Handling Of Pipette, Burets, Measuring Cylinder and

separator funnel

K.RAMALINGAM2015601510

TAMIL NADU AGRICULTURAL UNIVERSITY

THE SEPARATORY FUNNEL

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.

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.

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.

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.

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.

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.)

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

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

Your eye should be level with the top of the liquid

You should read to the bottom of the MENISCUS

Reading a graduated cylinder’s volume

From above meniscus WRONG!!!!!

From below meniscus WRONG!!!!!

Reading a graduated cylinder’s volume

From the side of the meniscus CORRECT!!!

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.

Practice Reading the Graduated Cylinder

What is this reading?

18.0 ml

Clamp the buret to the buret stand.

close stopcock

open stopcock

Rinse the 50.00 mL buret using a small quantity of the base solution (use the glass

funnel to avoid spillage).

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

solution.

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.

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).

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

Place the Erlenmeyer flask under the tip of buret and put

the Kimwipe under the Erlenmeyer flask (white

background).

Titrate to the end point.

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.

End point. Over titrated end point –

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

Pasteur Pipette

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.

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.

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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

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8 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).

Some Micropipettes have preset

volumes and cannot be adjusted. This pipette will only deliver 200μL.

Parts of the PipetteParts of the Pipette

Pipette tips

Pipette GripPipette Grip

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.

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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)

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278μL

- 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

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278μL

- Extra volume (? μL)

- Find all three positions.

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Volume Adjustment Knob:

Digital Volume Indicator:

Pipettors – 3 Volumes:

Step 1: Set the VolumeStep 1: Set the VolumeOperating the MicropipetteOperating the Micropipette

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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

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Settings can vary between models and manufacturer s.

This pipette’s range is 20-100 μL and is set to deliver 27.8μLHowever you might see the following readings for this exact setting on different pipettes:

Setting Volume

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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.

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Settings can vary between models and manufacturer s.

This pipette’s range is 1-10 μL and is set to deliver 2.78μLHowever you might see the following readings for this exact setting on different pipettes:

Setting Volume

2 7 8

2 .7 8

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Some companies will change the color of the plunger button.

Example of tip sizes:

Attaching the disposable tip

Step 2: Attach the Disposable TipStep 2: Attach the Disposable Tip

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

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.

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

 

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

Step 9: Withdraw the PipetteWith 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.

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8 Proper Use

Attaching tipBe 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

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Attaching tip

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

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Obtaining a Sample

STEP ONE – press plunger to first stop and hold.

HOLD

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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

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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

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

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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

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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

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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

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Delivering the Sample

STEP SEVEN – Allow the plunger to return home.

KEEP the pipette VERTICAL at all times

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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

Things to AVOID !!

278

Never use a pipette in anything but a vertical orientation.

Things to AVOID !!

278

Never use a pipette without a tip.

Accuracy and Accuracy and PrecisionPrecision 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

Cont…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

Pippetting Guidelines and PrecautionsPippetting Guidelines and PrecautionsFor 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