Experiment 3: Absorption, the Beer Lambert Law, and Fluorescence

Question 1

Wavelength (λ)

Answer 1

The distance between two consecutive peaks is called a wavelength

Question 2

Absorption (definition and how it can be explained)

Answer 2

The interaction of electromagnetic radiation with matter and is a quantum phenomenon.

Absorption can be explained by invoking the photon, which is the elementary particle responsible for carry electromagnetic radiation.

Question 3

What is a Jablonski diagram?

Answer 3

In a very simple manner it depicts the various energy states in which a hypothetical molecule can exist. The horizontal lines represent different energy levels, and each line represents a very specific energy state that is determine by the rules of quantum mechanics. (look at pg 34 for the pic). below is what it kind of looks like

^ S3 ____________
l ____________
l ____________
l S2 ____________
l ____________
l ____________
l ____________
l S1 ____________
l ____________
l ____________ V3
l ____________ V2
l ____________ V1
l S0 ____________
l ____________

BASICALLY, electrons can exist in many different energy states within a single molecule, and that these states exist at very specific and defined energy levels.

Question 4

What are two possible fates when a photon of light strikes a molecule?

Answer 4

1. it can either be scattered away from the molecule in some manner
2. it can be absorbed

Question 5

What determines if the photon is absorbed?

Answer 5

if the energy of the incoming photon exactly matches the energy difference between two distinctive energy levels, the photon can be absorbed and an electronic transition takes place whereby an electron in the ground state is excited to a higher energy orbital.

Question 6

How do spectrophotometers work?

Answer 6

diagram on pg 36.

light source –> white light –> monochromator –> slit (selected wavelengths get through) –> sample –> second slit –> detector

Question 7

What does the monochromator do?

Answer 7

Separate the light emitted from the lamp into as many different distinct wavelengths as possible. Done through either prisms or gratings

Question 8

What do the slits do?

Answer 8

Once the light emitted from the lamp has been subdivided, there has to be some way to select for the particular wavelength(s) needed for the particular application. One approach to achieving this is through the use of slits within a very narrow wavelength range.

Question 9

What does the detector do?

Answer 9

The function of detector is to gather and quantify raw intensity and sometimes the wavelength of the light reaching it.

Question 10

Why do we use blanks?

Answer 10

Blanks are reference solutions that have all the same components as the sample solution other than the particular analyte that you want to measure. This allows us to measure how much light was originally shining.

Question 11

What is incident light (I0)?

Answer 11

The intensity of the light reaching the detector for the BLANK is known as the incident light and is denoted as I0. and the intensity of the light when analyte is present is denotes as I.

Question 12

The formulation known as the Beer-Lambert Law recognizes that the variables affecting the absorbance of a sample are determined by three general considerations:

Answer 12

1. the wavelength used, and the probability that photons of that wavelength will be absorbed by the molecules in the sample.
2. the thickness of the absorbing material
3. the concentration of the absorbing material

Question 13

What is a calibration curve?

Answer 13

A calibration curve is a graph that depicts the response of an instrument to some analyte of interest under a specific set of conditions, so that you can determine the amount of the same analyte when its amount is unknown.

Question 14

What is an absorption spectrum?

Answer 14

If the absorption of light for a molecule is measured across a broad range of wavelengths, the absorbance values that are obtained can be assembled t/o into a graph known as an absorption spectrum.

Question 15

What are some factors affecting absorption?

Answer 15

1. solvents - they influence the creative stability of different electronic states in a molecule.
2. protonation - can have significant effects upon its absorption in certain regions of the spectrum.
3. redox state - many important biological processes involve the transfer of electrons from one chemical species to another.
4. interaction effects - the light absorbing properties of molecules can be changed when these molecules interact with other molecules.

Question 16

What are chromophores?

Answer 16

chromophores are structures within a molecule that absorb light.

Question 17

What makes fluorescence different then absorption?

Answer 17

What makes fluorescence different from absorption is what happens once the molecule is an excited state. When molecule fluoresces, some of the energy of the excited electron is dissipated by releasing a different photon of lesser energy than the photon originally absorbed. Absorption releases the energy of the excited electron as heat into the environment.

Question 18

What are some limitations of fluorescence?

Answer 18

1. scarcity of fluorescent molecules - relatively few molecules are fluorescentwhichmeans that one has to restrict one’e analysis to molecules that generate a useful fluorescent signal.
2. artefacts- to get around limitation 1: fluorescent problems can be attache to the molecule one is study but it raises the question of whether the attached fluorescent probe is affecting the properties of the molecule.
3. photobleaching - when fluorescent molecule is continuously exposed to the excitation light, a predictable percentage of of the fluorescent molecules will be destroyed.
4. toxic effects
5. cost

Question 19

What is one of the principle reasons that fluorescence is more sensitive than absorbance?

Answer 19

One principle reason for the differences in sensitivity is attributable to the fact that absorbance is based upon a difference between two signals, whereas fluorescence is based upon the absolute value of a signal compared to a theoretically zero background.