Analytical Chemistry

1. Analytical Chemistry
BS (HONS) Chemistry
University Of Gujrat
Hafiz Muhammad Athar Rizwan
Roll # 09050607-008
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2. Presentation Topic
Calibration of Glassware
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3. Basics Review and Calibration of Volumetric
Glassware
There are three types of containers used in lab to contain or deliver
liquids: volumetric, ordinary, and disposable glassware. Volumetric
glassware are containers that have been calibrated at a specific
temperature to deliver or contain very precise amounts of liquid.
Examples of volumetric glassware that we will use include
burettes, pipettes, and volumetric flasks. Ordinary glassware has
less precise volume calibrations and are used whenever the
volumes do not have to be measured as accurately. Examples
include beakers, Erlenmeyer flasks, and graduated cylinders.
Disposable glassware (or plastic ware) is used to transfer or hold
liquids temporarily and may not contain any volume markings.
Examples include medicine droppers and disposable pipettes.
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4. Volumetric pipette or flask, that is, you will experimentally determine
what volume a pipette or flask really delivers. You will also calibrate
a beaker or Erlenmeyer flask and a 50 ml burette. You will use these
calibrated values throughout the remainder of the quarter.
Care of glassware:
Glassware should be washed with a mild soap solution, rinsed with
tap water, and then rinsed with distilled water. If beads of water stick
to the inside walls, it be should be cleaned again. If glassware is to be
dried, allow it to drain or use paper towels. Never sling glassware to
remove water. Never tap glassware against the side of the sink. A
burette or pipette should be rinsed with a small amount of the solution
to be used to remove any water droplets from the glass walls.
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5. Burette:
Burette must be read to the hundredths of a milliliter. Note that the
liquid level markings begin at the top or open end. This is because the
burette is designed to deliver liquids. All volumes are measured as the
difference between an initial and final reading.
Pipette:
The volumetric pipette is designed to deliver a single, fixed volume of
liquid at a specific temperature. A graduated pipette is used in a similar
fashion to the burette to deliver specific amounts of liquid. Remember
to touch off the drop of liquid hanging outside the tip into the
transferred liquid (it is part of the delivered volume) but do not blow
out any liquid that remains inside the tip of the pipette. The pipette has
been calibrated to contain this last drop of liquid.
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6. Principles of Measuring the
Volume of Liquids
• Overview
– Volume
• The amount of space a substance occupies
• Liter
– Basic unit of volume
• Measuring devices
– Depends of amount to measure and accuracy
– Larger volumes
» Glass and plastic vessels
» Graduated cylinders
» Volumetric flasks
– Pipettes
» Preferred for volumes in 1-25ml range
– Micropipette devices
» Less than 1ml
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7. Basic principles of Glassware
Calibration
• Calibration up to manufacturer
– Capacity marks/graduation
• Lines marked on volume measuring devices that indicate volume
– Lines the way to make the measurements
– Determined by American Society for Testing and Materials
(ATSM)
• Meniscus
– Must be considered
– A curve formed by the surface of liquids confined in narrow
spaces (measuring devices)
» Lowest point of the meniscus used as point of reference
for calibration
» Hold at eye level to be entirely accurate
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8. Basic principles of Glassware
Calibration
• Liquid measuring calibrated to either
– To contain (TC)
• Will contain the specified amount when filled to the capacity mark
– Will not deliver that amount
– Adhesion to sides
– To deliver (TD)
• Marked slightly differently so that it does deliver the specified
amount, assuming the liquid is water at 20oC and it is poured using
specific techniques
– Volumetric flask
» Hold specific volume
» Essential for proper concentrations of buffers, etc
» Not good for “delivering” proper volume
» Use of TD device should be used
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9. Basic principles of Glassware
Calibration
• Small amount error in calibration inevitable
– Tolerance
• How much error is allowed in the calibration of a volume
measuring item
• Most accurately calibrated glassware termed volumetric
– Has narrowest tolerance allowed
• Two effects of temperature to consider in high accuracy measurements
– Expand or contract with changes in temperature
• Affects accuracy
– Volume of the solution changes as the temperature changes
• Devices calibrated at 20oC with water
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10. Glass and Plastic Labware used to Measure
Volume
• Beakers and Erlenmeyer flasks
– Hold liquids
– Not to measure
• Calibrated with tolerance of ± 5%
– 100mL line may be anywhere form 95-105mL
• Graduated cylinders
– Cylindrical vessels calibrated with sufficient accuracy for most volume
measurements
• ± 0.6mL tolerance
• Usually calibrated TD
• Can be obtained with different set of graduations, depending on need
• Not designed for mixing or storing
– Unstable/easily knocked over
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11. Glass and Plastic Labware used to Measure
Volume
• Burettes
– Long graduated tubes with a stopcock at one end that are used to
dispense known volumes accurately
• Volumetric Flasks
– Vessels used to measure specific volumes where more accuracy is
required than is attainable from a graduated cylinder
• Calibrated either TC OR TD a single volume
– Several disadvantages
• Relatively expensive
• Calibrated for only one volume
• Used only in situations where high accuracy volume measurements
are required
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12. Glass and Plastic Labware used to Measure
Volume
• Volumetric Flasks
– Proper use
• Choose the proper type of flask for the application
– TC or TD
– Class A or Class B
– Serialized or not
• Be sure the flask is completely clean before use
• Read the meniscus with eyes even with liquid surface
• If calibrated TD, then pour as follows:
– Incline the flask to pour the liquid; avoid splashing on the walls as
much as possible
– When the main drainage stream has ceased, the flask should be nearly
vertical
– Hold flask in vertical position for 30 sec or so and touch off the drop
of water adhering to the top of the flask by touching it to the
receiving vessel
Never expose volumetric glassware to high temperatures because heat causes
expansion and contraction that can alter it calibration
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13. Pipettes-Pipettes and Pipette-Aids
• Pipettes
– Hollow tubes that allow liquids to be drawn into one end and are
generally used to measure volumes in the 01.-25ml range
• Glass or plastic
• Disposable or multiple use
• Pre-sterilize disposable with cotton plugged tops
• Pipette-aids
– Devices used to draw liquid into and expel it from pipettes
• Mouth pipeting
– Used to be the norm
– Now banned by safety regulation
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14. Pipettes-Measuring Pipettes
• Measuring pipette
– Calibrated with a series of graduation lines to
allow the measurement of more than one volume
• Serological pipettes
– Usually calibrated so that the last drop in the tip needs to be
“blown out” to deliver the full volume to deliver the full
volume of the pipette.
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15. Pipettes-Measuring Pipettes
• Serological pipettes
– To use:
» Check that the pipette is calibrated to be “blown out” by
looking for the bands at the top
» Examine the pipette to be sure the tip is not cracked or
chipped
» Fill the pipette about 10mm above the capacity line
desired and remove any water on the outside of the tip by
a downward wipe with lint-free tissue
» Place the tip in contact with a waste beaker and slowly
lower the meniscus to the capacity line
» Deliver the contents into the receiving vessel by placing
the tip in contact with the wall of the vessel
» When the liquid ceases to flow, “blow out” the remaining
liquid in the tip with on firm “puff” with the tip in contact
with the vessel wall.
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16. Pipettes-Measuring Pipettes
• Mohr pipettes
– Calibrated “to deliver” but, unlike the serological
pipettes, the liquid in the tip is mot part of the
measurement and the pipette is not blown out.
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17. Pipettes-Volumetric (Transfer) Pipettes
• Volumetric (transfer) pipettes
– Made of borosilicate grass and are calibrated TD a
single volume when filled to their capacity line at
20oC.
• Most accurately calibrated pipette
• Can be serialized
– Calibrated
• “Delivery of the contents into the receiving vessel is
made with tip in contact with the wall of the vessel and
no after-drainage period is allowed.
– Not “blown out”
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18. Pipettes-Other Types
• Pasteur pipettes
– Used to transfer liquids from one place to another
• Not volume-measuring devices
– No calibration lines
– Good for transferring liquids
• Manual dispenser for reagent bottles
– Devices placed in a reagent bottle with a tube that extends to the
bottom of the bottle
– Dispenser has a plunger that is depressed to deliver a set volume of
liquid.
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19. Micropipetting devices-positive displacement
and air displacement micropipettors
• Micropipettors
– Devices commonly used to measure smaller volumes, in the
1-1000µL range
– Two types
• Positive displacement
– Include syringes and similar devices where the sample comes in contact
with the plunger and walls of the pipetting instrument
» Used for viscous and volatile samples
• Air displacement micropipettors
– Designed so that there is an air cushion between the pipette and the
sample such that the sample only comes in contact with a disposable tip
and does not touch the micropipettor itself
» Disposable tips-reduce chance of cross contamination
– Accurately measure volume of aqueous samples
» Most common instruments used in biotech
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20. Micropipetting devices-air displacement
micropipettors
• Obtaining accurate measurements
– Procedure for operation
• Plungers
– Control used by operator for the uptake and expulsion of liquids
» Different stop levels can be felt (fig 20.13, pg. 357)
– Factor affecting accuracy
• Operator’s technique
• Physical/chemical properties of the liquids
• Measurements affected by the environment in which they are
made
• Condition of the micropipettor
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21. Glassware
Laboratory glassware refers to a variety of equipment, traditionally made
of glass, used for scientific experiments and other work in science,
especially in chemistry and biology laboratories. Some of the equipment
is now made of plastic for cost, ruggedness, and convenience reasons,
but glass is still used for some applications because it is relatively inert,
transparent, more heat-resistant than some plastics up to a point, and
relatively easy to customize.0 ml.
• Glassware can be divided into 2 groups
– Non-volumetric glassware
• Beaker
• Flask
– Volumetric Glassware
• Volumetric Flask
• Graduated Cylinder
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22. Beaker
• Used for transferring liquid to another container or to transfer a
small amount of reagent for use in procedures.
• Volume is not accurate, just an estimate.
• Never place a reagent in another container without labeling the
container first.
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23. Erlenmeyer Flask
• Features a conical base, a cylindrical neck and a flat bottom.
• They are marked on the side (graduated) to indicate the
approximate volume of their contents.
• This is NOT used for ACCURATE measurement
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24. Graduated Cylinder
• For rapid measurement of liquid volume.
• They are generally more accurate and precise for this purpose
than flasks.
• This is a semi-accurate liquid measuring vessels.
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25. Reading the Volume
• 10 mL has approx 6.62 mL
• 100 mL 52.7 mL
• 25 mL has 11.5 mL
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26. 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.
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