1 Laboratory Activity Three

Title: “Acids, Bases, Buffers, Titrations” Word / Phrase Association:  H + ions, OH - ions.  pH, pOH.  Strong vs. weak acids & bases.  Measurement of H + & OH - ion concentration  Stabilization & control of pH.  Incremental adjustment of pH. Title: “Acids, Bases, Buffers, Titrations” Word / Phrase Association:  H + ions, OH - ions.  pH, pOH.  Strong vs. weak acids & bases.  Measurement of H + & OH - ion concentration  Stabilization & control of pH.  Incremental adjustment of pH. 2 2

Introduce you to, or review the theories and principles of...  pH, pH measurement, and pH control.  Amino acids as “amphoteric” molecules and “zwitterions”.  The detection & quantification Amino acids.  The titration of amino acids. Specific Activities:  Measure the pH of several solutions.  Perform the titration of an unknown amino acid.  Make a simple plant tissue extract.  Use the ninhydrin reagent for the detection & quantification of amino acids. Introduce you to, or review the theories and principles of...  pH, pH measurement, and pH control.  Amino acids as “amphoteric” molecules and “zwitterions”.  The detection & quantification Amino acids.  The titration of amino acids. Specific Activities:  Measure the pH of several solutions.  Perform the titration of an unknown amino acid.  Make a simple plant tissue extract.  Use the ninhydrin reagent for the detection & quantification of amino acids. 3 3

 Mathematically defined by S.P. Sorensen (1909): pH =  log 10 [H + ] Rationale for pH Scale:  Studied effects of [H + ] and [OH - ] on proteins.  Range in possible [H + ] and [OH - ] is extremely large.  Relevant changes in [H + ] and [OH - ] is very small.  Log scale eliminates need to work with decimal fractions.  Mathematically defined by S.P. Sorensen (1909): pH =  log 10 [H + ] Rationale for pH Scale:  Studied effects of [H + ] and [OH - ] on proteins.  Range in possible [H + ] and [OH - ] is extremely large.  Relevant changes in [H + ] and [OH - ] is very small.  Log scale eliminates need to work with decimal fractions. 4 4

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Strong Acid: HCl  H + + Cl - Weak Acid: CH 3 COOH  CH 3 COO - + H + DissociationEquilibriumExpression:HendersonHasselbalchEquation: (see Appendix IV) Strong Acid: HCl  H + + Cl - Weak Acid: CH 3 COOH  CH 3 COO - + H + DissociationEquilibriumExpression:HendersonHasselbalchEquation: (see Appendix IV) 6 6 [H + ][A - ] [H + ][A - ] K a = ––––––– [HA] [HA] pK a = - log 10 K a [H + ][A - ] [H + ][A - ] = - log 10 ––––––– = - log 10 ––––––– [HA] [HA] [A - ] [A - ] pH = pK a + log 10 –––– [HA] [HA]

 The amount/ratio of A - vs. HA can be adjusted by adding strong acid or strong base (called a “titration”).  At a certain point, the [A - ] = [HA] (i.e. the weak acid is 50% dissociated).  [A - ]/[HA] = 1  Log 10 ([A - ]/[HA]) = 0  pH = pK a  The amount/ratio of A - vs. HA can be adjusted by adding strong acid or strong base (called a “titration”).  At a certain point, the [A - ] = [HA] (i.e. the weak acid is 50% dissociated).  [A - ]/[HA] = 1  Log 10 ([A - ]/[HA]) = 0  pH = pK a 7 7 CH 3 COOH  CH 3 COO - + H + (HA) (A - ) [A - ] [A - ] pH = pK a + log 10 –––– [HA] [HA]

 Predict the pH of a weak acid solution (without a pH meter) when the concentrations of A - and HA are known.  Determine the ratio of [A - ]/[HA] in a weak acid solution by measuring its pH.  Experimental determination of the pK a of a weak acid (i.e. via titration).  An analytical technique used to determine the unknown concentration of substance by gradually reacting that substance with a known amount of a second reactant until the reaction is complete.  Predict the pH of a weak acid solution (without a pH meter) when the concentrations of A - and HA are known.  Determine the ratio of [A - ]/[HA] in a weak acid solution by measuring its pH.  Experimental determination of the pK a of a weak acid (i.e. via titration).  An analytical technique used to determine the unknown concentration of substance by gradually reacting that substance with a known amount of a second reactant until the reaction is complete. 8 8 [A - ] [A - ] pH = pK a + log 10 –––– [HA] [HA]

pK a  the pH where the buffer is 50% dissociated.  the pH where the strongest buffering occurs. 2. Useful pH range where most buffering occurs. 3. Buffering capacity  the amount of base (or acid) that can be added. 4. Total concentration of original acid. 5. The number of dissociable groups. 1. pK a  the pH where the buffer is 50% dissociated.  the pH where the strongest buffering occurs. 2. Useful pH range where most buffering occurs. 3. Buffering capacity  the amount of base (or acid) that can be added. 4. Total concentration of original acid. 5. The number of dissociable groups. (100 mL of 0.1M Acetic Acid)

Amphoteric - can act as an acid or a base (i.e. can release or accept H + ). Zwitterionic – can have both a positive and negative charge on one molecule. Amphoteric - can act as an acid or a base (i.e. can release or accept H + ). Zwitterionic – can have both a positive and negative charge on one molecule. 10 Amino Group Carboxyl Group “  ” Carbon IRIR C – C – O II O - H HN –HN – HHHH + Zwitterion (neutral pH) IRIR C – C – OH II O H HN –HN – HHHH + Acidic Form (acidic pH) IRIR C – C – O II O - H HN –HN – H Basic Form (basic pH) pK 1 pK 2

Important Points:  Two pK a ’s:  COOH  NH 3 +  Isoelectric point (pI):  Graphical determination.  Mathematical determination.  Titration Endpoint. Important Points:  Two pK a ’s:  COOH  NH 3 +  Isoelectric point (pI):  Graphical determination.  Mathematical determination.  Titration Endpoint. 11 pK 1 =2.3 (-COOH) pK 2 =9.7 (-NH 3 + ) pI=6.0 Endpoint

Important Points:  Student pH meter.  Inaccurate readings at extreme pH’s.  Two pK a ’s:  COOH  NH 3 +  Isoelectric point (pI):  Graphical.  Mathematical.  Endpoint. Important Points:  Student pH meter.  Inaccurate readings at extreme pH’s.  Two pK a ’s:  COOH  NH 3 +  Isoelectric point (pI):  Graphical.  Mathematical.  Endpoint. 12 mEq NaOH pH pK 1 pK 2 pI Endpoint

 Obtain an aliquot of amino acid “unknown”.  Measure pH of unknown.  Add 200 µ L 4 N NaOH (0.8 mEq).  Mix & re-measure pH.  Repeat until endpoint is reached.  Make titration curve & interpret results.  Obtain an aliquot of amino acid “unknown”.  Measure pH of unknown.  Add 200 µ L 4 N NaOH (0.8 mEq).  Mix & re-measure pH.  Repeat until endpoint is reached.  Make titration curve & interpret results. 13

The Ninhydrin Reaction:  Ninhydrin reacts with (consumes) the primary amino group of amino acids to form the blue/purple “di-ninhydrin” product.  The carboxyl group is released as CO 2.  The “R” group is released as the  -carbon aldehyde. The Ninhydrin Reaction:  Ninhydrin reacts with (consumes) the primary amino group of amino acids to form the blue/purple “di-ninhydrin” product.  The carboxyl group is released as CO 2.  The “R” group is released as the  -carbon aldehyde. 14 Blue-Purple ProductNinhydrinAmino Acid

The Ninhydrin Reaction: 15 Amino Acid Color ColorAlanineArginineAsparagine Aspartic acid CysteineGlutamine Glutamic acid GlycineHistidineIsoleucinepurpleblue-purplebrown-yellowblue yellow* purplepurplepurplebrownpurpleLeucineLysineMethioninePhenylalanineProlineSerineThreonineTryptophanTyrosine Valine Valinepurplepurplepurplepurpleyellowpurplepurpleblue/graypurplepurple Table II. Color Reactions of the Standard Amino Acids with Ninhydrin. *Cysteine normally does not react with ninhydrin unless the reaction is strongly acidified.

Procedure: 1. Make an aqueous extract of pea seedling tissues (roots, shoots, seeds, + one).  Homogenize in motor & pestle.  Centrifuge in microfuge.  Use supernatant. 2. Make a series of dilutions of 10 mM Glycine (0, 0.1, 0.2, 0.3, 0.4 and 0.5 µmoles/100 µL). 3. Dispense aliquots of various juices/drinks. 4. Dispense aliquots of selected amino acids. 5. Dispense aliquots of ? plant tissue extracts. 6. Add 2.0 mL ninhydrin reagent & heat 15 min. 7. Read ABS 570 ; make standard curve; estimate AA content. Important Note Important NoteProcedure: 1. Make an aqueous extract of pea seedling tissues (roots, shoots, seeds, + one).  Homogenize in motor & pestle.  Centrifuge in microfuge.  Use supernatant. 2. Make a series of dilutions of 10 mM Glycine (0, 0.1, 0.2, 0.3, 0.4 and 0.5 µmoles/100 µL). 3. Dispense aliquots of various juices/drinks. 4. Dispense aliquots of selected amino acids. 5. Dispense aliquots of ? plant tissue extracts. 6. Add 2.0 mL ninhydrin reagent & heat 15 min. 7. Read ABS 570 ; make standard curve; estimate AA content. Important Note Important Note 16

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