UV-

• Whats the wavelength of visible light?

  400-700nm
• Whats the wavelength of UV?

  200 - 400nm
• What type of molecules absorb UV/visible light?

  Only molecules with conjugated double bonds absorb UV/visible light

  The longer the conjugation the longer the wavelength absorption.

  
• What happens when you shine a beam of radiation through a sample?

  Some of the radiation is absorbed by the sample

  Some of the radiation is scattered when it hits larger particles.

  Some of the radiation will pass straight through - transmitted

  
• How do you measure the amount of radiation absorbed the sample?

  You'll do a bank sample that contains anything apart from the analyte and measure the amount of light that was transmitted.

  Repeat this but with the analyte included and measure the difference of intensity of the transmission.
• Why do you need to do a blank sample in order to measure the amount of radiation absorbed?


  To minimise the effects of scattering light
• Whats transmittance?

  is the amount of radiation which passes through the sample
• How is transmittance calculated?

  T = 𝐼 /I o X 100

  I = Intensity of light from sample solution - value should be lower than blank

  Io = intensity of light from blank sample - should be 100% transmittance

  Find the

  
  

  If you do log 10 of ( 𝐼 /I o) that will give you the absorbance

  
• What does 100% and 0% transmission mean?

  100% transmission = no absorption of sample so same amount of light thats been transmitted from sample and blank.

  0% transmission = sample is absorbing everything thats coming through
• What does transmittance depend on?

  Path length of the cell = distance of the sample thats passing through

  Concentration of the absorbing substance = the more molecules you have the more likely its going to interact with the light

  Nature of the substance = how strongly the substance interacts with light
• Whats the Beer-Lambert Law?

  The dependence of absorbance on concentration, and path length.

  A= acl

  A= absorbance

  a = absorbance coefficent (constant)

  l- path length
• What will be the units for absorption coefficient?

  It depends on the units of conc and path length.

  If path length is in cm

  Conc in mol dm-3

  Then a = dm3 mol-1 cm-1
• A 2.305 x 10-5 M solution of a substance had an absorbance of 0.497 in a 0.5 cm path length cell. Calculate the molar absorption coefficient for the substance.?

  43100 dm3 mol-1 cm-1

  
• Whats specific absorbance?

  Specific absorbance is used when the concentration is expressed as a mass percentage and path length is in cm.

  It represents the absorbance of 1% m/v solution in a cell of path length of 1cm

  C= % m/v (g/100cm3)
• The absorbance of a 0.00140% m/v solution of tolbutamide in methanol when measured in a 1 cm path length cell was found to be 0.715 at 228 nm. Calculate the specific absorbance?

  511

  This means the absorption of a 1% solution of a path length of 1cm is 511.

  You keep it as 0.00140 and not divide by 100 as the percentage means g/100cm3 so the value is already given

  
• What does absorption depend on?


  When choosing a specific wavelength you should choose one where only the analyte absorbs with no absorption from any impurities

  
  

  Absorption depends on the wavelength of light and you can plot this on a graph.

  You can choose a specific wavelength when designing an assay so that the absorption is strong = high peak - corresponds to absorption maximum

  
• Why should you aim for the wavelength to correspond to the absorption maximum?

  You choose the wavelength where the substance has its absorption maximum because that's when it absorbs light most efficiently. So, if you're doing dilutions and lowering the concentration of your substance, the detector can still pick up the absorbance, even if the concentration is very low.

  This provides better sensitivity for measuring low concentrations of the analyte.

  
  

  Also avoids any impurities from absorbing
• How can you convert the measured absorbance into a concentration?

  Plot a calibration plot using standard solutions of a pure substance and measure its absorbance and plot the graph and extrapolate the graph.

  Beer-lambert law with a reported absorption coefficent
• Whats a disadvantage of using the calibration plot over the beer lambert?

  This method can only be used if a pure sample of the analyte is available, which is not always the case
• A solution of methyltestosterone had an absorbance of 0.890 at 241 nm in a 1 cm path length cell. Given that A(1%, 1 cm) = 540 at 241 nm, calculate the concentration of the methyltestosterone in the solution?

  0.00165%

  
• How would you measure the conc of an unknown sample that has a mixture of components?

  Each component of the mixture will absorb light at different wavelengths.

  The absorbance at a given wavelength will be the sum of the absorbance of all the constituents in that mixture.

  
• What are chromophores?

  Parts of the molecule thats responsible for its ability to absorb light at a specific wavelength
• How will you measure conc if 2 substances in the mixture have dissimilar chromophores?

  Then they will absorb light at different maxima

  At wavelength 1 you can measure whatever molecule a is measuring at wavelength 1 + the same for molecule b at the same wavelength

  Repeat at wavelength 2

  Absorption coefficient can be detemined from measurements made on pure solutions solutions of known conc.

  
• The absorbance of a mixture of the amino acids tyrosine (Y) and tryptophan (W) is measured by UV spectroscopy at wavelengths 268 and 290 nm. Calculate the concentrations of tyrosine and tryptophan in this mixture from the data below?

  7.27 x 10-5M

  
• Whats the difference between precision and accuracy?

  Precision: how close measurements are to each other. Accuracy: how close measurements are to the true value.
• How would you see if you have a measurement error?

  Plot a U-shaped curve of percentage error of absorbance against true absorbance

  
• Interpret the graph

  An absorbance between 0 -0.1 % produces a higher percentage error

  An absorbance above 0.2% produces lower percentage error
• Whats the optimal range to get high precision?

  0.2-0.8 AU
• What happens if you have an absorbance outisde the range and what can you do to improve it?

  Higher percentage error

  You can either dilute or concentrate the sample
• What happens at low absorbance?

  Low precision because At low absorbance, most of the light passes through the sample, and very little is absorbed. This makes it harder to detect small changes, leading to low precision in measurements.

  For instance if from your reference sample you measure 100 +- 0.1 and then you measure light intensity from sample and get 98 + - 0.1 the difference is very small.

  Since the measured change is tiny compared to the total light passing through, small errors in measurement can have a big impact on the final result.
• What happens at high absorbance?

  Poor precision : Sample is absrobing a lot of light and very little reaches the detector. Small changes in this tiny amount of light can cause big errors

  For instance, if you have a reference that measures 100 and your sample measures 5 that's a 20% error, as there's not enough light going through it to give an accurate detection.
• Why is it that some calibration curves dont form a straight line?

  If you change conc the chemicals may change.

  At high conc the molecules may associate with each other which will change the interaction with light, absorption coefficient will change.

  Stray light may interfere: Light from outside the intended wavelength range (stray light) can affect readings,

  Non-monochramatic radiation- If the spectrophotometer does not use perfectly single-wavelength (monochromatic) light, different wavelengths may be absorbed differently,
• Whats scattering?

  Light deflected in all directions - happens in larger molecules
• Whats optical density?

  All the losses of light including scattering and absoption
• What does a high optical density?

  High concentration of particles - means that very little light passes through a sample because most of it is absorbed or scattered.
• Whats luminescence?

  Emission of light by molecules without heat
• Give types of luminescence?

  Fluorescence

  Phosphorescence (light of stars you put up in children room)

  Chemiluminescence: ( light emitted from chemical reactions

  
• Whats fluorescence?

  When molecules absorb UV or visible light, the absorbed energy by electron move onto a higher state, that energy gets dissipated through heat and used to collide with our molecules.

  However, some excited molecules are able to re-emit the absorbed energy as light, as the electrons move back down to a lower energy state - fluroscence.
• Whats chemi-luminescence?

  In some chemical reactions, The product molecules are left in the excited state, and when they return back to a lower-energy state, the light emitted is chemi-luminescence
• Whats bio-luminescence?

  A a form of chemiluminescence that occurs in living organism that emit light like glow worms