Week 1 practical - pH and buffers Flashcards
acidity
measure of H+ concentration
pH definition
pH = -log10[H+]
[H+]
[H+] = 10^-pH
[H+] of pure water
1.0x10^-7
why do we use pH to measure acidity as opposed to H+ concentration?
H+ is small and varies widely
acids
proton donators
bases
proton acceptors
what type of acids are carboxylic acids?
weak
what do pipettes give their maximum volume in?
micro litres
what do we use to set the volume on a pipette?
thumbwheel
how do we know which tip to use on a pipette?
match the tip colour with the colour of the push button
what would we set the pipette to to measure 2 micro litres?
P20
how many microlitres in 1ml?
1000
what is a P20 pipette used to measure?
2-20 microlitres
what is a P200 pipette used to measure?
20-200 microlitres
what is a P1000 pipette used to measure?
between 200-1000 microlitres
what is a P5000 pipette used to measure?
1000-5000 microlitres
on which pipette do we need to twist the tip to tighten?
P5000
extra step when using P5000 pipette
twist tip to tighten
what must we do when not using the glass electrode bulb?
keep it moist
how many microlitres in 1ml?
1000
steps to calibrating a pH meter
-place electrode in the pH 7 standard buffer first
-completely immerse glass electrode bulb for an accurate reading
-agitate probe for faster reading
-reading to within 0.02 units of the pH 7 using “buffer offset”
-rinse then place electrode in the pH 4 standard buffer - adjust reading to within 0.02 units of the pH of the chose buffer using “slope %”
-probe back in pH 7 and shake gently
= meter is calibrated
=reading within 0.05 pH units of 7 (repeat if not)
+ remember to replace the calibrated electrode in the storage solution (bulb dries out = damaged)
steps to taking a measurement with a pH meter
-rinse and blot electrode before using (to avid contamination of measured solution)
-completely immerse glass electrode bulb and shake
-stabilise to 0.02 units before recording
what do pipettes give their maximum volume in?
micro litres
how many pH units should a reading be within once it’s been calibrated?
0.05
on which pipette do we need to twist the tip to tighten?
P5000
what do we do before using the electrode on a pH meter and why?
rinse with distilled water to avoid contamination
what do we use to set the volume on a pipette?
thumbwheel
what must we do when not using the glass electrode bulb?
keep it moist
what type of acids are carboxylic acids?
weak
what would we set the pipette to to measure 2 microlitres?
P20
Ka equation + define symbols
Ka = [A-][H+]/[HA]
HA = weak acid
A- = conjugate base
H+ = proton
what’s the difference between a conjugate acid and conjugate base?
the presence of an additional proton
what is the only thing the Henderson-Hasselbalch equation can be used for?
for conjugate acid-base pairs
pKa equation
pKa = -logKa
can the henderson-hasselbalch equation be used for acids and bases that react together?
no, only for conjugate acid-base pairs
conjugate acid and base pair equation
acid –><– base + H+
Henderson-Hasselbalch equation
pH = pKa + log10 [A-]/[HA]
derivation of Henderson-Hasselbalch equation
Ka = [A-][H+]/[HA]
take logs of both sides
log Ka = log [A-][H+]/[HA]
rearrange
log Ka = log [A-]/[HA] + log [H+]
rearrange
-log[H+] = -logKa + log [A-]/[HA]
definitions of pH and pKa
pH = pKa + log [A-]/[HA]
Uses of Henderson-Hasselbalch equation
- describe how concentrations of conjugate acid and base in a buffer solution change at different solutions
- find pKa of a buffer (mix known amounts of conjugate acid + base + see how concentration changes)
-pH = pKa –> concentrations of conjugate acid + base are equal
-predict how a solution will respond to adding a strong acid or base
how do we know if the concentrations of conjugate acid and base are equal?
pH = pKa
what does it mean if pH = pKa?
the concentrations of conjugate acid and base are equal
what are biochemical reactions sensitive to?
pH
why is the pH of the body tightly regulated?
biochemical reactions are sensitive to pH
acidosis
when the pH of the blood plasma is less than 7.35
when the pH of the blood plasma is less than 7.35
acidosis
symptoms of acidosis
headaches, confusion, breathlessness, coma
what are there to control intracellular pH?
proteins that pump protons out of cells
buffers
major buffer in blood and interstitial fluid
bicarbonate buffer system
where is the bicarbonate buffer system found?
it’s a major buffer in blood and interstitial fluid
bicarbonate buffer system equation
CO2 + H2O –><– H2CO3 –><– HCO3- + H+
why is there CO2 in the bicarbonate buffer system equation?
produced in the tissues
carbonic acid
H2CO3
what happens to the carbonic acid in the bicarbonate buffer system?
it ionises immediately
bicarbonate ions
HCO3-
how do clinicians estimate the concentration of HCO3- in patient’s plasma?
from…
pH
[CO2]
HH equation
use a pKa of 6.1
name a buffer commonly used in biochemistry
phosphate
why is phosphate a buffer that’s commonly used in biochemistry?
3 ionisable groups = 3 pKas and 4 different chemical forms
ionisation
gain or lose electrons
how much of the ionic species is remaining at 2 pH units from the pKa and why?
1/100
pH is a log function
what happens to the phosphate buffer at different pHs?
different concentrations of the different forms of phosphate in the buffer
forms of phosphate in the buffer
H3PO4
H2PO4-
HPO4^2-
PO4^3-
at which pH is phosphate in the H3PO4 form in the buffer?
low pH
at which pH is phosphate in the PO4^3- form in the buffer?
high pH
what do we have when pH = pKa?
equal concentrations of conjugate acid and base
(careful with H2)
what is true when we have equal concentrations of conjugate acid and base?
pH = pKa
(careful with H2)
when is a buffer most resistant?
when [conjugate acid] = [conjugate base]
describe the buffer when [conjugate acid] = [conjugate base]
most resistant
what does a greater difference between the pH and the pKa lead to?
a greater difference between the relative concentrations of acid and conjugate base
when do buffers function best?
within one pH unit of their pKa
why do buffers function best within one pH unit of their pKa?
because pKa is the -log of Ka
when do we have a greater difference between the relative concentrations of acid and conjugate base?
when there’s a greater difference between the pH and the pKa
which buffer should we choose and why?
one with a similar pKa to the pH of the solution you want to use
buffers function best within one pH unit of their pKa
what gives a buffer a greater buffering capacity?
greater concentration of the buffer
buffer concentration often used by biochemists
10-100mmol.L-1
why are buffers used a lot?
near-physiological pKa
lack of toxicity
tend not to interfere with biochemical reactions
examples of buffers used + pKa
MES 6.1
phosphate 6.9
HEPES 7.5
Tris 8.1
What would we set the pipette to to measure 450 micro litres? Explain
P45
On P1000, bottom number is 10 micro litres
