Quiz 2 Flashcards
What is spectrophotometry?
is any technique that uses light to measure chemical concentrations
What is frequency as a property of light?
Is the number of complete oscillations that a wave of light makes per second. Denoted as u=oscillations/s
What do light waves consist of?
Perpendicular and oscillating electric and magnetic fields
What is wavelength as a property of light?
denoted as lambda, and is the crest to crest distance between waves.
What is a hertz?
Is one oscillation per second.
What is plane polarized light?
Light exists in two dimension that are perpendicular to each other (the magnetic and the electric) and therefore exist in the xy and xz axis. Plane polarized light is when light goes through an object that polarizes it and becomes 2D, as in light only travels in two axis (xy) for example rather than (xyz)
What is the mathematical relationship between frequency and wavelength?
frequency (u) times wavelength (l) equals c (speed of light) (3x10^8 m/s)
What changes when light moves between mediums with different refractive indexes?
The lights wavelength, but not it’s frequnecy
What is the equation for light in terms of energy?
Energy = Frequency (u) * Plancks constant (h)
What is Plancks constant?
it is h= 6.626 * 10^-34 J/s
What is the equation that relates frequnecy, energy, and wavelength?
E=hc/l or E=hc*ubar
What is ubar?
Is 1/lamda, and is called wavenumber, units in cm^-1
What happens when molecules absorb light in the x ray range?
Molecules break bonds and ionize
What happens when molecules absorb light in the ultra violet range?
electronic excitation
What happens when molecules absorb light in the infrared range?
They vibrate
What happens when molecules absorb light in the microwave range?
rotation
look at electromagnetic spectrum diagram and name the wavelengths for each type of light.
What is the ground state?
The lowest energy state of a molecules
What happens when light is absorbed by a molecule?
The energy of the molecule increases and the molecule is promoted to an excited state.
When light is absorbed by a sample what happen to the irradiance of the beam of light?
The irradiance of the beam of light is decreased.
What is irradiance?
Irradiance (denoted as I) is the energy per second per unit area of the light beam
Why does the irradience of a light beam decrease after it hits a sample? Explain with an example
For example if we have a light source which goes through a monochromator and then narrows the range of wavelengths to select one wavelength (Ltr), and then that wavelength of light hits our sample with a certain length, our sample will have absorbed some of the light so the irradiance of our light beam after it hits the sample (L0) is lower.
What is transmittance?
Transmittance (T) is defined as the fraction of the original light that passes through the sample, ie how much light passed through the sample from the original amount that hit it.
What is the eqn for transmittance?
T=Ltr/L0
What is absorbance?
A= log(L0/Ltr) = -log T
What is beers law?
It’s A =ebc, C is the concentration of the sample (in mol/l M), b is the pathlength of the sample 9in cm), and e is the molar absorptivity (M^-1 cm^-1). A is absorbance here.
What is molar absorptivity?
It tells us how much light is absorbed at a particular wavelength by a particular substance
How can beers law change if we measure how Absorbance or molar absorptivity depends on wavelength?
A=ebc becomes A(lambda)= e(lambda)bc
What is a chromophore?
The part of a molecule that absorbs light
How do we see colors in objects?
When white light (which contains all wavelengths of light in the visible spectrum) hits an object and is transmitted or reflected through it, the object absorbs certain wavelengths of the white light and our eyes detect the wavelengths that are not absorbed which is what make sit appear to have color
In what situations does beers law work?
Only works with monochromatic radiation and in dilute solutions (<0.01 M), by monochromatic radiation the bandwidth of the light must be smaller than the absorption band of the chromophore, and it also does not work samples in which the concentrations are dependent on an equilibrium as absorption differences between the two compounds can cause errors in beers law.
What is a cuvet?
Is a sample cell that has flat fused silica surfaces and contains liquid samples for spectroscopy, it has a common pathlength of 1.0 cm.
How do we use a cuvet?
We first measure the irradiance of light passing through a cuvet containing a pure solvent or reagent blank, and define that as L0 (so light before hitting sample). Then we measure the irradiance of light passing through through a cuvet with our sample in it and define that as LTr, knowing both L0 and LTR, we can get transmittance and absorbance values.
Why don’t we measure the incident irradiance (L0) directly? Why is it measured as the Ltr of our blank cuvet?
This is to account for any absorption, reflection, and scattering that can be done by the cuvet and solvent, so we get the exact absorbance and transmittance of our sample.
Are glass cuvets suitable for visible light? Uv?
yes they’re suitable for visible light but not uv because it absorbs uv,
What kind of cuvets are suitable for uv light?
UV light needs cuvets that are made of fused quartz
How do we measure the IR absorbance/transmittance of an unknown solid sample?
We take 1% wt mixture of the solid sample with KBr and grind it to a fine powder, then apply pressure (600 bar) which converts it into a translucent pellet, we do the IR spectroscopy and measure the absorbance of IR light (y axis) as a function of wavenumber.
Can you touch the clear faces of a cuvet?
No! This skews results of irradience from the sample and alters absorbance.
Why do we need longer sample cells (cuvets) for gasses? What size do we need?
We need longer pathlengths for cuvets (10 cm) when dealing with gasses because gasses are more dilute than liquids and therefore need a large surface area from the light to hit it.
Name 8 types of cuvets?
Standard 1 cm path
cylindrical
Micro cells
5 mm path
1 mm path
20 mm path
Flow
Thermal
Where should we measure absorbance from?
Measure absorbance at the wavelength of maximum absorbance, this is because the sensitivity of the analysis is the greatest at maximum absorbance, we get the maximum response for the analyte, and also because there’s less noise at the maximum absorbance so there’s little variation here if the monochromator drifts or if the width of the transmitted band changes slightly
What absorbance is most precise/reproducible in spectrometry? What should we do to our sample if we do not meet this abosrbance?
Absorbance values ranging from 0.3 to 2 are the most reproducible, as anything higher leads to too little light getting through the sample and anything lower leads to too much light getting through sample and therefore hard to differentiate between transmittance from the sample and that of the reference, you should try to adjust the sample conc so that it falls in this range.
What two things are needed for a compound to be analyzed by spectrophotometry?
The compound must absorb light, and this absorption should be distinguishable from that due to other substances in the sample.
Why do we usually have the visible region be where we do analysis by spectrophotometry?
This is because most compounds absorb UV radiation but specific compounds absorb visible light, if most compounds absorb UV radiation our results become inconclusive and we cannot tell which compounds absorption is which.
What is the first step in serum iron determination (measuring the iron content of transferrin in blood)?
Transferrin is protein in blood and has a binding site where Fe3+ binds.
You reduce Fe3+ to Fe2+ which allows the iron to be released from the protein, you do this by adding acid such as thioglycolic acid
What is the second step in measuring iron content of transferrin in blood?
You precipitate proteins by adding trichloroacetic acid, you then centerfuge the mixture to leave the iron in solution but remove the ppt proteins. You do this so ppt does not interfere with absorbance as light scattered by it could be mistaken for absorbance.
What is the third step in serum iron determination?
Transfer a measured volume of the supernatant liquid from step 2 to a fresh vessel, add buffer (to create a ph in which a complex can fully form) and add excess ferrozine to form a purple complex. Now measure the absorbance at the 562 nm peak.
Why should you measure a reagent blank before doing analysis of iron in serum?
Because you want to make sure all the absorbance from the sample is coming from the iron in the sample, you do this finding the absorbance of any iron impurities or uncomplexed ferrozine from the reagent blank and then subtracting it from your standard.
When creating calibration curves, what types of iron should we use to make our standard solutions?
Should use irons dissolved in acid like ferrous ammonium sulfate.
What is a problem in our iron serum determination procedure?
That results for iron will be around 10% too high because the blood serum also contains copper which also forms a coloured complex with ferroine, we address this by masking the copper through adding neocuprine/thiourea which form strong complexes with copper making them not want to interact w ferrozine.
What is spectrophotometric titration
Is where we monitor changes in absorbance during a titration to tell when the equivalence point has been reached.
Explain how spectrophotometric titration works with a solution of the iron transport protein transferrin?
Apotransferrin is transferrin without the iron molecules bonded to it and is colorless, each molecule of apotransferrin binds two Fe3+ ions, when fe3+ binds to it the protein turns into a red colour with an absorbance maximum at a wavelength of 465 nm. This absorbance is proportional to the concentration of Fe3+ bound to transferrin. As apo transferrin can only bind two fe3+ atoms and reaches its equivalence point here, we can use the maximum absorbance to determine the amount of iron it takes to reach the equivalence point and then use the stoich ratio of apo transferrin to two iron molecules to find he original amount of apotransferrin in the sample.
When titrating apo transferrin do we take the absorbances as is or do we correct them, if we correct them, why and how do we do that?
We correct them, this is because we have to take into account that when we add titrate to the sample we are also diluting the sample of apotransferrin and the volume is different at each absorbance. We correct absorbance so that it shows what the absorbance would be if the sample was never diluted in order to get accurate absorbance values, corrected absorbance = (tot volume/initial volume)(observed absorbance)
What happens when a molecule absorb light?
If a molecule absorbs light that has sufficient energy to cause electronic transition (ie vibrational and rotational transitions), then changes in the vibrational and rotational states of the molecule will occur.
Why are electronic absorption bands broad?
Because many different vibrational and rotational levels are available at slightly different energies (wavelengths)
What actually happens when a molecule absorbs energy?
When a molecule absorbs energy (like a photon) that promotes the molecule from it’s ground state (S0) to a vibrationally and rotationally excited electronic state (S1). Then from this S1 state a nonradiative transition occurs (R1) where the molecule goes through vibrational relaxation- giving it’s vibrational energy to other molecules through collision and it lowers down to it’s lowest vibrational level of S1. Converting some of the photons energy to heat.
What can happen once the molecule has gone through non radiative transition and reached the lowest vibrational level of S1?
- The molecule could go through internal conversion (IC) where is enters a high excited vibrational level of S0 which is at the same energy level as the lowest vibrational level of S1.
From this excited state the molecule can relax back to the ground vibrational state (S0) and transfer the energy lost to other particles through collision
(this process is R2). If the molecule follows this path (A-R1-IC-R2), the entire energy of the photon will have been converted into heat.
What other path (not A-R1-IC-R2) could the molecule go through?
After nonradiative transition (R1) the molecule can go through (ICS- intersystem crossing), where the molecule goes from S1 to T1 (triplet electronic state) go through another non radiative transition (R3) and then go through another ICS to go back to S0 followed by another non radiative transition (R4) to reach the lowest energy state in the ground electronic state. This process converts light to heat.
What is luminescence?
Is emission of light from an excited state of a molecule.
What path could a molecule go through if it is emitting a photon?
A molecule could relax from an excited state 9S1 or T1) to a ground state (S0) by emitting a photon.
What is fluorescence?
The radial transition from S1 to S0
What is phosphorescence?
The radial transition from T1 to S0
Whats the difference between a singlet sate and an triplet state?
In a single state the electron are still facing opp directions and are still paired, in a triplet stage electrons are facing opposite directions (and are unpaired).
What is SBS? How is it set up?
SBS is a single beam spectrophotometer, and has one light source, that hits one monochromator, and then one sample, and then hits the light detector.
