Spectrophotometry

Question 1

What is spectrophotometry?

Answer 1

The quantitative measurement of the reflection/transmission of a material as a function of wavelength

Question 2

In what units of measurement is wavelength measured?

Answer 2

ℷ - wavelength (nm)

Question 3

What state is the sample tested normally in?

Answer 3

Usually liquid form, can be solid or gaseous

Question 4

What is the average Abs or a DNA sample?

Answer 4

A²⁶⁰ of 1.0 = 50ugml⁻¹

Question 5

What is the function of the slit?

Answer 5

Selects the wavelength the light beam will pass through

Question 6

What is ℷmax?

Answer 6

Wavelength of the peak absorption

Question 7

What is the detector used?

Answer 7

A photodiode

Question 8

Which part of the wavelenght spectrum is spectrophotometry mainly used?

Answer 8

The visible spectrum - mainly in UV

Question 9

Why do nucleic acid abs. vary?

Answer 9

The abs value of nucleic acids varies with base composition

Question 10

A 10nM solution of diborubicin has an absorbance of 0.48 at 540nm (A⁵⁴º = 0.48). An unknown solution of the drug has an A⁵⁴º of 0.22.

Estimate the concentration of the unknown solution

Answer 10

Absorbance of 0.48 = 10nM

Absorbance of 1 = 10nM/0.48

Concentration of 0.22 = (10nM/0.48) x 0.22
= 4.58mM

Question 11

What is the role of the monochromator?

Answer 11

Splits light into different wavelengths and colours aka prism of difraction grater

Question 12

What is the relationship of absorbance to concentration?

Answer 12

Absorbance is additive -
so has a linear relationship with concentration
absorbance is proportional to concentration

Question 13

Why are plastic cuvettes a better choice than glass?

Answer 13

Glass cuvettes can absorb UV, therefore can alter readings

Question 14

What is the Beer Lambert Law?

Answer 14

A = 𝜺 x I x C

I = Path length (m)
C = concentration 
𝜺 = extinction coefficient (specific to each substance)

Question 15

Why are measurements usually observed at ℷmax?

Answer 15

Gives the maximum sensitivity
minimum slope
tiny errors are therefore insignificant

Question 16

What are the components of a spectrometer?

Answer 16

• light source
• lens
• monochromator
• wavelength selector/slit
• cuvette with sample solution
• detector
• digital display/meter

Question 17

What is the difference in absorbance spectra between molecules and atoms?

Answer 17

Atoms: have sharp spectra

Molecules: broad/multiple peaks as they absorb over a
range of wavelengths

Question 18

What is absorbance defined as?

Answer 18

Absorbance aka optical density

A = log₁₀(I₀/)
= log₁₀(T)

Question 19

What factors affect absorbance?

Answer 19

Wavelength (superscript nm)
Path length (subscript cm)
- absorbance is proportional to path length

Question 20

What does the Beer Lambert Law state?

Answer 20

The molar extinction coefficient (MEC) is the theoretical absorbance of 1cm of a 1M solution of the substance (M⁻¹ cm⁻¹ )

Question 21

How is transmittance defined?

Answer 21

T=I/I₀
(light in/ light out) x 100 = %

I₀ = intensity of incident light 
I = intensity of transmitted light

Question 22

A substrate has a molar coefficient of 3x10² M⁻¹ cm⁻¹ and the concentration is 1.5mM.

Calculate the absorbance

Answer 22

MEC = 3x10² M⁻¹ cm⁻¹

so 1M = 3x10² abs 
     1mM = 3x10²x10⁻³ abs 
     1.5mM = 3x10²x10⁻³ x1.5
                = 4.5x10⁻¹
                = 0.45

Question 23

Why is spectrophotometry useful in medicine?

Answer 23

Proteins, nucleic acids and many drugs absorb light in the visible spectrum or UV

Question 24

What controls can be added to ensure the spectrometer results are as accurate as possible?

Answer 24

• Use in the range of 0.1 - 1.5
• Use a homogenous solution
• Solution must be clear (suspended particles/dust give
  false readings)

Question 25

How is absorbance written?

Answer 25

e.g. A⁴⁵⁹ⁿ or A₁ₘ

Question 26

What are the major uses of spectrophotometry?

Answer 26

• Quantitation

- Checking DNA purity

Question 27

How can we use the beer lambert law to calculate molar concentration?

Answer 27

A = 𝜺 x I x C

so if I = 1cm
𝜺 = MEC
molar concentration = A/𝜺

Question 28

Why is the MEC only the theoretical absorbance?

Answer 28

Theoretical as solution isn’t entirely dilute

Question 29

Why is transmittance and absorbance unitless?

Answer 29

They are based on a ratio so have no units