-A spectrophotometer in an instrument that measures the amount of light at a specific wavelength which is absorbed by a sample. A spectrum is a plot relating the quantity of light absorbed to its wavelength (energy). The following plot is the spectrum of chlorophyll in the solvent diethyl ether. We can learn a

lot about thestructure of a compound

by studying its spectrum. Spectroscopic

techniquesare commonly use to identify

unknown compounds and their

concentrations in dilute solutions.

Furthermore, (in the absence of

photochemical reactions),

spectroscopic analysis does not alter

the identity of the compound while

Fig1. Spectrum of chlorophyll in diethyl ether

investigating it. This makes it an

invaluable tool in the study of chemical

reactions. In experiment 4a you will

create the spectrum of aqueous potassium permanganate.

Spectrophotometers are some of the most advanced equipment used in scientific research. The fundamental theory behind their operation is rather easy to understand. The Spec-20 is a single beam spectrometer and can be considered to consist of 4 major components,

a light source, a monochromator (which creates light of a single wavelength), a sample

cavity and a detector.

There are various types of detectors but they

can all be understood from the principles of

the photoelectric effect, which we studied in

the first semester. When a photon of light hits

the detector it has enough energy to release an

electron which causes a current to flow. The

amperage (flow) of the electric current is

proportional to the flux (flow) of photons and

thus the intensity of the current becomes

proportional to the intensity of light incident

upon the detector. In the Spec-20 this current

is used to create a magnetic field which

deflects a (galvometer) needle and the amount

of deflection becomes a measure of the intensity of light.

In the following procedures you will fill the sample cavity

with the solvent (the blank) and adjust the galvometer

needle to "100" % transmittance (0 absorbed) for a

specific wavelength of incident light. Then you will

place in the cavity the solute you are measuring (dissolved

in the same solvent) and any reduction in light transmitted

will be a result of light absorbed by the solvent . As the

solvent has an absorption spectrum you must "rezero" its

absorbance at each wavelength.

When you look at the scales of the Spec-20 you will see

2 different scales. The top scale reads from zero to 100%

transmittance (reading left to right) while the bottom scale

reads from infinity to zero absorbance (effectively right to

left in terms of increasing absorbance). The top scale

is a linear scale measuring the % transmittance while the

bottom scale is a logarithmic

scale measuring the

absorbance. Chemists are

usually interested in the absorbance

and not the transmittance as the

absorbance is proportional to the

concentration of the compound

absorbing the light.

2. Instrumental Operating Procedures:

100% Transmittance/Light

Control

-Turn Power On with Power/Zero Control Knob (red light goes on)

-Turn light control knob (right) counterclockwise and back up ½ turn to protect the phototube

-Wait 15 minutes

-Turn amplifier knob so the needle

points to zero on % transmittance scale

(0 is on left)

-Set wavelength scale to

desired value with wavelength

control knob

-Insert cuvette with solvent (balnk)

into sample holder

(see section on sample handling)

-Close top of sample holder

-Adjust %Transmittance to 100% with

the light control knob

-Remove blank and place the sample

into the compartment

and measure the %T or

absorbance at this wavelength.

NOTE: There are two models

in the lab: the Spec-20 and

Spec-20+.

The Spec-20+ has better optics

but requires a filter to be

properly set in either the

340-599 nm range or the

600-950 nm range.

3. Tips on using the Spec-20:

-Always read the scale

perpendicular to avoid

parallax errors. The best

angle is where you can not

see the shadow of the needle.

Parallax Error

Correct

-Tips of handling cuvettes

-All readings are used with

cuvette which are similar

to test tubes but optically

transparent in the UV-vis

region of the spectra

-Wash the outside of the

cuvettes with water and

dry with Kimwipes.

-Fingerprints and any external smudges can

give false absorbance readings

-Once cleaned always handle cuvettes by the top

-Rinse the inside of the cuvettes thoroughly with

the solution you are measuring.

-Be sure to avoid any bubbles. You can do this by

adding fluid with a Pasteur pipet and you can tap

the side to remove any bubbles which may form

-Fill cuvettes 2/3rd to

3/4ths with solution.

- Do not overfill as

you can spill samples

into the sample

compartment and d

amage the instrument.

-Always use the same orientation

when placing the cuvette in the

sample compartment.

-This can be done by aligning

the mark on the cuvette

with the raised nub on the front

of the sample holder.

-Never leave cuvettes in

the spectrometer after taking

a reading.

Derivation of Beer's Law:

Beers law is based on a few basic assumptions and its derivation requires calculus like many of the equations we will using this semester. Calculus is not a prerequisite for this class and you can memorize beer's law. To understand beer's law we ask ourselves what variables influence the amount of light absorbed by a solution. There are 3 variables for a given wavelength; the concentration (C) of absorbing compounds (chromophores), the distance the light travels (DX) in the solution (path length) and the intensity (I) of light itself (noting that intensity of light is related to the number of photons while the energy is related to the wavelength). Mathematically we use a proportionality constant (k) to state that the change in light intensity is proportional to these 3 factors.

The negative sign is a result of the fact that the intensity decreases as the light is absorbed.

The solution of this equation requires the mathematics of change, calculus, where dI represents an infinitesimally small change in the intensity correlated with an infinitesimally small change in the path length (dx): Once again we see why logarithms are so important in science. Note that in going from the natural logs of the calculus to the log base 10 of beers law we use the relationship lnX=2.303log x and so k = 2.303a.