Name: Gustavo Gick Dickel

Number: 300 985 690

Experiment #1: Gel permeation Chromatography (G. P. C.)

Objectives:
1) To separate hemoglobin and myoglobin based on their size.
2) To evaluate the amount of hemoglobin and myoglobin, that were separated, in the sample based on
volume x absorbance.
3) To compare the result with another group to verify the amount of hemoglobin diluted can be different
in both samples.

List of Chemicals:
- Sodium Chloride solution 10 g/L
- Solution of Hemoglobin/Myoglobin
- Methylene blue
List of Equipments:
- Chromatograph columns
- Beakers
- Spectronic 200D
- Sephadex G-25
- Pasteur Pipettle
- Graduated Cylinder
- 15 test tubes
- Curvettes
- Glass rod
Safety Procedure:
- Wear lab coat, gloves and goggle inside the lab.
- Do not eat inside the lab.
- If any glassware broke, I must place it in the appropriate container.
- Place the corvettes properly in the machine to avoid cause some damage there.
- Dispose the gel and products after the use in the right beaker or the right container.
- Be careful to do not spill methylene blue in the lab coat.
Proce- dure:

Swell the Sephadex in Place 20 ml of Pour carefully Sephadex

sodium chloride solu- Sephadex into a clean in NaCl using a glass
tion for 3 hours. beaker rod

Let it settle for 5 min- Repeat this procedure

Wash the column few
utes before apply the until reach 12 - 13 cm of
times
sample height
Drain the level of the Use Pasteur pipette to Place 10 ml graduated
liquid to the top add 3 drops of dark cylinder below the
Hemoglobin solution column

Keep the sodium chlo-

Add Sodium Chloride Open the stopper and
ride in the lever near
in the top of the col- allow the hemoglobin
to the top to increase
umn using a dropper flow into the gel
the speed

Record the amount of

Drain slowly the NaCl passed in the
Label 15 test tubes
sodium chloride into column when the first
with numbers
the graduated cylinder colour reach the bot-
ton

Collect the red solu-

Write down this value
tion as they leave the Discard this solution
as a dead volume
column in each 1 ml

After collect the last

Collect the red solu- Colect all clear liquid
red solution, place 10
tion as they leave the between red and blue
ml graduated cylinder
column in each 1 ml solution
below the column
 Collect the blue solu-
Dilute both samples if tion in 3 ml incre- Measure total volume
it is necessary ments placing in dif- and discard it
ferent test tubes

Set Spectronic 200D Adjust the display yo Place a colourless

to Transmittance mode read 0% sample in the machine

Measure the ab-

Change the display to Adjust the transmit-
sorbance of RED solu-
Absorbance tance to 100%
tion at 425 nm

If the absorbance is Pour all solution in the

higher than 1.5, dilute Measure the ab- drain and rinse the
it using the same sorbance of blue solu- corvettes and glass-
amount of sodium tion at 655 nm ware with water and
chloride soap

Complete the data

sheet and plot the
graph on Excel includ-
ing the initial and dead
volume