Buffer Systems Flashcards
pH and pKa
- Acid
Base
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+]
What is a buffer and what are its applications
-. 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
General Action of a Buffer
- 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
pH Balance maintained by
- main organs of excretion are lungs (volatile) and kidneys (nonvolatile)
- regulation of [H+]
- body fluids are supplied with buffer systems and act quickly
Henderson-Hasselbalch equation
note the acid and base equation
pH and pKa for buffers
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
Best buffering range
- 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
BUFFER SYSTEMS IN THE HUMAN BODY
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
Buffer systems: Bicarbonate
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
Buffer systems: Phosphate
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
Buffer systems: Protein
- 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
Buffer systems: Hemoglobiin
- 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
The isohydric principle
- 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
Transport of oxygen
Oxygen-hemoglobin dissociation curve
- 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
Shift of dissociation curve
What is a shift to right?
- 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
Shift to left
- 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)
Acid Base Balance
goal:
regulation:
compensation:
- 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
Acid Base Disorders: Respiratory
Respiratory regulation
*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
Respiratory deregulation
- excessive pulmonary ventilation reverses the process
respiratory alkalosis (pH > 7.45)
* rare
* voluntarily overbreathing
* psychoneurosis
* high altitude
* crying baby
Renal compensation
- 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
Correction of respiratory acidosis
- 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
Correction of respiratory alkalosis
- 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
Acid Base Disorders: Metabolic
acidosis and alkalosis
* primary disturbance is [HCO3-]
acidosis = decreased [HCO3-]
(= increased H+)
- alkalosis = increased [HCO3-] (= decreased H+)
Metabolic acidosis
and correction
- 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
Metabolic alkalosis
correction
- 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
pH meter/electrode
internal conductor electrode
inner buffer
internal conductor electrode - Ag-AgCl
inner buffer - KCL
Calibration of pH meter
- 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
Maintaining a pH electrode
For long term storage, keep electrode capped and/or stored in storage solution to prevent it from drying - KCL
Spectrophotometry
- Reflectometry: measure reflected light
- Nephelometry: measure light scatter
- Fluorometry: measure fluorescent light
- Immunoassay: measure chemiluminescent signal using a luminometer
Absorption Spectrophotometry
- 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
Transmittance (T)
- 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
Beer’s Law A= abc
-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
Standard Graph
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
