Handling of pipette ,buret,separatory funnnel, graduated cylinder

this chapter clearly explain the Handling of pipette ,buret,separatory funnnel, graduated cylinder during the experiment in the laboratory ..this are the common practices in the science lab...

1. Handling Of Pipette, Burets, Measuring
Cylinder and separator funnel
K.RAMALINGAM
2015601510
TAMIL NADU AGRICULTURAL UNIVERSITY

2. THE SEPARATORY FUNNEL

3. SEP FUNNEL STEP -1
Putthefunnelinaring.Makesurethe
stopcockisclosed!
Taketheprecautionofputtingaflask
underneathincaseyouforgettoclose
thestopcocklater,oritleaks,orthere
isanothermishap.

4. SEP FUNNEL STEP -2
Thereareseveralvariationsofthe
extractiontechniquethatallusea
separatoryfunnel.
Thispresentationshowstheextractionof
anorangesubstancefromanaqueous
solutionintoether.
Firstpourthesolutionintothefunnel.

5. SEP FUNNEL STEP 3
Pourintheether.Itwillformaseparatelayer.Becauseetheriscolorless,it
ishardtoseeinthispicture.Thetopoftheetherlayerisshownbythearrow
inthedetailbelow.
Ahelpfulguideline:Useaboutequalvolumesofthetwoliquids(herethe
watersolutionandtheether).Thetotalvolumeofliquidshouldbeabouthalf
to3/4thecapacityofthefunnel.

6. SEP FUNNEL STEP 4
Closethefunnelwithatightly-fitting
stopper.Graspthestopperfirmlyin
place,invertthefunnel,andimmediately
ventbyopeningthestopcock.
Therewillbearushofvaporandmaybe
asprayofliquidoutofthefunnelstem.
Forthisreason,besurenottopointthe
stematanyoneinthelab,yourself
included.

7. SEP FUNNEL STEP -5
Shake once or twice
and vent again.
Shake a few more
times and vent again.
Shake more
vigorously and vent
less frequently as
you proceed.

8. SEP FUNNEL STEP -6
Puttheseparatoryfunnelbackinto
thering,removethestopper,andlet
thelayerssettle.
Hereyoucanseethatmostofthe
orangesolutehasbeenextractedinto
theether,leavingbehindatracein
theloweraqueouslayer.(Most
organicmaterialsarecolorless,and
sobothlayerswillalsobecolorless.)

9. Drainoffthelowerlayer,takingcareto
makeascleanaseparationaspossible.
Pourtheupperlayeroutofthetopinto
anothercontainer.
Toobtainahigheryieldoftheorange
substance,youcanputtheaqueouslayer
backintothefunnelanddoasecond
extractionwithfreshether.
SEP FUNNEL STEP -7

10. Graduated Cylinder
Liquidsinglassandsomeplastic
containerscurveattheedges
Changingthediameterofthecylinderwill
changetheshapeofthecurve
ThiscurveiscalledtheMENISCUS

11. Your eye should be level with the top of the liquid
You should read to the bottom
of the MENISCUS

12. Reading a graduated cylinder’s
volume
From above meniscus
WRONG!!!!!
From below meniscus
WRONG!!!!!

13. Reading a graduated cylinder’s
volume
From the side of the meniscus
CORRECT!!!

14. Measuring volume of an irregularly-shaped
object
1.Putamoderateamountofwaterinagraduatedcylinder
andmeasurethevolume.
2.Placetheobjectinthegraduatedcylinderwiththewater.
3.Measurethevolumeofthewaterinthegraduatedcylinder
withtheobjectsubmergedinit.
4.Subtractthevolumeofjustthewaterfromthevolumeof
thewaterwiththeobjectsubmerged.Thisvaluetells
youthevolumeoftheobject.

15. Practice Reading the Graduated Cylinder
What is
this
reading?
18.0 ml

16. Clamp the buret to the buret stand.

17. close stopcock
open stopcock

18. Rinse the 50.00 mL buret using a small
quantity of the base solution (use the glass
funnel to avoid spillage).

19. Turn and rotate the buret so all inside
surfaces have come into contact with the base
solution.

20. Make sure that the tip of the buret
is rinsed!
Drain and discard the base solution into
the waste beaker. Rinse the buret three
times with a small quantity of this solution.

21. Next, fill the buret with the base
solution.
Remove the funnel and put the waste
beaker under the tip of the buret. Open the
stopcock and allow some solution to run
through (ensure that the buret tip is filled
with the base solution and contains no air
bubbles).

22. Close the stopcock and wait a
few seconds for drainage to
be complete, then note the
reading on the buret to two
decimal places (V
initial).
V
initial= 3.78 mL

23. Place the Erlenmeyer flask
under the tip of buret and put
the Kimwipe under the
Erlenmeyer flask (white
background).

24. Titrate to the end point.

25. When you are close to the end
point use the drop-by-drop
approach and rinse the last
drop from the tip of the buret at
the side of the Erlenmeyer flask
with the distilled water.

26. End point.
Over titrated end point –

27. Close the stopcock and wait a
few seconds for drainage to
be complete, then note the
reading on the buret to two
decimal places (V
final).
V
final= 18.20 mL

28. TYPES OF PIPETTES
Pasteur Pipette

29. Pour 20 mL of acid solution into the 50 mL beaker and rinse the
15.00 mL volumetric pipet three times with a small amount of it.

30. Fill the pipet with the acid
solution above the calibration
mark. Dry the outside of the
pipet with a Kimwipe.
Make sure to place
your index finger on
the end of the pipet.
To obtain an accurate reading,
you should have the calibration
mark (meniscus) at eye level;
i.e., your line of sight should be
parallel with the mark.

31. 200
-
1000
2 7 8

32. Introduction
Automaticpipettesareusedtoaccuratelytransfersmallliquid
volumes
Glasspipettesarenothighlyaccurateforvolumeslessthan1
milliliter(1ml),buttheautomaticpipettesarebothaccurateand
precise
Thesearecontinuouslyadjustabledigitalpipettes
Eachpipettecanbesettotransferanyvolumewithinitsownvolume
range

33. 200
-
1000
μ
L
2 7 8
ThisisaMicropipetteitisusedtoaccurately
transfersmallquantitiesofliquid.
Onthismodelthedesiredvolumecanbeadjusted
withintherangeofmicroliters(μL).

34. 200
μ
L
Some Micropipettes have preset volumes
and cannot be adjusted.
This pipette will only deliver 200μL.

35. Parts of the Pipette
Pipette tips

36. Pipette Grip

37. Howdoesitwork?
Itworksmuchlikeasyringethatwould
deliveraninjection.
Insidethereisaspringloadedpiston
thatmovesupanddown.
2 7 8

38. Theplungerhastwostops:
-attheFirstStopthepistonmoves
throughthevolumethatisseton
thepipette.(Inthiscase278μL)
2 7 8
2 7 8
278μL

39. -Thesecondstopisextratravelused
forcertaincircumstanceslike
evacuatingthetip,ordrawingup
morethanthevolumeindicatedon
thepipette
2 7 8
2 7 8
278μL
-Extra volume (? μL)

40. -Findallthreepositions.
2 7 8
2 7 8

41. Volume Adjustment Knob:
Digital Volume Indicator:
Pipettors –3 Volumes:
Step 1: Set the Volume
Operating the Micropipette

42. 200
-
1000
2 7 8
Set the correct volume on your
micropipette by turning the adjustment
knob (typically the plunger button).
This pipette’s range is 200-1000μL and is
set to deliver 278μL
Setting Volume
2 7 8

43. 20
-
100
2 7
8
Settings can vary between models and
manufacturer s.
This pipette’s range is 20-100μL and is set
to deliver 27.8μL
However you might see the following
readings for this exact setting on different
pipettes:
Setting Volume
2 7 8
2 7 . 8
2 7 8
The first two make the
decimal places obvious,
while the last one implies
the tenths place if you
know the range.

44. 1.00
-
10.002
7
8
Settings can vary between models and
manufacturer s.
This pipette’s range is 1-10μL and is set to
deliver 2.78μL
However you might see the following
readings for this exact setting on different
pipettes:
Setting Volume
2 78
2 .7 8
2 7 8
Some companies will change the color of the
plunger button.

45. Example of tip sizes:
Attaching the
disposable tip
Step 2: Attach the Disposable Tip

46. Step 3: Depress the Plunger to
the First Stop Step 4: Immerse Tip in Sample

47. Step 5: Draw up the sample
Toaspiratethesampleintothetip,allowthepushbuttontoreturnslowlyand
smoothlytothefullyextendedUPPOSITION.NEVERLETTHEPLUNGERSNAP
UP!Thisdrawstheexactcalibratedvolumeintothetipifthetipremainsbelowthe
liquidsurfaceduringwithdrawal.
Step 6: Pause
Waitafewsecondstoensurethatthefullvolumeofsampleisdrawnintothe
plastictip.WAITLONGERFORLARGERVOLUMES.WAITLONGER
FORMOREVISCOUS("SYRUP-LIKE")SUBSTANCES.

48. Step 7: Withdraw the Tip
Removethetipfromthesampleliquid.NoliquidshouldremainontheOUTSIDE
ofthetip.Wipeawayanydropletsontheoutsideofthetipwithalint-freetissue,suchas
KIMWIPES,butonlywipedropletsfromthesideofthetip.NEVERTOUCHTHETIP
OPENINGoryoumayabsorbpartofyoursample.
Proper Droplet Removal
WRONG Droplet Removal

49. Step 8: Dispense the Sample
To dispense the sample from the pipette:
a) Touch the tip end to the side wall of the receiving vessel and
b) Depress the plunger to the FIRST STOP.
c) Pause for at least one second--1-2 seconds for P-1000, 2-3 seconds for P-5000,
or longer for viscous liquids.
d) Press the plunger to the SECOND STOP (the second point, of greater resistance,
at the bottom of the stroke) to expel any residual liquid in the tip (like "blowing
out" a glass pipette).
(a) Start
Dispensing
(b) 1st Stop =
Dispense
(c) 2nd Stop =
Expel

50. Step 9: Withdraw the Pipette
Withtheplungerfullydepressed,withdrawthepipetfromthereceivingvessel
carefully,slidingthetipalongthewallofthevessel.Holdingthetipagainstthesideofvessel
isespeciallyimportantwhentransferringsmallvolumesofliquid.
Step 10: Release the Plunger
Gently allow the plunger to return to the UP position. DO
NOT allow it to SPRING BACK!
Step 11: Discard the Tip
Discardthetipbydepressingthetipejectorbutton,asshown
below.Afreshtipshouldbeusedforeachsampletoprevent
samplecarryover.
Press ejector button to discard tip.

51. 2 7 8
Proper Use 

52. Attaching tip
Be sure to choose the proper size tip.
0.5 –10 µl = White tips
20 –200 µl = Yellowtips
500 –1000 µl = Bluetips
> 1000 µl = White tips

53. 2 7 8
Attaching tip
Press the pipette into the tip
firmly to create an airtight seal.

54. 2 7 8
Obtaining a Sample
STEP ONE –press plunger to
first stop and hold.
HOLD

55. 2 7 8
Obtaining a Sample
STEPTWO–Inserttipinto
sampleonlyfarenoughto
ensureitstayssubmerged
butnottothebottomwhere
itwillgetblocked.
HOLD
KEEP the pipette VERTICAL at all times

56. 2 7 8
Obtaining a Sample
STEPTHREE–Allowplunger
toreturntothehome
positionSLOWLYsoyoudon’t
drawinairbubbles,orsplash
sampleupintotiporthe
pipetteitself.
KEEP the pipette VERTICAL at all times

57. Obtaining a Sample
Removed tip from sample
before the plunger was all
the way home
Likely allowed the plunger
to move to home too
quickly.
CORRECT

58. 2 7 8
Delivering the Sample
STEP FOUR –insert tip into
the area you wish to deliver
your sample. (in this case a
gel for DNA fingerprinting)
KEEP the pipette VERTICAL at all times

59. 2 7 8
Delivering the Sample
STEPFIVE–depressthe
plungerslowlytothefirst
stop,thencontinuetothe
secondstop,thiswill
evacuatetheentirecontents
ofthetip.AndHOLD
HOLD
KEEP the pipette VERTICAL at all times

60. 2 7 8
Delivering the Sample
STEP SIX –While still holding
the plunger at the second
stop. Withdraw the tip from
the well.
HOLD
KEEP the pipette VERTICAL at all times

61. 2 7 8
Delivering the Sample
STEP SEVEN –Allow the
plunger to return home.
KEEP the pipette VERTICAL at all times

62. 2 7 8
Discarding the Tip
STEP EIGHT –Place tip into
the opening of the waste
container, then depress the
tip ejector.
WASTE
Tip Ejector
Be sure to use a new
tip each time.
KEEP the pipette VERTICAL at all times

63. Things to AVOID !!
Never use a pipette
in anything but a
vertical orientation.

64. Things to AVOID !!
278
Never use a pipette
without a tip.

65. Accuracy and Precision
Accuracymeanstheclosenesswithwhichthedispensedvolume
approximatesthevolumesetonthepipette
Accuracyisspecifiedasmeanerror,theaveragedeviationofreplicate
measurementsfromtheexpectedsetvolume
Precisionisthe"scatter"orreproducibilityofindividualmeasurementsof
thesamevolume
Precisioncanalsobeexpressedasstandarddeviation

66. Cont…
Relative accuracies are generally about 1% or less
Precision is less than 0.5 % except when transferring the
smallest recommended volume for a given pipette model
Using the pipettes to transfer volumes which are below the
recommended range will introduce larger errors

67. PippettingGuidelines and Precautions
Foroptimalreproducibility,usethefollowingpipettingprocedures:
(1)ConsistentSPEEDandSMOOTHNESS whenyoupressandreleasethe
PLUNGER
(2)ConsistentpressureonthePLUNGERattheFIRSTSTOP
(3)ConsistentandsufficientIMMERSIONDEPTH
(4)NearlyVERTICALPOSITIONINGofpipette
(5)AVOIDALLAIRBUBBLES:Sincetheplasticpipetteshaftcanbe
damagedifliquidsaredrawnbeyondthetipintotheshaft
(6)NEVERlaythepipetteonitsSIDEnorINVERTthepipetteifliquidisin
thetip