is Beer’s Law?

Answer 10

A = εcl, where A is absorbance, ε is molar absorptivity, c is concentration, and l is path length.

Question 11

What does ‘A’ represent in Beer’s Law?

Answer 11

The absorbance.

Question 12

What does ‘l’ represent in Beer’s Law?

Answer 12

The path length (cm) of the sample holder or cuvette.

Question 13

How can the concentration of an analyte be determined?

Answer 13

By measuring absorbance and applying Beer’s Law or plotting a reference standard curve.

Question 14

What are the types of spectrophotometers?

Answer 14

Single beam and dual beam.

Question 15

What are the sample requirements for spectrophotometry?

Answer 15

Sample must be soluble in either aqueous or organic solvent.

Question 16

What are examples of aqueous solvents?

Answer 16

• Water
• Buffer
• Acids
• Bases

Question 17

What are examples of organic solvents?

Answer 17

• Methanol
• Chloroform
• Dichloromethane

Question 18

If ε = 10,000 M-1cm-1 and absorbance = 0.400, what is the concentration?

Answer 18

0.04 mM

Question 19

What is the equation for a linear regression plot in data analysis?

Answer 19

y = mx + c

Question 20

What does ‘y’ represent in the equation y = mx + c?

Answer 20

The absorbance.

Question 21

What does ‘m’ represent in the equation y = mx + c?

Answer 21

The slope.

Question 22

What is a Colourimetric Assay?

Answer 22

A method where a compound undergoes a chemical reaction to produce a coloured complex in solution.

Question 23

What is the relationship between intensity of colour and concentration in a Colourimetric Assay?

Answer 23

Intensity of colour is proportional to concentration.

Question 24

What are some applications of spectrophotometry?

Answer 24

• Pharmaceutical Analysis
• Forensic Science
• Biopharmaceutical
• Colourimetric Assay

Question 25

What are some limitations of spectrophotometry?

Answer 25

* Absorbance is cumulative * UV-Vis spectra often not specific enough * Chemical dissociation can alter absorption * Non-linear calibration curve at high concentrations

Question 26

True or False: Beer’s Law applies at all concentrations.

Answer 26

False

Question 27

How can the Concentration of a analyte be determined

Answer 27

– measuring the absorbance of a solution and directly applying Beer’s Law (if ε is known) – by plotting a reference standard curve of absorbance against concentration. The curve can be used to calculate the unknown concentration of our sample