Spectrophotometry Flashcards

1
Q

What is spectrophotometry?

A

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

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2
Q

In what units of measurement is wavelength measured?

A

ℷ - wavelength (nm)

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3
Q

What state is the sample tested normally in?

A

Usually liquid form, can be solid or gaseous

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4
Q

What is the average Abs or a DNA sample?

A

A²⁶⁰ of 1.0 = 50ugml⁻¹

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5
Q

What is the function of the slit?

A

Selects the wavelength the light beam will pass through

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6
Q

What is ℷmax?

A

Wavelength of the peak absorption

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7
Q

What is the detector used?

A

A photodiode

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8
Q

Which part of the wavelenght spectrum is spectrophotometry mainly used?

A

The visible spectrum - mainly in UV

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9
Q

Why do nucleic acid abs. vary?

A

The abs value of nucleic acids varies with base composition

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10
Q

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

A

Absorbance of 0.48 = 10nM

Absorbance of 1 = 10nM/0.48

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

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11
Q

What is the role of the monochromator?

A

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

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12
Q

What is the relationship of absorbance to concentration?

A

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

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13
Q

Why are plastic cuvettes a better choice than glass?

A

Glass cuvettes can absorb UV, therefore can alter readings

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14
Q

What is the Beer Lambert Law?

A

A = 𝜺 x I x C

I = Path length (m)
C = concentration 
𝜺 = extinction coefficient (specific to each substance)
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15
Q

Why are measurements usually observed at ℷmax?

A

Gives the maximum sensitivity
minimum slope
tiny errors are therefore insignificant

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16
Q

What are the components of a spectrometer?

A
  • light source
  • lens
  • monochromator
  • wavelength selector/slit
  • cuvette with sample solution
  • detector
  • digital display/meter
17
Q

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

A

Atoms: have sharp spectra

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

18
Q

What is absorbance defined as?

A

Absorbance aka optical density

A = log₁₀(I₀/)
= log₁₀(T)

19
Q

What factors affect absorbance?

A

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

20
Q

What does the Beer Lambert Law state?

A

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

21
Q

How is transmittance defined?

A

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

I₀ = intensity of incident light 
I = intensity of transmitted light
22
Q

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

Calculate the absorbance

A

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
23
Q

Why is spectrophotometry useful in medicine?

A

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

24
Q

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

A
  • Use in the range of 0.1 - 1.5
  • Use a homogenous solution
  • Solution must be clear (suspended particles/dust give
    false readings)
25
Q

How is absorbance written?

A

e.g. A⁴⁵⁹ⁿ or A₁ₘ

26
Q

What are the major uses of spectrophotometry?

A
  • Quantitation

- Checking DNA purity

27
Q

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

A

A = 𝜺 x I x C

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

28
Q

Why is the MEC only the theoretical absorbance?

A

Theoretical as solution isn’t entirely dilute

29
Q

Why is transmittance and absorbance unitless?

A

They are based on a ratio so have no units