Experiment 1 Introduction to basic
technique of biochemistry
experiment
(glass pipette, centrifuge,
spectrophotometry)
Rule of biochemistry’s lab
1.Wear your write gowns.
2.Each group has a set of apparatus.
3.If you break your apparatus, you must
pay for it.
4.Four students will be on duty each time.
5. Hand in your report after experiment.
6.Signature your names after experiment .
How to write the report？
⑴Title of experiment, Name, Date of
experiment.
⑵Objectives
⑶Principle
⑷Material and reagents
⑸Procedure
⑹Results and calculations: The section
will contain the calculation formula and
results.
⑺Discussion
Discussion is very important
a. Conclusions
b. Significance of your results
c. Was the original objective achieved?
(if not, why not?)
d. Discussion of possible errors
Part Ⅰ：Pipette
A pipette is used to measure small
amounts of solution very accurately, A
pipet bulb is used to draw solution into
the pipet。
⑴Recognition
⑵Select
⑶Draw solution
⑷Release
Exercise
Practice this with water until you
are able to use the pipette and bulb
consistently and accurately.
Part Ⅱ：Centrifugation
The theoretical basis of this technique
is the effect of gravity on particles
(including macromolecules) in suspension.
Two particles of different masses will
settle in a tube at different rates in
response to gravity. Centrifugal force
(measured as as xg, gravity) is used to
increase this settling rate in an instrument
called a centrifuge.
Simple centrifuges are used in biology
and biochemistry for isolating and
separating biocompounds on the basis
of molecular weight。
Balance the load in the opposite slot
with the same volume of fluid。
centrifuge caution
①The machine should be placed on a
stable and firm desk.
②Keep the centrifugation tube balanced
and symmetric.
③After turning on machine, increase
the rotation speed little by little.
④Regulate the speed you wanted and
record the time.
⑤At the end of centrifugation，turn
down the speed little by little.
Exercise
Take 1ml blood serum into a tube，then
add 10 drops of 10% acetate chlorine.
Centrifuge the sample at the speed of
3000r/min for 5 minutes. Observe the
liquid in the tube and record the change
happened after centrifugation.
Part Ⅲ：Spectrophotometry (spectrometer)
A spectrometer measures the amount of visible
light absorbed by a colored solution. This can be
read as Absorbance or % Transmittance.
Principle:
According to Lambert-Beer's Law, the
concentration of a sample could be calculated:
A(Absorbance)=K × C (concentration)
A（sample）/A（standard） = C（sample）/C（standard）
Fingure 1. The main elements of spectrometer
1. 721 spectrometer
Procedure:
① Setting up the spectrometer，Turn
on the power of the instrument ，
Allow 10 minutes for warming up. Set
the wavelength to the desired value using
the knob on the left.
② Calibrating the spectrometer
Add solution to the cuvette. Wiping the
cuvette with a dry Kimwipe to remove
drops of solution or finger prints. with
empty, opened sample compartment,
turn the knob to obtain a reading of 0%
T，Close the cover. Adjust the knob to
obtain a reading of T= 100% (A=0)
③Using the spectrometer
Lining up the mark on the cuvette in
the sample compartment. Insert sample
cuvette and record the value of A on the
scale.
2.722 spectrometer ( digital spectrometer)
Procedure:
①Setting up the spectrometer
Turn on the power of the instrument .
Allow 10 minutes for warming up. Set the
wavelength to the desired value using the
nm and nm buttons. The wavelength is
displayed on the LCD..
②Calibrating the spectrometer.
Fill a cuvette with your blank solvent
and dry the outside of the cuvette
carefully. Insert your blank, opened
cuvette in the sample compartment. Press
the A/T/C button to select Transmittance
mode and set the transmittance of the
blank to zero. Close the cover. Press 0
ABS/100% T to set the absorbance of the
blank to zero.
③Using the spectrometer
Lining up the mark on the cuvette in
the sample compartment.
Insert sample cuvette and the
absorbance value of the sample will now
be shown on the LCD.
Exercise
Use the spectrometer to measure
A500nm of 0.5%, 1.0%, 1.5%, 2.0% cobalt
sulphate, as well as an unknown
concentration of it. Adjust the wavelength
to 500nm. Draw a standard curve with the
X axis representing respectively the given
concentration of the liquid and the Y axis
the absorbance of the four tubes. Obtain
the concentration of the unknown cobalt
sulphate with two methods.