2069

Measuring pH/ Measuring the pH of a Sample

Exercise 1: Determination of pH of a Various Solution

Stir coco cola sample

well.

Rinse electrode with

distilled water.

Read samples and rinse

the electrode, replace
on stand and the base
of the electrode is
immersed in distilled
water/buffer solution.

Measure 7 ml of the
sample using measuring
cylinder.

Record reading when

stabilized for 5-7
seconds.

Repeat for 0.25 M NaOH

(save for exercise 2) and
10 % Acetic Acid.

Remove electrode from

storage solution and
rinse with distilled
water.

Pour solution into large

test tube and immerse
the electrode into
sample and swirl.

Shake gently to remove

excess water.

Remove electrode from

storage solution and
rinse with distilled
water. Shake gently to
remove excess water.

Practical Skills in Biochemistry

Dr. Adrian Lennon

2069
Exercise 2: Adjusting the pH of a solution.

Place the
electrode into
0.25M NaOH.

Use Pasteur
pipette
dropwise until
pH nears 6.87.2.

Adjust volume
to 10 mL with
distilled water.

When pH is 6.87.2, adjust pH

using 0.25 M
HCL to 6.

Use 0.1 M HCl

to adjust pH to
5.

Practical Skills in Biochemistry

Dr. Adrian Lennon

2069
Exercise 3: Using the micropipette
a) Small Volume Micropipettor

Label three 1.5 mL

tubes with A, B and
C.

Sharply tap the tube

bottom on the
bench top. Make
drops pool into one
drop at the bottom
of the tube.

Set the pipette to 10

L and withdraw
solution from each
tube.

Set micropipettor to
4 L and add
solution 1 to A, B
and C.

Pool and mix

reagents.

Rotate to displace
air.

Use fresh tip to add

volume of solution 2
L in clean spot on
Tubes A, B and C.

Close tops.

Use fresh tip to add

2 L of solution 3 to
Tubes A and C.

Use fresh tip to add

2 l of solution 4 to
Tubes B and C.

Practical Skills in Biochemistry

Dr. Adrian Lennon

2069

b) Large Volume Micropipettor

Label two 1.5 ml

reaction tubes D
and E.

Set the
micropipettor
1000L and
withdraw solution
from each tube.

Set micropipettor
and add volumes
of solutions 1-4
to D and E.

Use fresh tip to

add 2 l of
solution 4 to
Tubes D and E.

Use fresh tip to

add volume of
solution 2 l in
clean spot.

Use fresh tip to

add 2 l of
solution 3 to
Tubes D and E.

Practical Skills in Biochemistry

Dr. Adrian Lennon

Rotate to
displace air.

2069

Spectrophotometry & Calibration Curves

Measuring Samples

Remove lid, place

cuvette in holder.
Cover and press
blank.

Remove sample,
pour sample into
test tube, rinse with
water and place new
sample into cuvette.

Leave
spectrophotometer
for 5 minutes.

Open lid.

Place the cuvetter

into the holder,
cover and record the
reading.

Set desired
absorbance.

Pour sample into

clean cuvette, half
filled. Sample is at
room temperature
and no bubbles.

Rinse between each

sample.

Use cuvette with

clear sides.

Practical Skills in Biochemistry

Dr. Adrian Lennon

2069

Exercise 4: Protein Degradation by the Lowry Method

Preparation of the
Protein Calibration
curve

Read absorbance
against the blank at
750 nm.

Preparation of the
Protein Calibration
Curve.

Prepare a calibration
curve by pipetting 0,
0.1, 0.2, 0.3, 0.5, 0.8
and 1.0 mL.

Add 0.5 Ml of diluted

Folin-Cocicalteaus
solution, mix well and
leave the tubes
standing for 30
mintues at room
temperature.

Pipette 0.1. 0.2 and

0.5 Mll into clean test
tubes.

Make volumes up to
1.0 mL.

Add 5 mL of reagent
C and leave standing
for 10 minutes.

Bring the volumes to

1.0 ml with distilled
water and proceed
with Lowry Assay.

Practical Skills in Biochemistry

Dr. Adrian Lennon