GLASSWARES AND PIPETTES CONTENTS • GLASS-TYPES • GLASSWARES • PIPETTES GLASS Glass is a complex silicate compound Properties depends on silicate anion as well as its cation content Addition of metal ions BOROSILICATE GLASS Ingredients •Silica Ingredients •Boron Silica oxide •Sodium oxide Boron oxide •Aluminium Sodium oxide oxide • Aluminium oxide Percent 80.6 12.6 4.15 2.2 FEATURES High thermal resistance Low Alkali content Free from Zinc group elements and heavy metals Resists heat, corrosion and thermal shock Low coefficient of expansion FEATURES Thermal durability of borosilicate glass: Strain point 510 °C Annealing point 555 °C Softening point 820 °C USES Advantages: Can be heated upto 600 °C Brand names: Pyrex, Kimax Most lab equipment Beakers,flasks,pipettes COREX-ALUMINIUM SILICATE GLASS • Resist clouding due to alkali ,staining • Harder • For temperature more than250 c • 30000 PSI-6 times stronger than borosilicate • Thermometers,graduated cylinders,centrifuge tubes HIGH SILICA GLASS Made by removing all elements except silica from borosilicate glass High silica content – 96% Comparable with fused quartz in thermal endurance, chemical stability and electrical characteristics Radiation resistant Good optical qualities USES Used for optical reflectors, cuvettes , high temperature thermometers VYCOR Used in applications involving high temperatures, drastic heat shock and extreme chemical treatment Resistant to attack by all acids except hydrofluoric acid Uses: Ashing and ignition techniques BORON FREE GLASS Less than 0.2% boron High resistance to alkali Thermal resistance less Uses: strong alkali reactions LOW ACTINIC GLASSWARE Impart red or amber colour to glass • Uses: Provides highly protective laboratory glassware for handling materials sensitive to light in the 300 – 500 nm range • Eg: Bilirubin, Carotene, Vitamin A FLINT GLASS Soda lime glass – mixture of silicon, calcium, sodium oxides Lowest cost Poor resistance to high temperatures and sudden changes of temperature • Uses: Bottles, disposable pipette DISADVANTAGES • Breakable • May leak alkali in solution COLOURED AND OPAL GLASSES Made by adding small amounts of metallic oxides to the glass Uses: Filters and light bulbs OPTICAL GLASS Made of lead, sodalime and borosilicate High optical purity Uses: Prisms, lens, mirror COATED GLASS Has thin metallic oxide permanently firebonded to the surface of glass Conducts electricity • Uses: Heat shield to protect against infra red light Electrostatic shield to carry off charge GLASS CERAMICS High thermal resistance, chemical stability, and corrosion resistance Useful for making hot plates, heat exchangers RADIATION ABSORBING GLASS Made of soda lime and lead Useful in preventing the transmission of high energy rays (X-rays and γ rays) ASTM DESIGNATIONS • Class A glassware: Highest level of accuracy Borosilicate is used • Class B glassware: Same basic design as class A Sodalime is used Volumetric tolerances for class B – twice the specified range allowed for class A LABORATARY GLASSWARE • VOLUMETRIC • GRADUTED CYLINDERS • VOLUMETRIC FLASKS • BURETS • PIPETTES • NON VOLUMETRIC • FUNNELS • CUVETTES • TEST TUBES • DESSICATORS • SYRINGES • REAGENT BOTTLE • STIRRING ROD • PETRI DISH • WATCH GLASS,COVER SLIPS VOLUMETRIC FLASKS Flat-bottomed flask ,round lower portion Long thin neck Etched calibration line To prepare reagents single graduation line Class A • For accurate volumetric measurements • Pipette to be used in adding final drops TOLERANCE TABLE Capacity (mL) 25 50 100 250 500 1000 Tolerance for class A(mL) 0.03 0.05 0.08 0.12 0.20 0.30 Tolerance for class B(mL) 0.06 0.10 0.16 0.24 0.40 0.60 ERLENMEYER FLASKS • Wide bottom,evolves into smaller short neck • Chemicaly inert and heat resistant • Preparing solutions and titrations • Not accurate ROUND SHAPED FLASK • Round Shaped Flat Bottomed Flask: Often used to evaporate a sample to dryness Round Shaped Round Bottomed flask: Used in distillation Should not be heated Not to be used for reagent storage BURETTES Long graduated glass tubes with a stop cock at one end Used to deliver accurately, known amounts of liquid into the container BURETTE Standard burettes vary in size from 25 to 100 mL Stop cock – all glass or all Teflon at one end Micro processor controlled automatic burette with accuracy 0.1% Digital display - reading upto 0.001mL USES • For titrations • Known volume is dispensed BEAKERS Wide mouthed, straight sided, cylindrical vessels Volumes vary from 5mL to several liters The Griffin beaker - flat bottom, straight sides, and an opening as wide as the flat base, with a small spout in the lip. • Uses: General mixing Non volumetric liquid reagents preparation GRADUATED CYLINDERS Narrow long ,cylindrical tubes Circular or octagonal base Measure volume of liquids Not as accurate as volumetric flask Size vary from 10-2000ml FUNNELS • Filtering funnels: 58 – 60 ° angled funnels with either short or long thin stems • Powder funnels: For transferring solids Wide mouthed stem • Separatory funnels: Pear shaped or cylindrical Used for manual liquid – liquid extractions CUVETTES CUVETTES Special kinds of test tube with straight sides May be rectangular in shape Holds solutions intended for photometric readings Width determines the length of the light path through the sample Have two transparent sides opposite one another Costly Should not get scratched DESSICATORS Used to dry solids or liquids Dessicant is placed in the bottom A shelf is placed above the dessicant in which the material is placed Wide, flat ground glass lip on the top CENTRIFUGE TUBES SYRINGES Used for accurate volumetric work Injection of small volume of sample, • liquid or gas for chromatography REAGENT BOTTLES PIPETTES PIPETTE • Used to transfer a known volume of liquid from one container to another • creates a partial vacuum above the liquid holding chamber and selectively releases this vacuum to draw up and dispense liquid TYPES OF PIPETTES TC - TD PIPETTES • Difference is the amount of liquid needed to wet the interior surface of the ware • The amount of any residual liquid left in the pipet tip. • Most manufacturers stamp TC or TD near the top of the pipet to alert the user as to the type of pipet. • TC pipet holds or contains particular volume but does not dispense that exact volume, • TD pipet will dispense the volume indicated. TC PIPETTE Contains particular volume Doesn’t dispense the same volume TD PIPETTE • Dispenses the volume indicated BASED ON DRAINAGE CHARACTERISTICS • Self draining pipette • Blow out pipette BASED ON GRADUATION MEASURING / GRADUATED PIPETTE Mohr pipette Serological pipette MEASURING PIPETTE • Piece of glass tubing that is drawn out to a tip and graduated along its length • MOHR pipette-calibrated between two marks • SEROLOGICAL pipette-graduated to the tip MOHR PIPETTE SEROLOGICAL PIPETTE Smaller orifice Larger orifice Needs more controlled delivery Drains faster Blow out to deliver entire volume DIFFERENCE MOHR PIPETTE SEROLOGICAL PIPETTE MEASURING PIPETTE • Room for error • Only used to measure reagents TRANSFER PIPETTES • Designed to transfer known volume • Volumetric pipettes • Ostwald –folin pipettes VOLUMETRIC PIPETTES • To deliver accurately a fixed volume of aqueous solution. • Precision decreases with decrease in size • Cylindrical bulb joined at 2 ends to narrower glass tubes • Caliberation on upper suction tube • Delivery tube is tapered • To prevent uncontrolled delivery and incomplete drainage OSTWALD- FOLIN PIPETTE • Similar to volumetric pipette • The bulb is closer to the delivery tip • For accurate measurement of viscous fluid, blood,serum. • Etched mark to indicate blow out pipette • Slow drainage MICROPIPETTE • TO measure microliter volumes • Volume coating inner walls can cause error • TC pipettes • Rinsed with the final solution after delivering contents into the diluent • 1-1000ul FIXED VOLUME AND VARIABLE VOLUME PIPETTE MULTI CHANNEL PIPETTE TYPES-MECHANISM • Air-displacement pipettes relies on a piston for suction creation to draw the sample into a disposable tip • Positive-displacement pipettes Piston moves like in a hypodermic syringe POSITIVE DISPLACEMENT PIPETE • Direct contact with the liquid • Air cushion in pipette tip • Displaces the liquid USES • Viscous liquid • Volatile liquid • Hot or cold PIPETTING TECHNIQUE • Set the volume • Attach the tip • Depress the plunger to first stop • Immerse the tip into the liquid and draw liuid by releasing piston • Dispense the liqud-push plunger top • Withdraw pipette before releasing the piston • Remove tip by ejecting DONT’S WHEN PIPETTING • Never use pipette without pipette tip • Do not use pipette beyond volume limits • Never repeatedly jam tip into a pipette if it doesn’t fit • Don’t push past first stop while aspirating set volume • Release push button in a controlled manner • Always make sure tip is below liquid level while aspirating • Never place pipette horizontally without expelling liquid PHOTOMETRIC CALIBRATION • Method I : An aliquot of diluent is pipetted out The change in concentration will reflect the volume of the pipette • Method II : Comparing absorbances of dilutions of potassium dichromate using Class A volumetric glassware versus equivalent dilutions made with the pipetting device GRAVIMETRIC CALIBERATION • MATERIALS : • Pipette • 10 to 20 pipette tips • Balance • Weighing vessel • Type I water • Thermometer METHOD • Record weight of vessel • Note temperature and pressure • Place small volume of water in container and cover CALIBERATION • Set the balance to zero • Dispense specified amount by pipette to be tested • Record result • Repeat 9 more times after changing tips CALCULATION CALCULATE • VOLUME OF WATER MEAN WEIGHT • • Z-DENSITY OF WATER AT GIVEN TEMPERATURE AND PRESSURE CALCULATION • VOLUME ( ml or µl ) = WEIGHT( mg or µg ) x Z ACCURACY AND PRECISION • Accuracy is the proximity of measurement results to the true value; • precision is the degree to which repeated (or reproducible) measurements under unchanged conditions show the same results. …………….. ACCURACY & PRECISION ACCURACY TOLERANCES • CLASS A • Accuracies in mL of Manual pipettes Type of pipette 1.0 mL NBS standard 5.0 mL 10.0 mL 25.0 mL 0.01 0.02 0.025 Class A Volumetric 0.006 0.01 0.02 0.03 Mohr 0.01 0.02 0.03 0.10 Serological 0.01 0.02 0.03 0.10 SEMIAUTOMATIC AND AUTOMATIC PIPETTES • Programable devices • Dispensing alquots of liquid into multiple wells • Piston driven ELECTRONIC PIPETTES • Electronically dispenses predefined volumes(0.5-200ul) • Disposable plastic tips to contain • Retains less in inner walls • Avoids contamination • Improves reliability AUTOMATIC DISPENSING APPARATUS • Aspirates & dispenses preset volume of two different liquids • 2 motor driven syringes • Microprocessor controlled • Easily programmed and stored in memory PERSONAL COMPUTER CONTROLLED APPARATUS ROBOTIC LIQUID HANDLING WORK STATION • For clinical,genomic,proteomic applications • 96-384 well microtiter plates • Single or multi probed • 2 dimensional movement of probes • 0.5-1000ul in serial dilutions 4-16 channels ROBOTIC LIQUID HANDLING WORKSTATION ROBOTIC LIQUID HANDLING WORKSTATION BULBS TIPS Commonly available pipette volumes Min. volume (µL) Max. volume (µL) P2 0.2 2 Orange 10 P10 1 10 Red 10 P20 2 20 Lemon 200 P100 20 100 Salmon 200 P200 50 200 Yellow 200 P1000 200 1000 Blue 1000 P5000 500/1000 5000 Purple 5000 P10000 1000 10000 Sky 10000 Name Color on Gilson[clarification needed] tip size (µL) PIPETTING TECHNIQUES • Tip must be immersed in liquid to be transferred • Without touching the vessel wall • Pipet is held upright, not at an angle • Pipet bulb or similar device used to apply slight suction READING MENISCUS NBS • National bureau for standards-standard specifications for glass volumetric apparatus Class A Borosilicate volumetric glassware • Superior thermal and chemical resistance • Better suited - glassware for storage of solutions, e.g. flasks. • Accuracy is retained over a longer working lifetime than their soda-lime equivalents.