Mastering the micropipette
What is a micropipette?
How do you use a micropipette?
How can I determine if I’m pipetting correctly?
Micropipettes work by air displacement
3 kinds of micropipette
(various manufacturers)
Calibrated to deliver volumes
within a certain range
P-20 capacity of 20 µL
P-200 capacity of 200 µL
P-1000 capacity of 1000 µL
They’re expensive!!
Handle with care
Micropipettes are designed to be both accurate and precise
A
B
C
D
Precise
Accurate
Precise
Inaccurate
Imprecise
Accurate
Imprecise
Inaccurate
Accurate – the correct volume is delivered
Precise – there is minimal variation between the volumes delivered
You will do three trials with one micropipette to see how precise your
transfers are
Your data will be compared with the rest of the class to see how
accurate the measurements are
What is a micropipette?
How do you use a micropipette?
How can I determine if I’m pipetting correctly?
To begin, attach the appropriate tip to the micropipette
P-20 and P-200 use yellow or natural color tips; P-1000s use blue tips
Tips have been sterilized
Replace the lid after you
attach the tip
Dispose of used tips in
the appropriate waste
receptacle!
To fill the micropipette, depress the
piston to the first stop
Depress to the
second stop ONLY
when expelling the
contents
Fill stroke
Depress the plunger to the first stop
Insert the pipette a few millimeters into the
solution to be transferred
Slowly release the plunger up to the starting
position (tip will fill with fluid)
For the most accurate transfers, keep
the micropipette within 20 degrees of
the vertical axis
Delivery stroke
Place the tip of the micropipette against
the wall of the receiving tube
Press the plunger to the first stop
(a small amount of liquid may remain in
the tip)
Press the plunger to its full extent to
deliver any fluid remaining in the tip
What is a micropipette?
How do you use a micropipette?
How can I determine if I’m pipetting correctly?
Ideally, you would weigh a drop of water
transferred by the micropipette on an analytical
balance, using the specific gravity of water to
calculate the volume actually transferred
Since we don’t have enough analytical balances, we will use an indirect measure:
the absorbance of colored solutions measured with a spectrophotometer
Common elements of spectrophotometers
cuvette with 1 cm
path length
Detector
Adjustable monochromator allows
a single wavelength to pass
You will prepare dilutions of a bromophenol blue solution and
measure the absorbance of the dilutions at 590 nm
Absorbance spectrum of bromophenol blue
http://www.wellesley.edu/Chemistry/Chem105manual/Lab10/lab10.html
Beer-Lambert Law
Predicts a linear relationship between absorbance of a solution and
the concentration of a light-absorbing substance in the solution
A590 = (a590) x (b) x (c)
a
b
c
is a wavelength-dependent proportionality constant
is the path length through the absorbing substance
(our cuvettes have a standard 1 cm path length)
is the concentration of the light absorbing substance
Orient the cuvette so the light passes through its 1 cm path
Adjust
wavelength
Set for
absorbance
readings
Light path