preparation of biological buffers

Question 1

a buffer for biological purposes would ideally possess the following beneficial characteristics

Answer 1

impermeability to biological membranes
biological stability and lack of interference with biological processes
lack of significant absorption of ultraviolet or visible light
lack of formation of insoluble complexes with cations
minimal effect of ionic composition or salt concentration
limited PH change in response to temperature

Question 2

citric acid and phosphate buffers

Answer 2

can readily form insoluble complexes with divalent cations , while phosphate can also potentially act as a substrate , activator or inhibitor of several enzymes. TRIS buffer is often toxic to biological systems , by penetrating cell membranes and subsequently uncoupling electron transport reactions in whole cells and isolated organelles. In addition it is markedly affected by temperature , with a 10 fold increase in H+ concentration from 4 degrees to 37 degrees

Question 3

two factors to consider when getting the required PH

Answer 3

ratio of acid and conjugate base required to give the correct PH
the amount of buffering required : buffer capacity depends upon the absolute quantities of acid and base , as well as their relative proportions .

Question 4

using a micropipette

Answer 4

transfer distilled water into a 50ml beaker
using the P1000 micropipette, set the volume at the maximum 1000 ul , by holding the micropipette in one hand and turning the volume adjustment knob until the correct volume shows on the indicator.
attach a new disposable blue tip to the end of the pipette. press on firmly with a slight twisting motion to ensure a good seal.
push the plunger down with your thumb until you feel initial resistance . this is the first positive stop . this part of the stroke is the calibrated volume displayed on the indicator .
immerse the pipette tip into the beaker of distilled water to a depth of about no more than half way up the length of the tip , but enough to be able to take up the water without capturing any air bubbles
slowly release the push button in a controlled manner , allowing the water to gently rise up the pipette tip. check the tip to make sure that you have not captured any air bubbles. if this is the case ,carry on but you will need to repeat the exercise.
to dispense the water , place the end of the pipette tip against the inside wall of the beaker of distilled water and depress the plunger to the second stop, expelling any residual water in the tip. gently touch the pipette tip against the inside wall of the test tube to remove the last drop from the micropipette
withdraw the micropipette from the test tube and let the plunger return to the up position. do not repeatedly push the plunger down to get any last amount out of the pipette tip.

Question 5

measuring micropipette accuracy

Answer 5

to asses accuracy of your P1000 micropipette , you will require an analytical balance , a plastic weighing boat and the small beaker of distilled water from the previous activity
with the P1000 micropipette set to 1000 ul , Draw up distilled water into the tip and dispense into the pre-weighed plastic boat . record the new weigh in the table

Question 6

finding a volume or concentration

Answer 6

C1V1=C2V2

Question 7

definition of accuracy

Answer 7

the closeness of a measured value to a standard or known value.

Question 8

definition of precision

Answer 8

how close measurements of the same item are to each other