Source: Smaa Koraym at Johns Hopkins University, MD, USA
In the first part of this experiment, you will prepare a sodium phosphate solution buffered at pH 7.0. Monosodium phosphate is a weak acid with the conjugate base, disodium phosphate. Unadjusted monosodium phosphate solutions usually have a pH of about 4 - 6.
Buffers are most effective close to their pKa, which is 6.8 to 7.2 for monosodium phosphate. So, you will use NaOH to push the equilibrium towards the conjugate base without altering the overall composition of the buffer equilibrium.
| Molar mass of NaH2PO4 = 119.98 g/mol | |
| Solution volume (mL) | 200 |
| Molarity (mM) | 50 |
| Moles of NaH2PO4 (mol) | |
| Mass needed (mg) | |
| Initial volume of 1 M NaOH (mL) | |
| Final volume of 1 M NaOH (mL) | |
| Volume of NaOH used (mL) | |
| Volume of DI water needed (mL) | |
Buffers can be used to evaluate compounds at specific pH values. In this section, you will record the absorbance spectrum of the indicator neutral red in various buffers. The protonated form of neutral red is red, and the deprotonated form is yellow-orange, meaning that they absorb green and blue-violet light, respectively. Thus, the acidic and basic forms have distinct absorption wavelengths. Protein binding alters the properties of neutral red, changing both its absorbance and its pKa.
After measuring the absorbance spectrum of free neutral red at several pH values, you will add riboflavin-binding protein, or RP, to the solutions and measure the absorbances again. Absorbance intensity is related to concentration, so you will use the spectra to determine the pKa of free and bound neutral red after the lab.
| Cuvette # | Buffer pH | Abs. at λmax (Free NRH+) | Abs. at λmax (Bound NRH) |
| 1 | 5.0 | ||
| 2 | 5.5 | ||
| 3 | 6.0 | ||
| 4 | 6.5 | ||
| 5 | 7.0 | ||
| 6 | 7.5 | ||
| 7 | 8.0 | ||
| 8 | 8.5 | ||
| 9 | 11 | ||
| 10 | blank |
Now, let's analyze our absorbance data to determine the pKa's of neutral red.
| Free NRH+ | Bound NRH+ | |
| λmax (nm) | ||
| ΔA (nm) | ||
| Midpoint (nm) | ||
| pKa |
Click Here to download Table 3
In the first part of this experiment, you will prepare a sodium phosphate solution buffered at pH 7.0. Monosodium phosphate is a weak acid with the conjugate base disodium phosphate. Unadjusted monosodium phosphate solutions usually have a pH of about 4 to 6.
Buffers are most effective close to their pKa, which is 6.8 to 7.2 for monosodium phosphate. So, you will use sodium hydroxide to push the equilibrium towards the conjugate base without altering the overall composition of the buffer equilibrium. Before starting the lab, calculate the mass of monosodium phosphate that you will need to make 200 milliliters of a 50-millimolar solution.
This section of the lab uses sodium hydroxide, which is corrosive and toxic. Use caution when pouring and transporting sodium hydroxide. Now, let's get started.
First, put on a lab coat, splash-resistant safety glasses, and nitrile gloves. Next, fill a 250-milliliter plastic wash bottle with deionized water. Label two 100-milliliter beakers for neutral and basic aqueous waste, respectively.
Then, calibrate your pH meter using the provided buffers. Store the probe in its storage solution when you are done. Now, bring a 400-milliliter beaker to the analytical balance area to obtain the monosodium phosphate that you will need.
Tare a piece of weighing paper and use a clean spatula to measure out the required amount of monosodium phosphate according to your calculations. Monosodium phosphate absorbs moisture from the air, so work quickly to obtain an accurate measurement, and close the container tightly when you are done with it. Record the exact amount of monosodium phosphate that you measure out in your lab notebook.
Then, place the monosodium phosphate in the beaker and clean the spatula with a laboratory wipe. Throw out the weighing paper and the laboratory wipe before returning to your fume hood. Now, use a graduated cylinder to measure out 175 milliliters of deionized water.
Pour the water into the beaker of monosodium phosphate and add a magnetic stir bar. Stir the solution on a stir plate until the salt has dissolved completely and the solution appears homogeneous. This usually takes two to three minutes.
Then, measure 15 milliliters of deionized water with a graduated cylinder. Pour the deionized water into the beaker and continue stirring the solution until it appears homogeneous again, which usually takes one to two minutes. Then, turn off the stir motor.
Rinse the pH probe with deionized water, and then clamp it in the solution with the sensor above the stir bar. Now, bring a 10-milliliter graduated cylinder and watch glass to the dispensing hood, and measure out 10 milliliters of 1 molar sodium hydroxide. Cover your sodium hydroxide with the watch glass, and carefully bring it to your fume hood.
Note the exact volume in the graduated cylinder. Then, resume stirring the monosodium phosphate solution. While monitoring the pH reading, use a disposable pipette to slowly add sodium hydroxide to the stirring solution in a drop-wise manner.
Once the buffer pH reaches 7.0, return any sodium hydroxide still in the pipette to the graduated cylinder. Then, calculate the volume of sodium hydroxide that you added from the initial and final volumes in the graduated cylinder. Subtract that volume from 10 milliliters to determine how much deionized water you must add to your buffer to reach a total solution volume of 200 milliliters.
Measure the deionized water that you need with another 10-milliliter graduated cylinder and add it to the stirring solution to finish making the buffer. Rinse the pH sensor with deionized water, cover it with the storage solution filled cap, and unplug or turn off the probe. After that, label a 250-milliliter polyethylene bottle as 50 millimolar monosodium phosphate buffer, pH 7.0'Use forceps to retrieve the magnetic stir bar from the solution.
Then, place a funnel in the mouth of the bottle and pour the buffer solution into the bottle. Remove the funnel, and cap the bottle tightly. You are now ready to move on to the absorption spectroscopy section.
Buffers can be used to evaluate compounds at specific pH values. In this section, you will record the absorbance spectrum of the indicator neutral red in various buffers. The protonated form of neutral red is red, and the deprotonated form is yellow-orange, meaning that they absorb green and blue-violet light, respectively.
Thus, the acidic and basic forms have distinct absorption wavelengths. Protein binding alters the properties of neutral red, changing both its absorbance and its pKa. After measuring the absorbance spectrum of free neutral red at several pH values, you will add riboflavin-binding protein, or RP, to the solutions and measure the absorbances again.
Absorbance intensity is related to concentration, so you will use the spectra to determine the pKa of free and bound neutral red after the lab. Before starting this section, draw a table in your lab notebook, listing the cuvette number, buffer pH, absorbance at lambda max for free protonated neutral red, and absorbance at lambda max for bound protonated neutral red. Number the cuvettes 1 through 10 and list the nine buffer pH values that you will use.
The 10th cuvette will be a deionized water blank. Always hold cuvettes by the textured sides and wipe the transparent sides just before putting the cuvette in the spectrophotometer. Remember to align the transparent sides with the beam of light in the spectrophotometer.
Now, let's get started. Obtain ten 1.5-milliliter cuvettes and caps, and label the caps 1 through 10 to match the table in your lab notebook. Label a 25-milliliter beaker as DIH2O'and fill it with deionized water.
Then, flush your neutral aqueous waste down the drain, and relabel the beaker as aqueous buffer waste'Also, label a 400-milliliter beaker for used micropipette tips. Then, attach a tip to a 1-milliliter micropipette, and use it to dispense 1000 microliters of deionized water into cuvette 10. This will be your solvent blank.
Now, eject the tip, set the micropipette to dispense 925 microliters, and attach a new tip. Place 925 microliters of your pH 7.0 monosodium phosphate buffer in cuvette five. Eject the tip and cap the cuvette.
Next, bring the remaining empty cuvettes to the buffer table. Guided by the table in your lab notebook, dispense 925 microliters of each buffer into the appropriate cuvette using the labeled micropipettes. Be careful not to use the same pipette tip for different buffers.
Once you have finished, bring the buffers back to your workbench. There, set a 200-microliter micropipette to dispense 75 microliters and attach a tip to the micropipette. Now, obtain a vial or tube of neutral red solution, which may be shared with another student group.
Dispense 75 microliters of neutral red into each of the nine buffer cuvettes. Replace the pipette tip if it touches a buffer. Eject the pipette tip and cap the cuvettes when finished.
Now, invert each cuvette several times to thoroughly mix the solutions. Once you have mixed neutral red with every buffer, take a picture, or write down the colors of the solutions. Next, turn on a hand-held spectrophotometer and wait for the light source to warm up.
Once it is ready, create a new experiment to measure absorbance versus wavelength for the free neutral red. Then, insert the cuvette of deionized water. Acquire a spectrum of the deionized water, and set it as a solvent background or blank.
Then, remove the blank from the spectrophotometer and insert the cuvette of neutral red in the lowest pH buffer, which will have the highest concentration of protonated neutral red. Acquire a spectrum, which should show one strong peak. Identify the wavelength corresponding to the highest point of this peak, or the maximum.
Record this wavelength in your lab notebook as lambda max for protonated free neutral red. Save the data and remove the cuvette. Record the absorbance in your notebook then collect spectra for cuvettes 2 through 9 using the same procedure.
The spectra should all cross at a single point, called the isosbestic point. Record the wavelength of this point in your lab notebook. If a spectrum does not cross at that point, empty and clean the cuvette, prepare a fresh sample, and try again.
Once you finish collecting spectra for all 9 cuvettes, save and export the data. Then, create a new experiment to measure absorbance versus wavelength for the bound neutral red. Fit a new tip to the 200-microliter micropipette and obtain a shared container of riboflavin-binding protein.
Add 75 microliters of riboflavin-binding protein solution to each cuvette, including the blank. Eject the tip, secure the cuvette caps, and invert the cuvette several times to mix the solutions. Now, insert cuvette 10 into the spectrophotometer and set it as the solvent blank.
Then, acquire a spectrum of the lowest pH sample and identify the wavelength corresponding to the maximum of the most intense peak. Record it in your lab notebook as the lambda max of protonated-bound neutral red. After that, acquire spectra for cuvettes 2 through 9 in the same way as you did for the free neutral red samples.
Record the intensities at lambda max for protonated-bound neutral red in your table, along with the wavelength at the isosbestic point. When you are done, save your data, export it for later analysis, and shut down the spectrophotometer. Put away the micropipettes, the tip boxes, the pH meter, and the spectrophotometer.
Dispose of your used pipette tips in an approved waste container or trash bin. Now, empty the cuvettes into the aqueous buffer waste beaker and rinse the cuvettes into the beaker with deionized water. In your fume hood, empty the excess sodium hydroxide into the basic waste and rinse the graduated cylinder with water.
Flush the basic aqueous waste down the drain with copious tap water, along with the other aqueous waste. Wash your glassware, cuvette caps, and stir bar per your lab standard procedures. Lastly, clean your work surfaces with a damp paper towel and throw out used paper towels and lab wipes in the lab trash.
Now, let's analyze our absorbance data to determine the pKa's of neutral red. First, plot both sets of intensity data with absorbance intensity at lambda max on the y-axis, and pH on the x-axis, with the points joined by smooth lines. Then, for each data series, calculate the difference between the starting and ending absorbance intensities.
Use that to calculate the absorbance midway between the starting and ending absorbances for each series, or the absorbance midpoints. Now, find where the midpoint occurs on each line, and identify the pH value at that point. These pH values are the pKa's of free and bound neutral red.
Here, we see an increase in pKa of about 1 between free and bound neutral red, indicating that bound protonated neutral red is a weaker acid than free protonated neutral red by an order of magnitude.
