Buffer Systems Flashcards

1
Q

pH and pKa

  • Acid

Base

A

pH is a logarithmic measure of the hydrogen ion concentration of an aqueous solution it is affected by temperature

pKa
* dissociation constant of a buffering solution in equilibrium.
* negative log of the dissociation constant.
* pH= pKa
* [salt] = [acid]

  • Acid-when dissolved in water, an acid donates a hydrogen ion (H+)
  • Base: accepts a hydrogen ion (H+)
  • pH = – log [H+]
  • decrease in 1 pH unit represents a 10-fold INCREASE in [H+]
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2
Q

What is a buffer and what are its applications

A

-. When the pH doesnt change upon adding small amount of acid or base
* Weak acid and it’s corresponding salt = acidic buffer
OR
* Weak base and it’s corresponding salt= basic buffer

Applications of a buffer
* Maintain a constant pH in a reaction in the lab (e.g., clinical
tests such as enzyme tests)
* Maintaining the pH in microbiological media, tissue cultures
* Maintain pH of blood in the human body
* pH range 7.35-7.45

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3
Q

General Action of a Buffer

A
  • free ions that act to change the pH when and H+ or OH- is added
  • In a buffered solution:
  • Buffer component and the free ions combine to form molecule
    that stays undissociated in solution
  • Removes the excess free ions and results in only a slight change in pH
  • Buffer generally effective within ± 1 of the pKa
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4
Q

pH Balance maintained by

A
  • main organs of excretion are lungs (volatile) and kidneys (nonvolatile)
  • regulation of [H+]
  • body fluids are supplied with buffer systems and act quickly
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5
Q

Henderson-Hasselbalch equation

A

note the acid and base equation

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6
Q

pH and pKa for buffers

A

Direct relationship, pH of a buffer and its pKa
Max buffer capacity when [salt] and [acid] are equal concentrations
-pKa is a constant value, the pH will vary of a buffer because of [salt] and
[acid]

When [salt] > [acid], pH > pKa
When [salt] = [acid], pH = pKa
When [salt] < [acid], pH < pKa

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7
Q

Best buffering range

A
  • A buffer can function well when salt : acid ratio is 1:10 or salt : acid ratio is10:1

Look at slide for equation

  • Therefore, pH = pKa ± 1.0
  • If acetic acid/sodium acetate buffer, pKa = 4.8
  • Best buffering range, pH = 4.8 ± 1.0 = 3.8-5.8
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8
Q

BUFFER SYSTEMS IN THE HUMAN BODY

A

Primary EC buffer system
Phosphate, protein, and bicarb

Primary IC buffer systems
phosphate, protein , and hemoglobin

“open” buffer systems: phosphate, bicarbonate

“closed” buffer systems: protein, hemoglobin

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9
Q

Buffer systems: Bicarbonate

A

HCO3-/H2CO3-most important buffer pair in plasma

pKa of 6.1 cannot buffer at pH 7.4
* Chemically speaking bicarbonate is not a buffer in action

HCO3- - regulated by kidneys
PCO2 - regulated by lungs

open buffer system

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10
Q

Buffer systems: Phosphate

A

important intracellularly as organic phosphate (2,3-DPG in red cells)
* excretion of acids in the urine
* action similar to bicarbonate buffer system
* Open buffer system

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11
Q

Buffer systems: Protein

A
  • Most plentiful non-bicarbonate buffer of the body
    -most powerful
  • presence of both free acidic and basic radicals
  • can accept H or donate H as metabolism requires
  • Each albumin molecule contains 16 histidines
  • imidazole groups of histidines (pK 7.3)
  • H+ ion sequestered
  • Closed buffer system
  • protein buffers react much more slowly
  • concentration in mmol is lower than bicarbonate
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12
Q

Buffer systems: Hemoglobiin

A
  • primary intracellular buffer
    *2 buffer pairs: De/oxygenated
  • buffering of H+ and CO2 depends on Hb concentration of blood
  • CO2 is an acid
  • Closed buffer system

DO WE NEED TO KNOW THE SLIDE

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13
Q

The isohydric principle

A
  • the buffer systems all work together
  • H+ is common to all the systems
  • if [H+] changes, the balance of all
    systems change at the same time (the isohydric principle)
  • in other words, the buffer systems actually buffer each other
  • all four buffer systems act in concert
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14
Q

Transport of oxygen

Oxygen-hemoglobin dissociation curve

A
  • oxygen combines loosely and reversibly with Hb
  • basis for oxygen transport from lungs to tissues
  • increased PO2 causes oxygen to bind to Hb (lungs)
  • decreased PO2 causes oxygen to be released from Hb (tissues)
  • PO2 = partial pressure of oxygen
  • relationship seen on oxygen-hemoglobin dissociation curve

-the log going up is the reduced blood returning from tissues
and the plateau is the oxygenated blood leaving the lungs
*P50 affinity of Hb for oxygen
- partial pressure of oxygen (PO2) at which Hb is 50% saturated

  • P50 is increased when its more
    difficult for Hb to bind O2
  • curve shifts to the right
  • P50 is decreased when its easier for Hb to bind O2
  • curve shifts to the left
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15
Q

Shift of dissociation curve
What is a shift to right?

A
  • increased [H+] and decreased pH leads to decreased affinity of Hb for O2
  • increased PCO2 leads to decreased affinity of Hb for O2
  • increased temperature leads to decreased affinity

therefore
* increased 2,3-diphosphoglycerate (2,3-DPG) leads to decreased affinity
* promotes oxygen release to tissues in hypoxia

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16
Q

Shift to left

A
  • Hb F
  • greater saturation at a given PO2
  • oxygen delivery to fetal tissues
  • Carbon monoxide (CO)
  • binding of CO, increases the affinity of the remaining three binding sites (heme units) for O2 so
    much so that it is reluctant to give it up
  • tissues become oxygen-starved (anoxic/tissue anoxia)
17
Q

Acid Base Balance

goal:
regulation:
compensation:

A
  • goal is to maintain blood pH 7.35 - 7.45
  • controlled by pH regulation and pH compensation
  • regulation: (bicarbonate, hemoglobin, protein and phosphate buffer systems working with the respiratory and renal systems
  • compensation: intervention of the respiratory and renal systems to restore normalcy
  • variations in the acid-base ratio of 20:1 will result in
  • acidosis (pH below 7.35) too much acid (or too little base), or
  • alkalosis (pH above 7.45) too much base (or too little acid)
  • acid base imbalance is not a disease itself, but an indicator of disease

Acid base imbalances are:
* respiratory or metabolic in origin
* respiratory changes are change in PCO2
* metabolic changes are change in HCO3-

Compensation of acid base balances are
* respiratory compensation for a metabolic disorder
* metabolic compensation for a respiratory disorder

18
Q

Acid Base Disorders: Respiratory

Respiratory regulation

A

*Respiratory
*acidosis and alkalosis
*primary disturbance is [PCO2]
*acidosis = increased CO2 retention
*alkalosis = decreased CO2 retention

Respiratory regulation
* decrease rate of pulmonary ventilation causes decrease rate of CO2 expiration, increased CO2 in ECF, and decreased pH
therefore respiratory system controls [H+]

  • conversely, [H+] can control rate of pulmonary ventilation
19
Q

Respiratory deregulation

A
  • excessive pulmonary ventilation reverses the process

respiratory alkalosis (pH > 7.45)
* rare
* voluntarily overbreathing
* psychoneurosis
* high altitude
* crying baby

20
Q

Renal compensation

A
  • slow
  • normal pH can be restored in 1 to 3 days
  • however, it continues until pH is almost exactly normal
    *real value is its ability to neutralize completely any excess acid or alkali
21
Q

Correction of respiratory acidosis

A
  • Renal compensation
  • How do the kidneys readjust pH of extracellular fluid (ECF) when it becomes acidotic?
  • H+ excretion into the urine increases
  • HCO3- is reabsorbed with Na+
  • Henderson-Hasselbalch equation and the isohydric principle
  • all buffers are shifted in the alkaline direction
22
Q

Correction of respiratory alkalosis

A
  • How do the kidneys readjust pH of ECF when it becomes alkalotic?
  • HCO3 - excretion into the urine increases with Na+
  • H+ is retained
  • Henderson-Hasselbalch equation and the isohydric principle
  • all buffers are shifted in the acidic direction
23
Q

Acid Base Disorders: Metabolic

A

acidosis and alkalosis
* primary disturbance is [HCO3-]
acidosis = decreased [HCO3-]
(= increased H+)

  • alkalosis = increased [HCO3-] (= decreased H+)
24
Q

Metabolic acidosis

and correction

A
  • detected by measuring decreased plasma HCO3-
  • failure of kidneys to excrete metabolic acids
  • increased formation (intake) of acid
  • increased loss of base
  • diarrhea (increased loss of bicarbonate from GI tract)
  • vomiting of deep GI contents
  • methanol, salicylate poisoning
  • uremia of renal failure
  • diabetes mellitus

Respiratory compensation
* increased rate and depth of respiration to eliminate CO2
(hyperventilation)

Renal compensation (if possible)
* increased Na+-H+ exchange
* increased ammonia formation
* increased reabsorption of HCO3

25
Q

Metabolic alkalosis

correction

A
  • not common
  • increased ingestion of alkaline drugs, eg., antacids
  • vomiting of gastric contents
  • hyperaldosteronism (renal “wasting” of K+ and H+ in exchange for Na+)
  • licorice
  • bicarbonate-containing i.v. fluid therapy

Respiratory compensation
* increase pH depresses the respiratory center, causing retention of CO2

Renal compensation (if possible)
* decreased Na+-H+ exchange
* decreased ammonia formation
* decreased reabsorption of HCO3

lungs respond quickly, renal compensation occurs over
several days

26
Q

pH meter/electrode

internal conductor electrode

inner buffer

A

internal conductor electrode - Ag-AgCl

inner buffer - KCL

27
Q

Calibration of pH meter

A
  • Colour coded buffers, Buffer 4.0, 7.0 and 10.0
  • 2-3 point calibrations
  • The balance the system with the electrodes in a buffer with 7.0 pH
  • The balance or intercept control shifts the entire slope
  • Rinse with deionized water between each sample reading
  • If meter does not register the correct pH, amplification of the response changes the slope to
    match the predicted pH value
28
Q

Maintaining a pH electrode

A

For long term storage, keep electrode capped and/or stored in storage solution to prevent it from drying - KCL

29
Q

Spectrophotometry

A
  • Reflectometry: measure reflected light
  • Nephelometry: measure light scatter
  • Fluorometry: measure fluorescent light
  • Immunoassay: measure chemiluminescent signal using a luminometer
30
Q

Absorption Spectrophotometry

A
  • Measurement of intensity of light of a particular wavelength transmitted by a solution
  • Measures the amount of radiant energy transmitted when monochromatic light is
    directed through a solution
  • Amount of light transmitted through solution depends on its concentration
  • Light is the signal (measured by analyzer)
  • Signal is directly or inversely proportional to concentration of an analyte
31
Q

Transmittance (T)

A
  • ability of a substance to permit light to travel through
  • light that is not transmitted is absorbed by substance’s molecules
  • T = ratio of radiant energy transmitted thru a medium (I) divided by the radiant energy shone or incident to the medium (Io)

T = I/1o

100% T – all light transmitted (none absorbed)
-set at 100% by using a reagent blank so all reagents used in an assay but not the specimen

  • as absorbance (A) increases, %T decreases
32
Q

Beer’s Law A= abc

A

-Relationship between absorbance & concentration is directly proportional at a given wavelength

A = absorbance
a = molar absorptivity
* fraction of a specific wavelength absorbed by a given molecule
(a constant)
* varies with molecule’s structure, wavelength used, pH, temp.
b = length (cm) of light path through the solution
c = concentration of absorbing molecules [M]

  • absorbance (A), is directly proportional to concentration (c) and path length (b)
  • application of Beer’s Law involves
  • standardization (or calibration) with “knowns”
  • measurement of “unknowns”
  • conversion of signal to concentration
  • can be done graphically or mathematically
33
Q

Standard Graph

A

Absorbance on Y and concentration in mol/l on X

if the graph is linear use the formula
Cu = Au x Cs/As
Cu = [unknown]
Cs = [standard]
Au = A of unknown
As = A of standard