Post Lab Experiment 3 & 4
Proper Pipetting and Calibration of
Volumetric Apparatus
Objectives
Demonstrate proper technique of
handling and using a pipette
Identify the different types of pipettes
used in Clinical Chemistry
Enumerate the specific uses of each
pipette
Discuss primary standards, their
characteristics and examples
Outline steps in the preparation of
chromic acid
Proper Use
When filling a pipette, the tapered end is held
beneath the surface of the liquid at all times.
The liquid is drawn into the pipette by suction
until the level is equal to or greater than the
volume of liquid to be delivered.
Since serological pipettes are labeled with the
zero mark at the top of the pipette you will need
to subtract the amount you are going to pipette
from the total volume of the pipette to determine
the exact mark to fill the pipette to.
For example if you are going to pipette 6.5 mL
using a 10 mL pipette you would fill the pipette
to the 3.5 mL mark. To determine the total
volume of the pipette look near the top of this
type of pipette.
What volume does this pipet contain?
How much was delivered?
Proper Use
When reading the volume,
ALWAYS view the pipette
dead-on at eye level with
the pipette held vertically,
perpendicular to the
ground.
Pipettes are designed to
be used with a hand pump
or bulb, of which there are
many varieties.
Never use your mouth
with a pipet!
Pipets (Pipettes)
Laboratory instrument used to transfer a
measured volume of liquid.
May be classified as to graduation and as to
calibration
Mechanical or Automatic pipettes
As to graduation they may be classified as
(1) Volumetric Pipette (2) Measuring
Pipette
As to Calibration classification includes:
TC, TD, Between two marks, To Blow out
Kindly watch the following videos
• Drawing of liquid
• Preparing pipette for transfer
• Transferring of liquid after pipetting
•Indicate whether correct volume was drawn o
•Determine whether transferred amount is cor
•Identify and explain error committed if there
• Cleaning of pipette
Additional Videos
• Filling up of volumetric pipette
•Liquid transfer using a volumetric pipette
Pipettes as to Graduation
Volumetric Pipette
or transfer pipettes are designed to deliver
a single volume precisely (the volume will
be indicated near the top of the pipet (i.e.,
2 mL).
Volumetric Pipette
Above the bulb in the tube is an engraved
ring. Fluid must be drawn up the pipet to
above the ring and then released slowly until
the bottom of the meniscus is exactly at the
ring (the tip of the pipet should be touching
the wall of the sample vessel as fluid is
released). To transfer this volume to a second
container, touch the pipette tip to the inside
of the new container and allow the liquid to
drain out.
Volumetric
Designed to transfer a fixed amount
of liquid when filled to the mark,
e.g. 10 mL and only 10 mL.
There is generally only one "fill-line"
on a volumetric pipet.
For example a 5 mL volumetric
pipette has one marking on it. This
marking measures exactly 5 mL. of
liquid, no more, no less.
Volumetric
Designed to deliver a single volume precisely,
the volume will be indicated near the top of the
pipette
At the top of the pipette is an etched ring.
Fluid must be drawn up the pipette to above
the ring indicating the volume and then
released slowly until the bottom of the
meniscus is exactly at the ring.
To transfer this volume to a second container,
touch the pipette tip to the inside of the new
container and allow the liquid to drain out.
Measuring or Graduated Pipette
Mohr or measuring pipets are
graduated but stop at a baseline
before the pipet begins to narrow.
To accurately transfer fluid with this type of
pipet, the meniscus must be precisely on a
calibration mark both at the beginning and
at the end of a transfer.
Measuring or Graduated Pipette
Serological pipets are graduated to deliver (there
is no base mark).
The appropriate amount of fluid is drawn into the
pipet (with the meniscus precisely on the correct
mark) and the entire amount is transferred.
Those with a single painted or frosted ring at the top should
be allowed to simply drain with the tip placed against the
side of the receiving vessel. Those with double rings are
designed to be "blown out" by pushing a small amount of air
out of the pipet, completely emptying it.
Serological - TD
Pipets with double rings are designed to
be "blown out" by pushing a small
amount of air out of the pipet, completely
emptying it.
Serological Pipet
Since serological pipets are labeled with the zero
mark at the top of the pipet you will need to
subtract the amount you are going to pipet from the
total volume of the pipet to determine the exact
mark to fill the pipet to
For example if you are going to pipet 6.5 mL using a
10 mL pipet you would fill the pipet to the 3.5 mL
mark. To determine the total volume of the pipet
look near the top of this type of pipet.
According to Calibration
To Deliver Pipettes (TD)
Calibrated using water
Serological pipette, volumetric pipette
To Contain Pipettes (TC)
Calibratedusing liquid mercury
Mohr pipette
To Blow Out Pipette
Calibrated using water (serological pipette)
Between two marks pipette
Water as calibrating medium (Mohr pipette)
Pipets
Serological and Mohr pipets have different
markings to assist in identifying them as
they are used differently.
Serological pipets are TD = to deliver. To
accurately dispense the measured volume
the last bit must be blown out.
Mohr pipets are TC = to contain. These
pipets are designed to dispense the
correctly measured volume, so there will
be a minute amount of liquid left in the
tip.
Markings
TD pipets will have an etched or
colored ring at the top of the pipet.
TC pipets will have no rings
although there may be a colored
bar to indicate the volume.
Mechanical or Automatic
Mechanical pipets can be set to draw and
dispense different volumes or be preset to
deliver an exact volume.
Used to accurately deliver very small
volumes, microliters, of liquid.
Although 0.1 mL could be delivered by a
serological pipet most labs use
mechanical, or automatic pipets.
A volume of 0.1 mL is equal to 100
microliters
Mechanical or Automatic
Mechanical pipets are operated by depressing the plunger.
On the downward stroke of the plunger there are TWO
stops.
The first offers firm resistance, and the second is a hard
stop.
To take up a volume in the pipet, place a tip on the end of
the pipet.
Depress the plunger to the first stop and insert into the
sample to be transferred.
Draw the liquid into the pipet by SLOWLY releasing the
plunger.
To dispense the liquid from the pipet, place the tip of the
pipet into the opening of the well and slowly depress the
plunger all the way to the second stop.
When the liquid has been dispensed withdraw the pipet tip
from the well BEFORE releasing the plunger.
Reading the Volume
10 mL has approx 6.62 mL
100 mL 52.7 mL
25 mL has 11.5 mL
Volumetric Flask
A volumetric flask is used to measure very precisely one
specific volume of liquid (100 ml, 250 ml, etc., depending
on which flask you use).
This flask is used to prepare a solution of known
concentration.
To make up a solution, first dissolve the solid material
completely, in less fluid than required to fill the flask to
the mark.
After the solid is completely dissolved, very carefully fill
the flask to the mL mark.
The top is then sealed and the flask is inverted several
times to mix.
Primary Standards
• Used to calibrate other standards
• reliable, readily quantified substance
• Requirements of a good primary standard
• High purity
• Stability (low reactivity)
• Low hygroscopicity and efflorescence
• High solubility (if used in titration)
• High equivalent weight
• It should be nontoxic
• It should be readily available (inexpensive)
• It should be environmentally friendly
Examples of Primary Standards
Benzoic acid for standardization of waterless basic solutions:
ethanolic sodium and potassium hydroxide, and alkali
methanolates in methanol, isopropanol, or DMF
Potassium bromate (KBrO3) for standardization of sodium
thiosulfate solutions
Potassium hydrogen phthalate (usually called KHP) for
standardization of aqueous base and perchloric acid in acetic
acid solutions
Sodium carbonate for standardization of aqueous acids:
hydrochloric, sulfuric acid and nitric acid solutions (but not
acetic acid)
Sodium chloride for standardization of silver nitrate solutions
Sulfanilic acid for standardization of sodium nitrite solutions
Zinc powder, after being dissolved in sulfuric or hydrochloric
acid, for standardization of EDTA solutions
Chromic acid
Used in cleaning glassware
dissolve 140g of technical grade sodium dichromate
dihydrate in approximately 100 ml of water
Add two liters of technical grade 98% sulfuric acid to a 4-
5 liter glass beaker that is sitting in a cold water bath in a
fume cupboard.
Carefully stir the acid gently and pour a few mls of the
dichromate solution slowly into the acid.
Keep repeating the addition every few seconds - after the
previous dose has been dispersed
Allow to cool before storing in a glass-stoppered reagent
bottle. Always ensure that the stopper is sufficiently loose
to release any gas pressure. Never use a screw-capped or
similar types of sealed containers.
Secondary Standard
is a standard that is prepared in the laboratory for
a specific analysis. It is usually standardized
against a primary standard. NIST Standard
Reference Materials The National Institute of
Standards and Technology (NIST) provide a
wide variety of standard reference materials
(SRMs) for validating and calibrating analytical
methods.
a solution that has been titrated against a primary
standard.
A standard solution is a secondary standard
