Activity 1: Introduction to the Spectrophotometer

Introduction

The spectrophotometer you will use in this activity is an instrument that contains a light source (ultraviolet, visible, or infrared), a diffraction grating monochromator, and a detector. The light source produces light of all wavelengths within limits in the ultraviolet and visible regions of the spectrum. The diffraction grating monochromator disperses the light into a spectrum and allows successive narrow wavelength bands to pass through a slit to impinge upon a photoelectric detector. The detector measures the intensity of light coming from the monochromator.

The sample holder lies in the path of the light coming to the detector. When a sample is placed in the holder, the light passes through the sample to the detector. The sample determines whether all of the light, some of the light, or none of the light is transmitted to the detector. Light is absorbed at those wavelengths having the correct energy to cause electronic transitions in the molecules, atoms, or ions in the sample. By measuring the intensity of the light passing through the sample over a range of wavelengths, it is possible to plot the absorption of light vs. the wavelength. The resulting plot is the absorption spectrum of the sample.

Two kinds of information are obtained from the absorption spectra for substances. First, each substance has its own characteristic absorption spectrum. Thus, the spectrum may be used to identify the substance. Second, the intensity of the absorption can be related to the concentration of the substance in the sample. The Beer-Lambert Law, A = abc, allows the determination of the amount (concentration) of the substance in a solution. Here A = sample absorbance; a = absorptivity, a proportionality constant; b = path length; and c = sample concentration.

There are two parts to this activity, qualitative and quantitative spectrophotometry. In the former, the absorption spectrum of chromium(III) nitrate in the visible region (375-700 nm) of the spectrum will be determined. In the latter, a sample of chromium(III) nitrate with unknown concentration will be analyzed. The unknown concentration can be determined by measuring the percent (%) transmittance at the wavelength of maximum absorption and comparing it with a graph (called a concentration curve) of absorbance vs. concentration. The % transmittance (%T) is converted to absorbance (A) by the relationship: A=2- log (%T); because A is directly proportional to concentration, but %T is not.

Purpose

To determine the absorption spectrum of a solution containing chromium(III) nitrate, Cr(NO 3 ) 3 ; to determine the concentration of an unknown solution of chromium(III) nitrate.

Safety

  1. Wear protective goggles throughout the laboratory activity.

  2. Chromium(III) nitrate is toxic and is a skin irritant. Avoid contact with this compound and wash hands thoroughly after use.

  3. Handle reagent bottles carefully. Always replace the top or stopper after obtaining a reagent.

Procedure

Part I. Qualitative Spectrophotometry

Your teacher will demonstrate the operation of the spectrophotometer you will use. The procedure below is written for the Spectronic 20 " with a dial meter.

  1. Prepare a data table to record wavelength, percent transmittance data and absorbance. (It would be good to do this before coming to laboratory.) The data table should have three columns headed:

  2. If the spectrophotometer is off, then turn the instrument on and allow about 20 min for it to warm up. After warm up, with the sample holder cover closed, set the wavelength dial to 375nm and adjust the meter to read 0%transmittance with the amplifier control knob (left knob).

  3. Obtain two 13-mmx100-mm test-tubes made of borosilicate glass. The test-tubes must be clean and the outside must be dry and free of fingerprints or smudges. Use lint-free tissue to wipe the outside of the test-tubes. Check to be certain the test-tubes are clean. Put distilled water in both tubes to within 2cm of the top. In the next step, optical properties of the two tubes will be compared.

  4. Check to see that the meter reads 0%transmittance. If it does not, adjust the amplifier control to zero the meter. The test-tubes must always be oriented in the same direction. To insure this, use the frosted or painted area at the top of the tubes to align with the mark on top of the sample holder. Place one of the test-tubes in the sample holder pushing it down until it stops and close the cover. Use the light control knob (right knob) to set the meter to 100%transmittance. Remove the test-tube and read the meter. If it reads 0%transmittance, then you are ready to proceed. If it does not, then readjust the amplifier control knob so the meter reads 0% and then readjust the light control knob once again to read 100% with the test-tube in place. Repeat this step until stable 0% and 100% readings occur. Remove the test-tube and insert the other tube. It should read 100% also. If it reads more than 100%, use it as the blank tube and the first one as the sample tube. Mark the tubes B and S so as not to confuse them. Put the blank tube (B) in a test-tube rack and pour out the water from the sample tube (S).

  5. Rinse S twice with a small amount (about 1mL) 0.020MCr(NO 3 ) 3 solution. Discard the rinses into a 150-mL beaker, not into the sink! Add 0.020MCr(NO 3 ) 3 to the test-tube so that it is about three-fourths full. This same solution will be used for all subsequent readings made in Part I of the activity.

  6. Zero the instrument if the meter does not read zero. Use the blank tube with distilled water to set 100%transmittance. Remove the blank and insert the sample tube with the chromium(III) nitrate solution. Without adjusting any knobs, read the percent transmittance and record it along with the wavelength in the data table. Remove the sample tube from the holder and place it in a test-tube rack.

  7. Turn the wavelength dial to 400nm and zero the instrument. Place the blank tube in the holder and adjust the meter to 100%transmittance with the light control knob. Remove the blank tube and insert the sample tube again. Read and record the % transmittance in the data table for 400nm.

  8. Continue the procedure of setting 0% transmittance, setting 100% transmittance, and measuring the % transmittance of the chromium(III) nitrate solution at 405, 415, 425, 440, 455, 470, 490, 500, 520, 530, 540, 550, 570, 580, 600, and 625nm. Remember to record the data collected in the data table.

  9. Unless otherwise directed by your teacher, return the chromium(III) nitrate to the reagent bottle from which it was obtained. Follow directions from your teacher in this regard. Pour the small amount of chromium (III) nitrate used to rinse the test-tube from the 150-mL beaker into the receptacle designated by the teacher.

  10. Thoroughly wash your hands before leaving the laboratory.

Part II. Quantitative Spectrophotometry (Optional)

Specific directions will be given about whether this part of the activity will be done.

  1. Prepare a data table to record the wavelength, percenttransmittance, and absorbance for the unknown sample. The unknown is in a stoppered test-tube. Record the unknown identifier on the data table.

  2. Measurements on the unknown will be made at the wavelength where the maximum absorption (lowest % transmittance) occurred. This information may be obtained from the data table or furnished by your teacher.

  3. Measure the absorbance for the unknown in the same manner as in Part I. However, in this case only one measurement will be made. Record the wavelength and absorbance of the unknown.

  4. Pour the unknown back into the sample test-tube and return it as instructed by your teacher.

  5. Thoroughly wash your hands before leaving the laboratory.

    Data Analysis and Concept Development

    1. Convert all measured % transmittances to absorbance values in your data table using the conversion table supplied by your teacher.

    2. On a piece of graph paper, plot absorbance (A) on the vertical axis vs. wavelength (l) on the horizontal axis. Connect the points to make a smooth curve. This curve is the visible region absorption spectrum of Cr(NO 3 ) 3 .

    3. OPTIONAL: Use the provided calibration curve to determine the concentration of the unknown sample of Cr(NO 3 ) 3 . Report the concentration in the laboratory writeup.

    Implications and Applications

    1. At what wavelengths does Cr(NO 3 ) 3 absorb the maximum amount of radiation?

    2. To what colors of light do the wavelengths of the absorption maxima correspond? (It may be necessary to refer to a table of complementary colors or to a diagram of the visible light spectrum.)

    3. When a solution is red, does it absorb or transmit red light wavelengths?
    4. Give two uses of an absorption spectrum.

    5. Colored substances absorb light in the visible region of the spectrum. What is the cause of these absorptions?

    6. Instruments are often considered as extensions of human senses. In this sense, what does a visible spectrophotometer do?

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