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Polyprotic Acids

1Polyprotic Acids Have More than One Acidic GroupH2CO3 + H2O H3O+ + HCO3-pKa1 = 6.4HCO3- + H2O H3O+ + CO32-pKa2 = 7.2Carbonic acidpKa2 = 9.33pKa1 = 2.35

Tryptophan (W)Phosphoric acidH3PO4 + H2O H3O+ + H2PO4-pKa1 = 2.12H2PO4- + H2O H3O+ + HPO42-pKa2 = 7.21HPO42- + H2O H3O+ + PO43-pKa3 = 12.68Polyprotic Acids2

SmallNucleophilicHydrophobicAromaticAcidicAmideBasic

X-ray Crystal Structure of Collagenase Binding to Collagen.Amino Acids3Amino Acids

X-ray Crystal Structure of Collagenase Binding to Collagen.4pKa1pKa2H2AA2-

HA-Principle Species

In other words, what chemical species has the highest concentration at the given pH?Solution CompositionpHGeneral Polyprotic Acid5The isoelectric point is the pH at which a polyprotic acid has a net charge of zero.For a simple diprotic amino acid: pI = (pKa1 + pKa2)

For other higher polyprotic acids, the calculation is more complex.

Distribution of surface charge on:(A) AmyL (B) AmyLQS50.5 (pI10.0)(C) AmyLQS55.6 (pI5.0).Surface charge distributions were calculated using the GRASS software; negative charge is shown in red and positive charge is shown in blue. The conservation of negative charge in the active-site cleft explains why the enzymatic activity is conserved in AmyLQS50.5, despite its high overall pI.(A) AmyL(B) AmyLQS50.5(C) AmyLQS55.6Active SiteBiochem. J. (2000) 350 (3139) Isoelectric Point6Isoelectric Point

The near-neutral pI region has presumbably been depleted by selective pressure -- cytoplasmic proteins are least soluble at their isoelectric point.

Proteins may thus be evolutionarily trapped on one peak or the other. Isoelectric Point of Human Proteinshttp://www.mad-cow.org/00/annotation_frames/tools/genbrow/hgwdev.html7Isoelectric Focusing

A protein dissolved in buffer at its pI has no net charge and thus no net electrophoretic mobility.

Isoelectric focusing employs a pH gradient extending the length of an electrophoresis gel. A protein stops migrating when it enters the zone in which the surrounding pH equals its isoelectric point, pI. At any other point in the gradient, the protein acquires a charge (positive or negative) which causes it to migrate toward its pI (green and blue arrows). Negative ElectrodePositive Electrode

MWpHProteins from Human Retinal Pigment Epithelium (RPE) Cells2-D Gel Separation8Proteomics

Figure 1. (A) IE-MS electropherogram for a lysate from E. coli. The positive ESI mass spectra in (B)-(D) are from single scans under their respective peaks. The insets of (B) and (C) show the high-resolution spectra for a single charge state.

Unlike the genome, the proteome is exquisitely sensitive to cellular conditions and will consist of proteins having abundances dependent upon stage in the cell cycle, cell differentiation, response to environmental conditions (nutrients, temperature, stress, etc.), or disease state(s).

LC-MSJensen, P. K.; Pasa-Tolic, L.; Anderson, G. A.; Horner, J. A.; Lipton, M. S.; Bruce, J. E.; Smith, R. D.; Anal. Chem. 1999; 71; 2076-2084. 9In General Chemistry You Should Have Learned:

1) How to calculate the pH of a polyprotic acid solution HnASet up ICE table

2) How to calculate the pH of a diprotic salt solution NamHnApH ~ (pKa1 + pKa2) (will discuss more)

3) How to calculate the concentration of all species of a diprotic acidSet up two or more ICE tables

4) The relationships between Kas and Kbs for polyprotic acidsKa1 x Kb2 = Kw(diprotic acid)Ka2 x Kb1 = Kw

Ka1 x Kb3 = Kw(triprotic acid)Ka2 x Kb2 = KwKa3 x Kb1 = Kw

Polyprotic Calculations10pKa = 9.33pKa = 2.35

Example 1:Calculate the pH of 0.10 M tryptophan hydrochloride.

Example 2:Calculate the composition of all species in a 0.10 M tryptophan hydrochloride solution.

Example 3:Calculate the pH of 0.10 M monosodium tryptophan chloride.

Polyprotic Problems11Answer:pH = 1.68Answer:pH = 5.85Answer:[H2T+] = 0.081 M, [HT] = [H3O+] = 0.019 M, [T-] = 4.7 x 10-10 M

To calculate the pH at any step in the titration of a polyprotic acid:1) Use stoichiometry & amount of base added to compute stage of titration2) Focus on the principle species & its proton transfer equilibrium

Titrations of Polyprotic Acids12

H2CO3 + H2O H3O+ + HCO3-HCO3- + H2O H3O+ + CO32-

BufferRegionsStoichiometric PointsInitialCarbonic Acid

Diprotic Acids13

H3PO4 + H2O H3O+ + H2PO4-H2PO4- + H2O H3O+ + HPO42-HPO42- + H2O H3O+ + PO43-

Phosphoric AcidTriprotic Acids14Example:100 mL of 0.100 M phosphoric acid, H3PO4 was titrated with the following volumes of 0.100 M NaOH. Calculate the pH at each point.pKa1 = 2.12 pKa2 = 7.21 pKa3 = 12.68

(a) 50 mL of NaOH added(b) 100 mL of NaOH added(c) 175 mL of NaOH added(d) 350 mL of NaOH added

H3PO4H2PO4-HPO42-PO43-(a)(b)(c)(d)100 mL NaOH added200 mL NaOH added300 mL NaOH addedAnswers:(a) pH = 2.12(b) pH = 4.67(c) pH = 7.69(d) pH = 12.05

Sample Problem15