Computer practical 1 on UV/Vis absorption, FTIR absorption and

Raman spectroscopy

In this practical you will work with real spectral data and handle the data using the program
Spectragryph, which is available under the chemistry folder in the start menu or if you work on your
own computer can be downloaded from the Nestor course homepage (do not download it from the
official Spectragryph site as you will need to request a licence etc).

The spectra are provided in their native format so cannot be opened directly in excel. You need first
to open the spectra in the program spectragryph and then extract the necessary data

The data for each exercise can be found in the sub folder with the exercise number (Set 0-6)

UNZIP ALL CONTENTS OF THE FOLDER BEFORE STARTING!!!! 😊

0. How is an absorption spectrum recorded

In this exercise you will learn how an absorption spectrum is recorded. The spectra you need
are in the folder ‘set 0’.

An absorption spectrum is calculated by first measuring the spectral density of the output of
a lamp (i.e. the intensity of the light as a function of wavelength)
Open the spectrum labelled ‘blank’

And then overlay the spectrum labelled sample

Click on the file tab and copy the data to clipboard

In excel, paste the data as three columns

Then use excel to calculate the transmittance spectrum

And then the Absorbance spectrum

You should be able to generate the three graphs shown below

Compare the spectrum that you have calculated with the spectrum ‘abs.sif’ in the folder.
The following exercises use the relations A =  c l = -log10 (I/Io)

Where A is absorbance,  is molar absorptivity in M-1 cm, c is concentration in M, l is pathlength in

cm, I is the intensity with the sample and Io is the intensity without the sample. For these data sets
the path length is 1 cm.

Be careful!! Not all of the spectra provided are useful – you have to decide which spectra are
meaningful!

1. Determination of molar absorptivity of Fe(III) porphyrin-tetrasulfonic acid

sodium salt (Mw = 942.60 g mol-1)
Open the spectra in folder ‘set 1’ in Spectragryph
Determine the molar absorptivity at around 450, 355 and 630 nm, the wavelengths chosen
should either be the maxima or where the absorbance does not change dramatically with
wavelength.
Remember that some spectra are not run well and the baseline (absorbance at long
wavelength is not zero). This needs to be corrected for.
Determine the molar absorptivity (  ) at each of the chosen wavelengths from a graph of
Absorbance against concentration.

2. Determination of molar absorptivity of Cu(II) porphyrin-tetrasulfonic acid

sodium salt (Mw = 984.25 g mol-1)
Do the same as in part 1 for Cu(II) porphyrin-tetrasulfonic acid sodium salt (set 2) using the
same wavelengths.

3. Determination of molar absorptivity of Rose bengal (Mw = 1017.64 g mol-1)

Do the same as in part 1 for Rose Bengal (set 3) using the same wavelengths.

In each case identify the range of concentrations for which the Beer lambert law holds.

4. Calculate the concentration of each of the compounds in the mixtures

Compare the spectra in set 1-3 with those of mixtures in set 4. Calculate the concentration of each
component for each spectrum. You can this by using the molar absorptivities at two or more
wavelengths and solving the equations (or by using the ‘scaled subtraction’ function in Spectragryph
– but this is more challenging).

Save your work as word/excel files (you can copy spectra to the clipboard)
FTIR and Raman spectra are available in set 5 and 6 respectively
5. Building a spectral database of compounds with FTIR.
Open the spectra in folder ‘set 5’ in Spectragryph.

When preparing a spectral database it is important to correct for spectral errors such as a
poor baseline. It is essential however that all changes are made to an absorbance spectrum
and not the transmittance spectrum.
For example:

Drag and drop a spectrum into Spectragryph and select the y-axis dropdown as shown below

Select Absorbance to convert from transmittance to absorbance.

Then go to the process tab as shown below and select simple baseline.
Click on a point on the baseline where there are no bands e.g. at 2200 cm-1

Then save the spectrum as a jcamp.dx format (this is an industry wide format).

You can do this for all the spectra in one go

First convert to absorbance

Then select ‘simple baseline’ and immediately click at a peak free point on the spectra e.g.
2400 cm-1 in this case.
You can save these spectra as a series of processed spectra
Make a new folder: and then carry out a batch export as shown
Tick the legends as filenames checkbox!
 a) Open the ‘unknown spectra’ folder and use this spectral data base to identify the
unknown compounds by comparison. It is faster if you first look at the unknown
spectrum and identify what functional groups are present.
b) Open the spectrum mixture of ‘three solvents’ and work out by comparison and band
assignment what the three solvents are.

6. Identification of impurity in a bottle of acetonitrile

There are a sets of Raman spectra of common compounds shown in set 6. The names of the
compounds are given on the label.

We haven’t done any Raman theory etc yet so for now threat the spectra as upside-down IR
spectra, with sometimes different band intensities than you would expect. Use these spectra
to identify the contaminant in the acetonitrile labelled ‘contaminated acetonitrile)

7. Identification of the solvents present in a mixture

There are a three FTIR spectra of mixtures of common compounds. Use the database of
solvents and chemical intuition to work out which solvents make up the mixtures
responsible for each of the three spectra.