COLORIMETRY
Gary L. Bertrand
MissouriUniversity of Science and Technology
Equipment: (per pair of students)
4Berel Pipets, large drop
3Strip, 1x12-well
1Plate, 3x4-Well
1Spectronic20 Spectrophotometer
5Cuvettes
3Toothpicks
Part I
1.Fill the designated wells of the 3x4-well plate with the appropriate solutions, directly from the squirt bottles.
Well: Solution:
A1Red Dye Standard
C1Blue Dye Standard
B1Water
B2Water
A4Red or Blue Dye Unknown
C4Purple Unknown
Write the identifying codes for your unknowns on your data sheet.
2.Label 4 clean, dry Beryl Pipets: "Red", "Blue", "Purple", and "Water". Label 3 clean, dry 1x12-well plates: "I", "II", "III"; placing labels on the end of the strip which has a hole.
Drop Procedure: Hold a Pipet vertically about 0.5 to 1 cm above a well, and squeeze gently and smoothly until a drop breaks off of its own weight. The drop should not be shaken off, nor allowed to touch anything until it has broken free.
3.Place strip "I" on a white background with the label end to your right. Draw some standard solution of your assigned color from Well A1 or C1 into pipet "Red" or "Blue". Deliver 8 drops into the leftmost well (#1), 7 drops to the next (#2), etc., through adding 1 drop to the 8th cell.
4.Repeat the step above (3.) with strip "II". Compare strips "I" and "II" by placing them side by side on a white background, and looking down through them. Note that the intensity of the color diminishes smoothly from Well #1 to Well #9. Place one strip above the other, and hold then up so that you are looking through them through the sides of the strips. Note that the intensity of the color is the same in all of the cells of either strip, though there is some variation of intensity horizontally within each well.
If there are significant differences in the intensity of color between the two strips in any of the wells when viewed from above, or in the heights of the liquid when viewed from the side, one or both strips should be cleaned and dried, and prepared again.
5.Set strip "I" aside for the moment, and place strip "II" on a white background with the labelled end to the right. Fill pipet "Water" from Well B1. Make no addition to the leftmost well of strip "II", but add 1 drop of water to the next (#2), 2 drops of water to #3, etc., through adding 8 drops to the empty 9th cell. Starting with well #9, and working right-to-left, stir each solution by swirling a toothpick in the well, wiping against the edge of the well, touching to a towel, then moving to the next well.
6.View strip "II" against a white background, both from above and from the side. Note that the intensity of color now diminishes smoothly from well #1 to well #9 when viewed from either the top or the side.
7.Compare strip "I" to strip "II", viewing from above, and from the side. There should be no substantial differences in the intensity of color in the two strips when viewed from above. Why? (Answer this as the last part of the question below.)
8.Place strip "III" on the white background, with the labelled end to the right. Draw some unknown red or blue solution into pipet "unknown" from Well A4. Place 8 drops into the leftmost well (#1), skip 3 wells, 8 drops into well #5, skip 3 wells, and 8 drops into well #9.
9.Compare the individual wells in strip "III" to strip "I", viewing from above. Note on Data Sheet #1 the well on strip "I" which has an intensity of color closest to each of the wells of strip "III", interpolating if possible (5.5, if between 5 and 6, for instance).
10.Compare the individual wells in strip "III" to strip "II", both from above and from the side. Note the results on Data Sheet #1 for each comparison, as in Step 9 above.
11.Dump, clean, and dry strips "II" and "III". Change the label on one of these to "IIII". Place strip "IIII" on the white background with the labelled end to the right. Fill pipet "purple" with solution from Well C4. Add 8 drops of purple solution to cells #1, #5, and #9.
12.Change the label on strip "I" to "IA". Place strip "IA" on the white background, with the labelled end to the right. Using the red or blue pipet which has not yet been used (not the assigned color) and standard solution from the appropriate well (A1 or C1). Make no addition to the leftmost well of strip "IA", but add 1 drop of standard to the next (#2), 2 drops of standard to #3, etc., through adding 8 drops to the 9th cell. Stir these solutions with a toothpick, as in Step 5, again working from right-to-left. Note that the color of these solutions varies smoothly from red through purple or violet to blue, whether viewed from above or from the side. Note also that it is difficult to judge relative intensity between different colors.
14.Compare the individual wells in strip "IIII" to strip "IA", from above and from the side. Enter your "best" comparisons on Data Sheet #2.
Questions:
Why does the intensity of color vary along the strip(s) when viewed from above in step 4?
Why doesn't the intensity vary when viewed from the side?
Why do strips I and II have the same intensity of color when viewed from above in step 7?
DATA SHEET #1: Red or Blue Unknown # ______
Drops in Well
Strip # 1 23456789
"I" Red/Blue Standard 876543210
"II" Red/Blue Standard876543210
Water012345678
"III"Red/Blue Unknown88 8
Well# 1 2 3 4
"II" Dilution factor (df): 1.000 0.875 0.750 0.620
Well# 5 6 7 8
Dilution factor (df): 0.500 0.375 0.250 0.125
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COMPARISONS FOR CONCENTRATION:
"III"Red/Blue Unknown Well# 1 5 9
Matches from above:
"I"Red/Blue Standards Well#
"II" Well#
Matches from side:
"II" Well#
average match
average dilution factor (df) of standard
"Best" percentage: unknown/standard %
DATA SHEET #2: Purple Unknown # ______
Drops in Well
Strip # 1 23456789
"IA" Red/Blue Standard876543210
Blue/Red Standard012345678
"IIII" Purple Unknown888
Well# 1 2 3 4
Fraction of Red Dye: 1.000 0.875 0.750 0.625
Well# 5 6 7 8
Fraction of Red Dye: 0.500 0.375 0.250 0.125
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COMPARISONS FOR COLOR:
"IIII" Purple Unknown Well# 1 5 9
Matches from above:
"IA"Red + Blue StandardsWell#
Matches from side:
"IA"Red + Blue StandardsWell#
average matchWell#
average fraction Red Dye in standard
"Best" percentage: Red Dye in unknown _____%
Instructor Note: Dyes are from Rainbow Colors (203) 871-2033
Solutions are prepared as: Red Dye #3 1.4 ppt
Blue Dye #1 0.9 ppt
then diluted 1/100 to prepare stock solutions. There may be some deterioration of the stock solutions over a 6-month period.
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