Spectrophotometry Flashcards
What is spectrophotometry?
The quantitative measurement of the reflection/transmission of a material as a function of wavelength
In what units of measurement is wavelength measured?
ℷ - wavelength (nm)
What state is the sample tested normally in?
Usually liquid form, can be solid or gaseous
What is the average Abs or a DNA sample?
A²⁶⁰ of 1.0 = 50ugml⁻¹
What is the function of the slit?
Selects the wavelength the light beam will pass through
What is ℷmax?
Wavelength of the peak absorption
What is the detector used?
A photodiode
Which part of the wavelenght spectrum is spectrophotometry mainly used?
The visible spectrum - mainly in UV
Why do nucleic acid abs. vary?
The abs value of nucleic acids varies with base composition
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
Absorbance of 0.48 = 10nM
Absorbance of 1 = 10nM/0.48
Concentration of 0.22 = (10nM/0.48) x 0.22
= 4.58mM
What is the role of the monochromator?
Splits light into different wavelengths and colours aka prism of difraction grater
What is the relationship of absorbance to concentration?
Absorbance is additive -
so has a linear relationship with concentration
absorbance is proportional to concentration
Why are plastic cuvettes a better choice than glass?
Glass cuvettes can absorb UV, therefore can alter readings
What is the Beer Lambert Law?
A = 𝜺 x I x C
I = Path length (m) C = concentration 𝜺 = extinction coefficient (specific to each substance)
Why are measurements usually observed at ℷmax?
Gives the maximum sensitivity
minimum slope
tiny errors are therefore insignificant
What are the components of a spectrometer?
- light source
- lens
- monochromator
- wavelength selector/slit
- cuvette with sample solution
- detector
- digital display/meter
What is the difference in absorbance spectra between molecules and atoms?
Atoms: have sharp spectra
Molecules: broad/multiple peaks as they absorb over a
range of wavelengths
What is absorbance defined as?
Absorbance aka optical density
A = log₁₀(I₀/)
= log₁₀(T)
What factors affect absorbance?
Wavelength (superscript nm)
Path length (subscript cm)
- absorbance is proportional to path length
What does the Beer Lambert Law state?
The molar extinction coefficient (MEC) is the theoretical absorbance of 1cm of a 1M solution of the substance (M⁻¹ cm⁻¹ )
How is transmittance defined?
T=I/I₀
(light in/ light out) x 100 = %
I₀ = intensity of incident light I = intensity of transmitted light
A substrate has a molar coefficient of 3x10² M⁻¹ cm⁻¹ and the concentration is 1.5mM.
Calculate the absorbance
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
Why is spectrophotometry useful in medicine?
Proteins, nucleic acids and many drugs absorb light in the visible spectrum or UV
What controls can be added to ensure the spectrometer results are as accurate as possible?
- Use in the range of 0.1 - 1.5
- Use a homogenous solution
- Solution must be clear (suspended particles/dust give
false readings)
How is absorbance written?
e.g. A⁴⁵⁹ⁿ or A₁ₘ
What are the major uses of spectrophotometry?
- Quantitation
- Checking DNA purity
How can we use the beer lambert law to calculate molar concentration?
A = 𝜺 x I x C
so if I = 1cm
𝜺 = MEC
molar concentration = A/𝜺
Why is the MEC only the theoretical absorbance?
Theoretical as solution isn’t entirely dilute
Why is transmittance and absorbance unitless?
They are based on a ratio so have no units
