119 3 Sample Preparation Techniques to Isolate and Recover Organics and Inorganics Separation methods form the basis of chemistry, and the definition of a pure chemical substance ultimately depends on separative operations. —Arne Tiselius CHAPTER AT A GLANCE Sample prep for trace organics Liquid–liquid extraction (LLE) 121 Separatory funnel 131 LLE as cleanup 131 Mini 145 Micro 145 Continuous 148 Soxhlet liquid–solid extraction (S-LSE) 149 Conventional Soxhlet 149 Automated Soxhlet 150 Ultrasonic liquid–solid extraction (U-LSE) 153 Microwave accelerated extraction (MAE) 157 Accelerated solvent extraction (ASE) 160 Sample prep for volatile organic compounds (VOCs) 165 Mini-LLE 165 Static headspace 165 Hexadecane screening via LLE 178 Purge and trap 181 Extract cleanup 191 Adsorption column chromatography 192 SPE adsorption 194 Gel permeation chromatography 195 Supercritical fluid extraction (SFE) 200 Reversed-phase solid-phase extraction (RP-SPE) 211 © 2006 by Taylor & Francis Group, LLC 120 Trace Environmental Quantitative Analysis, Second Edition Matrix solid-phase dispersion 251 Solid-phase microextraction (SPME) 255 Stir Bar Sorptive Extraction 268 Sample prep for trace inorganics Categorization of sample prep methods for trace inorganics 276 Conventional approaches to sample prep for trace metals 276 Matrix modification in graphite furnace atomic absorption spectrophotometry 279 EPA’s microwave digestion approaches to sample prep for enviro- chemical trace metals 281 Clinical laboratory approaches to sample prep for enviro-health trace metals 283 Preconcentration of aqueous samples for ultratrace metals 284 Trace metal chelation and RP-SPE 292 Sample prep to determine trace mercury 305 Sample prep to determine trace cyanide 307 References 316 The importance of sample preparation to TEQA is clearly indicated in the following story. This author was once approached by a student during the era when it became apparent that in the 1970s polychlorinated biphenyls (PCBs) had contaminated the striped bass that migrate up the Hudson River in New York to spawn every spring. Once the student learned that a gas chromatograph (GC) is used to measure the extent that fish are contaminated with PCBs and noticed the instrument on the bench in the corner of the laboratory, the student was curious as to exactly how a fish the size of a striped bass could be put into the injection port of the GC. The diameter of the injection port of the GC was less than 1 mm, which, of course, is miniscule in comparison to the size of the fish. The student thought that all that was necessary was to find a way to get the fish into the injection port and the data, which at that time were displayed on a strip-chart recorder, would indicate the extent of this PCB contamination. The student speculated that it might be easier to cut the fish up and attempt to stuff it into the injection port on the GC. Ah, we see for the first time, in this student, a glimpse into the need for sample preparation. Indeed, the fish must be transformed in some manner prior to measurement by a determinative technique — in this case, by gas chromatography. Determinative The removal of the PCB from fish tissue (known as the sample matrix) to a form that is compatible with the determinative technique or particularly analytical instru- ment — in this case, the GC — is the basis for sample preparation. The GC requires the introduction of a solvent that contains the dissolved solute — in this case, PCBs. A gas can also be injected into the GC. However, it is much more convenient to get the PCBs from the sample matrix to the liquid state. The liquid is quickly vaporized under the elevated temperature of the GC injection port and undergoes GC separa- tion. The number of molecules of each chemically different substance now present © 2006 by Taylor & Francis Group, LLC techniques utilize instrumental analysis approaches and are discussed in Chapter 4. Sample Preparation Techniques 121 in the vapor causes a perturbation in the GC detector. This perturbation results in an electrical signal whose magnitude becomes proportional to the number of mol- ecules present in the liquid. This chapter introduces the various techniques that are commonly used to prepare environmental samples and animal and human specimens and comprises an impor- tant component of TEQA. The laboratory approach used to “get the striped bass into the machine” to achieve the utmost goal of TEQA (i.e., to isolate, identify, and quantitate the PCBs in the sample matrix) defines sample preparation. This chapter starts out with the most common and most conceptually simplistic form of sample preparation, whereby a liquid such as water or a solid such as a soil is placed in a beaker or equivalent container. To this container is added an organic solvent that is immiscible with water. The mixture is shaken and allowed to remain stationary for a period, such as 15 min. The analytes originally dissolved in the water or adsorbed onto soil particulates are partitioned into the organic solvent. The organic solvent that now contains the dissolved analyte as a solute is referred to as the extractant. After the principles of liquid–liquid extraction (LLE) are introduced and developed, the practice of LLE in its various forms will be discussed. In addition to LLE, there are two other major types of analyte isolation and recovery: solid-phase extraction (SPE) and supercritical fluid extraction (SFE). SPE refers to those techniques that isolate the analyte from a sample matrix and partition the analytes of interest onto a chemically bonded silica or polymeric surface. SFE refers to those techniques that isolate the analyte from a sample matrix and partition it into a liquid that has been heated and pressurized beyond its critical temperature and pressure. It is indeed overly simplistic to think that a striped bass can be stuffed into a GC as a means to conduct TEQA. 1. WHAT ARE THE PRINCIPLES UNDERLYING LLE? A good grounding in the basic principles of LLE is a useful way to begin a chapter that focuses on sample preparation for TEQA. LLE was historically the first sample preparation technique used in analytical chemistry. Organic chemists have used LLE techniques for over 150 years for isolating organic substances from aqueous solutions. A good definition of LLE has been given earlier in the literature and is stated here: A substance distributes between contacting immiscible liquids — water and a suitable organic solvent, for example — roughly in the ratio of its solubility in each if it does not react with either and if it exits in the same form in both. If, at equilibrium, its concentration is much greater in the organic solvent phase than in the aqueous phase, the distribution behavior may be put to analytical use in concentrating the substance into a small volume of the organic liquid and, more importantly, in separating it from substances that do not distribute similarly. 1 This definition of LLE is concise yet profound in that it covers all ramifications. The first sentence establishes two conditions: compounds that react with the extractant do not obey the rules, and the chemical nature of the compound needs to remain the same throughout the extraction. Mathematical relationships have also been developed to account for the fact that the chemical form may change. This has been called © 2006 by Taylor & Francis Group, LLC 122 Trace Environmental Quantitative Analysis, Second Edition secondary equilibrium effects, and this topic will also be introduced in this chapter. The second sentence implies that a concentration factor can be realized. The concentrating nature of LLE is most important to TEQA. The fact that different chemical sub- stances will distribute differently between immiscible liquids also forms the theo- retical basis for separation among two or more organic substances that might be initially dissolved in the aqueous solution. These differences are exploited in the design of sample preparation schemes as well as provide for the fundamental basis to explain analyte separation by chromatography. Aqueous solutions are of prime importance to TEQA because our sample matrix, if a liquid, consists of drinking water, surface (i.e., rivers) water, groundwater, or wastewater obtained from the environment. The fact that the chemical form can change during the extraction process can be exploited in analytical chemistry toward the development of new methods to separate and isolate the analyte of interest. To understand the most fundamental concept of liquid–liquid extraction, consider placing 100 mL of an aqueous solution that contains 0.1 M NaCl and 0.1 M acetic acid (HOAc) into a piece of laboratory glassware known as a separatory, or com- 2 process. Figure 3.1A shows this process just prior to mixing the two immiscible phases. Next, 100 mL of diethyl ether, a moderately polar organic solvent that is largely immiscible with water, is added to the funnel. Indeed, some ether will dissolve in water to the extent of 6.89% at 20°C, while some water dissolves in the ether to the extent of 1.26% at 20°C. 3 Upon shaking the contents of the funnel and allowing some time for the two phases to become stationary, the solute composition of each phase is depicted in Figure 3.1B. The lower layer is removed from the sep funnel, thus physically separating the two phases. Taking an aliquot (portion thereof) of the ether phase and separately taking an aliquot of the water phase while subjecting the aliquot to chemical analysis reveals a concentration of NaCl, denoted as [NaCl], at 1.0 × 10 –11 M, and that in water, [NaCl] aq = 0.10 M. Analysis of each phase for acetic acid reveals [HOAc] ether = [HOAc] aq = 5 × 10 –2 M. Upon combining both phases again, a second chemical analysis of the composition of each phase reveals exactly the same concentration of HOAc and NaCl in each phase. As long as the temperature of the two phases in contact with each other of the sep funnel remain fixed, the concentration of each chemical species in both phases will not change with time. A dynamic chemical equilibrium has been reached. The significant difference in the extent of partitioning of NaCl and HOAc between diethyl ether and water-immiscible phases can be explained by introducing a thermodynamic viewpoint. 2. DOES THERMODYNAMICS EXPLAIN DIFFERENCES IN NACL VS. HOAC PARTITIONING? For spontaneous change to occur, the entropy of the universe must increase. The entropy of the universe continues to increase with each and every spontaneous process. LLE represents an ideally closed thermodynamic system in which solutes originally dissolved in an aqueous sample taken from the environment can diffuse across a solvent–water interface and spontaneously partition into the solvent phase. These concepts are succinctly defined in terms of the change in Gibbs free energy, © 2006 by Taylor & Francis Group, LLC monly abbreviated as a sep funnel. Figure 3.1 shows a conceptually simplified LLE Sample Preparation Techniques 123 G, for system processes that experience a change in their enthalpy H and a change in the entropy of the system S. The criteria for spontaneity requires that the Gibbs free energy, G, decrease. In turn, this free-energy change is mathematically related to a system’s enthalpy H and entropy S. All three depend on the state of the system and not on the particular pathway, so a change in free energy at constant temperature can be expressed as a difference in the exothermic or endothermic nature of the change and the tendency of the matter in the system to spread according to This equation suggests that for spontaneous physical or chemical change to occur, the process proceeds with a decrease in free energy. As applied to phase distribution, equilibrium is reached when the infinitesimal increase in G per infini- tesimal increase in the number of moles of solute i added to each phase becomes equal. Hence, the chemical potential of solute t is defined as The chemical potential can also be expressed in terms of a chemical potential under standard-rate conditions µ 0 and the activity a for a solute in a given phase. FIGURE 3.1 Hypothetical distribution of solutes NaCl and HOAc between two immiscible solvent phases. Organic phase (ether) Organic phase (ether) Aqueous phase Aqueous phase NaCl HOAc NaCl NaCl NaCl (aq) HOAc (aq) NaCl (ether) HOAc (ether) HOAc HOAc A B Ether is being added to the aqueous phase that contains dissolved solutes e ether and aqueous phases have been in contact for some time and equilibrium has been established for the dissolved solutes between the two phases K D HOAc K D NaCl ∆∆ ∆GHTS= − µ= ∂ ∂       G n i TP, © 2006 by Taylor & Francis Group, LLC 124 Trace Environmental Quantitative Analysis, Second Edition Recognizing that a phase has an activity equal to unity (i.e., a = 1 defines the standard state at a given temperature and pressure), the equation for the chemical potential µ for an activity other than a = 1 is found according to (3.1) Once equilibrium is reached, the net change in µ for the transfer of solute i between phases must be zero, so that for our example of NaCl or HOAc in the ether/water-immiscible phase illustration, the chemical potentials are equal: (3.2) Hence, upon substituting Equation (3.1) into Equation (3.2) for solute i, which rearranges to (3.3) The change in standard-state chemical potential, ∆µ 0 , is usually expressed as the difference between the organic phase and the aqueous phase according to Solving Equation (3.3) for the ratio of solute activities gives Because ∆µ 0 is the difference of two constant standard-state chemical potentials, it must be a constant. The ratio of activities of NaCl or HOAc is fixed provided that the temperature and pressure are held constant. A thermodynamic approach has just been used to show what is important analytically; that is, LLE enables an analyte to be transferred from the sample to the extracting solvent and remain at a fixed concentration over time in the extractant. This ratio of activities is defined as the thermodynamic distribution constant, K 0 , so that (3.4) µµ=+ 0 RT aln µµ ether NaCl aq NaCl = µµ ether ether aq aq 00 +=+RT a RT aln ln RT a a ln ether aq ether aq       = −µµ 0 0 ∆µµ µ 00 0 = − etheraq a a e ether aq = −∆µ 0 K a a 0 ≡ ether aq © 2006 by Taylor & Francis Group, LLC Sample Preparation Techniques 125 3. WHAT ARE SOLUTE ACTIVITIES ANYWAY? A solute dissolved in a solvent such as water is only partly characterized by its concentration. Solute concentration can be expressed in one of any number of units. The most commonly used units include the following: moles solute per liter solution or molarity (M), moles solute/100 g water or molality (m), and millimoles solute per liter solution or millimolarity (mM). Those units that have greater relevance to TEQA include the following: milligrams of solute per liter solution or parts per million (ppm), micrograms of solute per liter solution or parts per billion (ppb), and picograms of solute per liter solution or parts per trillion (ppt). Note that TEQA relies exclusively on expressing solute concentration in terms of a weight per unit volume basis. The fact that equilibrium constants in chemistry depend not only on solute concentration but also on solute activities serves to explain why any discussion of distribution equilibria must incorporate solute activities. Solute activities are introduced in any number of texts. 4 * Activities become important when the concen- tration of an electrolyte in an aqueous solution becomes appreciable (i.e., at solute concentrations of 0.01 M and higher). The extent to which a solution whose concentration of solute i contributes to some physical/chemical property of this solution (i.e., its activity, a i ) is governed by the solute’s activity coefficient γ i according to 1. Neutral molecules dissolved in water do not affect ionic strength. 2. Very dilute aqueous solutions are most likely found. However, one aspect of TEQA that is strongly influenced by ionic strength, and hence provides an opportunity for activity coefficients to play a role, is the concept of salting out. The solubility of one chemical substance in another, like K 0 [Equation (3.4)] in LLE, is also governed by the need for the substance to lower its Gibbs free energy by dissolving in a solvent. Isopropyl alcohol (IPA) or 2-propanol is infinitely soluble in water, as is true for most lower-molecular-weight alcohols. However, for a solution that might consist of 50% IPA and 50% water, the alcohol can be separated out as a separate phase if enough NaCl is added to almost saturate the system. This is a direct influence of ionic strength in an extreme case. The fact that polar solvents can be separated as an immiscible phase opens up new sample preparation oppor- tunities. For example, Loconto and coworkers 5 recently demonstrated that the homol- ogous series of polar 2-aminoethanols could be efficiently partitioned into IPA from an aqueous sample of interest to wood chemists. The sample was saturated with NaCl, then extracted using IPA. Two important relationships must be discussed that relate activity coefficients to ionic strength. Ionic equilibria are influenced by the presence of all ions in an aqueous solution. The most useful indicator of the total concentration of ions in a * The concept of activity and activity coefficients is found in most physical and analytical chemistry texts that consider ionic equilibria. The texts listed in reference 4 are part of the author’s personal library. ac iii = γ © 2006 by Taylor & Francis Group, LLC 126 Trace Environmental Quantitative Analysis, Second Edition solution is the ionic strength, I. The ionic strength can be calculated if the concen- tration C i of an ion whose charge is z i is known according to (3.5) The summation is extended over all ions in solution. For example, consider two aqueous solutions, one containing 0.01 M NaCl and the other one containing 0.01 M K 2 SO 4 . Using Equation (3.5), the ionic strength for the former solution is calculated to be 0.01 M and that for the latter is 0.03 M. Assume that a solution is created that consists of 0.01 M in each salt. The ionic strength of such a mixture is calculated according to Equation (3.5) to be 0.04 M. Knowledge of a solution’s ionic strength enables a determination of the activity coefficient to be made. This can occur through the application of the Debye–Huckel equation according to where α refers to the size of the hydrated radius of the ion, and z is the charge of the ion. This equation gives good approximations for ionic strengths below or equal to 0.1 M. For ionic strengths less than 0.01 M, the following relationship suffices: 4. CAN THE DIFFERENCE BETWEEN K 0 VALUES FOR NACL AND HOAC BE SHOWN GRAPHICALLY? The thermodynamic relationship between standard-state chemical potential differ- illustrates what happens to the Gibbs free energy G when the solute is partitioned between an aqueous phase in contact with an immiscible organic phase, diethyl ether in this example. The hypothetical plots of G vs. the mole fraction, denoted by X i , of solute i dissolved in the ether phase, are superimposed for comparison. When there is no solute in the ether phase, a standard-state chemical potential, can be realized. In the other extreme, when 100% of all of the mass solute is in the ether phase (i.e., having a mole fraction X ether = 1), a standard-state chemical poten- tial, can also be defined. The situation at X ether = 1 is a hypothetical one in that for some solutes, 1 mol of solute cannot dissolve to that extent in an organic solvent like ether. This is particularly true for an ionically bonded substance such as sodium chloride. Imagine if this much NaCl could dissolve in ether. The free energy that would be required to dissolve as much as 1 mol NaCl in 1 L of ether would be expected to be extremely large indeed. Icz ii i = ∑ 1 2 2 log . γ α = − + 051 1 305 2 zI I/ log .γ = 051 2 zI µ aq 0 , µ ether 0 , © 2006 by Taylor & Francis Group, LLC ences and the position of chemical equilibrium can be shown graphically. Figure 3.2 Sample Preparation Techniques 127 Such is not the case when considering the free energy required for the dissolution of 1 mol HOAc in 1 L of ether. The mole fraction of solute partitioned into the ether at equilibrium is that point along the x axis where G is at a minimum, or in other words, the slope of the tangent line (i.e., dG/dX i ) is zero. It becomes quite evident when viewing this graphical display that the magnitude of standard-state Gibbs free energies are chiefly responsible for the position along the x axis where G reaches a minimum. At this position, the mole fraction of each solute becomes fixed as defined by Equation (3.3). Figure 3.2 shows that the Gibbs free energy is minimized at equilibrium for NaCl at a much lower mole fraction when compared to the value of the mole fraction for HOAc, where its Gibbs free energy is minimized. In other words, the value of X i where dG/dX i is minimized at equilibrium depends entirely on the nature of the chemical compound. If a third solute is added to the original function-of-X i plot and reach a minimum at some other point along the x axis. These concepts render Equation (3.3) a bit more meaningful when graphically represented. 5. CAN WE RELATE K 0 TO ANALYTICALLY MEASURABLE QUANTITIES? It becomes important to TEQA to relate the thermodynamic distribution constant, K 0 , to measurable concentration of dissolved solute in both phases. Because the chemical potential for a given solute must be the same in both immiscible phases FIGURE 3.2 Hypothetical plot of solute free energy, G, in ether and in water vs. solute i mole fraction, X i dissolved in ether for solutes NaCl and HOAC. For example: n = number of moles; T,P,X j#i ∂X i ∂G µ i = 0 X i ether 1 G 0,aqueous HOAc G 0,ether HOAc G 0,aqueous NaCI G 0,ether NaCI G Xn(n n NaCl ether NaCl ether NaCl aqueous NaCl eth =+ eer ). © 2006 by Taylor & Francis Group, LLC aqueous solution, as depicted in Figure 3.1, it too would exhibit its own G-as-a- 128 Trace Environmental Quantitative Analysis, Second Edition that are in equilibrium, Equation (3.2) can be rewritten in terms of activity coeffi- cients and concentration according to Upon rearranging and simplifying, we get This equation can be solved for the ratio of measurable concentration of solute in the ether phase to that of the water phase; this is shown by (3.6) If we define a partition constant K D as a ratio of measurable concentrations of solute in both phases, we get (3.7) Upon substituting Equation (3.6) into Equation (3.7), we obtained the relation- ship between the partition ratio and the thermodynamic distribution constant according to (3.8) Equation (3.8) is the desired outcome. In many cases, with respect to TEQA, the activity coefficients of solutes in both phases are quite close to unity. The partition ratio and thermodynamic distribution constant can be used interchangeably. For either NaCl or HOAc, or for any other solute distributed between immiscible liquids at a fixed temperature and pressure, provided that the concentration of solute is low (i.e., for the dilute solution case), K 0 can be set equal to the partition constant K D because activity coefficients can be set equal to 1. The partition constant or Nernst distribution constant in our illustration for acetic acid partitioned between ether and water can be defined as µ γ µ ether ether ether aq aq 00 ++=++RT C RT RT Cln ln ln lln γ aq RT C C RTln ln ether aq ether aq +=− γ γ µ∆ 0 C C e RT ether aq ether aq = − γ γ µ∆ 0 / K C C D ≡ ether aq KK D = γ γ ether aq 0 K HOAc HOAc D = [] [] ether az © 2006 by Taylor & Francis Group, LLC [...]... solid-phase extraction (RP-SPE).25 Scheme 3. 1 (the first of numerous schemes devised by the author to illustrate the steps and logic of sample prep) is a flowchart that utilizes U-LSE, coupled with RP-SPE, for up-front analyte extraction and cleanup The procedural details outlined in Scheme 3. 1 will be discussed in a subsequent section on RP-SPE techniques A second alternative to S-LSE is microwave-accelerated... 137 2CH3COOH Ether phase Aqueous phase (CH3COOH)2 H+ + CH3COO− CH3COOH FIGURE 3. 5 Distribution of HOAc between ether and water assuming dimerization in the ether phase the organic phase, is shown in Figure 3. 5 The fundamental basis for the partitioning of HOAc between ether and water as introduced by the Nernst law is not violated and still is given by KD The measurable concentrations [HOAc]ether and. .. LLE (C-LLE) is often more appropriate and convenient within which to conduct LLE To illustrate, if a 2-L wastewater effluent sample is to be extracted, C-LLE would be the technique of choice C-LLE requires a relatively large glass apparatus whereby the receiving pot can vary in size C-LLE can be performed using a lighterthan-water extractant or a heavier-than-water extractant Typical lighter-than-water... various lower-molecular-weight alkanes such as n-hexane, while typical heavier-than-water extractants include various chlorinated solvents such as methylene chloride (dichloromethane) The operational procedure for lighter-than-water C-LLE has been described from a manufacturer of C-LLE glassware as follows:20 The aqueous phase to be extracted and a stirring bar are placed in a 24/40 round-bottom flask... of the pot and continuing to reflux as shown in the sketch from EPA Method 35 41( 23) below: Condenser Thimble Glass wool plug Sample Aluminium beaker (cup) Hot plate Several SVOCs of environmental interest are given below, along with mean percent recoveries from spiked clay, using both S-LSE and AS-LSE techniques.21 Mean % Recovery (% RSD) Analyte δ-BHC (lindane) Endrin p,p'-DDT a S-LSEa AS-LSEb 65.6... difficult to estimate KD from the hyperbolic curve shown in Figure 3. 4 Equation (3. 13) can be rearranged by taking reciprocals of both sides and rewriting Equation (3. 13) in the form of an equation for a straight line of form y = mx + b, where m is the slope and b is the y intercept: © 2006 by Taylor & Francis Group, LLC 136 Trace Environmental Quantitative Analysis, Second Edition 1  Ka  1 1 = + D  K... LLC 148 Trace Environmental Quantitative Analysis, Second Edition liquid membrane that consists of n-octane confined to within a Teflon ring sits on top of 0.5 or 1 mL of an aqueous sample whose pH is approximately 13 and contains an ionizable analyte If an amine is dissolved in water and the pH adjusted to 13, the amine would remain unprotonated and therefore neutral A large KD would be expected, and the... flask size (up to and including the 5 L) is chosen so that it is not more than 2 /3 4/5 full of aqueous phase The flask is then filled with the lighter-than-water extracting solvent and gentle stirring is started The extractor and an efficient condenser are put into place and a small flask containing an additional portion of the lighter-thanwater extracting solvent is connected to the side-arm and the solvent... phase in very high yields and only a relatively small amount of extracting solvent need be used © 2006 by Taylor & Francis Group, LLC Sample Preparation Techniques 149 Heavier-than-water C-LLE designs are operated similarly:20 Some heavier-than-water extracting solvent and a stirring bar are placed in the flask that contains the aqueous phase to be extracted A good rule-of-thumb is that the flask should... 150 Trace Environmental Quantitative Analysis, Second Edition pot The pot is removed, and due to the large volume of solvent required, the analyst may have over 30 0 mL of extractant Common solvents used in S-LSE are, in general, those solvents that are moderate to nonpolar and possess relatively low boiling points Methylene chloride and petroleum ether are the two most commonly used to conduct S-LSE . Figure 3. 1, it too would exhibit its own G-as-a- 128 Trace Environmental Quantitative Analysis, Second Edition that are in equilibrium, Equation (3. 2) can be rewritten in terms of activity coef - cients. 200 Reversed-phase solid-phase extraction (RP-SPE) 211 © 2006 by Taylor & Francis Group, LLC 120 Trace Environmental Quantitative Analysis, Second Edition Matrix solid-phase dispersion 251 Solid-phase. undissociated FIGURE 3. 3 Distribution of HOAc between two immiscible phases. The aqueous phase is alkaline. CH COOH H H O CH COO OH 3 - 32 3 + → ←→ + − + O Ether phase Aqueous phase CH 3 COOH + OH − CH 3 COOH CH 3 COO − ©