colorimetry introduction.docx

8/14/2019 Colorimetry Introduction.docx

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Introduction to Colorimetry

A spectrophotometeris an instrument that passes a selected wavelength of

electromagnetic radiation through a solution and measures the amount of that

electromagnetic radiation that the solution absorbs (does not allow to pass) or

transmits (does allow to pass). Refer for a moment to theelectromagnetic

spectrumshown on the linked page, (from Life, The Science of Biology, by Purves

et al., 5thed., 1998). One type of spectrophotometer uses ultraviolet (UV)

wavelengths; those are the wavelengths that cause sunburn, for example. Another

type uses infrared (IR) wavelengths, which are most familiar as heat. You mayhave an opportunity to use one of these in another course. The third type of

spectrophotometer, which we will use in this course, uses visible wavelengths,

which we think of as the different colors of light, ranging from violet to red. Since

these are the familiar colors, this type of spectrophotometer is called a

"colorimeter." The particular model we will use is the Spectronic 20 colorimeter.

Observation of the visible spectrum inside the

Spectronic 20 colorimeter

1. Turn the power switchclockwise to turn on the instrument; you'll hear/feel the

on-click. The pilot lampshould glow, and you may hear the gentle sound of the

cooling fan inside. If the pilot lamp doesn't glow on one of these instruments, don't

be concerned. For the moment you may ignore the other uses of the two knobs on

the front of the instrument.

2. Refer to the illustration of the instrument below. Each solution that you will test

is placed into a small test tube, called a colorimeter tube, which is inserted into
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thesample holder.The beam of light, of whatever wavelength is selected, passes

through the tube in the holder from right to left.

3. In your test tube rack is a colorimeter tube containing a rectangular strip of

white paper that is a bit shorter than the length of the tube and just wide enough to

fit snugly into the colorimeter tube. Insert this tube into the holder (pushing it all

the way down); rotate the tube so that the faint light beam entering the tube from

the right will strike the paper's flat surface.

4. Cup your hand around the top of the open sample holder and look closely down

into the tube to see the faint light reflected off the paper. You will need to haveyour eye close to your cupped hand (near the top of the tube) because the light is

faint. If the room light is too bright the instructor will turn off the room lights for a

moment.

5. As you turn the wavelength control knob, you will see the light reflected by the

paper change from violet/blue (about 400 nm) to green to yellow to orange and

finally to red (about 700 nm). The lamp inside the instrument produces white light

(a mixture of all wavelengths), but the wavelength control knob screens all but the

one you select. In normal operation (i.e. without the paper in the colorimeter tube)

the light that you see reflected would enter the tube containing a test solution and

some portion of it would pass out the other (left) side of the tube to strike a

detector, which would convert that light into an electrical signal that registers on

thetransmittance and absorbance scales.

6. Remove the tube from the sample chamber and replace it in the test tube rack.

7. In normal operation the lid of the sample holder is always closed after inserting

the sample tube, before recording measurements, to shut out stray light.


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Standardization of the colorimeter for determining the

absorption spectrum of riboflavin, the vitamin

As light passes through a solution in a colorimeter tube, some small fraction of that

light may be absorbed by the glass of the tube and by the solvent in which the

molecule of interest is dissolved. Further, as you will see in your next lab exercise

(the biuret test for measuring protein in solutions), the solution in the tube may

contain other substances that are necessary to produce the color that you measure.

Since many types of molecules are not colored, they must be reacted with

something that will produce a color before you can use the colorimeter to measure

them. Anything that the light passes through may absorb some of the light and


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influence measurements. Since the point is to measure absorption of one, particular

type of molecule in a solution, it is necessary to correct for (to "subtract") light

absorption by everything else (the glass of the test tube, the solvent, any color-

producing reagents present). To do that you standardize the instrumentbefore

doing your measurements.

Riboflavin is a water soluble vitamin. The yellow solution in one of the tubes in

your rack contains riboflavin dissolved in water, 0.017 mg/mL; nothing else is

present. In order to get the most accurate possible data for determining the

absorption spectrum of riboflavin, you must standardize the colorimeter to correct

for light absorption by the glass and the solvent (water in this case). Therefore, you

have another tube containing only water, that is, everything except the molecule of

interest, riboflavin; this second tube is called the blank. You use the blank to

standardize the instrument. In effect you will be "telling the instrument to ignore"

light absorption by everything except the molecule of interest, riboflavin.

The steps in standardization are few and simple, but theymust be done correctly and carefully to obtain reliable data.

1. If the instrument were not already turned on, you would use the power switchto

turn on the instrument and let it warm up for about 15 minutes.

2. Use the wavelength controlknob to select the desired wavelength; the

wavelength values shown in the wavelength dialdisplay window are graduated in

nanometers (nm). NOTE WELL that in next week's work you will use only one

wavelength; today, though, you will measure light absorption at many wavelengths

in order to determine the absorption spectrum of riboflavin. So, we'll begin with

340 nm for the wavelength. Set that now.


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3. With the sample holder empty and its lid closed, use the "0" controlknob

(same as the power control knob) to set the indicator needle to zero percent on the

transmittance scale, 0 %T. Without a tube in the chamber, no light enters the

chamber (it's pitch black in there), so no light would strike the detector. In effect,

you're telling the instrument that if there were a tube with a solution in the

chamber, this is what you'd see if absolutely none of the light entering the tube

passed through to the other side, i.e. 0 % of the light got through.

4. Insert the blank tube into the holder and close the lid. The needle will sweep

upward on the %T scale. Wherever the needle stops, use the light controlknob

now to move the needle to 100 %T on the scale. In effect, you're telling the

instrument that with a sample in the chamber, but no riboflavin present, this is

what you'd see if all of the light (100 % of it) entering the tube passed through to

the other side. Since the glass and the solvent will also be present with your

riboflavin sample, this step tells the instrument to ignore light absorption by those

factors. Now, if you see any %T values less than 100%, that must be due to light

absorption by the riboflavin; with riboflavin present, less than 100% of the light

entering the tube will pass through.

5. Remove the blank from the sample holder and close the lid. The needle should

fall back to zero %T.

6. The instrument is now standardized for 340 nm wavelength. If you were going

to do measurements on many samples at this single wavelength, you would nottouch the light controlknob or the "0" controlknob hereafter.


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Determining riboflavin's absorption spectrum

An absorption spectrum plots absorbance(A), not % transmittance, on the y-axis,

as a function of wavelength on the x-axis. Absorbance is the other scale on your

colorimeter. Note that absorbance and %T scales run in opposite directions; than

means that they are reciprocally related; as one rises, the other falls. But note also

the spacings between divisions on the scales; %T has a linear scale whereas

absorbance has a logarithmic scale (common log, i.e. base 10 log). Specifically, A

= log (1 / T), where T is the decimal equivalent of %T. Example: if %T = 50%,

then T = 0.5 and (1 / T) = 2.0, and A = 0.301. Make sure you understand this

algebraic relationship.

To collect data for riboflavin's absorption spectrum now, you must record

absorbance values at many wavelengths, not just one, across the visible spectrum.

It will be sufficient to take measurements at 5 nm increments. Each time the

wavelength is changed, however, the 100 %T setting must be redone because the

glass and solvent may not absorb light to the same degree at different wavelengths.

But this extra step takes little time. So, proceed as follows.

1. With the colorimeter already standardized for 340 nm wavelength, insert your

riboflavin tube, close the chamber lid, and record the absorbance reading. Don't

change any knob settings yet. Then remove the riboflavin tube and close the lid.

The needle will fall back to 0 %T, which is A = "infinity" (the maximum

absorbance possible). You set the 0%T only once.

2. Reset the wavelength to 5 nm higher than the last setting. Insert the blank tube,

close the lid, and note that the needle doesn't go all the way back to 100 %T. That's

because the tube's glass and the water absorb different amounts of light at different


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wavelengths. So, reset the needle to 100 %T with the light controlknob now. The

machine is now standardized for the new wavelength.

3. Remove the blank tube, and see that the needle falls back to 0 %T (= maximum

absorbance). Insert the riboflavin tube into the sample holder, close the lid, and

record absorbance at the new wavelength. Don't adjust any knobs with the

riboflavin sample in the chamber. Then remove the riboflavin tube and close the

lid.

4. Now repeat steps #2 and #3: increase the wavelength in 5 nm steps, reset the 100

%T with the blank each time, and record a new absorbance value for theriboflavin solution. Your last wavelength setting will be 530 nm. This process

goes quickly, but it must be done carefully.

5. Return both the blank tube and the riboflavin tube to the test tube rack. Never

leave a sample in the instrument. Turn off the instrument by turning the power

switchcounter clockwise; you'll hear/feel the click.

6. Plot your data on graph paper that has both axes with linear scales: absorbance

on the y-axis and wavelength on the x-axis. What is riboflavin's absorption

maximum wavelength? How many "peaks" does the absorption spectrum have?

Check your results with your lab instructor.

colorimetry introduction.docx

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