acid base lecture 15-16 september 2014 agnes rinaldo...

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Acid Base lecture 15-16 September 2014Agnes Rinaldo-Matthis

1. Introduction to pH

2. Acid base concept -calculations

3. Buffert -calculations

4. Titration

5. Physiologic pH regulation -calculations

6. Measure pH?

-Read: Kemiboken från gymnasiet, Harvey & Ferrier, sid 6-9, Kompendiet Syra-Bas

Introduction to pH

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2M HCl -0.30.1 M HCl 1Coca Cola 2.3Schampo 5-6active/resting muscle 6.1/6.9Saliva 6.4-7Blood (venous) 7.32-7.38Blood (arteriell) 7.37-7.43

•The cell has low amounts of H+, around 10-7 M, =>pH=7 same as 0,0001 mM. (compare with [Na+]=140 mM in the cell)

Introduction to pH

pH describes the concentration of H+ or H3O+ in water solution.

The lower the pH, the greater the hydronium ion concentration.

Introduction to pH

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pH = -Log[H+]

It is easier to think in log of concentrations but it takes practice!!

Introduction to pH

Example : If the pH of a solution is 3.4, what is the hydronium ion concentration?

[H3O+] = 10-pH = 10-3.4 = 0.000398 M=3.98x10-4 M

• Neutral water has a tendency to ionizeH2O <-> H+ + OH-

• The free proton is associated with a water molecule to form the hydronium ion

H3O+

• High ionic mobility due to proton jumping

Introduction to pH

Where does the H+ come from?

Dissociation of H2O

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Proton Jumping

• Hydronium ion migration; hops by switching partners at 1012 per second

Introduction to pH Dissociation of H2O





4. Titration


6. Measure pH?

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Relationship between pH and [H+] / [OH-] concentration

2. Acids and bases

• Acidic if [H+] > 10-7 M• Basic if [H+] < 10-7 M

H2O <-> H+ + OH-

• Acid is a substance that can donate a proton.• Base is a substance that can accept a proton.• Conjugate acid base pair

HA + H2O H3O+ + A-/OH-

Acid1 Base2 Conjugate ConjugateAcid2 Base1

or

HA A- + H+

Acid1 Conjugate ConjugateBase2 Acid

2. Acids and bases Lowry definition

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– The stronger acids are those that loose their hydrogen ions more easily than other acids.

– Similarly, the stronger bases are those that hold onto hydrogen ions more strongly than other bases.

2. Acids and bases Relative strength of acids and bases

Strong acid undergoes complete dissociationEx. HCl

HCl + H2O Cl- + H3O+


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Strong acid undergoes complete dissociationEx. HCl


HCl H3O+ Cl-


Weak acid undergoes partial dissocationEx. Acetic acid

HA + H2O A- + H3O+


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HA HA

H+ A-

Weak acid undergoes partial dissocationEx. Acetic acid

HA + H2O A- + H3O+


A solution of strong acids and bases (ex HCl och KOH)

H+ + Cl- + K+ + OH- H2O + K+ + Cl-

If we take same amount of HCl as KOH it becomes neutral = neutralisation,

otherwise it becomes acidic or basic.

Neutralisation2. Acids and bases

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A weak acid is not completely proteolysed Equilibrium to the left.

around 1% of HAc will react.

The relative stability of the acid form as compared to its conjugated base formDetermines if it is a weak or a strong acid.

If the acid form is stable = the proton is not lost easily = weak acid(ex HAc)

If the base form is stable = the proton will easily be lost = strong acid (ex HCl)

HAc + H2O Ac- + H3O+

Proteolysis of a weak acid

Weak acids/bases2. Acids and bases

Determine pH in a solution



Strong acid, equilibrium shifted to the right,completely shifted as for HCl

Weak acid, at equilibrium there is still acid left ex Acetic acid HAc where 1% of HAc will react.

Easy!

Difficult!

How can we calculate pH in a solution of a weak acid?

Weak acids/bases2. Acids and bases

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• Law of mass action (massverkans lag): Therate of a chemical reaction is proportional to theconcentrations of the reacting substances.

and

• experimental values of Ka (acid base constant) is used to determine pH in a solution of a weak acid.

Law of Mass action

Calculate pH in a solution of a weak acid.We need:

2. Acids and bases

at equilibriumsame rates

2 delreaktioner:

Vid jämvikt är v1=v2 dvs Som kan omformuleras till

[Ac-] · [H3O+]k1

[HAc] · [H2O]k2

= där k1/k2 =Ka (syrakonstanten)

Ac- + H3O+HAc + H2O

1) HAc + H2O

HAc + H2O2) Ac- + H3O+

Ac- + H3O+ v1=k1 [HAc] · [H2O]

v2= k2 [Ac-] · [H3O+]

k1[HAc] · [H2O] = k2[Ac-] · [H3O+]

k1

k2

Law of Mass action2. Acids and bases

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2 reactions:

Vid jämvikt är v1=v2 dvs Som kan omformuleras till

[Ac-] · [H3O+]k1

[HAc] · [H2O]k2

= där k1/k2 =Ka (syrakonstanten)

Ac- + H3O+HAc + H2O

1) HAc + H2O


Ac- + H3O+ v1=k1 [HAc] · [H2O]

v2= k2 [Ac-] · [H3O+]

k1[HAc] · [H2O] = k2[Ac-] · [H3O+]

k1

k2



2 reactions:

At equilibrium v1=v2

[Ac-] · [H3O+]k1

[HAc] · [H2O]k2

= där k1/k2 =Ka (Acid constant)

Ac- + H3O+HAc + H2O

1) HAc + H2O


Ac- + H3O+ v1=k1 [HAc] · [H2O]

v2= k2 [Ac-] · [H3O+]

k1[HAc] · [H2O] = k2[Ac-] · [H3O+]

k1

k2


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Law of Mass Action

Ka betecknar syrakonstant och har ett specifickt värde för varje syra, ex. stort värde på Ka, stark syra

K = [H2O] = 55,6 M

Ka =

I utspädd lösning är H2O ungefär konstant ca 55M och kan inneslutas i jämvikts- konstanten och man får:

[Ac-] · [H3O+]

[HAc] · [H2O]

[Ac-] · [H3O+]

[HAc]


Ka is the acid constant and it has a specific value for each acid, ex. large value of Ka, strong acid

K = [H2O] = 55,6 M

Ka =

In water H2O is considered constant and can be included in the equilibrium constant K.

[Ac-] · [H3O+]

[HAc] · [H2O]

[Ac-] · [H3O+]

[HAc]

Law of Mass Action

Acid constant2. Acids and bases

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1. An acid with stable conj. Base has a tendency to loose its proton and its equil. Is shifted to the right and we get a large Ka.

1. A weak acid with a unstable conj. Base has a small tendency to loose its proton and equilibrium is shifted to the left – small Ka.

Ka = [Ac-] · [H3O+]

[HAc]


Acid constant, Ka

and analogous to the pH terminology the pKa = -lgKa

Acid constant, Ka2. Acids and bases

Which are the experimental values of Ka?


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Ka describes acid strengthHA + H2O A- + H3O+

[A-] · [H3O+]

[HA]Ka =

Syra Ka pKa

•HCl 1 · 107 -7•H2SO4 1 · 103 -3•H3PO4 6,3 · 10-3 2,2

•Mjölksyra 1,6 · 10-4 3,8•ß-OH-smörsyra 4,0 · 10-5 4,4•Acetic acid 1,7 · 10-5 4,76

stark

svag

(-lg Ka)


Base constant

Law of Mass action for bases

A strong base has a tendency to take up protons the equilibrium is shifted to the right and Kb is large. A small Kb is a weak base – lower tendency to take up a proton.

and analogous to the pH terminology the pKb = -lgKb

HAc + OH-Ac- + H2O

Kb = [HAc] · [OH-]

[Ac-]

Base constant, Kb2. Acids and bases

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Kb describes base strengthA- + H2O HA + OH-

[HA] · [OH-]

[A-]Kb =

Base Kb pKb

•OH- (lut) 8,7 · 101 -1,74•CO3

2- (soda) 2,5 · 10-4 3,7•NH3 1,74 · 10-5 4,76

•HCO3- (bikarbonat) 1,26 · 10-8 7,9

•acetate 5,75 · 10-10 9,24

strong

weak

(-lg Kb)

Base constant, Kb2. Acids and bases

For conjugated acid-base pair: pKa + pKb = 14

HAc / Ac- : 4,76 + 9,24 = 14

H2O / OH- : 15,74 + (-1,74) = 14

H3O+ / H2O : -1,74 + 15,74 = 14

H2CO3 / HCO3- : 6,1 + 7,9 = 14

Acid/Base constant2. Acids and bases

Kb can be calculated from its conjugated acid Ka value:Kb=Kw/Ka

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Now we know Law of Mass actionAnd some experimental values of Ka

So now we can calculate pH in a solution of a weak acid!

2. Acids and bases

Protolysis of a weak acidEx. 0.1 mol acetic acid (CH3COOH) in 1 liter H2O


2:a grade equation ....Make two approximations:1) X << 0,12) X >> 10-7

[Ac-] · [H3O+]

[HAc]Ka =

X · (X+10-7)

0,1-XKa =

Before equil. 0,1 noll 10-7

After equil. 0,1 - X X X + 10-7

Weak acids 2. Acids and bases

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Weak acids

[Ac-] · [H3O+]

[HAc]Ka =

X · (X+10-7)

0,1-XKa =

2. Acids and bases





Weak acids

[Ac-] · [H3O+]

[HAc]Ka =

X · (X+10-7)

0,1-XKa =

Ka = X · X Ka

HAc=1,7.10-5

0,1

2. Acids and bases

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Weak acids

[Ac-] · [H3O+]

[HAc]Ka =

X · (X+10-7)

0,1-XKa =

Ka = X · X Ka

HAc=1,7.10-5

0,1

1,74 · 10-5 = X2

0,1

2. Acids and bases





Weak acids

[Ac-] · [H3O+]

[HAc]Ka =

X · (X+10-7)

0,1-XKa =

Ka = X · X Ka

HAc=1,7.10-5

0,1

1,74 · 10-5 = X2

0,1

X = 1,32 · 10-3 M = [H3O+]

pH = 2,88

2. Acids and bases

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[H3O+] · [OH-]

[H2O] · [H2O]K =

H2O + H2O H3O+ + OH-

[H3O+] · [OH-]

10-14 M2 Kw= ion product of water

Kw =

Kw =

Waterconcentrationenis constant 55.5 M

K is the dissociation constantK becomes Kw

b) base H2O + H+ H3O+

a) acid H2O H+ + OH-

An amphoteric species is a molecule or ion that can react as an acid as well as a base. For ex water.

Amphoteric species2. Acids and bases

pKw = -lg 1x10-14 = pH + pOH = 14

Neutral water OH- = H3O+

Kw = (H3O+)(OH-) = (10-7)(10-7) = 10-14

Therefore the pH is 7 In pure water

pKw = pH + pOH

Kw = Ion product of water

-log10

Ion product of water2. Acids and bases

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a) HSO4- + H2O SO4

2- + H3O+

b) HSO4- + H2O H2SO4 + OH-

c) HPO42- + H2O PO4

3- + H3O+

d) HPO42- + H2O H2PO4 + OH-

e) H2PO4- + H2O HPO4

2- + H3O+

f) H2PO4- + H2O H3PO4 + OH-

g) HCO3- + H2O CO3

2- + H3O+

h) HCO3- + H2O H2CO3

2- + OH-

Ex. of other amphoteric species

Vätesulfat

Monovätefosfat

Divätefosfat

vätekarbonat

Amphoteric species2. Acids and bases





4. Titration


6. Measure pH?

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Observation

If you add 0.01 ml of 1M HCl to 1000 ml of water pH 7, the pH of the water drops from 7 to 5!!

i.e 100 fold increase in H+ concentration: Log = 2 change.

Problem:Biological properties change with small changes in pH, usually less than 1 pH unit.

How does a system prevent fluctuations in pH?

Buffer solutionsA buffer consists of corresponding pairs of weak acids and weak bases

HA H+ + A-

Weak acid proton conjugated base

A buffer solution is keeping the pH relatively constant when strong acid or base is added

3. Buffer

A buffer keeps the pH constant during dilution with water.

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• The buffer capacity is largest

at the pKa.

3. Buffer

By definition the pK is the pH where [HA] = [A-] : 50% dissociated

3. Buffer

pKa of Tris buffer is 8.08 the buffering range is 7.1 - 9.1

By definition the pK is the pH where [HA] = [A-] : 50% dissociated

Tris buffer

X

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1) Choose a buffer that can buffer at the pH interval.

Check pKa for conj. acid, should be close to desired pH.

Acetat buffer, can buffer at pH=4,76 ± 1,5


2) The higher conc. of ([HAc] + [Ac-]), the better buffering capacity.

3. Buffer Make a buffer

Example of different conjugate acid base pairs.

Fig 1-11pH 7


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How to make a buffer solution

1) Mix a weak acid with its corresponding weak base2) Mix weak acid with a strong base3) Mix weak base with stron acid

4) Or, first mix buffer as solid add water to desired volumeand then adjust the pH by adding acid or base to the pH you want



Exempel: Make 0,2 M acetatbuffert in 1L H2O .

Conjugate acid and base added in similar amounts –best buffering capacity at 50:50

3. Buffer

Take 0,1 mol 0,1 mol (same as 0.1 M in 1 L water)

Make a buffer

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How can we determine the pH in a buffer?Example: HAc + H2O Ac- + H3O+

3. Buffer

0,1 M 0,1 M

Buffer equation

We use the buffer equation:A variant of Law of Mass action

-Can only be used to calculate pH in buffers.

-Only valid if 1/30 < base/acid < 30

pH = pKa+ lg [Ac-]

[HAc]

3. Buffer Buffer equation

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Deduction of the buffer equationHAc + H2O Ac- + H3O+

[Ac-] · [H3O+]

[HAc]Ka =

lg Ka = lg [Ac-] + lg [H3O+] – lg [HAc]

-lg [H3O+] = -lg Ka+ lg [Ac-] – lg [HAc]

pH = pKa+ lg [Ac-]

[HAc]

3. Buffer

Taking the logaritm

Replacement

Law of Mass action

Buffer equation!(Hendelson-Hasselbach)

0,1 M 0,1 M

Buffer equation


Exempel: 0,2 M acetatbuffert (0,1 mol HAc, 0,1 mol Ac-, 1 liter H2O)

pH = pKa+ lg 0,1

0,1

pH = 4,76

Tillför 1 liter vatten

0,1 0,1


0,05 0,05

pH = pKa+ lg 1

pH = pKa+ lg 0,05

0,05

pH = 4,76

pH = pKa+ lg 1

3. Buffer

How does pH change in a buffer upon dilution

Buffer equation

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pH=4,28

HAC/Ac- 50:50

x

3. Buffer

Vid pKa är HAC/Ac- 50:50

AfteradditionHCl

x

ekvivalens-punkt

When the buffer isbuffering, the equilibrium is changed:

Buffer equation

Ac-

x ekvivalens-punkt

When the buffer is buffering, the equilibrium is shifted:

HAc + OH- Ac- + H2O

x

x

EftertillsatsHCl

EftertillsatsOH-

3. Buffer Buffer equation

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4. Titration


6. Measure pH?

Titration is a common laboratory method to determine the unknown concentration of an identified analyte.

Take a weak acid HAc, add or titrate NaOH while monitoring pH (x-axis) amount NaOH added (y-axis).

4. Titration

Buffer capacity: highest at pKa

The buffer effect can be seen in a titration curve.

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pH=4.76

Ac-

HAC/Ac- 50:50

x equivalens-point

pKa-punkt

Best buffertkapacitet Half-equivalenspointpKa ± 1,5

Titration of a weak acid with a strong base.

4. Titration

At the equivalenspointamount base added = amount mole acid at start

At pKa HAc/Ac 50:50

Distribution curves for acetate and acetic acid

pKa = 4.76Best buffer capacityHAc/Ac 50:50

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Ex. Titration curve for the amino acid alanine

2 groups on alanine that can be deprotonated, 2 pKa´s

-COOH / -COO- pKa1-NH3

+ / -NH2

pKa2

4. Titration

Often characteristic for an acid -good analytical method.

Ex. 20 ml HCl is titrated with 16 ml of 0.1 M NaOH to the equivalenspoint. What is the concentration of HCl?

An acid with one proteolytic step is titrated against a strong base. At the equivalenspoint , the n(acid) in the solution is the same as the amount (n) of base added.How much base do we have? We use n=cv => 0.1M x 0.016 L = 1.6 x 10-3 mol = 0.0016 molAt the equivalenspoint the n(acid) = n(base) => Which means that n(acid)=0.0016 mol. What is the concentration of acid? c = n/v = 0.0016 mol / 0.02 L = 0.08 M.

Answer: The concentration of theHCl solution is 80 mM.

4. Titration

equivalenspoint

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4. Titration


6. Measure pH?

6 7 8[H3O+]:

[OH-]:

10-6M 10-7M 10-8M

10-8M 10-7M 10-6M

Life

normal,arteriellblood

4. Physiologic pH regulation

Normal value blood pH 7.4Blood (venous) 7.32-7.38Blood (arteriell) 7.37-7.43

How can we keep the pH so constant?

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Our body uses 3 different systems to keep the pH constant:

1) Buffers in body fluids (the first minutes)Phosphatbuffer (in the cell)Proteins (albumin,Hemoglobin) in the cellBicarbonate buffer (blood, plasma)

2) Regulate breathing (adjust pCO2)3) Long term regulation via kidneys

5. Physiologic pH regulation Buffers in body fluids


1) Buffers in body fluids (the first minutes)Phosphatbuffer (in the cell)Proteins (albumin,Hemoglobin) in the cellBicarbonate buffer (blood, plasma)

2) Regulate breathing(justera pCO2)3) Long term regulation via kidneys


Bicarbonate most significant buffer in bloodFormed from gaseous CO2

Buffers in body fluids

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Buffers in body fluids

H2PO4- + H2O HPO4

2- + H3O+

korr. syra korr. bas

Phosphatebuffert

CO2 + 2H2O H2CO3 + H2O HCO3- + H3O+


Bicarbonatebuffer

protH + H2O prot- + H3O+


Proteins, (ex. albumin, Hb) buffrande förmåga -Histidin

Buffering at pH= 7


H3PO4 + H2O H2PO4- + H3O+


!Two of the ions are amphoteric species!

Can loose protons 3 times= 3 proteolytic steps

H2PO4- + H2O HPO4

2- + H3O+


HPO42- + H2O PO4

3- + H3O+


5. Physiologic pH regulation Phosphate buffer

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Phosphate buffer

H3PO4 + H2O H2PO4- + H3O+

At pKa best buffer capacity

H2PO4- + H2O HPO4

2- + H3O+

HPO42- + H2O PO4

3- + H3O+


What kind of phosphate ions do we have in the cell?


Many phosphate groups on molecules in the, t ex ATP, ADP, AMP, P-creatine.

Phosphate buffer

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When a protein acts as a buffer, only a few amino acids areresponsible

Ex of proteins that can act as a buffer: Albumin och Hemoglobin

5. Physiologic pH regulation Proteins as a buffer

Amino acids that can buffer:



+

-

When the amino acid is buffering, the charge will change!


Histidin

Cystein

Proteins as a buffer

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When Histidin-side chain buffer, it will take up a proton.

It becomes positively charged.

The new + gives a new electrostatic interaction with a neighbouring -

Structure will change.

Maybe even the function of the protein?

The protein structure canchange due to pH

+

H3O+


pH sensitive protein is protected by otherproteins, and other buffers

ProteinXexposed

ProteinX

ProteinX

H3O+

ProteinX

ProteinX

H3O+

ProteinProtein

ProteinProtein

Protein

ProteinProtein

ProteinProtein

ProteinProtein

Protein

H2PO4-/HPO4

2-

ProteinX

ProteinX

H3O+

ProteinProtein

ProteinProtein

Protein

ProteinProtein

ProteinProtein

ProteinProtein

Protein

H2PO4-/HPO4

2-

H2PO4-/HPO4

2-

H2PO4-/HPO4

2-

H2PO4-/HPO4

2-

H2PO4-/HPO4

2-

H2PO4-/HPO4

2-

H2PO4-/HPO4

2-ProteinXprotected


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Good with overlapping buffersOne buffer Mix of buffers

Buf

fert

kapa

cite

t


Bicarbonate buffer

H2CO3 + H2O HCO3- + H3O+

Conjugate acid. Conjugate base

First proteolytic step important.

2 proteolytic steps

HCO3- + H2O CO3

2- + H3O+

Conjugate acid Conjugate base


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H2CO3 + H2O HCO3- + H3O+

Conj. Acid conj.. base

Dihydrogen carbonate (kolsyra) (H2CO3) can exist in waterbut will quickly become CO2 bubbles.

The acid in bicarbonate buffer consists of H2CO3 and CO2(d) together.

CO2(d) + 2H2O H2CO3 + H2O HCO3- + H3O+

conj. acid conj. base

In body fluids

5. Physiologic pH regulation Bicarbonate buffer

The concentration of dissolved CO2, is proportional to the partial pressure for CO2 (pCO2).

Water with dissolved , dihydrogen carbonateand CO2

H2CO3

H2CO3

H2CO3

H2CO3

CO2

CO2

CO2

CO2CO2CO2 is dissolved in water

[CO2] + [H2CO3] = 0,23 · pCO2kPa, mM


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Buffer formula for bicarbonate buffer

CO2 + 2H2O H2CO3 + H2O HCO3- + H3O+

conjugate acid Conjugate base

pH = pKa+ lg [base]

[acid]

pH = pKa+ lg [HCO3

-]

[CO2] + [H2CO3]

[CO2] + [H2CO3] = 0,23 ·pCO2

pH = pKa+ lg [HCO3

-]

0,23 · pCO2

Henderson Hasselbalch´s formula



1) Buffers (the first minutes)Phosphatbuffert (in the cell)Proteiner (albumin,Hemoglobin) in the cellCarbonate buffert (blood, plasma)

2) Regulate breathing Acids are formed during metabolism. In tissues CO2 forms - transported to lungs as HCO3

-. CO2 can be released in lungs. If problems with lungs: can get acidosis.

1) Long term regulation via kidneys

5. Physiologic pH regulation Regulate pH by breathing

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Gas transport

O2 is transported from the lung to the tissues and CO2 formed in tissues is transported back to the lung. The transport of these gases is coupled to pH:

.

Gases affecting pH

O2

O2

O2

O2Hemoglobin transport O2.It has 4 Heme groups


pH, CO2, O2 and BPG (2,3-bisfosfoglycerat) attenuates Hb´s affinity for O2

Relaxed form Tense formBinds O2 releases O2

+[H+]

+[CO2]

+BPG

Low O2 affinityHigh O2 affinity

Low pH

Hemoglobin5. Physiologic pH regulation

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Bohr effekt:An increase of CO2 and a decrease in pH results in

reduced affinity of Hb for O2.

Tissue= Low pH, High CO2

O2 affinity decrease andis released to tissuesShift right

Lungs: High pH, Low CO2 and O2

affinity increases, Shift left



1) Buffers (the first minutes)Phosphatbuffert (in the cell)Proteiner (albumin,Hemoglobin) in the cellCarbonate buffert (blood, plasma)

2) Regulate breathing Acids are formed during metabolism. In tissues CO2 forms - transported to lungs as HCO3-. CO2 can be released in lungs. Problems with lungs: can get acidosis.

1) Long term regulation via kidneys


During normal metabolism of food – we produce acids - excreted via urine. The kidneys excrete/retain H+ and HCO3

-. If you are acidotic, your kidneyswill try to excrete H+ and retain HCO3

-.

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4. Titration


6. Measure pH?

pH paper: acid base form have different light absorbance

pH meter:

Measures the voltage produced by the solution, compares it with the voltage of a known standard solution, and uses the difference in voltage to calculate the pH.

In vivo: measure in plasma or urine. Difficult to measure inside the cell, microelektrodes.

6. Measuring pH

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Approximate pH values of various body fluidsBoron & Boulpaep

Compartment pH

•Gastric secretions 0.7

•Lysosome 5.5

•Cytosol, typical cell more acidic 7.2

•Arterial blood plasma 7.35-7.45 7.4

•Mitochondrial inner matrix 7.5

•Secreted pancreas fluid 8.1

6. Measuring pH

END of lecture

acid base lecture 15-16 september 2014 agnes rinaldo...

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