Instrumental Analytical Methods

Lab Report 2013

Anastazija Ristovska

Experiment 2
Turbidimetric Quantitative Analysis of Casein in Solution

The purpose of this experiment was to determine the wavelength at which casein best absorbs in
solution using spectrophotometric analysis. Solutions with different weight percent (wt%) casein
concentrations were prepared that were then analyzed at three different wavelengths. The data was
analyzed using linear fir analysis, and the wavelength for which the best linear fit could be constructed
with the highest coefficient of determination i.e. the highest r-squared value was the wavelength at which
casein absorbs best spectrophotometrically.

at 550 nm
wt%
abs.
0,1
0,009
0,2
0,007
0,3
0,012
0,4
0,017
0,5
0,019
1
0,043

at 600 nm
wt%
abs.
0,1
0,001
0,2
0,003
0,3
0,012
0,4
0,02
0,5
0,023
1
0,037

at 650 nm
wt%
abs.
0,1
0,015
0,2
0,016
0,3
0,017
0,4
0,016
0,5
0,022
1
0,035

At 550nm the model explained for 96,68% of the variance in the system. Even though there was
an outlier value at 0,1 wt%, this value could be rejected from the model and see if an improvement is
noticed. The slope of the linear fit is 0,0406, and the linear fit passes almost exactly through the origin,
indicating that at a concentration of zero, the absorbance would also be zero. The readings at 650 nm
need to be rejected because of the fact that, our results indicate that Casein most likely does not absorb at
650nm as well enough as it absorbs in the 550-600 range. From our results we concluded that Casein
absorbs best at 550 nm and at concentrations at 0.2 wt% or higher. Nevertheless, we recommend that
this experiment be repeated because of a possibility for an internal procedure error, and that there still
does exist some likelihood that 600 nm might be the wavelength at which Casein absorbs best but weve
been for some reason unable to detect this in our results.

0,05
0,045

0,043

y = 0,0406x + 0,0009
R = 0,9668

0,04

Abs. (A)

0,035
0,03
0,025

Series1

0,02
0,017

0,015

0,019

550 nm

0,012

0,01

0,009

0,005

0,007

0
0

0,2

0,4

0,6

0,8

1,2

wt% concentration of casein (%)

0,045

y = 0,0409x - 0,001
R = 0,9291

0,04
0,037

0,035

Abs. (A)

0,03
0,025

0,023

0,02

Series1

0,02

600 nm

0,015
0,012

0,01
0,005
0,001

0
0

0,2

0,003
0,4

0,6

0,8

1,2

wt% concentration of casein (%)

0,04
0,035

0,035

y = 0,0232x + 0,0105
R = 0,9264

0,03

Abs. (A)

0,025
0,022
0,02
0,015

0,015

0,016

0,017

Series1
0,016

650 nm

0,01
0,005

0
0

0,2

0,4

0,6

0,8

1,2

wt% concentration of casein (%)

When we reject the first reading from the data, we find that the model improves its R2 and now it
explains for 99.56% of the variance in the system. Its y-intercept is nonetheless set farther away from
zero. Its slope increased.
0,05
0,045

0,043

y = 0,0445x - 0,0018
R = 0,9956

0,04

Abs. (A)

0,035
0,03
0,025

Series1

0,02
0,017

0,015

0,019

550 nm

0,012

0,01
0,007

0,005
0
0

0,2

0,4

0,6

0,8

1,2

wt% concentration of casein (%)

We noticed that the more concentrated the solution was, the greater its sensitivity was at 550 nm.
The 1wt% solution displayed greater sensitivity than the 0.5% solution. Casein had maximum absorbance
at 550nm, as can be seen from the plots in the example of the 1wt% probe across all three wavelengths.
UV-vis absorption, turbidimetry, and nephelometry are three similar techniques. Turbidence is
analogous to absorbance and is unitless since its calculated by the formula S=log(P 0/Pt) where P0 is the
incident light and Pt is the amount of light that passed directly through the solution. Thus indirectly the
amount of scattered light can be determined. Alternatively, if we had wanted to use nephelometry, a
technique similar to turbidimetry, the light detector would had been set at a particular angle of a uniquely
shaped cuvette, at 45, 90, or 135 relative to the incident-transmitted light axis. Sometimes
nephelometry is used over turbidimetry in order to improve sensitivity at low concentrations. Indeed, as
we have noticed, in our absorption technique we did not notice a very good sensitivity at 0.2wt% casein
in the solution of lowest concentration. In such a case, using nephelometry over turbidimetry or
absorption would have improved the sensitivity of the general technique.
Even without rejecting the 0.1wt% sample reading at 550nm, the 550nm measurement gave the
best coefficient of determination, R2, 96.68% including the first sample reading, and 99.56% excluding it.
The coefficient of correlation is very similar for the 550nm and 600nm readings, 0.0406 and 0.0409
respectively, however, when rejecting the first reading at 550nm increases the coefficient of correlation in
that model to 0.0445, meaning in the 550nm model the correlation between the absorbance and casein
concentration is highest; this correlation is lowest at 650nm with 0.0232 coefficient of correlation.
I believe that the absorption method of analysis in cases as this is the best and most widely used,
presumably allowing for best linearity of measurement.