PHENOLSULFONPHTHALEIN OR PHENOL RED EXPERIMENT
INTRODUCTION:
Watch the videotapes 849 and 850 before you come to lab.
Bring this written experiment with you. You may have to study
the written version again after you have seen the videotapes.
This experiment has three parts, which are demonstrated and explained in
the videocassettes VC 849 and 850. The first part, Part A, uses the UVVisible spectrophotometer to analyze the spectrum so that the absorbtion
characteristics between 350 nm end 650 nm and the wavelength where maximum
absorption (λ max) occurs can be determined. The second part, Part B,
quantitatively determines the concentration of phenol red in an unknown
sample. The third part, Part C, determines the pK of phenol red
spectrophotometically. The theory for each of these parts follows the
"RESULTS" section of Part C, near the end of this experiment.
PART A; SPECTRUM ANALYSIS OF THE ACIDIC AND BASIC FORMS.
Phenolsulfonphthalein displays a yellow color in acidic solutions and a red
color in basic solutions. These characteristics make it beneficia1 to use as
a pH indicator in acid-base titrations and as a dye to detect renal tubular
damage in a patient. The chemical species responsible for each color are
shown below:
O
O
OH
O+
O
SH
OO
phenolsulfonphthalein
Acidic Species (HA)
Yellow Color
O
SH
OO
phenolsulfonphthalein
Basic Species (A-)
Red Color
H+
Part A PROCEDURE:
1. Take three clean preferably dry 16 mm x 150 mm test tubes
and label B,HA, and A-. Place them in a rack.
2. To tube B pipette in 2.0 mL of water( 2 Shots ), followed by 8 mL
of acetate buffer, pH 5.8 ( 1 shot ). Mix the contents by vortexing.
3. To tube HA pipette in 1.0 m1 of water, followed by 1 mL of phenol red,
followed by 8.0 ml of acetate buffer. Mix the contents by
vortexing.
4. To tube A- pipette in 1.0 ml of water, followed by 1 mL of phenol
red, followed by 8.0 ml of borate buffer, ph 9.8. Mix the contents
by vortexing.
Transfer the data from steps 2-4 into the table below:
Tube
Phenol Red
H2O
(mL)
(mL)
Acetate
Buffer
PH 5.8
(mL)
Borate
Buffer
PH 9.8
(mL)
B
HA
A5. Take the rack of tubes to the UV-Visible spectrophotometer and
pour some of the solution from tube B into a rectangular cuvette,
leaving about 1/8 to 1/4 inch space between the solution and the top
of the cuvette. Wipe all sides of the cuvette with a KimWipe to
remove moisture.
6. P1ace the cuvette into the cell well, with the frosted side facing
you.
7. Push the “SCAN ” button on the instrument and set the maximum and
minimum wavelengths you wish to scan.
8. Scan the blank sample by pushing the “BASELINE” button.
9. Pour some of the solution from tube HA into another rectangular
cuvette, leaving some space between the solution end the top of the
cuvette, and wipe all of its sides with a KimWipe.
10.Place the cuvette into the cell well, again with the frosted side facing
you. Scan the HA sample by depressing the “START SCAN” button. Once the
scan is completed, the screen will display the message “Storing to file
#…”, where # is a number between 0 and 9. Note the location of the saved
file.
11. Repeat steps 9 & 10 for the A- sample.
12. Once this scan is complete, the A- sample spectrum will be on the
instrument screen.
13. At this point, hit the “RECALL” button, on the left side of the keypad.
14. Scroll down to the location of your saved HA spectrum and press the
“ENTER” key.
15. The screen will now refresh, displaying both spectra.
16. Press the “PRINT” button, on the left side of the keyboard.
Next, press the “PRINT GRAPH” button.
RESULT:
Record the maximum and minimum absorptions of each curve in your notebook.
The reason for overlaying the two spectra is that each species (acidic and
basic) has unique absorption characteristics, and viewing them together
helps to identify the compound. Such a procedure is used for drug
identification. Once the drug has been identified, it is quantified in the
manner shown in Part B of this experiment.
PART B: DETERMINATION OF AN UNKNOWN CONCETRATION OF PHENOL RED
In the preceding section you saw that the red form of phenol red gave a
narrower and higher λ max than did the yellow form. This means that
assaying phenol red in the red form will give a more sensitive assay than if
you attempted to essay it in the yellow form. The lambda max of the red form
was approximately 550 nm while the yellow form gave almost the minimum at
550 nm. Some of you may have seen the lambda λ max at 560 nm on your graph,
but for the Spectronic 20, the bandwidth is larger than on the UV_VIS
spectrophotometer so 550 nm is close enough. It is also stated in the video
presentation.
PROCEDURE:
The fastest way to perform the assay is to follow the procedure as outlined
in the table below:
Tubes
B
1
2
4
6
8
10
Unk
H2 O
(mL)
2.0
1.9
1.8
1.6
1.4
1.2
1.0
1.0
Phenol Red
(mL)
0
0.1
0.2
0.4
0.6
0.8
1.0
0
Unknown
(mL)
0
0
0
0
0
0
0
1.0
Ph 9.8 Buffer
(mL)
8.0
8.0
8.0
8.0
8.0
8.0
8.0
8.0
1. First, record your unknown number in your notebook.
2. Those students using Spectronic 20’s:
Check to see which kind of phototube is present in your instrument.
There may be a note on the top of your machine that tells you which
one is present. Check with the instructor to see if you need to change
phototubes and add/remove f11ters. Set the wavelength to 550 nm then
turn it on, if it is not on already.
Those students using the UV-VIS Spectrophotometer:
Set the “FIXED” wavelength to 550 nm.
3. Obtain 8 CLEAN AND DRY test tubes.
of the table above.
Label these tubes as in column 1
4. Hand-pipette into each tube the appropriate amounts of solutions,
as specified in the above table.
by vortexing.
Mix the contents in each tube
Those using spectronic 20’s, will need eight 13 x 100 mm cuvettes,
labeled as in column 1 of the above table.
Those using th UV-VIS spectrophotometers will need eight of the 1 cm
square cuvettes used in Part A, labeled as in column 1 of the above
table.
5. Fill the tubes/cuvettes 4/5 full with the appropriate solution.
Record your data in tabular form using the following format:
Tube
B
1
2
4
6
8
10
Unk
Concentration x10-4
Absorbance
% Transmittance
1
2
4
6
8
10
?
Those using spectronic 20’s
6. With sample we1l door closed and sample well empty, turn the left hand
control knob so that the needle/display points to/reads 0%T and ~ A.
7. Wipe tube B with a KimWipe to remove any moisture that may be on
the outside of the tube and place it in the sample well, making sure that
the frosted or white mark on the tube is aligned with the mark on the lip
of the sample well. Close the lid. Turn the right hand control knob
clockwise until the needle or digital meter reeds 100%T and 0 A
8. Remove the tube containing the blank solution and close the lid.
Check to see if the needle returns to 0%T and ~ A. If it does not,
repeat steps 6 end 7. If the needle does return to 0% T and ~ A,
go to step 9.
9. The remaining tubes contain various concentrations of your sample.
Wipe the outside of each one with a KimWipe to remove any moisture then
read and record the Absorbance and the Percent Transmittance of each one
with the lid closed and the frosted or white mark on the tube aligned
with the mark on the lip of the sample we1l. While you will use the
absorbance in your calculations and graphs, It is wise to record the
Percent Transmittance as well because you can easily make e mistake
reading the Absorbance since you have to read 1ts scale from right to
left.
10.When you are done with your assay, remove the last sample tube, close
the lid, end turn off the spectrophotometer by turning the left hand
control knob counter clockwise.
Those using the UV-VIS spectrophotometers
6. With the “FIXED” wavelength set to 550 nm, the sample B in the we1l and
the sample door closed, press the “BLANK” button. The spectrophotometer
display will read “.000 ABS”.
7. The remaining tubes contain various concentrations of your sample.
Wipe the outside of each one with a KimWipe to remove any moisture then
read and record the Absorbance and the Percent Transmittance of each one
with the lid closed and the frosted side of the tube facing you. While
you will use the Absorbance in your calculations and graphs, It is wise
to record the Percent Transmittance as well.
8. When you are done with your assay, remove the last sample tube, close
the lid, and turn off the spectrophotometer by flipping the switch at the
back of the unit, on the left.
RESULTS:
Take the data from your table and graph the resu1ts. Your graph should be a
straight line with your unknown absorbance and concentration marked on the
graph. In all assays in clinical and biochemistry, your unknown has to have
a concentration somewhere between the values of the lowest standard and
highest standard, If it turns out that your unknown is below the lowest
standard, you have to make a standard whose Absorbance is below that of your
unknown. If your unknown value is above that of the highest standard, you
either have to make a standard whose absorbance is above the unknown, or you
have to dilute your unknown by a known amount and re-assay it.
In this assay, once you have plotted the values of your standards on the
graph, draw a line point to point from the origin to the point of the
highest standard. The result1ng curve should be linear or almost linear. If
your point to point line is linear or almost 1inear, draw a smooth, best fit
line through as many of the standard points as possible. Now find the
absorbance of your unknown on the ordinate and draw a light line from it
horizontally to the linear curve, then draw a light line vertically from the
curve to the abscissa and read off the concentration of your unknown.
Remember that you diluted your unknown 1:10 before you assayed it, so you
will report both the diluted concentret10n end the undiluted concentret1on
in you lab report. In addition, you will write your unknown number again,
this time in the RESULTS.
PART C: SPECTROPHOTOMETRIC DETERMINATION OF pK
This part of the experiment is not demonstrated in the videotape.
The explanation or theory for th1s part of the experiment will come after
the procedure. Suffice it to say that 1n this part of the experiment each
tube has some of the acidic species and some of the basic species in it.
PROCEDURE:
If you are performing Part C on a different day from when you did Part B,
you will have to make fresh standards of part B, read their absorbances and
% Transmittances after setting the meter to 100%T with tube B, and draw
another graph of the results. The total phenol red concentration 1n each
tube is 1 x 10-5.M, which also means 10 x 10-6 M. Part of that
concentration is in the acidic or yellow form and part of it is in the basic
or red form. You have to determine how much of each spec1es is in each
tube.
Tube
1
2
3
4
5
6
7
H2O
Phenol
Buffer
Buffer
Buffer
Buffer
Buffer
Buffer
(mL)
Red
(mL)
PH 7.2
(mL)
PH 7.5
(mL)
PH 7.8
(mL)
PH 8.0
(mL)
PH 8.4
(mL)
PH 8.8
(mL)
10
1
1
1
1
1
1
0
1
1
1
1
1
1
8
8
8
8
8
8
1. Obtain 7 clean and nearly dry large test tubes and label each one
with a number between 1 and 7.
2. To each tube use the repipettor to pipette the correct amount of
water, shown in the table above.
3. To each tube use the repipettor to pipette the correct amount of phenol
red as shown in the table above.
4. Pipette the correct amount of buffer into each tube as is shown in the
table above.
Mix each tube by vortexing.
5. Perform the assay as in Part B.
RESULTS:
Use the table below as a guide.
pH
7.2
7.5
7.8
8.0
8.4
8.8
A- ABS.
0.140
o.160
0.200
0.240
0.260
0.300
Conc A-x
3.5
4.0
5.0
6.0
6.5
7.5
Conc HA
6.5
6.0
5.0
4.0
3.5
2.5
A-/HA
0.538
0.667
1.000
1.500
1.857
3.000
Log A-/HS
-0.269
-0.176
0.000
0.176
0.269
0.477
1. In the first two columns are the laboratory data. Column 3 is read from
the graph of the standard solutions that were made up from the table in
Part B. You look up the absorbances that are in co1umn 2 of the table
above and following the instructions for reading the Part B Concentration
Graph, obtain the concentration of the red species. Record each
concentration in co1umn 3.
2. Next, calculate column 4 by subtracting column 3 FROM l0x 10-6 M ie.
10x 10-6 -[A-]; in order to obtain the concentration of HA, which is the
yellow species.
3. The pK shou1d be around 7.8; however yours may be different due to the
type of equipment that you have.
THEORY:
1.In Part A the buffer at pH 5.8 converted phenolsu1fonphthe1ein completely
to the acidic species.(ye11ow co1or) You recorded its behavior at
wavelengths from 350 nm through 650 nm. The buffer at pH 9.8 converted
the pheno1sulfonphthe1ein completely to the basic species (red color). you
recorded its behavior at wave1engths from 350 nm through 650 nm. These
scans of Absorbance against weve1ength help identify the compound. If you
had an unknown compound and thought it was phenlsu1fonphthe1ein, you
could compare the scans of your unknown with those of authentic
phenolsu1fonphthele1n. You were also ab1e to determine the lambda max for
each species in order to decide which form would be best to use for an
assay. The use of this kind of procedure in research and commercially is
given to you in the “RESULTS” section of Part A end in the assay of
Part B.
2. Part B is the norma1 way to assay a compound. All of the pheno1 red is
converted to the Basic species because the most sensitive essays of it can
be performed when it is 1n that form.
3. In Part C, you are using not only the Beer-Lambert law but a1so the
Henderson-Hasselbe1ch equation which helps you calculate the concentration of the acidic and basic species of a compound at e certain
pH, or the pH if the pK and concentrations are known, or the pK if
everything else is known. This equation is used mostly to help calculate
the amount of each sa1t to be used in making up buffers (see problem 4 in
the “PROBLEM” section at the end of this experiment). The equation is pH =
pK + 1og[A-l/[HA}. The derivation comes from the acid-base chemical
equation:
HA = H+ + AK = H+ x (A-/HA)
log K = log H+ + log [A-]/[HA]
-log H+ =-log K + log[A-]/[HA], but -log H+ = pH and -log K = pK, so
pH = pK + log [A-]/[HA]
If [A-] =[HA] then [A-]/[HA] =1, its log = 0 and pH = pK.
PRACTICE PROBLEMS:
1.
Calculate the pH of a phenol red solution if 0.8 ml of a
1.00 x 10-4.M solution was di1uted to 10.0ml with buffer, the
resulting basic species was found to have e concentration of
6.00 x 10-6 M, and the pK = 7.50
ANSWER: pH = 7.98
2. Calculate the concentration of each species of phenol red in a
1.24 x 10-4!1solution jf the pH = 7.20 and the pK = 7.80
ANSWER: HA =9.90 x 10-5!1 and A- = 2.5 x 10-5 t1
3. What is the pH of en acetate buffer if 500 ml of it contains 0.1.00 mole
of acetic acid and 0.07 mole of sodium acetate. The pK of acetic acid is
2.76. The term acetate buffer means the acetate concentration is the sum
Of acetic acid plus sod1um acetate. The terms phosphate buffer, borate
buffer, etc. have similar meanings.
pH = 4.60
4.The M.W. of sodium acetate is 82.0 g/mole. The M,W. of acetic acid is
60.1g/mole, its density is 1.05 g/ml. and its pK = 4.76. Assuming that
concentrated acetic acid is 1 00 %, what weight, in grams of sodium
acetate and what volume in ml of acetic acid would be needed to make
500 ml of 0.200 M acetate buffer at pH 4.90?
ANSWER: 4.76 gams of sodium acetate and 2.4 ml of acet1c acid.
5. Write the chemical structures of sodium acetate, acetic acid, and
phenol red (acidic and basic species).
ANSWER: Look in this experiment and any general chemistry textbook or
Handbook of Chemistry end Physics.
6. Derive the Henderson-Hasselbalch equation from HA = H+ + A- end the
latter from the Henderson-Hasselbalch equation.
ANSWER: See this experiment
7. Derive Beer's Law from I/I0 =e-kt or from -dl/dc=KI
ANSWER: See the Spectrophotometry Experiment
REFERENCES:
1. Rendina,George;EXPERIMENTAL METHODS IN BIOCHEMISTRV; W.B.
Sounders Co. (Golden Series); Philadelphia, Penn 1971;
pp51-56