Lab #3: Spectrophotometry
concentration, the absorbance of the solution
Background
One of the key functions of the homeostatic
mechanisms of the human body is to maintain
the chemical composition of the fluid
environment in which the cells of the body live.
The ability of the body to do so is really quite
remarkable when one considers how the flow of
different materials into and out of the body as
well as the diverse array of chemical reactions
that occur as a result of metabolism are
constantly altering the composition of body
fluids. Yet when physiological systems are
functioning properly, the concentrations of
various metabolites, nutrients, waste materials,
etc. are maintained within very narrow ranges.
Deviations in the concentrations of different
substances in body fluids, therefore, suggest a
problem with physiological mechanisms that
normally regulate those substances. Therefore,
abnormal levels of specific substances in the
body fluids can be indicative of specific
pathologies, and thus can be used to diagnose
particular ailments.
There are numerous methods for measuring
the concentrations of specific substances within
body fluids. One commonly-used method is
called spectrophotometry (also called
“colorimetry”). Spectrophotometry works on a
very basic principle—that if your solution
contains a solute that absorbs light, then the
more concentrated that solute is in the solution
the more light should be absorbed by that
solution and the less light will pass through that
solution. The mathematical relationship
between solute concentration and light
absorbance is described in Beer’s Law*:
(1) [X] α A
where [X] is the concentration of solute X and A
is the amount of light absorbed. Incidentally, α
means “proportional to” (i.e., that [X] multiplied
by some constant number will give you the
value of A). This describes a direct linear
relationship between the concentration and light
absorbance (i.e., per each unit increase in
Lab #3: Spectrophotometry
p. 1
increases a specific amount) (Figure 3.1).
Beer’s Law also describes the mathematical
relationship between solute concentration and
light transmittance by a solution (i.e., how much
light passes through the solution).
1__
(2) [X] α log T
where T is the percentage of light transmittance
(i.e. the percentage of the light entering the
solution that passes through the solution). This
equation describes an inverse curvilinear
relationship between solute concentration and
light transmittance (as concentration increases,
light transmittance decreases, but there is
progressively less of a decrease in transmittance
per unit concentration as the concentration
increases) (Fig 3.1).
Because there is a mathematical relationship
between solute concentration and light
absorption/transmittance, we can use
spectrophotometry to determine the
concentration of a substance in a sample of body
fluid (or any other solution where we do not
already know the substance’s concentration) by
comparing its light absorbance or transmittance
with a that of a solution that has a known
concentration of that substance (called a
reference, or a standard solution). For example,
let’s say we wanted to determine the
concentration of glycerol in a sample of blood
plasma. We react the glycerol with a reagent
that changes that absorbs light at a specific
wavelength when it reacts with the glycerol (this
allows us to identify light absorbance by
glycerol as opposed to that by other substances
in the solution). We then determine the light
absorbance of the solution by placing the
solution in a
*referring to the German mathematician and physical scientist
August Beer (1825-1863) who first described this relationship, not
after the alcoholic beverage (although scientists often make great
insights over a pint at the local brew pub). This mathematical
relationship is also called the Beer-Lambert Law or the Boulguer-
Beer Law. The mathematical proportionalities here are simplified
versions of this principle, and assume a constant light path length
and a constant molar extinction coefficient.
 1.2 We can then rearrange the equation to solve for
[blood] by multiplying both sides of the
1
equation by Ablood
Absorbance 0.8
(4) Ablood_
0.6 [blood] = [standard] ×
Astandard
0.4

0.2 We now fill in the values we have measured and

0 solve for [blood].
0 5 10 15 20 25
Concentration (g/dL) 0.090
[blood] = 250 mg/dL ×
100 (5) 0.300
= 75 mg/dL
80
% Transmittance

60
40
20
0
0 5 10
Concentration (g/dL)
Fig. 3.1. Examples of (left) the linear (direct
proportional) relationship between solute
concentration and light absorbance and of (right)
the inverse logarithmic relationship between
solute concentration and light transmittance as
predicted via Beer’s Law.
spectrophotometer (an instrument that measures
light absorbance at specific wavelengths). We
determine the absorbance our blood plasma
sample (Ablood) to be 0.090. We also run the
same procedure on a glycerol solution of known
concentration ([standard]) of 250 mg/dL, and
determine its absorbance (Astandard) to be 0.300.
So given these values, how can we
determine the concentration of glycerol in the
blood plasma sample ([blood])? Remember that
according to Beer’s Law the concentration of a
solution is proportional to its absorbance. That
proportionality (i.e., how much light is absorbed
per unit of concentration) will be the same both
in the blood plasma sample and in the standard
solution. So if that proportionality is the same,
we can describe it through the following
equation:
(3)
[blood]
=
[standard]
Ablood Astandard
Experimental Error and Standard Curves
Typically, we determine the concentration of
some solute of interest by reacting it with
another substance to produce a substance that
15 20 absorbs light at a particular wavelength, and
comparing it to a second solution with a known
concentration. When we do so, we are trusting
that we have conducted the experiment
correctly, and that the results accurately reflect
the actual concentration of the substance. But is
this a valid assumption?
The potential for error is always present in
any measurement. Here we are referring to error
as any deviation in our measured value away
from the true value for that parameter. In that
respect, all measurements contain some degree
of error—there is no way you can perfectly
measure anything down to the last molecule, to
the exact temperature, etc., and conditions that
you cannot control can change the reading you
obtain for a particular measurement. Despite
this, there are ways of minimizing experimental
error: by controlling extraneous conditions that
might affect your results (e.g., temperature), by
carefully measuring out quantities, and by
repeating the measurements when possible to
see if similar results are obtained.
When determining concentrations using a
spectrophotometer, there is certainly a potential
for experimental error. For instance, in the
example above where we measured plasma
glycerol, samples of blood plasma and the
standard solution were mixed with a reagent to
produce a substance that absorbed light at a

Lab #3: Spectrophotometry

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particular wavelength. There could be slight
differences in the amount of plasma or standard
solution used, the amount of reagent used, the
specific concentration of the standard solution,
etc., that could influence the absorbance
readings. Indeed, this could be especially
problematic if we do not account for possible
error in the readings obtained for the standard
solution. If only one standard value is used,
calculations of the unknown based on the
absorbance of that standard will reflect any
errors made in the preparation of the standard.
This could have serious effects on the results.
For example, let’s say you had very
carefully conducted the glycerol assay in the
example above. Thus the results should fairly
accurately reflect the true glycerol levels in the
plasma. Now pretend that you conducted the
experiment again, but were not quite as careful
with your pipetting, incubation, and other
aspects of preparation. In this case you obtain
an absorbance for the blood plasma solution of
0.100 (a bit higher than before, but not too
terribly off). However, because you were not
paying attention, you pipetted in only half as
much standard solution as you were supposed to,
and ended up obtaining an absorbance reading of
0.145 for the 250 mg/dL standard. The glycerol
concentration for the blood plasma would be
calculated to be 172 mg/dL (way too high)!
Replication of an experiment is one means by
which we can we account for experimental error
(either due to preparation mistakes or simple
random variation between one reading and
another) to get accurate estimates of the
concentrations.
Another potential problem is that a light-
absorbing solution may adhere to the Beer’s law
relationship between light absorbance and
concentration only over a certain range of
concentrations, beyond which there may be a
curvilinear relationship. This problem can be
avoided by referencing the measurements for the
test solutions against multiple standards, some
of which are higher in concentration than the test
solution and some of which are lower.
Lab #3: Spectrophotometry
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Typically, a researcher will prepare several
standard solutions of different concentrations in
order to determine the solution of an unknown.
The absorbance values of these solutions are
measured and plotted against the concentration
of the standards. A “best fit” line (e.g., a linear
regression line) would be drawn through the
points to produce a standard curve (Fig 3.2).
Note that even in the best of experiments not all
the individual points fit on the line—some are
above and some are below. The line effectively
balances out variation and provides an average
value of absorbance for a solution of a given
concentration. We can then determine the
concentration of the unknown by extrapolation.
First we find the value for absorbance on the
vertical (y) axis, and draw a straight horizontal
line from the y-axis to the best-fit line. We then
draw a straight vertical line from the point where
the horizontal line meets the best-fit line straight
down to the horizontal (x) axis. The value on
the x-axis where this vertical line intersects it is
the concentration of the unknown.
1.4
1.2 y = -0.03 + 0.027x R= 0.996
1
Absorbance
0.8
0.6
0.4
0.2
0
0 10 20 30 40 50
Concentration
Fig. 3.2. An example of a standard curve generated by
measurement of absorbance from multiple standard
solutions of different concentrations. The best fit line
helps to even out experimental error, and helps with
identification of samples where there might have been
some procedural mistake. The dashed lines provide an
example of extrapolation (e.g., a solution with an
absorbance of 1.000 would have a concentration of ~38).
The line slope equation derived from linear regression
could also be used.
 Fig 3.3. The Spectronic Spec 20 spectrophotometer.

Experiment: Determining the Concentration of Glucose

In today’s experiment we will be determining the concentration of monosaccharide glucose in various

solutions using the spectrophotometer to measure light absorbance and comparing the absorbance of
unknown solutions with those of standard solutions. The procedure used is one used in clinical settings
for measurement of plasma glucose. Samples of blood plasma are mixed together with a reagent solution
containing o-toluidine (a purplish substance). When glucose reacts with o-toluidine, it forms a greenish
complex that absorbs light optimally at 620-650 nm. Thus the more glucose present in the sample, the
more of the complex will be formed and the more light will be absorbed by the solution within that range
of wavelengths.

Note of Caution!
The reagent solution we will be using contains concentrated acetic acid and o-toluidine.
o The concentration of acetic acid is caustic: the vapors can irritate the eyes and mucus membranes, and
it will damage the skin and eyes if they come into direct contact. Wear eye protection and gloves, and
do not wear open-toed shoes. Also, I recommend wearing clothes you do not care too much about to
this lab, just to be safe.
o o-toluidine is a suspected carcinogen. Again, avoid getting the reagent on you.
o Clean up any spills immediately with LOTS of water

Procedures

1. Obtain four clean test tubes and label them with masking tape

2. Add 5.0 ml glucose reagent to each tube with an automatic pipetter

Lab #3: Spectrophotometry

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3. Add the following to the tubes:

Tube #1 – 0.1 ml distilled water (will serve as a “blank” solution for calibrating the
spectrophotometer)

Tube #2 – 0.1 ml blood serum (from some domesticated animal)

NOTE: Be very careful to remove only plasma from the sample. Avoid contaminating the plasma by
disturbing the packed cells in the bottom of the microtube. Do not add contaminated (red tinted) plasma to
your tube.

Tube #3 – 0.1 ml unknown #1

Tube #4 – 0.1 ml unknown #2

4. Immerse tubes 2, 3 and 4 in 100°C water bath for 10 min

5. While the tubes are in the hot water bath, calibrate the spectrophotometer:

o Turn on the machine with the Power Knob (front left)

o Set wavelength to 620-650 nm with the Wavelength Adjustment Knob (top right)
o The instrument should have nothing in the sample chamber, the sample chamber lid should be
closed, and the mode should be set to “Transmittance”. If it is not set to transmittance, push
the Mode Button until the mode indicator light is next to “Transmittance”.
o Set the transmittance to read “0.0” with front left knob.
o Transfer the blank solution (Tube #1) into a clean cuvette. Place the cuvette in the sample
chamber and close the lid. The readout on the instrument should read “100.0”. If it does not,
use the Zero Knob (front right) to adjust the value to 100.0.
o Push the Mode Button to switch the mode to “Absorbance”. The readout should change to
“.000”. The instrument has now been calibrated.

5. Remove Tubes #2-4 and immediately immerse in ice bath for 3 min.

6. Record absorbance values for remaining three tubes. Place the tube in the sample chamber, close the
lid, and record the absorbance value provided.

NOTE: The biggest mistake students make with this exercise is improperly calibrating the instrument. Usually,
they forget to switch modes between absorbance and transmittance at the proper times. Make sure that your
readings are for Absorbance. At the end of the calibration procedure your blank solution should give you a
reading of “.000”. All other measurements should have three digits to the right of the decimal, and should not
exceed 1.400. If you have only one digit to the right of the decimal point, or if your readings are between 2 and
100, your spec is set to the wrong mode.

7. Once complete, dispose of your solutions in the waste bottle under the fume hood, and clean out the
test tubes and cuvettes per the instructions posted by the sink.

8. Calculate the concentration of glucose in each of your three samples using your absorbance readings
and the concentration and absorbance of a standard solution (provided by your instructor). Record
these values on your data sheet and on the table on the instructor’s station. We will see whose
measurements are the closest to the actual concentrations for Tubes #3 and 4.

Lab #3: Spectrophotometry

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