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New Mexico State University
College of Arts and Sciences
Department of Chemistry and Biochemistry
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Spectronic 21 Spectrophotometer
  

Spectronic 21The Spectronic 21 Spectrophotometer is an update of the Spectronic 20 Colorimeter (spectrophotometer) The operation is very simple and similar to that of the Spec 20. At the top of the instrument is a meter that displays both absorbance and percent transmittance of the sample being measured. On the left side is the compartment for holding the sample to be measured. On the top right are the wavelength selector and indicator. On the lower front left is the knob that controls the 100% position on the %T scale. On the lower front right is the switch that turns on the power. There is also a swith to turn on the UV lamp and a know below the instrument to select the correct detector. Quartz cuvettes are required for the analysis of sample in the ultraviolet. A special sample holder is used for the 1-cm square quartz cells.

Here are the steps to follow when using a Spec 21 in the visible region.

  • Turn the instrument on using the switch at the lower right front of the instrument. Allow about 5 minutes for warm up when first turned on.
  • Select the appropriate wavelength for the sample to be measured.
  • With the Spec 21 no adjustment is necessary to set 0%T, this is done automatically.
  • Fill a tube half full with water. This is called a blank. Place it in the sample holder and close the cover.
  • With the blank in the sample holder and the cover closed, adjust the meter needle all the way to 100%T using the light control knob on the lower left front of the instrument.
  • Remove the blank and place the sample to be measured in the sample holder and close the cover.
  • Read absorbance value (or %T) from meter.
  • Repeat this step with additional known samples if making a calibration curve or verifying proportionality (Beer's Law).
  • Repeat this step with a solution of unknown concentration, so that its absorbance can be compared to the absorbance of a known solution.

Additional details on operation of the spectronic 21

  • SET WAVELENGTH
    The wavelength selector is on the top right side of the instrument. To set the wavelength correctly, view the dial from directly above. Otherwise, the correct wavelenght will not be set. Such 'parallax' errors occur if the line of sight is NOT Perpendicular to the face of the dial. A second adjustment is required for the photomultiplier for UV readings.
  • CUVETTES
    All readings are done in cuvettes, which resemble small glass test tubes, but are made from higher quality glass.
    Two cuvettes are required usually to take the readings -- one to hold the water blank and one to hold sample solutions.
  • RINSE CUVETTES
    In case the cuvettes are not clean and dry before using, rinse them thoroughly with the test solution. Several small rinses are preferred to using one big rinse in order to coat the inside of the cuvette.
  • FILLING VOLUME
    When pouring a liquid into the cuvette, the solution must fill the cuvette to a sufficient height so that the internal light beam passes through the solution in the cuvette, and not through air.
    The Spec 21 cuvettes have a horizontal index mark to show the minimum required filling volume.
  • Kimwipes
    It is important to clean the outside, lower portion of a cuvette before taking any readings. Fingerprints, liquid droplets, and smudges on the cuvette surface can give false light absorbance readings.
    The proper procedure for cleaning the surface of a cuvette is to use laboratory tissues, called Kimwipes.
  • CLEAN WITH KIMWIPES
    Wipe the cuvette first with a damp Kimwipe and then with a dry tissue.
    After cleaning the cuvettes, handle them by their tops. Don't touch the lower portion of the glass.
  • AIR BUBBLES
    Even after cleaning the cuvette errors may still occur in a reading if air bubbles are present in the solution.
    Before reading a sample of even a blank, Remove Air Bubbles
    Removing air bubbles can be done by tapping the bottom of the cuvette to dislodge the bubbles.
    Remove all air bubbles.
  • REMOVING AIR BUBBLES
    If tapping does not work, then cover the top of the cuvette with Parafilm (a stretchy plastic covering) and slowly invert the cuvette several times until all the bubbles are removed.
  • SAMPLE HOLDER
    Once the sample or blank is free from bubbles and in a clean cuvette, it can be inserted into the sample holder.
    The sample holder is located on the left, top surface of the Spec 21. It is fitted with a cover, which must be closed before taking readings.
  • INSERTING A CUVETTE
    When inserting a cuvette into the sample chamber, GENTLY push the cuvette into its position. Hard pushing could damage the instrument.
  • VERTICAL INDEX MARK
    To assure reproducible positioning in the sample chamber, the cuvette has a vertical index mark near its top.
    When inserted properly, the vertical index mark on the cuvette must be exactly aligned with the small nub on the top of the sample holder.
  • FINAL STEP
    Close the cover to the sample chamber. Stray light can enter and give false readings

SUMMARY 1

  • Rinse the inside several times with solution.
  • Fill the cuvette above the horizontal index mark.
  • Remove any air bubbles by tapping or inversion.
  • Clean the outside surface with Kimwipes.
  • Insert gently into sample chamber.
  • Align the vertical index mark on the cuvette in holder.
  • Close the chamber cover before taking a reading.
  • ADJUSTING THE BLANK
    The blank solution is used to calibrate the instrument so that the internal light beam passes through the cuvette to the light-sensing device (photomultiplier). This is indicated by a dial reading of 100% Transmittance or zero Absorbance.
    With the water blank properly in the sample holder with the chamber cover closed, now adjust the light control knob to the right until the readout meter on the face of the Spec 21 reads exactly 100%T. The light control switch is on the lower left of the instrument.
  • AVOID ERROR
    To avoid parallax error when reading the meter, look directly in front of the needle. When positioned correctly, see if there is a reflection of the needle in the mirror.
  • REMOVE CUVETTE AFTER READING
    Always remove the cuvette from the sample holder as soon as the necessary adjustment or reading has been completed. Leaving the cuvette in the sample holder for an extended period can damage the light-sensing device.
  • MATCHING CUVETTES
    To reduce reading errors due to imperfections or scratches on the cuvettes, the sample and blank cuvettes should be matched to one another.
    This entails finding two cuvettes, which give exactly the same reading when containing the same solution.
    An alternative to using matched cuvettes is to read all solutions, both blanks and samples, in a single cuvette. This is cumbersome, but it assures accuracy.
    If the second cuvette reads within 1% of the blank cuvette, then they may be considered as matching.
  • LEARNING TO READ THE METER
    Before beginning to work with the a sample solutions, learn how to read the Spec 21's scale for light absorption.
    The meter simultaneously indicates Absorbance (the amount of light absorbed by the sample) on the lower scale and Percent Transmittance(the portion of light passing through the sample) on the top scale.
  • HOW TO READ THE METER
    The top scale (%T), which is divided into increments of constant size, must be read from left to right. This is an easy scale to read.
    The bottom absorbance scale has increments between tick marks, which vary across the scale, making for some difficult readings. Also, the scale is read from right to left, the opposite of the %T scale.
  • PRACTICE TAKING ABSORBANCE READINGS
    • With two matching cuvettes and have learned to properly read the Spec 21 absorbance scale,begin taking absorbance readings of the copper sulfate solutions.
    • Select one of the three concentrations of CuSO4 to work with. Remember to handle the chemicals carefully, avoiding skin contact.
    • The Spec 21 must first be adjusted to 100% T using the blank cuvette. Verify that the Spec 21 is set to 100% T when the blank cuvette is inserted properly into the chamber.
    • Now prepare the sample cuvette. Pour the water out of the second, matched cuvette, rinse it with the selected blue solution several times, fill it to above the mark, wipe the outside clean, remove any bubbles, gently insert it into the sample chamber, and close the cover.

SUMMARY 2

  • First 'blank' the Spec 21 to 100% T (zero A)
  • Rinse the inside of the cuvette with sample solution
  • Fill cuvette to above horizontal index mark
  • Remove all air bubbles by tapping or inversion
  • Clean outside of cuvette with when, then dry, tissue
  • Insert GENTLY into sample chamber
  • Align vertical index mar on cuvette in holder
  • Close cover on sample chamber before reading
  • Read the %T and/or the absorbance on meter
  • Remove cuvette immediately after taking reading
  • COMPARE Read both the absorbance and %T for the sample and check its accuracy with the value on bottle containing the standard.
  • CHANGE THE WAVELENGTH
    After determining the absorbance and %T at a particular wavelength, change the wavelength and read the values for Absorbance and %T again for the same sample of copper sulfate solution. Use the same cuvette to take the reading.
    However -- and this is important to remember -- every time the wavelength is changed, readjust the Spec 21 using the blank solution.
  • BLANKING
    Insert the blank and adjust the light control so that the meter reads 100% T (also zero absorbance) before reading the sample at the new wavelength. You may not see much change in the 'blanking' adjustment for this wavelength change; but in may parts of the spectrum, the change is dramatic. Forgetting to 'blank' the instrument can give erratic data.
  • CHECK ABSORBANCE AT ALL WAVELENGTHS
    You should determine the absorbance and %T for the sample at all three suggested wavelengths, checking the accuracy of all the readings. For more practice using the Spec 21, try a different concentration of copper sulfate sample.

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