Pipetting Post Lab
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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) Calibrated
using 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 45 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
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) Calibrated
using 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 45 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
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