Cybertory™ Virtual Molecular Biology Laboratory

# pH, Dilutions, and Buffers

## Summary

By mixing acids, bases, and buffers together, and diluting them with water, you will learn how to handle solutions in the Virtual Lab. We will also review fundamental principles of pH, and the mathematics used for simple pH calculations.

## Objectives

• Learn to handle solutions in the virtual laboratory. Be able to obtain, arrange, inspect, and label tubes. Learn to use the micropipettor, the pH meter, and the felt marker.
• Practice the math skills necessary to perform serial dilutions, and to mix solutions with desired concentrations of ingredients.
• Be able to use logarithms.
• Review concepts of pH. Be able to calculate the pH of solutions of strong acid or base, and to estimate the pH of solutions of weak acids or bases.

## Background

Acidity is a fundamental characteristic of solutions. It is a measure of the chemical activity of hydrogen ions, which can range from about 10 M to less than 0.00000000000001 M in solutions that might be encountered in a biochemistry lab, with micromolar concentrations and below being quite common. To avoid writing so many zeros past the decimal point, we use the negative logarithm of the hydrogen ion concentration, so the range above becomes -1 to 14.

Hydrogen ion concentration is closely related to hydroxide ion concentration because these two ions react together to form water. Since this reaction is reversible, it is characterized by an equilibrium constant. The dissociation constant of water (the product of hydrogen and hydroxide ion concentrations) is about 1.00E-14. Some reflection on the meaning of logarithms should convince you that this means that pH + pOH = 14, where pOH is the negative logarithm of the hydroxide ion concentration.

Hydrogen ions participate in many chemical reactions, particularly those that release and consume hydrogen ions. Substances that release hydrogen ions are called acids, and those that consume them are called bases. An acid that gives up its hydrogens compeletely in water, such as hydrochloric acid (HCl) is called a strong acid, while one that reaches an equilibrium where its hydrogens are partially dissociated is a weak acid. Acetic acid (CH3CO2H) is an example of a weak organic acid. Conversely, bases that completely dissociate in water, such as sodium hydroxide (NaOH) are strong bases.

Weak acids and bases can serve as reservoirs for hydrogen and hydroxide ions, and act to stabilize the concentration of these ions if they are being released or consumed in a solution. Substances that tend to stabilize pH are called buffers.

The hydrogen ion concentration also affects the ionization state of many proteins, which contain weak acid and base groups on the side chains of certain amino acids, as well as at their amino and carboxy termini. Changing the ionization state of these weak acid and base moieties can dramatically affect the function and even the three dimensional structure of enzymes. Most enzymatic reactions require pH within fairly stringent limits, and thus require solutions to be appropriately buffered for pH.

## Equipment

Read the user interface section of the FAQ for clues on how to use the Virtual Lab.

Below is a list of the various pieces of furniture and equipment used in this experiment. Each item links to its entry in the Simzymes catalog, where you may find further information about that item and how to use it.

## Protocol

You may want to print this protocol so you don't have to keep flipping back and forth between windows.

1. Note the tube racks on the benchtop. The upper one is labelled 'acid and base'; zoom in to read the tube labels, and see that the rack has one tube of 1M HCL, one tube of 1M NaOH, and two tubes of water. The lower rack is labelled 'dilutions', and is filled with empty tubes labelled 1 through 12.
2. Put 45 µl of water in each of tubes 1 through 12. There are more tubes of water in the first drawer if you need them.
3. Add 5 µl of 1M HCl to tube 1.
4. Take 5 µl of the solution from tube 1. Transfer it to tube 2.
5. Transfer 5 µl from tube 2 to tube 3.
6. Transfer 5 µl from tube 3 to tube 4.
7. Continue transferring 5 µl from each tube to the next highest numbered tube, until you reach the last tube (12).
8. Take 5 µl from tube 12, and throw it away by disposing of the pipette tip in the waste can. Each tube should now contain 45 µl of HCl solution, in varying concentrations.
9. Calculate the concentration of HCl in each tube. Write these expected concentrations in your notebook.
10. Based on the concentration of HCl, estimate the pH you expect to find for each tube.
11. Find the pH meter in the second drawer (next to the felt marker). Measure the pH of each tube by dragging the pH meter until the tip of the probe is over the tube, then drop it into the tube. It will display the pH of the solution.
12. Record your results in a table with columns for tube number, concentration of HCl, expected pH, and measured pH.

## Discussion Topics

• Do you notice a suspicious correlation between tube number and pH? How do you explain this? Does it hold for all values of pH? Why (or why not)?
• On the shelf is a rack containing six unknown samples labelled 'U1' through 'U6'. Which are acids, and which are bases? Can you design a titration experiment to measure the number of equivalents of each? How could you determine their pKa values using just what you have in the lab?
• You will find a variety of acids and bases in the drawers of the second bench unit. Can you think of other pH-related experiments you could do using these chemicals?
• What aspects of the virtual lab are different from what you would expect in a real lab?

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