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
Put the funnel in a ring. Make sure the
stopcock is closed!
Take the precaution of putting a flask
underneath in case you forget to close
the stopcock later, or it leaks, or there
is another mishap.

4. SEP FUNNEL STEP - 2
There are several variations of the
extraction technique that all use a
separatory funnel.
This presentation shows the extraction of
an orange substance from an aqueous
solution into ether.
First pour the solution into the funnel.

5. SEP FUNNEL STEP 3
Pour in the ether. It will form a separate layer. Because ether is colorless, it
is hard to see in this picture. The top of the ether layer is shown by the arrow
in the detail below.
A helpful guideline: Use about equal volumes of the two liquids (here the
water solution and the ether). The total volume of liquid should be about half
to 3/4 the capacity of the funnel.

6. SEP FUNNEL STEP 4
Close the funnel with a tightly-fitting
stopper. Grasp the stopper firmly in
place, invert the funnel, and immediately
vent by opening the stopcock.
There will be a rush of vapor and maybe
a spray of liquid out of the funnel stem.
For this reason, be sure not to point the
stem at anyone in the lab, 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
Put the separatory funnel back into
the ring, remove the stopper, and let
the layers settle.
Here you can see that most of the
orange solute has been extracted into
the ether, leaving behind a trace in
the lower aqueous layer. (Most
organic materials are colorless, and
so both layers will also be colorless.)

9. Drain off the lower layer, taking care to
make as clean a separation as possible.
Pour the upper layer out of the top into
another container.
To obtain a higher yield of the orange
substance, you can put the aqueous layer
back into the funnel and do a second
extraction with fresh ether.
SEP FUNNEL STEP - 7

10. Graduated Cylinder
 Liquids in glass and some plastic
containers curve at the edges
 Changing the diameter of the cylinder will
change the shape of the curve
 This curve is called the MENISCUS

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.Put a moderate amount of water in a graduated cylinder
and measure the volume.
2. Place the object in the graduated cylinder with the water.
3. Measure the volume of the water in the graduated cylinder
with the object submerged in it.
4. Subtract the volume of just the water from the volume of
the water with the objectsubmerged. This value tells
you the volume of the object.

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 (Vinitial).
Vinitial = 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 (Vfinal).
Vfinal = 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
278

32. Introduction
 Automatic pipettes are used to accurately transfer small liquid
volumes
 Glass pipettes are not highly accurate for volumes less than 1
milliliter (1 ml), but the automatic pipettes are both accurate and
precise
 These are continuously adjustable digital pipettes
 Each pipette can be set to transfer any volume within its own volume
range

33. 200-1000μL
278
 This is a Micropipette it is used to accurately
transfer small quantities of liquid.
 On this model the desired volume can be adjusted
within the range of microliters (μL).

34. 200μL  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. How does it work?
 It works much like a syringe that would
deliver an injection.
 Inside there is a spring loaded piston
that moves up and down.
278

38. The plunger has two stops:
- at the First Stop the piston moves
through the volume that is set on
the pipette. (In this case 278μL)
278278
278μL

39. - The second stop is extra travel used
for certain circumstances like
evacuating the tip, or drawing up
more than the volume indicated on
the pipette
278278
278μL
- Extra volume (? μL)

40. - Find all three positions.
278278

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

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

43. 20-100
278
Settings can vary between models and
manufacturer s.
This pipette’s range is 20-100 μL 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. 1.00-10.00
278
Settings can vary between models and
manufacturer s.
This pipette’s range is 1-10 μL and is set to
deliver 2.78μL
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
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
To aspirate the sample into the tip, allow the pushbutton to return slowly and
smoothly to the fully extended UP POSITION. NEVER LET THE PLUNGER SNAP
UP! This draws the exact calibrated volume into the tip if the tip remains below the
liquid surface during withdrawal.
Step 6: Pause
Wait a few seconds to ensure that the full volume of sample is drawn into the
plastic tip. WAIT LONGER FOR LARGER VOLUMES. WAIT LONGER
FOR MORE VISCOUS ("SYRUP-LIKE") SUBSTANCES.

48. Step 7: Withdraw the Tip
Remove the tip from the sample liquid. No liquid should remain on the OUTSIDE
of the tip. Wipe away any droplets on the outside of the tip with a lint-free tissue, such as
KIMWIPES, but only wipe droplets from the side of the tip. NEVER TOUCH THE TIP
OPENING or you may absorb part of your sample.
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
With the plunger fully depressed, withdraw the pipet from the receiving vessel
carefully, sliding the tip along the wall of the vessel. Holding the tip against the side of vessel
is especially important when transferring small volumes of liquid.
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
Discard the tip by depressing the tip ejector button, as shown
below. A fresh tip should be used for each sample to prevent
sample carryover.
Press ejector button to discard tip.

51. 278
Proper Use 

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

53. 278
Attaching tip
Press the pipette into the tip
firmly to create an airtight seal.

54. 278
Obtaining a Sample
STEP ONE – press plunger to
first stop and hold.
HOLD

55. 278
Obtaining a Sample
STEP TWO – Insert tip into
sample only far enough to
ensure it stays submerged
but not to the bottom where
it will get blocked.
HOLD
KEEP the pipette VERTICAL at all times

56. 278
Obtaining a Sample
STEP THREE – Allow plunger
to return to the home
position SLOWLY so you don’t
draw in air bubbles, or splash
sample up into tip or the
pipette itself.
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. 278
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. 278
Delivering the Sample
STEP FIVE – depress the
plunger slowly to the first
stop, then continue to the
second stop, this will
evacuate the entire contents
of the tip. And HOLD
HOLD
KEEP the pipette VERTICAL at all times

60. 278
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. 278
Delivering the Sample
STEP SEVEN – Allow the
plunger to return home.
KEEP the pipette VERTICAL at all times

62. 278
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
 Accuracy means the closeness with which the dispensed volume
approximates the volume set on the pipette
 Accuracy is specified as mean error, the average deviation of replicate
measurements from the expected set volume
 Precision is the "scatter" or reproducibility of individual measurements of
the same volume
 Precision can also be expressed as standard deviation

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. Pippetting Guidelines and Precautions
For optimal reproducibility, use the following pipetting procedures:
(1) Consistent SPEED and SMOOTHNESS when you press and release the
PLUNGER
(2) Consistent pressure on the PLUNGER at the FIRST STOP
(3) Consistent and sufficient IMMERSION DEPTH
(4) Nearly VERTICAL POSITIONING of pipette
(5) AVOID ALL AIR BUBBLES: Since the plastic pipette shaft can be
damaged if liquids are drawn beyond the tip into the shaft
(6) NEVER lay the pipette on its SIDE nor INVERT the pipette if liquid is in
the tip