Colorimeter

The Colorimeter is a computer-interfaced probe designed to determine the concentration of a solution from its color intensity. The color of a solution may be inherent or derived by adding another reagent to it. Monochromatic light from a LED light source passes through a cuvette containing a solution sample, as shown in Figure 1. Some of the incoming light is absorbed by the solution. As a result, light of a lower intensity strikes a photodiode.

Transmittance and Absorbance

The amount of light that penetrates a solution is known as transmittance. Transmittance can be expressed as the ratio of the intensity of the transmitted light, It, and the initial intensity of the light beam, Io, as expressed by the formula:

T = It / Io

The Colorimeter produces an output voltage which varies in a linear way with transmittance, allowing a computer to monitor transmittance data for a solution. The transmittance of the sample varies logarithmically (base ten) with the product of three factors: a, the molar absorptivity (extinction coefficient), b, the cell or cuvette width, and c, the molar concentration.

log(l/T) = abc

In addition, many experiments designed to use a colorimeter require a related measurement, absorbance. At first glance, the relationship between transmittance and absorbance would appear to be a simple inverse relationship. That is, as the amount of light transmitted by a solution increases, the amount of light absorbed might be expected to decrease proportionally. But the true relationship between these two variables is inverse and logarithmic (base 10). It can be expressed as:

A = log(1/T)

Combining the two previous equations, the following expression is obtained:

A = abc

This formula states that the light absorbed by a solution depends on the absorbing ability of the solute, the distance traveled by the light through the solution, and the concentration of the solution. For a given solution contained in a cuvette with a constant cell width, one can assume a and b to be constant. This leads to the equation:

A = k C (Beer's law)

where k is a proportionality constant. This equation shows absorbance to be related directly to concentration and represents a mathematical statement of Beer's law. In this guide and in our computer programs, transmittance is expressed as percent transmittance or %T. Since T= %T/100, the formula can be rewritten as:

A = log (100/%T) or A = 2 - log %T

Beer's Law

In general, absorbance is important because of its direct relationship with concentration according to Beer's law. This linear relationship is obeyed only at the maximum absorbance (l_max) of the compound. The l_max of the compound being analyzed should correspond very closely to the available wavelengths of the LED's which are 470, 565 and 635 nm. Beer's law should be verified by using several standards (solutions of known concentration) and their absorbance values determined using a colorimeter. A graph of absorbance versus concentration is then plotted. A solution of unknown concentration is placed in the colorimeter and its absorbance measured. When the absorbance of this solution is interpolated on the Beer's law curve, as shown in the graph in figure 2, its concentration is determined on the horizontal axis. Alternatively, its concentration may be found using the slope of the Beer's law curve.

Absorbance and Transmittance Ranges for the Colorimeter

For the best results, the absorbance or transmittance values should fall within these ranges:

Percent Transmittance: 28% - 90%

Absorbance: 0.050 - 0.550
Beer's law curves start to lose their linearity at absorbance values above 0.550 (percent transmittance values less than 28%). If you have a solution that transmits such a low level of light, dilute the solution so that it falls within this range. Try to design experiments so that absorbance or transmittance values of solutions are in this range.

We have found that Beer's law curves started to lose their linearity at absorbance values above 0.550 (percent transmittance values less than 28%). If you have a solution that transmits such a low level of light, consider diluting the solution so that it falls within this range. Try to design experiments so that absorbance or transmittance values of solutions are in this range.

Wavelength Ranges

You can select three LED light colors with the Vernier Software Colorimeter Blue (470 nm or 4700 A), green (565 nm or 5650 A) and red (635 nm or 6350 A). You can select one of these three nearly monochromatic colors using the wavelength selection knob on the top left of the colorimeter (see figure 3). There are several ways you can decide which of the three wavelengths to use. Look at the color of the solution. Remember that the color of a solution is the color of light which passes through it. You probably want to use a different color of light that will be absorbed, rather than transmitted. For example, with a blue CuSO₄ solution, use the red LED (635 nm).

Another quick method is to place a cuvette containing the solution in question in the Colorimeter and check to see which of the three wavelengths yields the highest absorbance (or lowest transmittance).

Calibration of the Colorimeter

It is recommended that you do a new calibration any time you perform a new colorimetry experiment or change the wavelength within an experiment. Though it is possible to save a calibration for future use, you will certainly see immproved results if you recalibrate prior to doing a new experiment.

Texas Instruments Calculator-Based Laboratory (CBL) System

Connect the I/O ports of the CBL and the TI-92 calculator with double ended cable. Make sure to push both plugs in firmly. Using a CBL-DIN adapter, connect the sensor to any of the Analog input ports on the top or left side of the CBL unit (CH1, CH2 or CH3). In most cases, CH1 is used.

Calibrating the CBL

Any time that you want the Calculator-Based Laboratory System to read values other than voltages, such as transmittance, you need to calibrate the sensor.

The calibration process involves two steps. In the first step, close the lid of the Colorimeter, set the knob on the Colorimeter to 0%T, and allow the reading on theCBL to stabilize. Press the [Trigger] button on theCBL and enter 0 when asked to "ENTER REFERENCE:." Now set the knob on the Colorimeter to a wavelength and insert a blank cuvette. Allow the reading on the CBL to stailize and, again, press the [Trigger] button on the CBL. Enter 100 at the second "ENTER REFERENCE:" prompt. When calibration is complete, the CBL will be set up with the conversion equation, and you can now perform an experiment.

The general method for doing a 2-point calibration with the Colorimeter is similar to that used with most other colorimeters and spectrophotometers. A zero percent calibration is done with no light passing through a cuvette. The wavelength knob on the colorimeter is turned to "0% T" (see Figure 3). In this position the computer can read data from the Colorimeter, but the light source is turned off. Since the light is off, it makes no difference if a cuvette is in the cuvette slot. A 100% calibration is done with the wavelength knob turned to select one of the three LED wavelengths. This turns on the red, green, or blue LED. A blank is placed in the cuvette slot. The blank is a cuvette containing the solvent used in the solution being studied, usually distilled water. The blank acts as a control by taking into account the small amount of light absorbed by the solvent and by the walls of the cuvette.

The wavelength knob is turned to the "0%T" position. The lid of the cuvette slot must be closed (with or without the "blank" cuvette in place). When the voltage value shown on the computer monitor stabilizes, the user is asked to enter a value. This value will be "O" (for 0% transmittance).

To do a 100% calibration, the blank is placed in the cuvette slot and the lid of the colorimeter is closed. The wavelength knob of the colorimeter is turned to one of the three LED colors. When the voltage value stabilizes, the user is asked to enter a transmittance value. This value will be "100" (for 100% transmittance).

Using Cuvettes with the Colorimeter

The Colorimeter is designed to use polystyrene cuvettes. The cuvettes have a volume of approximately 4 mL. The cuvette slot of the colorimeter is designed to give a snug fit to the cuvette and ensure that it is always in precisely the same position between the LED light source and photodiode. Two opposite sides of the cuvette are ribbed and are not intended to transmit the light from the LED. The two smooth surfaces are intended to transmit light. It is important to position the cuvette correctly in the colorimeter. This should be done as shown in Figure 4, with the ribbed edges facing away from and toward you, and the smooth edges facing left and right. The light travels from left to right from the LED through the cuvette to the photodiode. It should also be noted that there is often a small variantion in the amount of light absorbed by the cuvette if it rotated 180^o between trials. To avoid this, you should use a water-proof marker to make a reference mark on the right side of the top edge of the cuvette s shown in Figure 4. This reference mark should be aligned with the white reference mark on the top right side of the colorimeter each time they insert a cuvette.

Just like most spectrophotometer sample tubes, individual plastic cuvettes vary slightly in the amount of light they absorb. You may choose to ignore these differences. For most lab exercises, this variation will not have a noticeable effect on experimental results.

For best results, variation in light absorbed by individual cuvettes can be controlled by using the same cuvette for all trials of a particular experiment. If you use five trials for a Beer's law experiment, the five standard solutions can be transferred to the same cuvette for each trial. This requires that the cuvette be clean and dry after each trial or rinsed sevel times with the solution that will be added to it. This method takes very little time and successfully controls a potential variable. It also eliminates concerns over possible scratches that may eventually develop on a cuvette. The effect of the same small scratch is eliminated using the 100% calibration.

It is very important that solutions be added to a cuvette to the proper depth. A "safe level" is between 2.2 and 3.5 mL of solution. When the cuvette is filled to the brim, its total volume is about 4.1 mL. Since the inside diameter of the cuvette is about 1.0 cm x 1.0 cm, this safe level can also be measure on the outside of the cuvette as 2.2-3.5 cm from the inside bottom of the cuvette. These levels are shown in Figure 5.