Flashcards for the First Midterm
What is the purpose of the phosphate buffering system and the bicarbonate buffering system?
- phosphate: in cytoplasm and helps cells maintain neutral pH
-bicarbonate: maintains equilibrium between H2CO3 in plasma and CO2 in the lungs.
(Lab 2)
What is the formula for pH when given [H+]?
pH=-log(10)[H+]
-remember this is only for acids so for bases have to subtract the pH from 14 or divide the concentration by 10^-14M^2
-pH does not change based on the amount of added H+/OH-
(Lab 2)
What is the Ka value and how do you determine the Ka and the pKa?
The Ka is a dissociation constant for weak acids (does not dissociate fully)
- ratio of activities of products to reactants
Ka=[H+][A-]/[HA] (at equilibrium)
pka=-log(Ka)
(Lab 2)
What are the properties of Buffers?
- pH function of ratio of salt [A-] to acid [HA]
- weak acids and its conjugate base
use up excess H+ and/or OH- ions - [A-]=[HA] when the pH of solution reaches the pKa
- means buffer resists changes in pH - buffering region +- 1 pH of the pKa
-buffering region depends on the dissociation of the buffer
(Lab 2)
For a titration curve, what is a zwitterionic form?
Have + and - charges
Lab 2
For a titration curve, what is a pI?
the pH when a molecule has a net charge of 0 (the isoelectric point)
(Lab 2)
For a titration curve, what are equivalents?
equivalents are molar equivalents of base and acid titrates that change the pH
(Lab 2)
How can temperature affect the pKa? (give formula)
- pKa is related to Gibbs Free Energy so temperature dependent
delta G=2.303RTpKa and delta G=deltaH-(Tdelta S)
-stronger the acid the lower its pKa
(Lab 2)
What is a buffer’s dilution factor?
the change in pH with equal volume of pure water
- + delta pH(1/2)= pH increases with dilution
- - delta pH(1/2)= pH decreases with dilution
- buffers can resist pH with dilution so have values below 0.3
(Lab 2)
What are the differences between making a buffer using the Henderson-Hasselbalch equation compared to the making of buffers using a conjugate acid or a conjugate base?
Henderson-Hasselbalch:
- need stock solutions of conjugate acid and conjugate base
-rarely gives exact pH (deviation due to errors)
Conjugate acid and conjugate base:
-only need 1 form of salt (either conjugate acid OR conjugate base), pH adjusted using strong acid or base
-add all components in 80-90% of final water then the remaining after
-uses dry reagent
(Lab 2)
What is the intrinsic difference between strong acids vs. weak acids?
- strong acids dissociate completely
- weak acids dissociate to limited extent
(Lab 2)
What is the definition of pKa?
the pH where the ionizable groups exist equally in ionized and non-ionized forms
(Lab 2)
Based on the Henderson-Hasselbalch equation, why can buffers resist changes in pH?
-buffers do dissociate in water but because the ratio of A- to HA is negligible, then it can resist the pH better than strong acids and bases. An addition of a base (OH-) would react with the acid component (HA) (this is the opposite for a added acid).
pH=pKa + log[product]/[reactant]
(Lab 2)
What is the purpose of a pH meter and how does it work?
- measures voltages between 2 electrodes when in solution
-the borosilicate glass is permeable to hydrogen ions but not other cations or anions (measures the the electric potential across the glass)
-use calibration reference solution
(Lab 2)
What does B-galactosidase hydrolyze and what makes a yellow colour during the effect of pH on enzyme assay? What is the wavelength of the spectrophotometer?
B-galactosidase hydrolyzes lactose into glucose and galactose. The yellow product is o-nitrophenol (ONP) and it is formed when ortho-nitrophenyl B-D-galactopyranoside (ONPG) is cleaved by B-galactosidase (from E. coli). It is read at 420 nm.
(Lab 2)
What is the molecular weight of the functional B-galactosidase and how many subunits and the pI? What is the optimal pH?
The molecular weight is 465,396 Da with four equal subunits. The pI is 4.61 and the optimum pH is from 6-8.
(Lab 2)
How are hydrogen ions kept in solution?
They are hydrated to hydronium ions (H3O+)
Lab 2
What are titration curves used for?
Show the pKa of weak acids
-can be the pH (Y) against the amount of NaOH added (X)
(Lab 2)
How can buffers work in biological systems?
the molecules on which they act have ionizable groups
Lab 2
In the B-galactosidase assay, what is used as the blank?
The blank used was everything in each of the test tubes, you blanked the test tube against itself to determine the activity of B-galactosidase. It contained: -B-gal assay buffer -B-galactosidase assay tube -ONPG (Lab 2)
What makes a great buffer?
-good buffers has a pKa similar to the maintained pH of the experiment
- non-specific buffer effects on the enzyme
-interactions with substrates and metals
(Lab 2)
What pH were the buffers made up to? What are the pKas of phosphate and TRIS buffers?
The buffers were made up to a pH of 7.00.
Phosphate has 3 ionizable groups: 2.15, 6.82 and 12.33 *only use the second pKa
TRIS buffer pKa: is 8.30
