Spectrophotometry

Jonas Steinhauser, pharmazeutische Wissenschaften, 4. Semester,

eMail: Jonasste@student.ethz.ch

Alain Haeller, Biologie, 4. Semester,

eMail: salain@student.ethz.ch

Assistant: Lee Kwang Geol

The experiment consists of four parts.

ˆ We measured a UV/VIS-spectogram while variating the parameter box-
carwidth. This parameter sets the number of detected points. Therefore
it’s defining the spectral resolution.
ˆ Observation of false-light effects and resulting non-linearity. Using a
series of diluted solutions of a concentrated standard dye solution we
obtained spectra with which we examined the influence of flase light at
different degrees of absorbance. It became visible that a absorbance >
1.5 may correlate with non-liniarity. That means Beer’s law cannot be
applied in a range like this.
ˆ Using MLRA-method we calculated the relative concentration of two
different substances of unknown concentration(which were available for
spectra in pure form).
ˆ Examining the influence of temperature on a system of cobalt(II)chloride
we found that the substance exists in two different complexes which ab-
sorb at different wavelenghts and therefore appear in different colors.

Zürich, May 26, 2010 J.Steinhauser, A. Haeller

 J. Steinhauser,
Spectrophotometrics A. Haeller

Contents

1 Introduction 3
1.1 Beer’s law . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 False-light effects and nonliniarity . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 Multi linear regression analysis (MLRA)-Method for bianry mixtures . . . 4
1.4 Temperature dependent spectra . . . . . . . . . . . . . . . . . . . . . . . . 5

2 Experimental section 5
2.1 Used chemical substances . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2 Setting, procedure and apparture . . . . . . . . . . . . . . . . . . . . . . . 7
2.2.1 Setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.2.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3 Experimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3.1 Measurement parameters . . . . . . . . . . . . . . . . . . . . . . . 9
2.3.2 False-light effects and Nonlinearity . . . . . . . . . . . . . . . . . 9
2.3.3 MLRA methods for binary mixtures of coloring materials . . . . . 9
2.3.4 Temperature dependent spectra . . . . . . . . . . . . . . . . . . . . 9

3 Discussion 10
3.1 Measurement parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2 False-light effects and Nonliniarity . . . . . . . . . . . . . . . . . . . . . . 11
3.3 MLRA for binary mixtures of coloring materials . . . . . . . . . . . . . . 13
3.4 Temperature dependent spectra . . . . . . . . . . . . . . . . . . . . . . . . 16

4 Possible sources of error 16

4.1 Error reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

5 Attachments* 17
5.1 Measurement Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
5.2 False-Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
5.3 MLRA methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
5.4 Temperature dependent spectra . . . . . . . . . . . . . . . . . . . . . . . . 20

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 J. Steinhauser,
Spectrophotometrics A. Haeller

1 Introduction
1.1 Beer’s law
Light consists of photons which are arranged in a quantized energy order. There is a
relation between the frequency ν, wavelength λ and energy for radiation (h = 6.626 ∗
10−34 Js ; c = 2.998 ∗ 108 m
s) :
νλ = c (1)
E = hν (2)
Let’s take a look on molecules:
The energetic state of a molecule(Etot ) can splitted in elektron-(Eel ), vibration-(Evib )
and rotation-(Erot ) states:
Etot = Eel + Evib + Erot (3)
If light meets a molecule energy can be converted into these states. One can assume
that an electron becomes elevated onto a higher energylevel (namely from Em to En ).
In this case we get:
Em = Em,el + Em,vib + Em,rot (4)
En = En,el + En,vib + En,rot (5)
(6)
∆Emn = En − Em (7)
= hνmn (8)
for the energy needed to transfer one electron. One can easily imagine that light looses
more energy the longer it crosses a certain medium. This loss of intensity can be ex-
pressed in a formula. Where I(λ; x) is the intensity of a monochromatic light-beam
with wavelength λ which declines (ingoing intensity = I0 (λ)) along the direction of it’s
movement x. The decline is proportional to the concentration of the medium c with a
thikness dx and a sbstance-specific value: α(λ):
dI(λ; x) = −α(λ)cI(λ; x) dx (9)
Through integration we get the amount of light which passed through our sample, the
transmitted intensity :
IT (λ) = I0 (λ)e−αcd (10)
The relation between incoming and outgoing intensities is called transmission T:
IT (λ)
T (λ) = (11)
I0 (λ)
The absorbance A is defined as:
A(λ) = −log1 0T (λ) (12)
= (λ)cd (13)
expression (13) is also called Beer’s law.

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Spectrophotometrics A. Haeller

1.2 False-light effects and nonliniarity

Looking at practical daily-work one notices that calculations with Beer’s law don’t match
measured values any more. Seemingly at high absorbance the detector not only detects
intentionously emitted light but also light from different bands of the spectrum. The
real measured absorbance then is:
Io0
A0 = log (14)
I0
I0 + If If
= log ||δ = (15)
T I0 + If I0
1+δ 1+δ 1+δ
= log = log −A = log −cd+δ (16)
T +δ 10 + δ 10
From practical observations one can tell the absorbance at a high value approximates

A0 = −log(δ) (17)

which is dependent on the rate of false-light and the wavelength.

1.3 Multi linear regression analysis (MLRA)-Method for bianry mixtures

n
X n
X
A(λ) = A(λ) = d k (λ)ck (18)
k=1 k=1

Equation (18) is true in a solution of n independent absorbing substances k. To analyse

the concentrations c1 and c2 of substances 1/2 we need to know the absorbances:

A1 (λ) = 1 (λ)c1 d (19)

A2 (λ) = 2 (λ)c2 d (20)

using (18) we get the absorbance of our mixture Am (λ):

Am (λ) = 1 (λ)c1 d + 2 (λ)c2 d (21)

(21) divided by (19) 1,2 substracted:

Am (λ) cm,1 cm,2 A2 (λ)

= + (22)
A1 (λ) c1 c2 A1 (λ)

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Spectrophotometrics A. Haeller

1.4 Temperature dependent spectra

The blue anhydrous complex of cobalt(II) chloride and the pink hexahydrate-complex
can be made visible and converted in another by several procedures. E.g. by heating.
The human eye catches the beams reflected by an object and converts them to vision.
If a complex appears e.g. blue it absorbs the other wavelengths in the light. Therefore
we expect an absorbance at λ = 500 − 750nm (comp. fig. 1)

Figure 1: Trivial names of light (colors) corresponded to wavelength λ 8

2 Experimental section
2.1 Used chemical substances

Table 1: Used chemical substances

R und S -Sätze
S 45 In case of accident or if you feel unwell, seek medical
advice immediately (show the label where possible).
S 53 Avoid exposure – obtain special instructions before use.

R 36/38 Irritating to eyes and skin..

Benzol 6 R 45 May cause cancer.
R 11 Highly flammable..
R 23/24/25 Toxic by inhalation, in contact with skin and if
swallowed.
R 46 May cause heritable genetic damage.
R 48 Danger of serious damage to health by prolonged ex-
posure.
R 65 Harmful: may cause lung damage if swallowed.

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Spectrophotometrics A. Haeller

Table 2: Used chemical substances

R und S -Sätze
S 24 Avoid contact with skin.
S 37 Wear suitable gloves.
S 61 Avoid release to the environment. Refer to special in-
structions / Safety data sheets.

Acridin 4 R 22 Harmful if swallowed.

R 36/37/38 Irritating to eyes, respiratory system and skin.
R 50/53 Very toxic to aquatic organisms, may cause long-
term adverse effects in the aquatic environment.

Table 3: Used chemical substances

R und S -Sätze
S 9 Keep container in a well-ventilated place.
S 26 In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice.
HCl 7 S 36/37/39 Wear suitable protective clothing, gloves and
eye / face protection.
S 45 In case of accident or if you feel unwell, seek medical
advice immediately (show the label where possible).

R 23 Toxic by inhalation.
R 35 Causes severe burns.

Table 4: Used chemical substances

R und S -Sätze
R 36 Irritating to eyes.

Acridon 5 S 26 In case of contact with eyes, rinse immediately with

plenty of water and seek medical advice.
S 39 Wear eye / face protection.

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 J. Steinhauser,
Spectrophotometrics A. Haeller

2.2 Setting, procedure and apparture

2.2.1 Setting

Figure 2: Setting for further Information see 5

Table 5: Used technical apparature

Instrument Producer Type
Laptop Dell Latitude D610
Operating system Microsoft Windows XP
Analysis software Ocean optics Spectra suite
Cooler Lauda E 003
Thermometer Lauda E 100
UV/VIS-spectometer not known USB4F03453
Lightsource Micropack DH-2000
Cuvette not known fused quartz glass 10mm3

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 J. Steinhauser,
Spectrophotometrics A. Haeller

Figure 3: Two-beam spectrophotometer Type PERKIN ELMER λ5. 2 p.206

Coming from a deuterium lamp (DL) the beam is dispersed in it’s spectral components
and fokussed onto the layer of the exit-crack (SA) via a turnable concave fence (G1).
The crack filters a certain bandwidth by this determinig the sepctral resolution of the
apparature. The filtered monochromal beam is either reflected by a rotating mirror
(C) into the sample-gait or let through into the reference-gait. After passing through
the sample the two beams get onto the detector(Photomultiplier, PM) with different
intensities I0 (Reference) I(Sample). The signals from the detector are converted into
digital and processed. 2 p.206

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Spectrophotometrics A. Haeller

2.2.2 Procedure
For every measurement one cuvette was filled with approx. 9 ml of the sample, while
the other was filled with the solvent(mostly water) for the baseline measurement. For
measuring the actual substances we used a deuterim-lamp. Before refilling the cuvette
they were cleaned with distilled water then washed out about four times with the new
sample.

2.3 Experimentation
2.3.1 Measurement parameters
Take absorption spectra of a broad absorbing species (organic dye solution: Acridin) and
of a narrow absorbing species (e.g. gas phase Benzene). Use different boxcar settings
from 0 to 20 (step size=4).

2.3.2 False-light effects and Nonlinearity

Use a series of appropriately diluted solutions of a the concentrated standard dye solution
to obtain absorption spectra and to check Beer’s law over a broad concentration range.
Material: Acridin (C13 H9 N ) Solvent : in water Spectrometer parameters: 460-260
nm scan range Dilute the solutions using the precision balance (read to 0.01 g). Take
spectra of at least 10 different solutions.

2.3.3 MLRA methods for binary mixtures of coloring materials

Using the standard solutions below prepare two different mixtures of exactly known
compositions (use the precision balance):

- Material 1 : Acridin (C1 3H9 N ) or p-Vanilline

- Material 2 : Acridon (C1 3H9 N O) or o-Vanilline (CH3OC6H3-2-(OH)CHO)
- Solvent : in water or 0.01 M NaOH

Measure the abosrption spectra of the two standards and of the two mixtures.

2.3.4 Temperature dependent spectra

First of all Measure the temperature dependent absorption spectra of an alcoholic aque-
ous solution of cobalt(II) chloride. Follow the procedure D outlined in Shakhashiri 3 .
Take different spectra between room temperature and approx. 60°C, reading the true
solution temperature with the digital thermometer and the fine thermoelectric couple.

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Spectrophotometrics A. Haeller

3 Discussion
3.1 Measurement parameters

Figure 4: Absorbance at λ = 220 − 270nm measured with Benzol(g) in a 1 cm cuvette.

Comparing boxcarwidth= 0 with boxcarwidth = 20

The features seen between λ = 200 − 300nm are distinctive for the Benzene molecule
(comp.fig. 12). Increasing the boxcarwidth(∆λ in nm) results in a reduction of measured
points along the wavelength λ (comp. fig.4). Thus the outcoming spectrum is less precise.
The areas under the plots are not compareable any more. Distinctive features e.g. the
two lefthanded outer peaks of Boxcar=0 are getting lost. This trend develops as ∆λ is
getting bigger (comp.fig. 4, or 11).

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Spectrophotometrics A. Haeller

3.2 False-light effects and Nonliniarity

Figure 5: Ansorbance of Acridin in aqarous solution at different (relative) concentra-

tions.). One can see the noisyness of the plot increasing at high absorbance
(>2)

In the spectra (comp fig. 5) we choose ”interesting wavelengths” which have a high
absorbance. If there is flase light affecting our results it will show up at high absorbance
where it’s relativ error is reasonably high.

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Spectrophotometrics A. Haeller

Figure 6: Observating false-light-error at three different wavelenghts (λ =

300; 357.5; 404nm.)

In fig.6 we examined the actual effect of flase light.

At λ = 300nm the absorbance is quite low(comp. fig5). Therefor we expect the values to
follow Beer’s law. Which they do how the results of the linear regression are showing 1 .
Analysing the data we realised the fitted regression crosses the Y-Achsis at a negative
level. Therefore the values are rejected and not taken into account(fig. 13)

At λ = 404nm the linear regression shows again that our measured values follw Beer’s
law pretty good.
Through the slope(m400nm ) of our regression (fig. 6 ; fig. 13) we can calculate 

m400nm 0.00909 ∗ 100 1

400nm = = = 0.909 (23)
d 1 cm
At λ = 357.5nm the absorbance in fig.5 was >2 we expect the false-light to have a
big affect. Looking at the nonlinear regression this suspicion gets confirmed: For an
absorbance > 1.5 the observed values don’t follow a linear coherence(fig. 6) ). For the
linear fraction of fig. 15 of the graph λ = 357.5nm we agian can calculate 357.5 :

m400nm 0.0545 ∗ 100 1

357.5 = = = 5.45
d 1 cm

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Spectrophotometrics A. Haeller

The false-light parameter

IF alselight
δ= (24)
I0

represents the fraction of flase-light above light emitted by our lighsource. . For λ =
357.5nm :

A0max = −log(δ) (25)

−Amax
δ = 10 (26)
−2.3228
= 10 = 0.0048 (27)

3.3 MLRA for binary mixtures of coloring materials

Figure 7: MLRA-method: Mixture(m) of o-Vanilline(1) and p-Vanilline(2). Spectra are

overlapping fully.

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Spectrophotometrics A. Haeller

Figure 8: MLRA-Analysis in the range between 250-300 nm. Calculation with Ap > 0.2
in order to minimize error through small values

Due to (22), whereas Am = AMixture ; A1 = Ap-Vanilline A2 = Ao-Vanilline .

Am (λ) cm,1 cm,2 A2 (λ)

= +
A1 (λ) c1 c2 A1 (λ)

we now have (through linear regression comp. fig 8)

Am (λ) cm,1 cm,2

= + ∗0 (28)
A1 (λ) c1 c2
cm,1
= crossing graph(regression fig 8) and Y-achsis. (29)
c1
= 0.51662 (30)
(31)
cm,2
= slope (regression fig. 8) (32)
c2
= 0.47587 (33)

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Spectrophotometrics A. Haeller

this corresponds to a relative concentration of:

cm,1 c1
= (34)
cm,2 c2
0.51662
= (35)
0.47587
= 1.08 (36)

Seemingly our mixture contained p-Vanillin and o-Vanillin in eaqual parts.

Figure 9: MLRA-Analysis in the range between 250-300 nm. Calculation with Ap > 0.2
in order to minimize error through small values

As already done with fig. 8 we calculate the same for fig. 9 (comp. 23 - 29) :
cm,1
= 0.59879 (37)
c1
cm,2
= 0.55804 (38)
c2

corresponding to
c1
= 1.08 (39)
c2
The unknown mixture seemingly was made from equal parts o-Vanilline and p-Vanilline.

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Spectrophotometrics A. Haeller

3.4 Temperature dependent spectra

Figure 10: Absorbance of octahedral (pink) at 21.4 °C and tetrahedal(blue) at 69.1 °C

commplex

As pointed out in the introduction and in the original document 3 there are two com-
plexes which absorb in different wavelenghts. The thermodynamical more stable pink
complex absorbs around 500nm. By adding energy as heat we turn the octahedral com-
plex into a tetrahedral one. Visible by the blue color/respectively the absorbance around
700nm which develops under heat.

4 Possible sources of error

4.1 Error reproduction
Because we only took one spectogram per experiment it is not sensable to use the law
of errorreproduction. We evaluate our results by general conclusions about techniques.
Propable errors are:

ˆ impurities in measured solutions

ˆ unprecisely mixed concentrations

ˆ wrong calibrated apperature

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Spectrophotometrics A. Haeller

5 Attachments*
*(due to the fact that the numerical data received directly from the experiments takes
a huge amount of space, this report appears in a reduced version. The original data can
be obtained any time via Jonas Steinhauser (Jonasste@student.ethz.ch))

5.1 Measurement Parameters

Figure 11: Spectra of Benzol wavelenght λ = 220 − 260nm Boxcarwidth-variation from

0 till 20 in steps of four

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Spectrophotometrics A. Haeller

Figure 12: Referenc spectogram Benzene 6

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Spectrophotometrics A. Haeller

5.2 False-Light

Figure 13: Data Sheet linear regression fig. 6

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Spectrophotometrics A. Haeller

Figure 14: Data Sheet non-linear regression fig. 6

5.3 MLRA methods

5.4 Temperature dependent spectra

References
[1] A. Haeller J. Steinhauser. Datenblatt regressionen. Datenauswerungsblatt Regreeio-
nen.

[2] E. Meister. Grundpraktikum Physikalische Chemie. vdf Hochschulverlag AG an der

ETH Z.

[3] B.Z. Shakhasniri. Chemical Demonstrations Vol 1, chapter Chloro and Thiocyanato
Complexes of Cobalt(II), publisher =.

[4] Wikipedia. Acridin — wikipedia, die freie enzyklop.

[5] Wikipedia. Acridon — wikipedia, die freie enzyklop.

[6] Wikipedia. Benzol — wikipedia, die freie enzyklopädie, 2010. [Online; Stand 29.
März 2010].

[7] Wikipedia. Chlorwasserstoff — wikipedia, die freie enzyklopädie, 2010. [Online;

Stand 29. März 2010].

[8] Wikipedia. Lichtspektrum — wikipedia, die freie enzyklopädie, 2010. [Online; Stand
31. März 2010].

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Figure 15: Data Sheet linear regression fig. 6

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