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1.3: Water and Buffers

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    Source: BiochemFFA_1_3.pdf. The entire textbook is available for free from the authors at

    When it comes to water, we’re literally drowning in it, as water is by far the most abundant component of every cell. To understand life, we begin the discussion with the basics of water, because everything that happens in cells, even reactions buried deep inside enzymes, away from water, is influenced by water’s chemistry.

    The water molecule has wide ‘V’ shape (the HO-H angle is slightly smaller than 109°) with uneven sharing of electrons between the oxygen and the hydrogen atoms (Figure 1.3.1). Oxygen, with its higher electronegativity, holds electrons closer to itself than the hydrogens do. The hydrogens, as a result, are described as having a partial positive charge (typically designated as δ+) and the oxygen has a partial negative charge (written as δ- ). Thus, water is a polar molecule because charges are distributed around it unevenly, not symmetrically.

    Water as a solvent

    Water (Figure 1.3.1.) is described as a solvent because of its ability to solvate (dissolve) many, but not all, molecules. Molecules that are ionic or polar dissolve readily in water, but non-polar substances dissolve poorly in water, if at all. Oil, for example, which is non-polar, separates from water when mixed with it. On the other hand, sodium chloride, which dissociate, forms ion-dipole interaction, while ethanol, which is polar, forms hydrogen bonds with water. Both compounds dissolve in water. Ethanol’s solubility in water is crucial for brewers, winemakers, and distillers – but for this property, there would be no wine, beer or spirits. As explained in an earlier section, we use the term hydrophilic to describe substances that interact well with water and dissolve in it, and the term hydrophobic to refer to materials that are non-polar and do not dissolve in water. Table 1.3 illustrates some polar and non-polar substances. A third term, amphiphilic, refers to compounds that have both properties. Soaps, for example are amphiphilic, containing a long, non-polar aliphatic tail and a head that ionizes.

    Table 1.3 Image by Aleia Kim


    Figure 1.3.1 .Arrangement of atoms in water Image by Aleia Kim


    The solubility of materials in water is based in free energy changes, as measured by ΔG. Remember, from chemistry, that H is the enthalpy (heat at constant pressure) and S is entropy. Given this,

    \[ΔG = ΔH - TΔS\]

    where T is the temperature in Kelvin. For a process to be favorable, the ΔG for it must be less than zero.

    Figure 1.3.2.  Structure of a Soap

    From the equation, lowered ΔG values will be favored with decreases in enthalpy and/or increases in entropy. Let us first consider why non-polar materials do not dissolve in water. We could imagine a situation where the process of dissolving involves the “surrounding” of each molecule of the nonpolar solute in water, just like each sodium and each chloride ion gets surrounded by water molecules as salt dissolves.


    Hydrogen bonds in biological systems

    The importance of hydrogen bonds in biochemistry (Figure 1.3.4) is hard to overstate. Linus Pauling himself said,

    “ . . . . I believe that as the methods of structural chemistry are further applied to physiological problems it will be found that the significance of the hydrogen bond for physiology is greater than that of any other single structural feature.”

    According to IUPAC, hydrogen bond is an attractive interaction between a hydrogen atom from a molecule or a molecular fragment X–H in which X is more electronegative than H ( X=F, O, N), and an atom or a group of atoms in the same or a different molecule".

    The role of hydrogen bonds does not apply to solubility (Figure 1.3.4) of polar substances in water, but it is crucial to understand protein structure and DNA double-helix formation (Figure 1.3.5).  Hydrogen bonds also play roles in binding of substrates to enzymes, catalysis, and protein-protein interaction, as well as other kinds of binding, such as protein-DNA, or antibody-antigen.

    Figure 1.3.4 - Hydrogen bonds between water molecules Image by Pehr Jacobson

    Table 1.3.2 Image by Aleia Kim


    Figure 1.3.5 - Hydrogen bonds in a base pair of DNA Image by Aleia Kim

    As noted, hydrogen bonds are weaker than covalent bonds (Table 1.4) and their strength varies form very weak (1-2 kJ/mol) to fairly strong (29 kJ/mol). Hydrogen bonds only occur over relatively short distances (2.2 to 4.0 Å). The farther apart the hydrogen bond distance is, the weaker the bond is.

    The strength of the bond in kJ/mol represents the amount of heat that must be put into the system to break the bond - the larger the number, the greater the strength of the bond. Hydrogen bonds are readily broken using heat. The boiling of water, for example, requires breaking of H-bonds. When a biological structure, such as a protein or a DNA molecule, is stabilized by hydrogen bonds, breaking those bonds destabilizes the structure and can result in denaturation of the substance - loss of structure. It is partly for this reason that most proteins and all DNAs lose their native, or folded, structures when heated to boiling.

    Image by Aleia Kim Table 1.5

    For DNA molecules, denaturation results in complete separation of the strands from each other. For most proteins, this means loss of their characteristic three-dimensional structure and with it, loss of the function they performed. Though a few proteins can readily reassume their original structure when the solution they are in is cooled, most can’t. This is one of the reasons that we cook our food. Proteins are essential for life, so denaturation of bacterial proteins results in death of any microorganisms contaminating the food.


    Acids vs bases

    Water can ionize to a slight extent (10-7 M) to form H+ (proton) and OH- (hydroxide). We measure the proton concentration of a solution with pH, which is the negative log of the proton concentration.

    pH = -log[H+]

    In pure water, the proton concentration is [H+]= 10-7 M, then the pH is 7. We could just as easily measure the hydroxide concentration with the pOH by the parallel equation,

    pOH = -log[OH- ]

    In pure water, dissociation of a proton simultaneously creates a hydroxide, so the pOH of pure water is 7, as well. This also means that

    pH + pOH = 14

    Now, because protons and hydroxides can combine to form water, a large amount of one will cause there to be a small amount of the other. Why is this the case? In simple terms, if I dump 0.1 moles of H+ into a pure water solution, the high proton concentration will react with the relatively small amount of hydroxides to create water, thus reducing hydroxide concentration. Similarly, if I dump excess hydroxide (as NaOH, for example) into pure water, the proton concentration falls for the same reason.

    Chemists use the term “acid” to refer to a substance which has protons that can dissociate (come off) when dissolved in water. They use the term “base” to refer to a substance that can absorb protons when dissolved in water. Both acids and bases come in strong and weak forms. (Examples of weak acids are shown in Table 1.5.) Strong acids, such as HCl, dissociate completely in water. If we add 0.1 moles (6.02x1022 molecules) of HCl to a solution to make a liter, it will have 0.1 moles of H+ and 0.1 moles of Cl- or 6.02x1022 molecules of each . There will be no remaining HCl when this happens. A strong base like NaOH also dissociates completely into Na+ and OH- .


    Weak Acids

    Weak acids and bases differ from their strong counterparts. When you put one mole of acetic acid (HAc) into pure water, only a tiny percentage of the HAc molecules dissociate into H+ and Ac-. Clearly, weak acids are very different from strong acids. Weak bases behave similarly, except that they accept protons, rather than donate them. Since we can view everything as a form of a weak acid, we will not use the term weak base here.

    Figure 1.3.6 - Dissociation of a weak acid. Image by Aleia Kim

    Students are often puzzled and expect that [H+] = [A- ] because the dissociation equation shows one of each from HA. This is, in fact, true ONLY when HA is allowed to dissociate in pure water. Usually the HA is placed into solution that has protons and hydroxides to affect things. Those protons and /or hydroxides change the H+ and A concentration unequally, since A- can absorb some of the protons and/or HA can release H+ when influenced by the OH- in the solution. Therefore, one must calculate the proton concentration from the pH using the Henderson Hasselbalch equation.

    \[pH = pKa + log ([Ac- ]/[HAc])\]

    Image by Aleia Kim Table 1.6

    You may wonder why we care about weak acids. You may never have thought much of weak acids when you were in General Chemistry. Your instructor described them as buffers and you probably dutifully memorized the fact that “buffers are substances that resist change in pH” without really learning what Clearing Confusion - this meant. Buffers are much too important to be thought of in this way.

    Weak acids are critical for life because their affinity for protons causes them to behave like a buffer in certain pH ranges, providing (or absorbing) protons as needed. Weak acids thus help to keep the H+ concentration (and thus the pH) of the solution they are in relatively constant.

    The importance of buffers: Buffered vs non-buffered solutions

    To understand how well a buffer protects against changes in pH, consider the effect of adding .01 moles of HCl to 1.0 liter of pure water (no volume change) at pH 7, compared to adding it to 1.0 liter of a 1M acetate buffer at pH 4.76. Since HCl completely dissociates, in 0.01M (10-2 M) HCl you will have 0.01M H+. For the pure water, the pH drops from 7.0 down to 2.0 (pH = -log(0.01M)).

    By contrast, the acetate buffer’s pH after adding the same amount of HCl is 4.74. Thus, the pure water solution sees its pH fall from 7 to 2 (5 pH units), whereas the buffered solution saw its pH drop from 4.76 to 4.74 (0.02 pH units). Clearly, the buffer minimizes the impact of the added protons compared to the pure water.



    1. apollo_12/experiments/surveyor/
    2. Arunan, Elangannan; Desiraju, Gautam R.; Klein, Roger A.; Sadlej, Joanna; Scheiner, Steve; Alkorta, Ibon; Clary, David C.; Crabtree, Robert H.; Dannenberg, Joseph J.; Hobza, Pavel; Kjaergaard, Henrik G.; Legon, Anthony C.; Mennucci, Benedetta; Nesbitt, David J. (2011). "Definition of the hydrogen bond". Pure Appl. Chem. 83 (8): 1637–1641. doi:10.1351/PAC-REC-10-01-02

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