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 Precise Imprecise Imprecise

Accurate Inaccurate Accurate 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 Depress to the
piston to the first stop 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 is a wavelength-dependent proportionality constant

b is the path length through the absorbing substance

(our cuvettes have a standard 1 cm path length)

c is the concentration of the light absorbing substance

 Orient the cuvette so the light passes through its 1 cm path

Adjust Light path

wavelength

Set for
absorbance
readings