# Module 3.1: Introduction to pH and Acid Base Behavior - Biology

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learning objective

• Understand the relationship between the structure and acidity of an acid.
• Calculate the amount of an acid that is protonated at any given pH.

## PH

In solution one water molecule can donate a proton to another. In doing so the first becomes a negatively charged hydroxide ion and the other becomes a positively charged hydronium ion. To simplify things, the hydronium ion is often abbreviated as a bare proton, (H^+). The concentration of hydrogen ions in solution can be changed by the addition of an acid (e.g. hydrochloric acid), which will increase the amount of H+. The amount of (H^+) can also be decreased by the addition of a base to the solution, such as ammonia.

pH is the measure of the concentration of positively charged hydrogen ions in an aqueous solution. pH stands for (-log[H^+]). At low pH, the concentrations of protons is high and the solution is acidic, and, at high pH, the concentrations of protons is low and the solution is basic, due to an abundance of hydronium ions, (OH^-). Neutral pH is 7.0. At this pH there are an equal number of (H^+) and (OH^-) ions in solution. ([H^+]=10^{-7} M).

pH of various compounds.

On the left are biological compounds and on the right are some foods and cleaning products.

## Acids and Bases

• Acid: can donate protons
• Base: can accept protons

The following describes ionization or dissociation of the proton from the acid:

The acid (HA) donates its proton to a water molecule, generating the conjugate base of the acid ((A^-)) and a hydronium ion. Although the released proton ((H^+)) is associated with a water molecule, it is often represented in a simpler way as just a free hydrogen ion, (H^+).

[HA +H_2O ightleftharpoons A^- +H_3O^+ onumber]

A compound can have one or more ionizable groups, each with different acid strengths. Here are examples of mono-, di-, and triprotic acids.

Monoprotic Acid

Diprotic Acid

Triprotic Acid

## General Equilibrium Reactions:

Consider first a very simple reaction and its equilibrium features.

[A underset{k_{2}}{overset{k_1}{ ightleftharpoons}} B onumber]

Kinetics gives the following rate equations:

[ frac{d[A]}{d t}=-k_{1}[A]+k_{2}[B] quad frac{d[B]}{d t}=k_{1}[A]-k_{2}[B] onumber]

• (k_1) is the rate (sec(^{-1})) at which A is converted to B. It is often referred to as the forward rate constant. The overall rate that A goes to B is (k_1[A]), giving units of moles/sec.
• (k_2) is the rate (sec(^{-1})) at which B is converted to A. It is often referred to as the reverse rate constant. Likewise, the overall rate of the conversion of B to A is (k^{-1}[B]), which also has units of moles/sec.

At equilibrium there is no change in the average concentration of A or B, therefore:

[egin{array}{l}{frac{d[A]}{d t}=frac{d[B]}{d t}=0} {0=-k_{1}[A]_{E Q}+k_{2}[B]_{E Q}} {K_{e q}=frac{k_{1}}{k_{2}}=frac{[B]_{E Q}}{[A]_{E Q}}=frac{[p r o d u c t s]_{E Q}}{[ ext {reactants}]_{E Q}}}end{array} onumber]

The ratio of [B] to [A] at equilibrium is called the equilibrium constant, (K_{eq}). It is a constant that is reached at equilibrium regardless of the starting amounts of (A) or (B). The equilibrium constant depends on the relative energy difference between A and B, which may depend on the conditions of the reaction (e.g. temperature)

## Characterization of Acid Strength Using (pK_a)

When a proton dissociates from the protonated acid (HA), it becomes bound to a water molecule, generating a hydronium ion, in addition to the deprotonated acid ((A^-)):

[H A+H_{2} O ightleftharpoons A^{-}+H_{3} O^{+} onumber]

Since the concentration of water is essentially constant, it can be ignored and we can write a modified equilibrium reaction that just focuses on the species of interest:

[H A ightleftharpoons A^{-}+H^{+} onumber]

and write the equilibrium constant for the dissociation.

[K_{a}=frac{left[H^{+} ight]left[A^{-} ight]}{[H A]} onumber]

The equilibrium constant for this reaction is given a special name, the 'K-a', or 'K-acidity'. The acidity constant, (K_a) is a fundamental property of the acid, it does not depend on the pH of the solution. However, it does depend on the chemical structure and environment of the acidic group. Since the pH scale is used to characterize [(H^+)], it is useful to express the acidity constant in the same way, by taking its negative log:

[mathrm{pK}_{mathrm{a}}=log mathrm{K}_{mathrm{a}} onumber]

Strong acids are completely dissociated in water and typically have (pK_a) values less than ~ -2. Weak acids do not completely dissociate and have (pK_a) values that are equal or greater than 2.0. Examples of strong acids include hydrochloric acid (HCl), and example of a weak acid is acetic acid.

## Prediction of Protonation State:

In many cases only one of the two species (protonated or deprotonated) may be biologically active. Given a pH and (pK_a) of the ionizable group, we would like to calculate the following:

 The fraction that is protonated: (mathrm{f}_{mathrm{HA}}=[mathrm{HA}] / mathrm{A}_{mathrm{T}}) The fraction that is deprotonated: (mathrm{f}_{mathrm{A}-}=[mathrm{A}] / mathrm{A}_{mathrm{T}})

because this would allow one to predict the biological activity of the system at different pH values. For example, if the protonated species is active, then the activity will be proportional to the fraction that is protonated.

The relationship between the pH, strength of the acid, and the fraction protonated/deprotanated can be obtained from the Henderson-Hasselbalch equation, which is derived below:

[egin{array}{l}{K_{a}=frac{left[H^{+} ight]left[A^{-} ight]}{[H A]}} {-log K_{a}=-log left{frac{left[H^{+} ight]left[A^{-} ight]}{[H A]} ight}} {-log K_{a}=-log left[H^{+} ight]-log left{frac{left[A^{-} ight]}{[H A]} ight}} {p K_{a}=p H-log left{frac{left[A^{-} ight]}{[H A]} ight}} {p H=p K_{a}+log left{frac{left[A^{-} ight]}{[H A]} ight}}end{array} onumber]

Note: when pH=(pK_a) the concentration of the protonated and deprotonated species are equal (([A^-] = [HA])), when the pH = (pK_a) the acid is 50% protonated.

The Henderson-Hasselbalch equation can be used to easily find the fraction protonated and deprotonated. Beginning with defining R=[A-]/[HA] and then writing equations for fraction deprotonated ((f_{A-})) and fraction protonated ((f_{HA})):

[egin{array}{l}{R=frac{left[A^{-} ight]}{[H A]}} {f_{A^{-}}=frac{left[A^{-} ight]}{[H A]+left[A^{-} ight]} quad f_{H A}=frac{[H A]}{[H A]+left[A^{-} ight]}} {f_{A^{-}}=frac{left[A^{-} ight] /[H A]}{[H A] /[H A]+left[A^{-} ight] /[H A]} f_{H A}=frac{[H A] /[H A]}{[H A][H A]+[A-] /[H A]}} {f_{A^{-}}=frac{R}{1+R} quad f_{H A}=frac{1}{1+R}}end{array} onumber]

The value of R is obtained from the given pH and the known (pK_a):

[egin{array}{l}{R=frac{left[A^{-} ight]}{[H A]}} {p H=p K_{a}+log left(frac{left[A^{-} ight]}{[H A]} ight)} {p H=p K_{a}+log (R)} {p H-p K_{a}=log (R)} {10^{(p H-p K a)}=R}end{array} onumber]

Example Calculation: Calculate the fraction protonated for the side chain of histidine, given that the (pK_a) is 6.0

Histidine sidechain ionization

pH(R=10^{(pH-pK_a)})(F_{HA}=1/(1+R))
4(R=10^{(4-6)}=10^{-2})(F_{HA}=1/(1+0.01)=0.99)
5(R=10^{(5-6)}=10^{-1})(F_{HA}=1/(1+0.10)=0.91)
6(R=10^{(6-6)}=10^{0})(F_{HA}=1/(1+1)=0.5)
7(R=10^{(7-6)}=10^1)(F_{HA}=1/(1+10)=0.1)
8(R=10^{(8-6)}=10^2)(F_{HA}=1/(1+100)=0.01)

learn by doing

To further explore the relationship between pH and (pK_a) and protonation state, complete the following learn by doing.

Let’s look at the relationship’s that exist between the original HA concentrations, the pH of the acid, and the acid’s K value.

1. Using the information provided in the chart to the right as the HA original concentration increases what happens to the K value?

a. increases

b. stays the same

c. decreases

hint

Look at the middle column of the box for the K values.

b. (the ratio of products to reactants does not change for a given substance at equilibrium.)

2. Using the information provided in the chart to the right as the HA original concentration increases what happens to the pH value?

a. decreases

hint

Look at the right column of the box for the pH values.

c (as the HA concentration increases more H+ ions are created making the solution more acidic.)

3. In the previous simulations you determined that the pH does not affect the equilibrium constant for the ionization of a weak acid. In this exercise you will determine how pH affects how much of the weak acid is ionized.

Using the equation for fraction protonated shown on the right, you are to select a given pH then calculate the fraction protonated using the concentrations of the various species given in the boxes. Enter your answer into the appropriate location in the data table. Do all five pH values.

Time saver: The total concentration of the weak acid, [HA]+[A-] doesn't change with pH, and is 1000 in this exercise.

As the pH increases

a. [HA] gets smaller as pH gets larger.

b. [HA] gets larger as pH gets larger.

hint

Using the formula above the data chart, select each pH value and calculate the fraction protonated by filling in the values for [HA] and [A-]

a.

4. Does your graph match the theoretical graph?

a. Yes

b. No

hint

Go back to the "fraction protonated calculation"

a. (Good Job!)

5. At what point does the fraction protonated equal 0.5?

a. pH 1.

b. pH 4.

c. pH 6.

hint

Look at how the graph changes with pH and how this changes the fraction.

b. (when the curve is at its midpoint the fraction protonated is .5. This also means the fraction deprotonated is also .5.)

6. What does the midpoint of the curved graph tell you about the acid?

a. (pK_a) of the acid

b. Concentration of the HA

hint

Remember that at this point you have a 50-50 mix of protonated and deprotonated.

a. (the (pK_a) of the acid is the point that you have a 50-50 mix of protonated and deprotonated.)

Now that you have completed the previous activity on weak acids, try applying what you have learned to this real life example.

did i get this

1. Aspirin will more rapidly cross membranes when it is uncharged (protonated). This learn-by-doing explores the effect of pH on aspirin uptake.

The structure of aspirin (acetylsalicyclic acid) is shown above (left). The carboxylate group has a (pK_a) of 4.0.

The pH of the stomach is 2.0 what is the fraction of aspirin that is protonated in the stomach (the right-hand equations may be helpful)?

a. 0.99

b. 0.90

c. 0.09

hint

The right-hand equations may be helpful

a. (pH=2, (pK_a)=4, (R=10^{2-4}=10^{-2}space f_{HA}=frac{1}{(1+0.01)}=0.99))

2. (the (pK_a) of the acid is the point that you have a 50-50 mix of protonated and deprotonated.)

3. (the (pK_a) of the acid is the point that you have a 50-50 mix of protonated and deprotonated.)

4. (the (pK_a) of the acid is the point that you have a 50-50 mix of protonated and deprotonated.)

## Chemical Structure and Acidity:

The equilibrium constant, Keq, for any reaction is related to the energy difference between reactants (e.g. [HA]) and products (e.g. [A-]), by the well-known formula:

[egin{array}{l}{E_{A^{-}}-E_{H A}=Delta G^{0}=-R T ln K_{a}} {K_{a}=e^{-left(E_{A-}-E_{H A} ight) / R T}} {K_{a}=e^{-Delta G^{0} / R T}}end{array} onumber]

(R = gas constant, 8.31 J/mol-K, RT=2.5 kJ/mol @300K).

The following table explores the relationship between structure and acidity.

Dissociation ReactionAcid Strength/AA typeExplanation of Acidity

Hydrochloric Acid, (pK_a) = -20Strong acid because ions interact with water. Complete dissociation.

Ethanol, (pK_a)= 14. (Amino acids serine & threonine)Very weak acid. Little ionization of the -OH group.

Acetic acid, (pK_a) = 4. (Amino acids aspartate & Glutamate)Weak acid, but stronger than ethanol due to the charge delocalization over carbonyl group.

Glycine, (pK_a) = 2. (All amino acids)Weak acid, but stronger than acetic due to charge delocalization over carbonyl group and positively charged nitrogen.

did i get this

1. The structure of trifluoroacetic acid (TFA, (pK_a)=0.5) and acetic acid ((pK_a)=4.8) are shown below. Which is the stronger acid, TFA or acetic acid?

a. TFA is the stronger acid

b. Acetic acid is the stronger acid

hint

(pK_a)=-log(K_a). Small (pK_a) values indicate large or small (K_a) values?

A stronger acid ionizes more completely, indicating a larger acidity constant, (K_a).

a. (The acid with the lower (pK_a) is the stronger acid.)

2. Explain why TFA is a stronger acid than acetic acid.

hint

How do fluorine atoms differ from hydrogen atoms?

When either of the two acids deprotonate, a negative charge is generated on the COOH group. The more electronegative fluorine atoms withdraw, and therefore delocalize, some of that charge, which lowers the energy of the deprotonated state, enhancing ionization.

## Review Quiz

did i get this

1. Upon losing a proton, an acid becomes

a. highly reactive

b. its conjugate acid.

c. its conjugate base.

d. a hydronium ion.

e. a hydroxide ion.

c.

2. One factor affecting the strength of an acid is

a. frequent exercise.

b. the electronegativity of atoms near the acidic group.

c. the number of double bonds.

d. the number of protons released.

e. none of the above.

b.

3. If the (pK_a) of trifluoracetic acid (TFA) is 0, and the (pK_a) of acetic acid is 4.0, the (pK_a) of trichloroacetic acid (TCA) is:

a. -1

b. 1

c. 3

hint

Try the exercise on the page comparing TFA to acetic acid.

b. (Because chlorine is less electronegative than fluorine, TCA is a weaker acid than TFA and the (pK_a) of TCA will be closer to TFA.)

4. When the pH is lower than the (pK_a), then

a. The concentration of [HA] exceeds [A-].

b. The concentration of [HA] is less than [A-].

c. The concentration of [HA] is equal to [A-].

d. The ratio of [HA] to [A-] cannot be determined.

e. The concentration of the acid is required to calculate the ratio of [HA] to [A-].

hint

The (pK_a) marks the pH that will 1/2 protonate the acid. How will lowering the pH affect the hydrogen ion concentration?

a. (The (pK_a) marks the 1/2 point in the effect of proton concentration of an acid. If the pH is less than (pK_a), then [HA]>[A-], if the pH > (pK_a), then [HA] is less than [A-].)

Originally the terms acid and base referred to taste. The practice of classifying substances according to their acidic (sour) or basic (alkaline or bitter) properties dates back to ancient times. An acid was something with a sour taste, such as lemon juice, and a base was something with a bitter taste, such as tonic water. Today there are three additional categories of taste: sweet, salty and umami. The newest, umami, is specific to mono sodium glutamate (MSG).

It is no coincidence that the acid&ndashbase properties of compounds are related to taste. Human taste receptors coupled with smell receptors have evolved to interpret certain molecular features as different tastes. Compounds formed from combinations of acids and bases taste salty and are referred to in chemistry as salts. Sweet compounds have characteristics of both acids and bases in the same molecule.

We will explore the relationship between molecular structure and acids&ndashbases, and consider water solutions of acids and bases. In water or a water solution, the solution is acidic if the hydrogen ion concentration is greater than the hydroxide (OH - ) ion concentration, the solution is basic if the hydroxide ion concentration is greater than the hydrogen (H + ) ion concentration, and the solution is neutral when the concentrations are equal. Thus the properties of an acid solution are due to the relatively high concentration of hydrogen ion, and the properties of basic solutions are due to the high concentration of hydroxide ions.

As do many of the fundamental ideas in chemistry, the acid&ndashbase concept dates back to ancient times and derives from everyday observations about substances people encountered. It was centuries later, however, before molecular interpretations were given to these real-life observations. The acid&ndashbase concept is a system of classifying chemical substances which permits both the organization as well as the prediction of a vast number of chemical reactions.

A substance may be assigned to one our four conceivable categories. It may be an acid or a base, but in addition, it may be both an acid and a base or it may be neither an acid nor a base. Early chemists realized that even among acids and bases, some acids were stronger (more sour) or more basic (more bitter) than others. Thus acids may be further classified as strong acids and weak acids, and bases as strong bases and weak bases.

## Bicyclic Systems With Bridgehead (Ring Junction) Boron Atoms☆

### 7.2 MIDA boronates

Boronic acid groups are commonly used in various synthetic reactions, for example, Suzuki coupling. However, the reactivity of organoboranes can be problematic when doing advanced multistep synthesis. One common resource is the N-methyliminodiacetate (MIDA) protecting group 100. First produced in 1986, this protecting group has proved to be very useful as it fully engages with the boron's sp 3 -hybridised form preventing it from interacting with other molecules ( Schemes 13 and 14 ). 48

This protecting group is resistant to many commonly used chemical reactions including Heck, Negishi, Sonogashira, Stille and anhydrous Suzuki-Miyraura couplings. 49 The Burke group in 2009 showed that MIDA boronate esters slowly release free boronic acid in the presence of mild aqueous base to allow for more effective Suzuki cross-coupling reactions. 50 These compounds have been effectively used as building blocks for the iterative assembly of complex molecules. 51 The MIDA boronate group is also resistant to oxidants, reductants, mild acids, anhydrous bases, electrophiles and soft nucleophiles. In addition, the MIDA moiety increases the solubility of boronic acids in protic solvents. For these reasons, the MIDA moiety has become a widely-used protecting group for boronic acids.

## Resistance mechanisms

### PH homeostasis

pH homeostasis is the regulation of the pH inside and outside the cell and is an important indicator of the physiological state of cells in an acidic environment (Baker-Austin and Dopson 2007). It is critical for cell growth and metabolism, influencing the absorption and utilization of nutrients, the degradation of substrates, and the synthesis of proteins and nucleic acids (Guan et al. 2013). As illustrated in Figs. 1 and 2, the maintenance of pH homeostasis is a result of interactions among multiple transport systems. Electrogenic proton pumps expel protons from cells, generating a membrane potential and a pH gradient. The interconversion of these is regulated by cation and proton transfer via secondary transporters (Călinescu et al. 2014).

Acid tolerance mechanisms associated with cell membranes and ion transport systems. Microbial cells maintain pH homeostasis by restricting the inward flow of protons through highly impermeable cell membranes (I) and modulating the size of membrane channels (II), deflecting the influx of protons through generating chemiosmotic gradients via potassium ATPases (III), pumping excess protons out from the cytoplasm through proton pump (IV), and maintaining the integrity and fluidity of cell membranes by modulating fatty acid composition (V)

Enzyme-based acid tolerance mechanisms

Different strategies to withstand acid stress by sustained pH homeostasis have evolved in microbes (He et al. 2017 Jain et al. 2013 Liu et al. 2016b Lu et al. 2013 Miller and Maier 2014 Sohlenkamp 2017). Some yeast and bacteria maintain a relatively stable and neutral intracellular pH (pHi) in the presence of constantly changing extracellular pH (pHex) and generate unfixed proton gradients (Siegumfeldt et al. 2000). However, a constant pH gradient is more favorable to most acid-tolerant microbes. This is because a large amount of energy must be consumed to maintain neutral pHi, which severely restricts the growth and metabolism of microbes (Sun 2016). The pHi of these acid-tolerant microbes decreases with acidification of the environment, but is maintained at a higher level than pHex. Once the acid reaches a certain concentration, the pHi declines sharply, and the pH homeostasis is destroyed. This results in protein and DNA damage, with the cells ultimately withering (Wu et al. 2012a). Therefore, sustaining pH homeostasis is essential for microbes to survive in acidic environments.

#### Restriction of proton permeation

Proton motive force (PMF) is a measurement of the energy state of the cell membrane generated by a charge separation between the cytoplasm and external milieu created by membrane potential and pH gradient across the membrane (Baker-Austin and Dopson 2007). It is a common indicative reference for controlling pH homeostasis, which is mainly served by pH gradient in the study of acid resistance (Lee and Kang 2016). It is sustained by the balance between the inflow and outflow of protons.

Protons travel into the cytoplasm through the plasma membrane and are restricted by the proton permeability and channel size of the membrane (Sohlenkamp 2017). Acid-tolerant microbes are generally equipped with less permeable membranes to reduce the entry of protons into the cells (Sohlenkamp 2017). It is suggested that several factors contribute to this feature, including the tough structure of the monolayer, the bulky isoprenoid core, and a unique lipid composition such as tetraether lipids (Macalady and Banfield 2003). Modulating the size of membrane channels is another important strategy adopted by some acid-tolerant microbes to maintain pH homeostasis. Expression of the outer membrane porin of Acidithiobacillus ferrooxidans increased in response to acid, attempting to control the size of the porin gateway by forming a large L3 loop (Amaro et al. 1991). Consequently, the influx of protons was limited to only the outer membrane (Guiliani and Jerez 2000).

The influx of protons can also be reduced in acid-tolerant microbes using a chemiosmotic gradient generated by a Donnan potential, and the difference in electric potential formed between two solutions separated by an ion-exchange membrane without any current flow through the membrane (Baker-Austin and Dopson 2007). Many cation transporters were discovered in acidophiles, and they are presumed to be involved in the generation of a Donnan potential (Fütterer et al. 2004). Potassium transporters are reported to be the most efficient in generating chemiosmotic gradients, through which a reverse membrane potential is generated, and the inward flow of protons is restrained (Suzuki et al. 1999). It was also observed that potassium ions participate in the respiration-linked proton pump in Sulfolobus spp. (Schäfer 1996). In addition, cation ATPases (such as K + -ATPase) are involved in the maintenance of pH homeostasis by exchanging H + and K + (Macpherson et al. 2005).

An interesting acid resistance mechanism of some bacteria is the formation of biofilms. It is a group behavior which involves cell to cell communication (Li et al. 2001). Biofilms protect microbial cells against acid shock through wrapping the cells in the innermost part. Hence, cell density, which is related to the formation of biofilms, is also a factor affecting the acid resistance of microorganisms (Liu et al. 2015c).

#### Enhancement of proton pumps

The PMF-dependent proton pump is one of the most important acid tolerance systems in bacteria in the maintenance of pH homeostasis, through which excess protons are pumped out from the cytoplasm (Jain et al. 2013). Several proton pumps have been shown to promote proton efflux, such as the H + -ATPase, symporter, antiporter, and secondary transporter (Sun 2016). Protons are reported to be exported from cells through H + -ATPase in bacteria, a process that consumes ATP (Sun 2016). Consequently, higher H + -ATPase activity and more energy accumulation enhance the ability of cells to regulate pHi homeostasis.

Normally, ATP is generated via FoF1ATPase when extracellular protons cross the cell membrane into the cytoplasm through a pH gradient (Sun 2016). However, the accumulation of H + leads to a sharp decrease in pHi under low pHex, and proton pumps begin ATP consumption (Fig. 1). Consequently, the energy available for cells is depleted, and the survival of the strain is inhibited (Zheng et al. 2011). Therefore, elevating the energy levels is an effective strategy to enhance proton pumps. Substrate-level and oxidative phosphorylation are the two ways in which microorganisms produce ATP the latter can be enhanced by adding auxiliary energy substrates (Zhou et al. 2009). It is reported that citrate is of significance in some prokaryotic microorganisms as an auxiliary energy cosubstrate, promoting ATP regeneration (Drici et al. 2010 Kang et al. 2013). Zhou et al. were able to increase ATP supply to Candida glabrata by adding citrate to the medium and increasing pH gradient of the system, thus improving its acid tolerance during pyruvic acid production (Zhou et al. 2011). In short, balancing proton transport and ATP metabolism forms the core of the proton pump mechanism. Besides for bacteria and yeast, Rhizopus oryzae has also been reported to resist acid stress through FoF1ATPase (Liu et al. 2015b).

#### Consumption of protons

In addition to controlling the transmembrane proton transport, some microorganisms have developed several acid tolerance mechanisms based on the consumption of excessive cytoplasmic protons to sustain pH homeostasis in acidic environments. The enzyme systems of cells that generate alkaline products play key roles in these mechanisms, as illustrated in Fig. 2.

The urease system is known to neutralize H + by producing ammonia, which helps resist low pH during the culture of bacteria such as Helicobacter pylori (Mols and Abee 2011 Zanotti and Cendron 2010). Three models of urease have been proposed to regulate pH homeostasis. Originally, it was believed that urea is catalyzed by cell-associated extracellular urease and yields ammonia, which neutralizes protons around the cells (Hazell 1991). However, urease was later found to be a cytoplasmic enzyme that is released via cell lysis (Scott et al. 1998). According to the second model, ammonia produced from urease combines with H + in the periplasm and the intracellular microenvironment is maintained by increasing pH of the same. The current generally accepted mechanism is that urease transforms urea into ammonia and CO2, directly neutralizing protons and regulating pHi in the cytoplasm (Miller and Maier 2014). Vollan et al. found the role of H. pylori outer membrane phospholipase A in acid tolerance based on urea influx and ammonia efflux. This was later found to be involved in the transporting of NH4 + into periplasm (Vollan et al. 2017).

Amino acids render several microorganisms acid-tolerant by raising the pHi during metabolism (Senouci-Rezkallah et al. 2011). Such systems have been termed amino acid-dependent acid tolerance systems. The arginine deaminase (ADI) system has been identified as an important defense mechanism in several bacteria against damage by acid (Liu et al. 2015c Shabayek and Spellerberg 2017). Three steps are involved in this system (Fig. 2). First, arginine transported into cells by ArcD is converted to citrulline and ammonia by ADI. Next, ornithine carbamoyltransferase (OTC) catalyzes the phosphorolysis of citrulline to ornithine and carbamoyl phosphate. The former is subsequently transported out of the cell, while the latter is finally converted to carbon dioxide and ammonia by carbamate kinase (CK), during which ATP is generated from ADP. Consequently, protons are neutralized by ammonia and carbon dioxide formed by the system, and the ATP produced is available to extrude protons through H + -ATPase (Guan et al. 2013). Meanwhile, an arginine-agmatine antiporter AdiC and arginine decarboxylase AdiA comprise the other branch of the arginine-dependent acid tolerance system (Kanjee and Houry 2013). Arginine passes into the cell through AdiC and is converted to agmatine and carbon dioxide through catalysis by AdiA, consuming intracellular protons in the process.

The glutamate-dependent acid tolerance system is also recognized as critical for bacteria to survive in acidic environments. The function of the glutamate decarboxylase (GAD) system in acid resistance is similar to that of arginine decarboxylase (Fig. 2). Glutamate decarboxylase catalyzes the decarboxylation of glutamate, yielding γ-aminobutyric acid (GABA) and carbon dioxide, accompanied by proton consumption (Reeve and Reid 2016). The specific amino acid antiporter GadC, which is also known to transport glutamine, transports extracellular glutamate and intracellular GABA (Laroute et al. 2016 Ma et al. 2012). Another system, comprising GadC and the glutaminase YbaS, is found in Escherichia coli (Lu et al. 2013). After being transported into the cytoplasm, glutamine is converted to glutamate and ammonia by acid-activated YbaS, following which the GAD system is initiated. Formation of alkaline products (ammonia and GABA) and the reduction of intracellular protons are the net consequences of this glutamate-related metabolism. Besides arginine and glutamate, the lysine-dependent system also plays a role in acid tolerance of cells via the decarboxylation of lysine (He et al. 2017) (Fig. 2). In addition, some other amino acids such as aspartate and citrulline are involved in the maintenance of pHi homeostasis by releasing ammonia during metabolism (Cusumano and Caparon 2015 Hu et al. 2010).

### Alteration of cell membranes

The primary target of environmental stress is cell membranes, which assist in sustaining cellular activities under acidic conditions in several ways. In addition to restricting proton permeation by adjusting channel size, membrane bioenergetics and lipid physiology are also closely related to the stress response in microorganisms (Yang et al. 2014). As mentioned above, the membrane-bound H + -ATPase regulates pHi of cells by pumping protons out of the cytoplasm. Therefore, higher levels of H + -ATPase and its activity result in higher acid tolerance capacity (Zhang and Yang 2009). Modulation of the integrity, fluidity, and lipid composition of cell membranes are also important mechanisms that protect bacteria against the deleterious effects of acids (Yan et al. 2016).

Cell membranes provide a constant intracellular environment for cell growth and metabolism (Sohlenkamp 2017). Maintenance of proper membrane structure and function is a prerequisite for all cellular metabolic activities. Low pH usually leads to morphological changes in cells, which is a consequence of the damaged lipoidal cell membrane and decreased fluidity (Streit et al. 2008). The viability of cells under stress conditions is regulated by membrane status cell membranes confer acid tolerance to cells through maintenance of their integrity and fluidity because of acid adaptation (Sohlenkamp 2017). Membrane fluidity is an integrated reflection of chain conformation, lateral and rotational diffusion, and resistance to sheer forces, and these characteristics are determined by the fatty acyl chain and head-group composition (Denich et al. 2003).

Some microbes regulate membrane fluidity by modulating fatty acid composition, since the bilayer structure can be modified by changing the distribution of fatty acids (Lindberg et al. 2013 Yang et al. 2014). The ratios of unsaturated to saturated, cis to trans unsaturated, and branched to unbranched fatty acids are all related to the acyl chain structure of glycerophospholipids. Altering the unsaturation ratio is a common mechanism employed by bacteria to control membrane fluidity. This depends on fatty acid synthesis by fatty acid synthases of the anaerobic pathways and desaturase enzymes of the aerobic pathways (Denich et al. 2003). It has been reported that higher unsaturation ratios of membrane fatty acids contribute to cell survival at low pH (Wu et al. 2012b). Isomerization of unsaturated fatty acids from cis to trans conformation also affects fluidity of the bacterial membrane (Tan et al. 2016). It is an energy-efficient post-synthesis lipid modification process, which occurs only in inactive cells (Diefenbach et al. 1992). Additionally, altering either the proportion or type of branching is another way in which cells modulate membrane fluidity (Kaiser et al. 2016 Sen et al. 2015). Specifically, membrane cyclopropane acyl chains were shown to be critical factors in acid tolerance in bacteria (Chang and Cronan 1999 Yang et al. 2015), where strains lacking such fatty acids were more sensitive to low pH (Kim et al. 2005). In addition, fatty acid chain length also plays a vital role in the response to acid stress. Strains reduce acid-mediated damage to their cell membranes by lengthening their fatty acid chains (Wu et al. 2012b).

### Metabolic regulations

Microorganisms have developed complex metabolic regulatory mechanisms to improve their acid tolerance during adaptation to acid environments. They upgrade their precursors, cofactors, and redox factors for survival, growth, and metabolism under acidic conditions by strengthening the glycolytic pathway (Guan et al. 2014). In a previous study, the glycolytic rate increased by 70% from pH 6.6 to 4.7 (Even et al. 2003), through changing enzyme concentrations and metabolic regulation of enzyme activities. The increase in enzyme activity compensates for the inhibition imposed by diminished pH, and rescues normal metabolism. Simultaneously, the transcription of central metabolic pathway genes is regulated and transcript stability increases. The increase in the enzyme pool and decrease in mRNA concentrations indicate that translational regulation plays a major role in enhancing enzyme concentrations by controlling ribosome activity (Even et al. 2003).

Glycolytic rates increased by 70%, and biomass synthesis was 80% less efficient at low pH, suggesting that the energy required in maintaining the metabolism of strains increased (Even et al. 2003). A portion of the energy that is consumed assists proton pumps in the maintenance of pHi by extruding protons out of the cells. However, the available metabolic energy is limited since the rate of energy synthesis decreases upon cytoplasmic acidification. Thus, endogenous RNAs are catabolized to provide bases and ribose for the synthesis of carbon chains and energy (Siegumfeldt et al. 2000). Furthermore, amino acid catabolism is enhanced by fivefold when pH decreases from 6.6 to 4.7. The generation of NH3 and the consumption of intracellular H + via deamination and decarboxylation, respectively, are considered key mechanisms in bacterial resistance to acidification (Lu et al. 2013 Xiong et al. 2014). Similarly, the metabolism and accumulation of cellular polyamines are also enhanced to promote cell survival in acidic pH (Fujihara and Yoneyama 1993).

Except for the protective mechanisms against protons, acid-resistant mechanisms based on anions from the dissociation of organic acids have also been developed. The consumption of acetate has been found to enhance acetic acid tolerance of S. cerevisiae (Geng et al. 2017). Through expression of genes in acetate degradation pathway, resistance of S. cerevisiae to acetic acid was improved during fermentation (Ding et al. 2015b). That is, anions may improve acid tolerance by involving in certain metabolic pathways and influencing the metabolism of acids.

### Protection and repair of macromolecules

An acid response mechanism that depends on protein synthesis has been widely observed in microorganisms (Liu et al. 2015c). Specific proteins are usually induced by acid stress to protect or repair macromolecules such as DNA and proteins. Several chaperones have been recognized as important acid tolerance factors, which are important during the synthesis, transport, folding, and degradation of proteins (Nicolaou et al. 2010).

In the periplasm of Gram-negative bacteria, the enzymes, transporters, and transmembrane antiporters encounter more severe acid stress because they lack the protection of the inner membrane. This leads to their denaturation and aggregation (Hong et al. 2012). HdeA and HdeB are two periplasmic chaperones that have been identified to protect enteric bacteria from damage by gastric acid, while HdeA also protects bacteria against acid stress due to accumulated organic acids (Mates et al. 2007). HdeA prevents the acid-induced aggregation of proteins by binding to them at an acidic pH, which is the condition in which the chaperone is activated (Tapley et al. 2009). HdeA is also involved in protein resolubilization and renaturation (Malki et al. 2008 Tapley et al. 2010). These proteins include transport proteins, metabolic enzymes, chaperones, lipoproteins, and proteases. Chaperones such as DegP and SurA can assist HdeA to protect proteins at low pH (Hong et al. 2012). They assist the recovery of protein activity by facilitating refolding during renaturation. HdeB is also an acid stress chaperone with the same functions as HdeA, although the optimum pH is different (Kern et al. 2007). HdeA and HdeB were recognized as the molecular chaperones that function specifically in acid tolerance (Hong et al. 2012).

Lo18 is a small membrane-associated heat shock protein that was characterized in Oenococcus oeni (Delmas et al. 2001). It improves the acid tolerance of bacteria through effectively suppressing protein aggregation, and it functions as a molecular chaperone to stabilize membrane and envelope proteins under acidic conditions (Weidmann et al. 2017). Ffh is a 54 kDa homolog of the signal recognition particle (SRP) complex, which is an essential component of the protein translocation pathway involved in membrane and extracellular protein transport (Gutierrez et al. 1999). It is part of the acid tolerance response system, and its transcription is regulated by pH. The lack of Ffh in Streptococcus mutans was found to lead to reduced H + -ATPase activity against a pH 5.0 shock (Kremer et al. 2001). In addition, several other chaperones such as DnaK, DnaJ, GrpE and HrcA, GroEL and GroES, Clp proteases, and EF-Tu have been shown to facilitate the repair of proteins as molecular chaperones during acid stress (Shabayek and Spellerberg 2017).

Depurination and depyrimidination of DNA can occur because of intracellular acidification, since protonation of a base can lead to cleavage of the glycosyl bond (Calhoun and Kwon 2011). DNA repair systems have been identified in microbial cells to survive DNA damage against low pH. recA encodes a multifunctional enzyme involved in synapsis, during which the paired DNA exchange strands (Adikesavan et al. 2011). The enzyme participates in DNA recombinational repair in E. coli, Bacillus subtilis, and H. pylori, along with RecN and AddAB (exonuclease V) (Ansari and Yamaoka 2017 Cardenas et al. 2014). The nucleotide excision repair system functions on damaged DNA produced from base modification, single-strand break, and abasic sites, and are considered the most important DNA repair system (Kisker et al. 2013). UvrABCD, DNA polymerase, and DNA ligase support the repair of acid-induced DNA damage, performing damage recognition, base excision, and gap filling (Das et al. 2015). UvrA overexpression enhanced the acetic acid tolerance and fermentation of Acetobacter pasteurianus, which is a widely used vinegar-brewing acetic acid bacteria (Zheng et al. 2018). In conclusion, the repair of damaged proteins and DNA is widely used by microbes to resist acid stress.

These mechanisms are mostly shared by various types of microorganisms. Additionally, the difference in cellular structure between prokaryotic and eukaryotic cells introduces diversity in acid-tolerant mechanisms. As eukaryote-specific organelles, mitochondria, vacuole and nucleus all play roles in acid tolerance of S. cerevisiae (Peng et al. 2017). Acid-tolerant mechanisms utilized by different microorganisms were summarized in Fig. 3 and listed in Table 1, respectively.

Acid stress responses in microbial cells

## How Do You Measure the pH of a Solution?

The pH of a liquid or solution is often an important piece of information in science. Measuring pH can be done simply and quickly using pH test paper, pH indicator sticks, or a pH meter. pH test paper and indicator sticks are pieces of paper or stiffer sticks that contain pH indicators (chemicals that change color depending on how acidic or basic a solution is). To measure pH, a piece of pH test paper or an indicator stick is dipped into the liquid. The color of the dipped paper/stick is then matched to a color key that comes with the container of pH test paper or indicator sticks. Each color on the key represents a different pH. An example of a used pH indicator stick and the corresponding color key is shown below in Figure 1. pH meters are electronic devices that used to measure pH. They consist of a probe that is dipped in a solution, and a digital readout. pH meters are even more precise than pH test paper or indicator sticks. Table 2 below discusses what types of pH measuring devices are best for different science project applications, and offers a quick link to purchasing different pH test papers and indicator sticks.

## Key Concepts and Summary

Chemistry deals with the composition, structure, and properties of matter, and the ways by which various forms of matter may be interconverted. Thus, it occupies a central place in the study and practice of science and technology. Chemists use the scientific method to perform experiments, pose hypotheses, and formulate laws and develop theories, so that they can better understand the behavior of the natural world. To do so, they operate in the macroscopic, microscopic, and symbolic domains. Chemists measure, analyze, purify, and synthesize a wide variety of substances that are important to our lives.

### Chemistry End of Chapter Exercises

1. Explain how you could experimentally determine whether the outside temperature is higher or lower than 0 °C (32 °F) without using a thermometer.
2. Identify each of the following statements as being most similar to a hypothesis, a law, or a theory. Explain your reasoning.

(a) Falling barometric pressure precedes the onset of bad weather.

(b) All life on earth has evolved from a common, primitive organism through the process of natural selection.

(c) My truck’s gas mileage has dropped significantly, probably because it’s due for a tune-up.

(a) The pressure of a sample of gas is directly proportional to the temperature of the gas.

(b) Matter consists of tiny particles that can combine in specific ratios to form substances with specific properties.

(c) At a higher temperature, solids (such as salt or sugar) will dissolve better in water.

(a) The mass of a lead pipe is 14 lb.

(b) The mass of a certain chlorine atom is 35 amu.

(c) A bottle with a label that reads Al contains aluminum metal.

(d) Al is the symbol for an aluminum atom.

(a) A certain molecule contains one H atom and one Cl atom.

(b) Copper wire has a density of about 8 g/cm 3 .

(c) The bottle contains 15 grams of Ni powder.

(d) A sulfur molecule is composed of eight sulfur atoms.

## 4. Conclusions and perspectives

Advances in biochemistry and materials science have sparked interest in the development of various biocompatible materials for bioanalytical and biomedical applications. Based on their high biocompatibility, high flexibility, low toxicity and highly tunable nature, hydrogels are promising materials in bioanalytical and biomedical applications. On the other hand, functional nucleic acids (FNAs), including aptamers, DNAzymes, i-motif structures, siRNAs and CpG motifs, provide additional molecular recognition, catalytic activities, and therapeutic potential, affording potential applications for diagnosis and therapeutics. Incorporation of FNAs into hydrogel systems can be manipulated with additional properties, significantly expanding the applications of DNA-based hydrogels, as demonstrated by the selected examples described in this review. Thus far, FNA-based hydrogels have been implemented to design sensors, separation platforms, cell adhesion platforms, controlled drug delivery and targeted cancer therapy methods. In addition, it should be noted that the use of FNA-based hydrogels for novel applications, such as molecular logic gates, mechanical actuators, and portable detection devices, has been achieved. Although these studies are still in the preliminary stage, they represent promising potential uses of these hydrogels.

However, to further move the applications of FNA-based hydrogels forward, several challenges must be addressed. First, colorimetric visual detection methods based on hydrogel phase changes provide simple and rapid detection of various targets, but the problems of sensitivity and quantification have limited their applications. Incorporation of enzyme-catalyzed signal amplification mechanisms (e.g., amylase/iodide system), cascade reactions (e.g., trienzyme cascades based on β-Gal/GOx/HRP) and other signal amplification strategies can improve sensitivity. Furthermore, the detection of targets in real samples remains challenging, given the complexity of virtual environments. Therefore, development of simple and sensitive sensors capable of analyzing targets in complex biological samples should be a prospective direction for hydrogel-based sensors. Second, some FNA-based hydrogel systems lack efficient and precise release mechanisms, leading to premature release. The design of dual-responsive hydrogels can be used for precisely controlled cargo release. For example, incorporation of pNIPAM into polyacrylamide gel will give the hydrogel additional thermosensitive property. In addition, light-sensitive property can be regulated by incorporating azobenzenes, and pH-sensitive property can be achieved by i-motif structure. Other types of stimuli, such as NIR irradiation and magnetic fields, can also be explored. In dual-responsive hydrogels, hydrogel transitions can be triggered only in the presence of both stimuli, thus leading to more accurate operation of FNA-based hydrogel systems. Third, the bulky size of some FNA-based hydrogels curtails their biomedical applications. The development of methods for preparation of nanohydrogels and effective delivery of nanohydrogels into cells will expand the scope of in vivo applications.

Finally, although FNA-based hydrogel systems appear to hold promise for controlled release and targeted cancer therapy, some issues remain to be resolved, such as poorly understood pharmacokinetics, long-term toxicity and off-target effects. To realize the full potential and overcome the challenges of such hydrogels, it is necessary to perform more stringent in vivo studies to further understand the behavior of these hydrogels. Furthermore, in-depth studies in animal models for the evaluation of safety and efficacy of these hydrogels will lay the foundation for further clinical applications. Future efforts should focus on improving these hydrogels for clinical use. Through collective efforts, we believe that the integration of further developments in materials science and nanotechnology will promote the development of FNA-based hydrogels for a variety of practical bioanalytical and biomedical applications.

## Normal Cell Division

The outer layer of skin (epidermis) is about 12 cells thick. Cells in the basal layer (bottom row) divide just fast enough to replenish cells that are shed. When a basal cell divides, it produces two cells. One remains in the basal layer and retains the capacity to divide. The other migrates out of the basal layer and loses the capacity to divide. The number of dividing cells in the basal layer, therefore, stays about the same.

## DENTAL HYGIENE

DH 101 Preventive Oral Health Care I : 4 Credits

This course introduces the student to the dental hygiene process. Fundamental concepts, assessment skills and preventive techniques are emphasized. Principles of communication, education and motivation provide a firm foundation for patient education. The laboratory component of this course provides the student with hands-on experience in learning and applying instrumentation techniques utilizing manikins and student partners. Related skills including dental unit operation and patient and operator positioning strategies are also addressed. (Three hours lecture/7 hours laboratory) Prerequisites:CH 101 this course is open to students enrolled in the Dental Hygiene

DH 102 Preventive Oral Health Care II : 5 Credits

This course focuses on transition into clinical practice. Development of clinical skills continues with consideration of periodontal assessment and treatment planning and the introduction of ultrasonic instrumentation, polishing pit and fissure sealant application, instrument sharpening procedures and pain control techniques. Students are also familiarized with the scope of dental specialty areas and common procedures performed in prosthodontics, endodontics, oral surgery, pedodontics and orthodontics. In the entry level clinical component of this course, the student applies principles and techniques learned in didactic and pre-clinical laboratory courses to actual clinical practice. Students render dental hygiene services to patients in a clinical setting. Assessment, diagnosis and planning skills are cultivated, as well as basic instrumentation skills. (Three hours lecture/8-9 hours clinic) Prerequisites:This course is open to students who have attained a passing grade of “C” or better in all attempted dental hygiene didactic courses, and a “Pass” in pre-clinic laboratory.

DH 103 Oral Radiology : 3 Credits

This course introduces the student to radiological technology to assure that dental professionals who expose patients to radiation for diagnostic purposes meet radiological health standards. Emphasis will be placed on radiation physics, biological effects of radiation, function of dental x-ray equipment, quality and interpretation of x-ray films and darkroom techniques. Students will be taught techniques for producing dental radiographs of acceptable diagnostic quality. Technical skills will be developed on manikins before students demonstrate competence in a clinical setting. (Two hours lecture/two hours laboratory.) Prerequisites:CH 101. This course is open to students enrolled in the Dental Hygiene Program.

DH 104 Oral Histology and Embryology : 2 Credits

This course provides the student with an overview of the development and function of cells, tissues and organs on both the macroscopic and microscopic levels. Embryonic development of the head and neck and the morphodifferentiation of the face and oral structures is presented. The emphasis of this course is to familiarize the student with the parts of oral histology and embryology that are pertinent to clinical dental hygiene practice. Prerequisites:This course is open to students who have attained a passing grade of “C” or better in all attempted dental hygiene didactic courses and a “pass” in pre-clinic laboratory.

DH 106 Dental Anatomy : 2 Credits

This course will provide the student with a comprehensive study of the form, function, and characteristics of the human dentition and supporting structures. Eruption sequence of the primary and permanent dentitions, as well as the occlusion and position of individual teeth will be reviewed. Students will learn pertinent terminology as it relates to dental anatomy. Various activities and exercises will be utilized in the course to enhance the student’s knowledge. (Two hours lecture.) Prerequisites:CH 101. This course is open to students enrolled in the Dental Hygiene Program.

DH 107 Dental Materials : 2 Credits

This course introduces the student to materials used in dental practice. Lectures, demonstrations, readings and laboratory activities will assist the student in developing an understanding of the properties, uses and manipulation of amalgam, composite resins, cements, impression materials, gypsum products, waxes, bleaching materials, porcelain and gold. Physical and biological properties will be emphasized and clinical applications will be shown in the laboratory portion of the course. (One hour lecture/2 hours laboratory) Prerequisite:CH 101. This course is open to all students enrolled in the Dental Hygiene Program.

DH 108 Oral Pathology : 2 Credits

This course presents a study of disease processes occurring in the oral cavity. Diagnosis and treatment of common lesions, inflammation and repair and the immune system will be studied in depth. Oral manifestations and systemic problems encountered with neoplastic lesions will be examined as well as the distinction between benign and malignant tumors. Systemic diseases with significant oral manifestations and complications will be covered. (Two hours lecture) Prerequisite:This course is open to students who have attained a grade of “C” or better in all attempted dental hygiene courses.

DH 109 Periodontics I : 2 Credits

This course is designed to teach students about the normal, healthy periodontium in order to understand the various stages of periodontal disease and its treatment. A study of the clinical and histological characteristics of both the healthy and the diseased periodontium is presented. (Two hours lecture) Prerequisite:This course is open to students who have attained a grade of “C” or better in all attempted dental hygiene courses.

DH 110 Medical Emergencies : 1 Credit

This course will examine a variety of medical emergencies that can and do occur in the dental office. Students will learn basic information necessary to prevent, recognize and manage medical emergencies as an effective member of the dental health care team. (One hour lecture) Prerequisite:This course is open to students who have attained a grade of “C” or better in all attempted dental hygiene courses.

DH 201 Preventive Oral Health Care III : 5 Credits

The lecture portion of this course focuses on advanced treatment planning, dietary analysis and counseling, and further consideration of pain control techniques. The management of patients with developmental, medical, physical, sensory and psychological impairments is discussed with emphasis on normalization of care, adaptation of oral care techniques and access to care. In intermediate level clinic, students continue to integrate preventive, educational and therapeutic care as they treat patients in a clinical setting. Emphasis is on the expansion and refinement of skills through the treatment of patients with moderate to advanced periodontal involvement. (Three hours lecture/12 hours clinic) Prerequisite:This course is open to students who have attained a grade of “C” or better in all attempted dental hygiene courses, and a “Pass” in entry level clinic.

DH 202 Preventive Oral Health Care IV : 5 Credits

Lecture, discussion and group activities will focus on ethical and legal issues and controversial topics relating to the dental hygiene profession. Alternative practice settings and job procurement strategies will be explored. In advanced level clinic, students continue to apply knowledge and skills learned in didactic and clinical courses. Emphasis is on efficiency and proficiency in all dental hygiene processes as students prepare for licensure examination and transition into private practice. (Three hours lecture/15 hours clinic) Prerequisite:This course is open to students who have attained a grade of “C” or better in all attempted dental hygiene courses, and a “Pass” in intermediate level clinic.

DH204 Head and Neck Anatomy : 1 Credit

An in depth study of the head and neck is presented in this course. The focus will be on identification of important anatomical structures of all major systems in this region including, but not limited to: bones, muscles, blood vessel, nerves, etc. Prerequisites: DH101, DH103, DH106, DH107 For Dental Hygiene students only.

DH205 Local Anesthesia : 2 Credits

This course is a study of basic and current concepts in the administration of local anesthetics, systemic effects, and tissues diffusion. Assessment of the patient's health, apprehension and pain threshold will be addressed in determining the indications and contraindications of pain control and alleviation. Emphasis will be placed on the selection and administration of appropriate anesthetic agents and evaluation of proper techniques. (lecture/ lab) prerequisite: this course is open to students who have attained a grade "C" or better in all attempted dental hygiene courses.

DH 209 Periodontics II : 2 Credits

This course is a continuation of Periodontics I. There is a strong emphasis on the different types of periodontal therapy and the reason for their use on periodontal involved patients. (Two hours lecture) Prerequisite:This course is open to students who have attained a grade of “C” or better in all attempted dental hygiene courses.

DH 212 Pharmacology : 3 Credits

Pharmacology introduces the hygiene student to the study of drugs and how they affect biological systems. This course will provide the student with a base of knowledge in the principles of pharmacology and the drugs used in the current therapy of disease states, as well as a solid foundation in the terminology and vocabulary that is associated with pharmacology. Special emphasis is given to those drugs administered or prescribed in the dental practice, as well as those drugs whose actions, side effects, or interactions with other drugs may impact dental healthcare. (Three hour lecture) Prerequisite:This course is open to students who have attained a grade of “C” or better in all attempted dental hygiene courses.

DH 215 Community Dentistry : 2 Credits

This course introduces the student to the role of dentistry and dental hygiene practice as it relates to community-based oral health promotion and prevention approaches. Students are introduced to health education methods, basic principles of research and the socioeconomic, demographic and epidemiological trends of oral disease. The course provides an opportunity for an active partnership between various community groups and the student by completion of a major project. The student will apply the principles of community dental health as they develop and evaluate a community-based oral health presentation. (Two hours lecture) Prerequisite:MH 203 this course is open to students who have attained a grade of “C” or better in all attempted dental hygiene courses.

## Characterization of Native and Modified Starches by Potentiometric Titration

1 Laboratorio de Polímeros y Reacciones, Escuela de Ingeniería Química, Facultad de Ingeniería, Universidad del Zulia, Sector Grano de Oro, Avenida 16 (Guajira), Ciudad Universitaria Dr. Antonio Borjas Romero, Edificio Petróleo y Química, Maracaibo 4011, Venezuela

#### Abstract

The use of potentiometric titration for the analysis and characterization of native and modified starches is highlighted. The polyelectrolytic behavior of oxidized starches (thermal and thermal-chemical oxidation), a graft copolymer of itaconic acid (IA) onto starch, and starch esters (mono- and diester itaconate) was compared with the behavior of native starch, the homopolymer, and the acid employed as a graft monomer and substituent. Starch esters showed higher percentages of acidity, followed by graft copolymer of itaconic acid and finally oxidized starches. Analytical techniques and synthesis of modified starches were also described.

#### 1. Introduction

Titration is an analytical technique commonly used in many research and industrial chemistry applications. This involves the measured addition of a solution of known concentration of chemical (titrant) to determine the concentration of another chemical (analyte) in a second solution. The chemical in the titrant reacts in a known manner with the analyte material. When the reaction of these chemicals/materials is complete, a surplus of the titrant is detected as a specific end point marking the end of titration. The end point can be determined by several methods: indicators of pH, redox indicators, potentiometry, conductometry, isothermal calorimetry, spectrophotometry, and amperometry [1].

Analytical techniques for this research included potentiometric titration. Potentiometric titration, based on the measurement of pH changes, is a versatile technique with a wide range of applications. It is a well-established analytical method always effective for simple acid-base systems [2, 3]. For over 70 years it has been applied to study macromolecules, whose early use was limited to the analysis of the behavior of proteins. At that time, the application for studying acid synthetic polymers was applied almost exclusively to poly(acrylic acid) and poly(methacrylic acid) [4]. Nowadays it is still used to investigate the dissociation behavior of poly(acrylic acid) [5] but has expanded to study poly(itaconic acid) [6, 7], copolymers of maleic acid with various olefins [5], styrene [8], and ionization amphiphilic diblock and triblock copolymers [9]. In this study the use of potentiometric titration for the characterization of native and modified starches technique is highlighted.

The soluble natural polymers include polynucleotides, polypeptides, and polysaccharides such as starch, cellulose, and chitosan. Due to increased interest in the use of polysaccharides for a wide range of practical applications, potentiometric titration has become a standard method to analyze specific properties of polyelectrolytes in this group. The technique has been widely used to determine the amylose content in the starch [10–14], the degree of deacetylation of chitosan [15, 16], and the degree of protonation of cellulose derivatives [17], among other applications.

Starch is the main storage carbohydrate in plants. It is stored as granules in most plant cells and in this state is called native starch. Native starches from different botanical sources vary widely in structure and composition, but all granules are mainly formed by two molecular components, amylose (20–30%) and amylopectin (70–80%) [18]. It is a food and an important basic engineering building product widely applied in various branches of the food industry (milk, meat, canned goods, and pastries) and in nonfood technologies, such as paper, textiles, adhesives, and pharmaceutical [19]. Industrial use is based on the adhesive and thickening properties, the ability to form films and gels, as well as its low cost and quality control [20–22].

However, this polysaccharide has unfavourable properties such as low shear strength, ease of thermal decomposition, and high tendency for retrogradation (crystallization and aging of gels), limiting its use in other applications. These properties can be overcome by chemical and/or physical modification [23]. The structure and properties of polysaccharides, such as starch, can be modified through grafting reactions, oxidation, etherification, esterification, and crosslinking, among others [24].

Starch oxidation using KMnO4, grafting, or esterification with organic acids, such as itaconic acid, generates structural changes in the starch by incorporating carboxyl groups –COOH [25], which gives the starch superhydrophilicity and acidity [24]. The presence of –COOH groups allows for the characterization of these starch derivatives via potentiometric titration.

In this study modified starches were synthesized. The polyelectrolytic behavior of oxidized starches (thermal and thermal-chemical oxidation), a graft copolymer of itaconic acid (IA) onto starch, and starch esters (mono- and diester itaconate) was compared with the behavior of native starch, the homopolymer, and the acid employed as a graft monomer and substituent.

#### 2. Experimental

##### 2.1. Materials

Food grade corn starch supplied by Alfonzo Rivas & Cía hydrochloric acid, HCl (37%) nitric acid, HNO3 (65%) silver nitrate, AgNO3 (>99.9%) potassium permanganate, KMnO4 (99%) from Fisher Scientific ammonium hydroxide, NH4OH (95%) iodine, I2 (99.9%) potassium iodide, KI (100.5%) from J.T. Baker hydroxylamine hydrochloride, NH2OH·HCl (99%) ethanol, C2H5OH (99.9%) acetone, CH3(CO)CH3 (99.9%) ammonium persulfate (APS), (NH4)2S2O8 (≥98.5%) itaconic acid (IA), C3H4(COOH)2 (≥99%) sodium hydroxide, NaOH (99%) from Merck sodium bisulfite, NaHSO3 (Mallinckrodt Baker, 66.9%) and potassium bromide, KBr (Riedel-de-Haen, 99.5%), were all used as received.

##### 2.2. Sample Preparation
###### 2.2.1. Oxidized Starches

Oxidation by hydrothermal treatment involved the preparation of an aqueous dispersion of 10% m/v (10 g dry basis corn starch, equivalent to 0.062 moles of anhydroglucose units (AGU) in 100 mL of distilled water). The dispersion was heated to 75°C for 15 min with gentle shaking to promote starch gelatinization. Once formed, the starch paste and 200 mL of distilled water were added and cooled to the reaction temperature (60°C). 300 mL distilled water were added and the starch was oxidized by heat treatment (Ht-St) after 3 h.

For the thermal-chemical oxidation, after cooling the slurry to 60°C, 0.63 g (4 × 10 −3 moles) of KMnO4 (oxidizing initiator) and 1.04 g (0.01 moles) of NaHSO3 (reducing activator) were added and kept at 60°C for 10 min in order to preoxidize the starch. The volume of distilled water was immediately made up to 500 mL and allowed to react for 3 h to obtain the oxidized starch by thermal-chemical treatment (Ox-St).

In both oxidations, the product obtained was cooled to room temperature and precipitated with ethanol. The oxidized starch was washed with a mixture of 50% v/v ethanol/water and dried at 40°C to constant weight.

###### 2.2.2. Poly(itaconic acid)

Poly(itaconic acid) (PIA) was synthesized thermally using a modification of a classic method developed by Marvel and Shepherd in 1959 [26]. The reaction was conducted in a 100 mL Schlenk containing 50 mL of degassed distilled water by three alternating cycles of vacuum and nitrogen supplies. Then, 2.1 mL of 12.02 moles/L HCl (37% m/m), 10 g (0.0768 moles) of IA, and 0.5020 g (2.2 × 10 −3 moles) of APS (thermal decomposition initiator) were quickly added. The loaded Schlenk was immersed in a thermal bath of silicone oil, at 60°C (controlled temperature) for 44 h. After this time the solution was cooled to room temperature and slowly poured into excess acetone for precipitating the polymer. The precipitate was redissolved in water and dried on a lyophilizer to remove both free and bound water. After lyophilization, it was washed with acetone for three days, changing the solvent every 24 h to remove residual monomer. Finally, the homopolymer was dried under vacuum to constant weight and stored over silica gel for further characterization.

###### 2.2.3. Graft Copolymer of Itaconic Acid onto Starch

The same thermal-chemical oxidation procedure was used but after the preoxidation of the starch, the monomer (IA) previously dissolved in 180 mL of distilled water was added. The volume was immediately made up to 500 mL with distilled water. This time was regarded as the initial time of reaction (0.18 moles/L IA). After 3 h, the reaction was stopped, and the product (St-g-IA) was precipitated with ethanol. Subsequently, it was washed with a mixture of 50% v/v ethanol/water to remove any residual monomer, homopolymer, and fragments of soluble starch in cold water. Washing was performed until iodine testing (detection of starch) and Baeyer testing (detection of residual monomer) were negative and the pH of washing water was equal to or very close to that of the original washing mixture. Finally, samples were dried in an oven at 40°C to constant weight.

###### 2.2.4. Itaconic Acid Starch Esters

The esterification reactions were carried out using a combination of procedures in the literature [27, 28]. For obtaining itaconate starch semiester or starch semi-itaconate (SI), 200 mL of distilled water and 20 mL of 0.5 moles/L NaOH were added in a 3-neck flask containing 6.25 g starch dry basis (0.1754 moles/L AGU) and gelatinized for 10 min at 80°C, with pH monitoring (2.9). 16.0386 g of esterifying agent (0.5604 moles/L IA) was added and heated at 80°C for 4 h.

The same procedure was used for the itaconate starch diester or starch di-itaconate (DI) but a solution of 150 mL (0.2269 moles/L AGU and 0.7252 moles/L IA) in water was used. Then after the first 4 h of reaction, 18.75 g of starch on a dry basis dispersed in 50 mL of water (0.1875 moles/L AGU) was added and heated at 80°C for another 4 h (0.7015 moles/L AGU and 0.5604 moles/L IA). The reaction product was precipitated with ethanol the precipitate was filtered and washed with ethanol until the Baeyer test was negative. The samples were dried in an oven at 40°C until constant weight was reached.

##### 2.3. Characterization of Samples
###### 2.3.1. FTIR

In all cases, the formation of the desired starch derivative was confirmed by infrared Fourier transform spectroscopy (FTIR). The spectra were taken on a Shimadzu IR Prestige spectrophotometer in the range of 4000–400 cm −1 , using KBr pellets.

###### 2.3.2. Potentiometric Titration

In this section the experimental procedure used for the potentiometric titration of different starch derivatives (Ht-St, Ox-St, St-g-IA, SI, and DI) of native corn starch (St), the homopolymer (PIA) and itaconic acid (IA), is detailed. All solutions used were prepared using distilled water. The NaOH was titrated with 0.1 moles/L HCl before use. Titrations were carried out with gentle shaking at room temperature controlled at 22°C using a pH meter BOECO BT-500. Stabilizing the dispersion was allowed for 2 min between each addition of titrant to ensure equilibrium. Titration curves were obtained by analyzing the gel fraction of the product separated by leaching with hot water. For this, the samples were placed in filter paper bags and immersed in beakers with 400 mL of distilled water at 60°C the change of leaching water was performed every 24 h until iodine test was negative.

(1) Potentiometric Titration of Itaconic Acid and Poly(itaconic acid). A volume of 25 mL of 0.5 moles/L IA solution and another of PIA were prepared with similar mass concentrations, since it was not possible to determine the molar mass of the PIA. In both cases the titrant was 0.1 moles/L NaOH.

(2) Carboxyl Content. The carboxyl content of oxidized starch was determined according to the modified procedure of Chattopadhyay et al. [29]. About 0.2 g of starch sample was mixed with 2.5 mL of 0.1 moles/L HCl, and the slurry was stirred occasionally for 30 min with a magnetic stirrer. The slurry was then vacuum-filtered and washed with 40 mL of distilled water. The starch cake was then carefully transferred into a 50 mL beaker, and the volume was adjusted to 30 mL with distilled water. The starch slurry was heated in a boiling water bath with continuous stirring for 15 min to ensure complete gelatinization. The hot starch dispersion was then adjusted to 45 mL with distilled water and titrated to pH 8.3 with standardized 0.01 moles/L NaOH. A blank test was performed without sample. Carboxyl content was calculated as follows: