Group 4 Project: Colorimetric Analysis for Phosphate Content

Determination in Water

Background
Scientists use a variety of instruments, sensors and techniques to analyze water quality.
Each technique relies on some physical or chemical property of the particular analyte being
measured to determine its presence or concentration. For example, scientists trying to identify
the presence of Pb2+(aq) in water might take advantage of the fact that it precipitates with I-(aq)
to create a bright yellow color. That yellow precipitate can be dried and measured to obtain the
quantity of lead present.
A common instrument in water quality analysis is the colorimeter. This device transmits
a specific wavelength (color) of light, known to be absorbed by the analyte in question, through
the water sample to be analyzed and then measures how much of that light was absorbed or
scattered by the water sample. It does this by detecting the amount of light that gets
transmitted through the water sample with a sensor on the other side. By comparing the
absorbance of a sample against the absorbance of a sample of pure water, you can obtain a
relative idea of the concentration of the analyte being measured.
The amount of light absorbed by the sample is determined by 3 variables, a relationship
known a Beers Law:

Molar absorbance is different for every analyte and for every wavelength. A particular
analyte may only absorb a small range of wavelengths of light, and of those wavelengths it does
absorb, it will absorb some better than others. Solutions get their color based on their light
absorbance. For instance, a blue solution is blue because it absorbs red light and transmits blue.
Your Task
In this project you will work together with other science students to build a colorimeter
and then use it to test the phosphate content of a water source. To do this you will be using a
phosphate testing chemical that changes color in the presence of phosphate. You will be graded
based on your ability to work together to design your colorimeter, collect data, and reach a
conclusion.
Understanding Solution Spectroscopy: Beers Law
Every solution will absorb light differently. If you can determine that exact relationship,
called the extinction coefficient or molar absorbance, then you can make determinations
about solution concentration from colorimetric data. Complete the following online simulation
to gain a better understanding of Beers Law before proceeding. Read and complete this
tutorial carefully. It will help you understand your project. Each group member should turn in a
completed answer sheet:

PhET Simulation - Beers Law

Go to: http://phet.colorado.edu/en/simulation/beers-law-lab

Warm up -- collecting and maintaining a sample:

Start with the Concentration tab, as shown at the right.
Drag the concentration meter into the solution and complete the
table:

What impact does each variable have on the concentration of a solution?

Adding a Adding a Adding Draining Allowing
Variable: solid liquid more tap off some water to
pollutant pollutant water water evaporate.
Impact on
concentration:
(increase, decrease,
unchanged)

Once a sample has been collected from the environment, it is important to preserve the
concentration and get an accurate measurement later in the lab. Does the size of the sample
matter? What steps should the researcher take to ensure the concentration measurement
doesnt change?

Part 1 Transmittance and Absorbance.

Now click on the Beers Law tab, as shown at the right.

Reset all and turn on the light source.

The % of light that is transmitted through a sample depends upon four

variables. First, just play around a bit. Manipulate these variables to
see what their impact on % transmittance is.
The % of light transmitted will simultaneously tell us the amount of light absorbed. For
example, what is the absorbance when the transmittance is 1, 100%? _______________

For this investigation we will examine all measurements of light in terms of Absorbance.
Their relationship is mathematically expressed according to A = - log10 T

Remember: if you are testing/manipulating a variable and want to see the change it causes,
then you must keep the other variables unchanged.
What impact does each variable have on the measured concentration of a solution, as given by Absorbance?
Length of Sample to Type of Pollutant
Concentration,
go through, examined, Wavelength of
c
Variable: (maintain the same pollutant
and the same Length of sample to
l .
(maintain the same pollutant and
light used?

go through) (maintain the same concentration and
the same Concentration) (maintain all other variables)
length of sample)
For all of these tests maintain the default fixed wavelength of light!

Relationship of this
variable to
Absorbance:
(direct both go up/down OR
indirect one moves in the
opposite direction as the other
OR Random unique to each

Part 2 Beers Law.

According to your observations, the measured absorbance will increase if
you increase either the actual concentration or the sample cell length.

In fact, these measurements are directly proportional and should

produce a straight line when graphed!

The rate of absorbance also depends upon the slope (type of pollutant
examined), which is described by the Molar Absorbance, .

A substance that absorbs a lot of light will result in a steeper slope

when graphed because of the greater Molar Absorbance, .

This also means that if you are looking at a particular (unchanging) type of pollutant, , using a
particular(unchanging) sample length, l, the slope, l, is constant.

Following the equation for a linear line, y = m x we get A = l c. This is Beers law.
This equation can be applied to determine the concentration of
almost any pollutant through its absorbance.

For example: if the following plot and equation of A vs c was

obtained, then what would be the concentration of pollution
for an unknown sample that has an absorbance of .55? ________
(hint: plug in .55 for the Absorbance value in the equation shown )

Based on your earlier observations, it shouldnt surprise you that the graph above only applies
to a particular wavelength of light, 410 nm. Although patterns of wavelength vs absorbance
can be useful in identifying the substance, the pattern is so unique and random it is impractical
for Beers Law.

Although Beers law can only applied to a fixed unchanging wavelength, it is a good idea to
first determine which wavelength will be most suitable and then to conduct a Beers Law
analysis with that wavelength.

Which wavelength would be the best to apply if you were to graph the A vs c for each of these
substances?
Building Your Colorimeter
Equipment and materials (minimum)
LEDs, various colors Cuvettes
Wires 6x16 Lego Plate (Optional)
Various battery types (9V, AA, Coin Etc) 2x2 Lego Bricks (2) (Optional)
Various resistors 2x1 Lego Brick with hole (Optional)
Your task is to build a colorimeter to analyze your water sample. It must meet the
following criteria:

1.) The LED light source must be powered reliably in a way that can be turned off and on easily
2.) The cuvette needs to be in the path of the light, with the detector (another LED) on the
other side to measure transmitted light.
3.) Nothing on the apparatus is allowed to be held in place by your hands. Everything must be
secure except for the voltmeter (which can be held).
4.) The apparatus must prevent light contamination from sources other than the cuvette from
reaching the detector LED. This includes unfiltered light from the emitting LED as well as
outside light.

One suggestion for holding your LED and cuvette in place is to use Legos. There are Lego
blocks that can hold both nicely:
 You will need to engineer how to wire your spectrometer with the correct resistor, block
out light pollution, hold the battery in place etc
As a detector, you will use another LED on other
side of the cuvette. LEDs produce light by exciting
electrons, using a voltage, into higher energy bands called
conduction bands, and then letting them fall back down to
their normal energy level called valence bands. When they
fall, energy is emitted in the form of light. The bigger the
gap between the valence band and conduction band, the
more energetic the light emitted. The process can be
reversed as well, with light being absorbed and causing a
resultant voltage based on the intensity of the light
absorbed by the LED. You will measure the transmittance
in your colorimeter by recording the voltage put out by the detector LED in your colorimeter.
Remember that the LED that you choose as your detector should have a band gap (determined
by the color of the LED) that is smaller or the same as your light source LED.
Wiring your light source LED
LEDs operate at different voltages. Check the LED you have for the forward voltage or
voltage drop. This is the optimal voltage required to light your LED, and the amount that the
voltage in your circuit will drop because of the LED. Choose a power source that supplies at
least that voltage.
When wiring your LED to a power source, you must adjust
the current so that the LED doesnt get burned out. Check the LED
specs for the max forward current and calculate the resistor that you
need based on your voltage using Ohms law:
I=V/R
I= Current in amps
V= Voltage of circuit
R=Resistance in ohms

You will need to make one modification to Ohms law, and that is that you cannot just
use the voltage of whatever battery you are using to power your LED, because the LED actually
subtracts voltage from the circuit itself as it runs. This is called the voltage drop or forward
voltage of the LED. So the voltage you would use when plugging values into Ohms law would
be the Voltage drop of your LED subtracted from the voltage of your battery. (V Supplied - VLED).
Here are some tips for wiring your LED:
1. The longer prong is the positive terminal on the LED.
 2. Choose a battery with a voltage higher that the forward voltage of your LED
3. After calculating the required resistance for your resistor, choose the closest resistor
without going over. For instance, if you need a 380, but you only have 360 and 400,
choose 400.
4. You can create higher voltage by wiring batteries together. The effect is additive, so 2
1.5V batteries would give you a 3V power supply.
5. You might notice the resistors arent labeled. You can tell the resistance of a resistor in
ohms by interpreting the colored bands on the resistor. Use the following chart:

Analyzing Your Water Sample

1. LEDs take time to warm up. Give your LED 5 minutes to warm up before using in order to
get consistent readings.
2. Place a cuvette with just distilled water in your colorimeter and record the record the
resulting voltage from your detector as a baseline. You cuvette should be wiped clean and
free of air bubbles. This voltage represents 100% transmittance. *To prevent fingerprint
smudges, only handle cuvettes by the ribbed edges *
3. Your LED sensor has a limit to how much light it can detect. If too much light passes through
the solution then the detector will reach a saturation point, at which point the voltage will
no longer change in proportion to the light detected. If this happens you will need to make
modifications to correct the issue. You can decrease the light intensity (modify the voltage),
increase the concentration, increase the path length, or modify the detector to have a
higher saturation point.
4. Prepare your sample by adding the contents of a Phosver 3 powder pillow to the
appropriate amount of water indicated on the powder pillow. Allow at least 2 minutes to
the color change reaction to take place.
5. Place your sample cuvette in your colorimeter and record the voltage at the detector. The
voltage is directly proportional to your transmittance. You can use your calibration voltage
 with your sample voltage to determine %transmittance as a function of
concentration, where Vsolution is the voltage measured when the solution (sample) is used
and Vwater when distilled water is used.
6. From the transmittance results, calculate and plot absorbance (A= -logT).
7. Use standards with known concentrations of phosphate to develop a Beers Law plot that
precisely maps the relationship of your analyte concentration to absorbance (not
transmittance). It should be linear. Standards can be obtained from Mr. Stewart.