USING AN AUTOMATIC PIPETTE
indicator, and a plastic shaft.
 Many laboratory procedures,
especially biological and
biochemical ones, require the
precise transfer of small
quantities of liquids.
 Glass pipettes, which are
commonly used in chemistry
labs, are simply not accurate
when working with volumes that
are less than 1 milliliter.
 The best method of transferring
small volumes of liquids involves
the use of an automatic pipette.
Automatic pipette
- consists of a piston that
displaces air to create a vacuum
when its plunger is pressed.
- The tip of the automatic pipette is
then placed into the sample vial
and the plunger is slowly
released.
- This action draws liquid into the
tip.
- Finally, this liquid is expelled into
the desired container by slowly
depressing the plunger a second
time.
- As shown, a variety of automatic
pipettes exist. This video will
demonstrate automatic pipetting
technique using the Pipetman.
STEPS IN USING PIPETMAN:
 Using good technique will ensure
accuracy, which is defined as
the closeness of the dispensed
volume to the set volume, and
precision, which is defined as
the reproducibility of individual
measurements between your
samples.
Pipetman
- consists of a plunger button, a tip
ejector button, a volume
adjustment knob, a volume
- are available in six different sizes.
Five of them are depicted here.
- The number on the plunger
button indicates the maximum
volume in microliters that each
Pipetman is capable of
transferring.
- For example, the P20 has a
possible volume range of 0 to 20
microliters.
 Reminder: Transferring volumes
at the low end of the range is not
recommended because the
standard deviation is too high to
ensure precision.
- Thus the minimum amount of
liquid that can be transferred in
microliters is 0.1 for the P2, 0.5
for the P10, two for the P20, 10
for the P100, 50 for the P200,
and 100 microliters for the
P1000.
 Now that we've covered what an
automatic pipette is and how it
works, we'll take a detailed look
into how the Pipetman is used in
the laboratory.
- Let's say, for example, your
procedure requires you to
transfer 153.5 microliters of
sample.
1. Selection of the Pipetman with
an appropriate volume range.
- In this example, a couple of
choices exist. The P200 has a
recommended volume range of
50 to 200 microliters while the
P1000 has a recommended
range of 100 to 1,000 microliters.
 Reminder: The lower end of a
volume range always has a
higher standard deviation.
 Therefore, the P200 is the better
choice.
2. Selection of the proper
disposable plastic tip.
- Three tip sizes
o Large blue tips - which fit
the P1000
o medium yellow tips - which
fit the P200, P100
o small white tips - which fit
the P10 and P2.
- Because we're using the P200,
we're going to need the yellow
tips.
 Reminder: Using the proper tip is
essential for preventing leaks and
promoting accurate delivery of
liquids.
3. Setting the desired transfer
volume.
- For the smaller Pipetman, P2 to
P200, the volume set on the
indicator translates exactly into
the volume that will be delivered.
- This P200 is set to deliver a
volume of 125.9 microliters.
- For the P1000, the volume set on
the indicator must be multiplied
by 10 to determine the actual
volume that will be transferred.
- This P1000 is set to deliver a
volume of 1,259 microliters.
- To correctly adjust the volume
setting, turn the volume
adjustment knob to one third of a
revolution past the desired
volume. Then slowly move the
dial back down to the desired
setting.
 Reminder: Always approach the
desired volume by dialing
downwards to prevent
mechanical backlash and to
ensure precise volume transfer.
- When adjusting the volume of a
Pipetman, take care not to rotate
the dial past the maximum. You
cannot transfer volumes above
the range of the Pipetman.
4. Transfering your sample.
- You will need your appropriately
sized Pipetman set to the desired
volume, the correctly sized tips, a
rack, the new container, a chem
wipe, and a waste container.
- Attach a disposable tip to the
Pipetman by pressing its plastic
shaft firmly into the tip with a
twisting motion.
- This action should ensure an
airtight seal and prevent leaks
from occurring.
- Always use a new sterile tip for
each sample and never let the tip
touch anything except sample.
a. To draw a sample into the
plastic tip, depress the
plunger until resistance is felt.
b. Hold the Pipetman vertically
and immerse it into the
sample.
c. After the tip is immersed,
slowly release the plunger to
draw liquid into the tip. Wait a
few seconds to ensure that
the full volume of sample is
drawn into the tip.
 Reminder: Wait one second for
the P2 to P200 Pipetman and 2
to 3 seconds for the P1000.
 Reminder: The larger the volume
or the more viscous the sample,
the longer you should wait.
d. Remove the Pipetman from
the sample and carefully wipe
excess liquids from the sides
of the tip.
e. To dispense the liquid in the
Pipetman, touch its tip to the
side wall of a new container
and slowly depress the
plunger to the first stop.
f. Wait a few seconds to ensure
complete sample flow, then
press the plunger to the
second step to expel any
remaining liquid.
g. With the plunger fully
depressed, carefully remove
the tip from the container by
sliding it up the container wall.
The importance of sliding the
table along the wall increases
as the volumes become
smaller.
h. Finally, allow the plunger to
return to the up position.
- If you are done working with a
particular sample, press the tip
ejector button to release the tip
into the proper waste receptacle.
GUIDELINES TO ENSURE
ACCURACY AND PRECISION WHEN
TRANSFERRING LIQUIDS:
1. Use a fresh tip for each sample to
avoid contamination.
2. Pre-rinse each new tip. Most
liquids leave a film on the inside
wall of a new tip, which affects
the actual volume that can be
transferred. By pre-rinsing a tip,
 these volume variances are
eliminated.
- To pre-rinse a tip, simply draw
solution into the tip and expel it
into a waste container.
3. Check the immersion depth.
- If the immersion depth is too
deep, liquid may get into the
plastic shaft and damage the
mechanics of the Pipetman.
- If the immersion depth is too
shallow, air may be drawn into
the tip, which greatly affects
accuracy.
- As a general rule, immerse the
weight tips to a depth of 1 to 2
milliliters, the yellow tips to a
depth of 2 to 3 milliliters, and the
blue tips to a depth of 2 to 4
millimeters.
- If you do find an air bubble in the
tip, dispense the sample into a
waste container, check your
immersion level, and pipette
more slowly.
- If you still see an air bubble,
discard your current tip and use a
new one.
4. Release the plunger button
slowly. Do not let the plunger
snap up.
- This violent action may force
liquid into the plastic shaft.
5. When wiping excess liquid off the
sides of the tip, avoid touching
the tip opening because some of
the sample may be lost.
6. When liquid is in its tip, never
allow the Pipetman to tilt more
than 20 degrees from vertical and
never lay the Pipetman on its
side.
BE CONSISTENT:
 For optimum reproducibility, it is
important to be consistent with
your samples.
 Reminder: Use a consistent
speed and smoothness when
pressing and releasing the push
button.
 Reminder: Use consistent
pressure at the first step.
 Reminder: Immerse the tip to a
consistent depth.
 Reminder: Hold the pipetman at a
consistent angle that is less than
20 degrees from vertical.
TROUBLESHOOTING:
If you are still seeing errors with
accuracy or precision, the following
troubleshooting guidelines may fix your
problem.
1. Calibration:
- To ensure that your Pipetman is
actually delivering the desired
volume, you should periodically
calibrate it.
- To calibrate a Pipetman:
a. determine how many grams of
your liquid equals 1 milliliter
from its density.
- For example, if water is the
solvent, then 1 milliliter of water
equals 1 gram of water.
b. Tare a small container on an
analytical balance and set the
Pipetman at a volume near
the top of its range.
c. Transfer the set volume of
liquid to the tared container on
the balance and note the
mass of the liquid.
- Repeat this step five more times
and determine the mean weight
and standard deviation from your
measurements.
d. Repeat the entire process
with the Pipetman set at a
volume near the bottom of its
range.
- If these values deviate from what
you expect, adjust the pipetman
accordingly when you transfer
liquid.
2. Checking for leaks:
- Occasionally, the Pipetman will
begin to leak due to improperly
fitted tips or worn o-rings.
a. To check for leaks, put a fresh
tip on the Pipetman.
b. Pre-rinse the tip and draw up
liquid.
c. Keep the Pipetman vertical
and wait about 20 seconds.
d. If you see a droplet, then the
Pipetman is leaking.
 Reminder: Inaccuracies of the
Pipetman results from:
a. below 4 or
b. above 70 degrees Celsius
and
c. using the Pipetman with acids
and corrosives
- If you absolutely must pipette an
acid or corrosive, then you should
disassemble the Pipetman
immediately to inspect and clean
the piston, shaft, seal
assemblies, and o-rings.
SUMMARY – KEY POINTS
To summarize, using proper technique
with an automatic pipette is essential for
ensuring accuracy and precision.
1. Be consistent with plunger timing
and pressure as well as
immersion time and depth.
2. Use a fresh, pre-rinsed tip for
each new sample.
3. Check for air bubbles and leaks
during each transfer.
4. And calibrate the automatic
pipette periodically.
5. In addition, take care not to drop
the expensive and easily
damaged Pipetman.
6. Do not allow the tip to touch
anything except sample.
7. Do not introduce liquid into the
plastic shaft by letting the plunger
snap up or by laying the
Pipetman on its side.
8. Finally, avoid using the Pipetman
under extreme temperature
conditions or with acids and
corrosives.
When used with consistency and care,
an automatic pipette is an excellent
means
of transferring microliter quantities of
liquid.
USING A PH METER
 ph meters can be small or hand
held larger stationary
 video used larger stationary
PARTS:
1. meter
2. arm – allows electrode to easily
move up and down into a beaker
with the sample to be tested
3. electrode
 Party use a ph meter should be
calibrated using color coded
nuffers of fixed ph (4,7,10 are
standard)
STEPS:
1. Remove the protective cap from
the electrode.
- Electrode need to be stored
properly
- Cup should be immersed in
stored solution preventing them
to dry out
2. Rinse the electrode using distilled
water
- Various ph meter will have diff
ways for calibration (read the
manual)
- Begin the calibration using the
standard with ph 7:
o Once the standard has
been calibrated, you need
to remove the sample and
rinse the electrode with
distilled water.
o Repeat the process again
with the ph of 4.
o Once the standard has
calibrated, remove the
sample and rinse
electrode with dw.
o Then calibrate with ph of
10. Rinse off again the
electrode with dw to
ensure the accuracy of
measurement.
3. Measure the ph of unknown
solution
- Place the unknown sol in
electrode
- Sample has 5.57 and temp of
23.9 deg C.
4. Remove the sample after reading
5. Rinse the electrode with dw
6. Replace the protective cap for
storage
FUME HOOD SAFETY: Dos and
DON’Ts
- Fumehood safety should be
taken very seriously. You should
regularly review the
recommended safe practices
listed on your lab fume hoods
corner post. Don't be like
Scooter.
 When using substances that
produce hazardous levels of
airborne chemicals such as gas
fumes vapors and dust, make
sure the selected enclosure is
appropriate for the work you
intend to do.
- Consult your safety officer for a
reference on what type of safety
equipment is required.
- The safety tips in this video
applied to chemical fume hoods
specifically.
 Scooter works with dangerous
chemicals and in appropriate
enclosures. Don't be like scooter.
 Do not place your head inside the
hood. The fume hood interior
should be considered a opening possible when in use.
contaminated space at all times.
 Don't lean in to the contaminated
fume hood like scooter.
 Minimize drafts and sudden
movements in front of the hood
because turbulence generated in
front of a chemical fume hood
can cause loss of containment.
 Locate hoods away from heavy
foot traffic. Don't be like scooter.
 Work a minimum of 6 inches
inside the hood. Potential loss of
containment increases
significantly at distances within 6
inches of the plane of the sash.
 Place all hazards beyond this
threshold. Don't keep your work
dangerously close to the front of
the work surface like scooter is
livid on the edge.
 Elevate equipment above the
work surface elevated apparatus
is much less obstructive to the
airflow through the hood.
Equipment left flat on the work
surface interferes with air flow
needed below to efficiently clear
contaminants.
 Don't block airflow with
equipment like scooter.
 Keep sill and baffle unobstructed.
The sill or airfoil of the hood is
critical to containment, so avoid
blocking it.
- All of the airflow through the hood
eventually moves through the
baffles at the back. Obstructing
these baffles can cause uneven
face velocities and potential loss
of containment.
 Don't block the airfoil with notes
reminders and other obstructions
like scooter.
 Do not use the hood for storage.
Because hoods work more
efficiently without obstructions
keep unneeded objects to a
minimum.
 chemicals should be capped and
stored in the appropriate
chemical storage cabinet
 scooter keeps things that he
needed long ago and things he
might someday need in his fume
hood and that thing.
 adjust the sash to the smallest
locate the sash at a comfortable
height just above the elbows and
look into the hood through the
glass. the sash glass should be
used as a physical barrier to
protect the user's breathing area
whenever possible.
 scooter likes to keep the sash
wide open says it gives him
elbow room.
 close the sash when the hood is
unattended. Close the sash
completely protects passers-by in
the event of a projectile reaction
inside the hood.
 additionally in a variable air
volume or Vav mechanical
system this improves energy
efficiency.
 scooter shows blatant disregard
for his lab mates.
 do not remove any of the hood
components. multiple
components of a chemical fume
hood are designed to manipulate
air flow into and through the
hood. these components
typically work together in
harmony and a single
modification can result in severe
loss of containment.
 Scooter fancies himself
something of a fume hood
engineer and he likes to make
changes on the fly. don't be like
scooter.
 do not place flammable solvents
near heat flame or sparks. if
flammable materials and heat
sources must coexist in the same
hood, take great care and work
practices and separate them as
much as possible to avoid an
accidental fire.
 scooter recklessly places
flammable solvents near heating
devices.
 do not evaporate large amounts
of flammable liquids especially in
a low air volume situation such as
with the sash closed on a Vav
 mechanical system. minimize the
evaporation rate of flammables
by keeping the vessels covered
whenever possible.
 scooter is a sucker for
convenience so he leaves
flammable agent containers wide
open see you tomorrow scooter if
the lab is still here.
 wipe up spills immediately
spilling a solvent increases its
surface area evaporation rate
and probability of exposure.
 Routinely validate air flow. Proper
air flow through the fume hood is
critical to safe operation.
 Annual inspection of the face
velocity with a calibrated
instrument should be minimum
protocol. Into complete ASHRAE
110 containment assessment is
strongly recommended.
 Scooter doesn't know if anyone
has ever done that and he's not
about to start caring now.
 If the ventilation system
malfunctions or if the airflow
alarm indicates an unsafe
condition close the sash,
discontinue hood operation
immediately and call for help.
 Do not use the hood with bio
hazards or perchloric acid.
Remember your chemical fume
hood is designed for a very
specific family of hazardous
substances.
 Working with biological hazards
requires the use of an enclosure
or cabinet classified for biological
applications. The use of
perchloric acid requires a fume
hood designed and dedicated to
the use of this specific very
hazardous chemical.
 Don't be like scooter. Take
protecting your laboratory
environment seriously and pay
attention to recommended
practices when using any
chemical fume hood.