TITRATION OF AMINO ACIDSHapan | Hernandez M | Hilario | Icaro | Jacinto | JanierC1-2019

INTRODUCTION

TITRATION Etymology: Greek Titulus (Title); French Titre (Rank) Used to determine the unknown concentration of a known reactant. Process of analyzing composition by measuring the volume of one solution needed to completely react with another solution

DEFINITION OF TERMS Titrant standardized substance reacted with analyte to determine analyte concentration Analyte substance being analyzed Indicator used to mark end point ; dye or pH meter Equivalence Point point when amount of added standard reagent is exactly equivalent to amount of analyte End point point in titration when physical change associated with condition of chemical equivalence occurs

TYPES OF TITRATION Acid-Base

neutralization Complexometric / Chelatometric volumetric analysis colored complex as endpoint Oxidation-reduction redox reactions Precipitation ionic compounds of limited solubility silver nitrate

TITRATION CURVE A plot of pH vs. the amount of titrant added Typically the titrant is a strong (completely) dissociated acid or base Such curves are useful for determining endpoints and dissociation constants of weak acids or bases.

ACID-BASE TITRATION neutralization reaction acid/base of concentration (titrant) reacts with acid/base of unknown concentration (analyte)Analyte + Titrant Products

IONIC PROPERTIES OF AMINO ACIDComponents:

amine group carboxyl group R group in aqueous solution

side chain which ionize depending on the pH can behave as acid & base

IONIC PROPERTIES OF AMINO ACID Isoelectric Point (pI)

pH at which net charge on a molecule is zero (zwitterions) average of two pK values pK = pH

TITRATION OF AMINO ACIDS When an amino acid is dissolved in water it exists predominantly in the isoelectric form. Upon titration with acid: acts as a base Upon titration with base: acts as an acid Amphoteric compound can act as either an acid or a base is known as an

GLYCINE smallest of the amino acids Ambivalent can be inside or outside of the protein molecule Exist predominantly as the zwitterion in aqueous solution at or near neutral pH Chemical property: Aliphatic Physical property: Non-polar

LYSINE Essential amino acid; has a positively charged -amino group Lysine is basically alanine with a propylaminesubstituent on the carbon. -amino group has a significantly higher pKa (about 10.5 in polypeptides) than does the -amino group. Chemical property: Basic Physical property: Polar (positively charged)

ASPARTIC ACID Alanine with one of the hydrogens replaced by a carboxylic acid group pKa of the carboxyl group of aspartic acid in a polypeptide is about 4.0 Has -keto homolog (oxaloacetate) Chemical property: Acidic Physical property: Polar (charged)

OBJECTIVES To determine the acid base behavior of the amino acid during titration with an alkali and acid To determine the effect of formaldehyde on the titration curve of the amino acid To plot the titration curve using the pH values obtained from the experiment

Materials/Reagents Needed 0.1 N NaOH 0.1N HCl 0.1M glycine solution 0.1M lysine solution 0.1M aspartic acid solution Neutralized formaldehyde

Materials/Reagents Needed0.1 N NaOH 0.1M glycine solution

Materials/Reagents Needed0.1 N Aspartic acid 0.1M lysine solution

Materials/Reagents Needed0.1 N Aspartic acid 0.1M lysine solution

Materials/Reagents NeededNeutralized Formaldehyde0.1N HClDistilled water

PROCEDURE

PROCEDURE0.1N HCl

0.1N NaOH10 mL of the amino acid solution Measure the resulting pH

Titrate with Hcl or NaOH, adding 2.0 mLat a time Determine pH after each addition until 10 mL or 20 mL is reached

Plot the pH vs. the equivalent acid/base

One mL of acid/base=0.1 mEqof acid/baseRepeat, but add first 5.0 mL of neutralized formaldehyde to the amino acid solution

Plot on the same graph and solve for Pi and pK values of the

Results and Discussion (GLYCINE)

Glycinew/o HCHO w/ HCHO

mL In 1 M HCl In 1 M NaOH In 1 M HCl In 1 M NaOH mEq0 4.77 4.88 6.01 6.36 02 2.93 6.23 2.96 9.18 .24 2.53 6.69 2.55 9.61 .45 2.39 6.88 2.40 9.74 .56 2.28 7.12 2.29 9.88 .68 2.11 7.53 2.10 10.17 .8

10 1.96 8.19 1.96 10.51 1

02

4

6

8

10

12

1 0.8 0.6 0.5 0.4 0.2 0 0 0.2 0.4 0.5 0.6 0.8 1

pH

mEq

Glycine

w/ HCHOw/o HCHOpK1 2.37

pK2 7.11

pK2 9.85

pK1 2.38

pI 4.74

pI 6.12

Results and Discussion (ASPARTIC ACID)

Aspartic acid (HCl) Aspartic acid (NaOH)mL w/o HCHO w/ HCHO w/o HCHO w/HCHO mEq0 4.64 4.59 4.67 4.62 02 4.30 4.07 8.62 7.21 .24 4.07 3.70 9.27 7.88 .45 3.97 3.53 9.46 8.10 .56 3.88 3.36 9.63 8.36 .68 3.71 3.00 9.95 8.70 .810 3.53 2.68 10.35 9.23 112 3.36 2.46 11.07 10.15 1.214 3.18 2.29 11.40 10.67 1.415 3.08 2.23 11.44 10.80 1.516 2.99 2.17 11.53 10.90 1.618 2.83 2.08 11.67 11.05 1.820 2.67 2.00 11.74 11.16 2

02

4

6

8

10

12

14

2 1.8 1.6 1.5 1.4 1.2 1 0.8 0.6 0.5 0.4 0.2 0 0 0.2 0.4 0.5 0.6 0.8 1 1.2 1.4 1.5 1.6 1.8 2

Aspartic acid

w/o HCHO w/ HCHO

Results and Discussion (Lysine)

w/o HCHO w/ HCHO w/o HCHO w/ HCHO0 0 9.98 6.52 0 9.41 6.492 0.2 8.89 6.37 2 9.73 6.924 0.4 8.41 6.23 4 10.04 7.425 0.5 8.1 6.13 5 10.18 7.756 0.6 7.75 5.98 6 10.32 8.148 0.8 3.93 4.01 8 10.61 8.9610 1 3.15 3.18 10 10.93 9.5912 1.2 2.89 2.94 12 11.2 10.1614 1.4 2.72 2.77 14 11.4 10.615 1.5 2.66 2.71 15 11.47 10.7316 1.6 2.61 2.66 16 11.53 10.8118 1.8 2.53 2.58 18 11.61 10.9620 2 2.48 2.51 20 11.67 11.06

pHpHmEq0.1 N HCl (mL) 0.1 N NaOH (mL)

02

4

6

8

10

12

14

1 2 3 4 5 6 7 8 9 10 11 12 13

pH

Milliequivalence (mEq)

Lysine

w/o HCHO (Acid) w/ HCHO (Acid) w/o HCHO (Base) w/ HCHO (Base)

02

4

6

8

10

12

14

2 1.8 1.6 1.5 1.4 1.2 1 0.8 0.6 0.5 0.4 0.2 0 0 0.2 0.4 0.5 0.6 0.8 1 1.2 1.4 1.5 1.6 1.8 2

pH

Milliequivalence (mEq)

Titration of Lysine

Lysine without HCHO Lysine with HCHOMilliequivalence of H+(mEq) Milliequivalence of OH- (mEq)

pK1=2.75

pK2=8.80pK3=10.40

pK1=2.74

pK2=6.25pK1=6.5

Guide Questions

What can account for Sorensens discovery that the endpoint of titration between an amino acid and a standard alkali is not reached?The free amino group at the alpha-carbon actsas a base and interferes with the end point ofthe titration using a standard alkali.Formaldehyde in excess is needed to modify thebasic free amino group to modify it to a neutralgroup, a dimethylol derivative, which allows forthe endpoint to be reached.

Compare the values obtained when the amino acid was titrated with HCl both in the absence and presence of formaldeyde. How do you account for this?- In the presence of formaldehyde, titration curve is slightly lower- This is due to the fact that formaldehyde forms the dimethlol and monomethylol group increasing the basicity

At which pH will an amino acid exert its maximum buffering capacity? Why? Where in your graph is the buffering region for your amino acid?- Maximum buffering capacity(pink) when pH = pKa +/- 1- Nearly equal amounts of protondonors and acceptors- Least buffering capacity at the pIbecause both the amino andcarboxyl group are protonatedand deprotonated- Therefore any minute additionof H+ or OH- will result in a largechange in pH.

From the titration curve of an amino acid, can you determine the nature of its R group, i.e., basic, acidic or neutral?