Measuring Volume

Biotech Ipg. 301 to 323 in Basic Laboratory Methods

Volume

Liquid volumes are measured in Liters (L), milliliters (mL) or microliters (L)

A liter is slightly more than a quart A microliter is 1/1000 of a mL or smaller

than a tiny drop Depending on the volume to be

measured, 3 options: graduated cylinder, pipet or micropipet

B S Rule

Bigger units left Smaller units right Move decimal point to the right if

converting big units to small units Move decimal point to the left is

converting small units to big units

B S Rule

1.25 L is equal to how many mLs? 1250 mL

How many liters is 60 mLs? 0.06 L

250 uL is how many L? 0.00025 L

Small to Medium Volumes

Typically 0.1 mL to 25 mL Plastic disposable or glass pipets Pipette roller or pipette bulb is needed Commonly used pipettes are:

1mL, 5mL, 10mL, and 25 mL Pipets are named by the maximum they deliver

Pipetting Terminology

Graduations: lines marked on volume measuring devices that indicate volume

Meniscus: a curve formed by the surface of liquids confined in a narrow space, such as in a measuring device

Pipetting Terminology

TC: To Contain; will contain the specific amount when filled to the capacity mark. It will not deliver the amount if the liquid is poured out because some of the liquid will adhere to the sides of the container.

Pipetting Terminology

TD: To Deliver; marked so that it will deliver the specified amount, assuming the liquid is 20C and is poured out.

Note: plastic containers are considered non-wetting which means water does not stick to them so there is no difference between TD and TC

Pipetting Terminology

Tolerance: how much error is allowed in the calibration or a measuring device.

Volumetric: the most accurately calibrated glassware

The “art” of Pipetting

Learning how to pipette takes practice and determination

Patience and careful lab technique will ensure your results are accurate and reliable

Steps to Proper Pipetting

Determine the proper pipette size Determine the proper pipette roller or bulb

Blue: 1 mL pipette Green: 5 or 10 mL pipette

Draw up past your required volume and dispense to your exact fill volume required

Pay attention to the meniscus

Pipetting Lab: Precautions

Never mouth pipette Hold the bottom of the roller and the top

of the pipette when you are assembling them

Always keep the pipette vertical when there is fluid in it

Always keep the tip of the pipette sterile and take care to not touch surfaces

Pipetting Lab: see handout

See your hand out for lab practical using disposable pipettes

Pipets with a TD on them indicate they are designed “To Deliver” and that the tiny amount left in the bottom of the pipet after dispensing should NOT be blown out.

Really Small Volumes

Most QC and R&D laboratories measure very small volumes.

Micropipets are used to measure microliter quantities. What is the symbol for microliter? L

Micropipets are expensive instruments which must be handled carefully

Getting to Know your micropipet

We have 2 types of pipets in our lab P20: for 2 to 20 L P200: for 20 to 200 L

Let’s take a look at your micropipet diagram handout. Keep this handout out for the micropipet

lab exercise

Micropipetters

Getting to Know your micropipet

There are 4 parts to a micropipet Plunger button Ejector button Volume display (setter) Dispensing Tip

Getting to Know your micropipet

Plunger button Typically there are 2 stops

The first evacuates the air in the micropipet The second stop evacuates the volume plus

another 50% or so. Practice to feel the difference

Picking the proper size micropipet

Look at your plunger It will give you the min. and max. that it

can deliver 3 numbers will be displayed

The top # is the digit for the maximum I.e. 2 is the top # on the P200

Interpret the following settings

How many microliters is this

on a m10? on a m100 on a m1000

3 5 0

3.50 uL or 0.00350 mL

3 5 0

35.0 uL or 0.0350 mL

3 5 0

350.0 uL or 0.350 mL

Setting your required volume

To set your volume you need to turn the adjusting knob. Rotate slowly to the desired volume

setting At no time should the knob be turned

past its upper or lower limits. Do not force the settings, the knob will

turn easily

Sources of Error for micropipet

Bad pipet tip: leaks, clogged tip Damaged pipetor Wrong size tip for pipet size Bubbles in your tip due to poor

technique Liquid not at room temperature or it

may be viscous

Multi channel pipets

In order to increase efficiency, pipets have been designed to hold 4-16 channels at a single dispense.

Most commonly used when running ELISA assays 8 channel pipet

Multi Channel pipet

Electronic or Automated pipets

Refer to your handout for a picture of an electric pipet. Typically they are battery operated Press a button to pick up, press button to

dispense Use to increase or decrease volume

settings For large volume labs, automated pipetting

instruments are used. Multi channel heads

Burettes

Long graduated tube with a stop cock at one end which is used to dispense known volumes accurately.

See your handout for a picture. There was one out during the scavenger hunt

Micropipetter Precautions

These are expensive and precise pieces of equipment. DO NOT play with them Handle them with care Keep them clean

Micropipet Lab Exercise

Micropipet operating instructions Choose the proper pipet for the desired

volume, choose proper pipet tip Set the volume Depress the plunger to first stop and draw

up required volume Wipe tip gently with Kimwipe, do not draw

out liquid Depress plunger to expel liquid. Go to the

second stop to blow out all of the liquid.

Calibration

Definition: adjustment of dispensing devices so they dispense accurate volumes.

In the case of pipets we are checking the accuracy and precision of the pipet.

Calibration

Accuracy: to deliver the exact quantity required Mean measured volume divided by

Nominal or required volume Precision: the ability to deliver that

exact quantity time after time The amount of variation between the

each volume measured 1.01, 1.01, 1.02

Calibration Procedure

Pre Calibration Activities Be sure the Micropipetter is clean Be sure the balance is calibrated and

working properly (Verification) Be sure the water you are using is 22 to

25C Ensure the scale is free of drafts

Place a wt. on and verify it provides a consistent reading

Calibration Procedure

Set the Micropipetter to deliver 2 uL on a 2 to 20 uL pipetor or 25 uL on the 20 to 200 uL pipetor

Place a weigh boat on the calibrated balance

Add a few drops of water to the weigh boat Tare the balance by pressing the zero

button

Calibration Procedure

Pipet the appropriate volume and dispense it into the weigh boat.

Record your reading Zero the balance (good practice to change tips each time)

Pipet the appropriate volume and dispense it into the weigh boat.

Record your reading Repeat until you have 5 readings

Calibration Procedure

Set the Micropipetter to 20 uL for 2-20 uL pipet or 200 for 20 to 200 uL pipet

Zero the balance Pipet the appropriate volume and

dispense it into the weigh boat. Record your reading Repeat until you have 5 readings

Calibration Procedure

Calculations: Calculate the mean weight of water

Density of water at 22C = 1.0031 g/mL 1 uL would weigh how many mgs?

1.0031 mgs

Convert the mean water weight to the mean volume weight Mean Volume of water = Mean weight /

density

Calibration Procedure

Mean of 5 weights / 1.0031 mgs i.e. if the weights of 2 uL setting were

2.001, 2.001, 2.003, 2.002, 2.002 mg What is the mean weight?

2.018 What is the mean volume of water?

2.018/1.0031 = 2.006 What is the accuracy of the pipetor?

2.006 / 2.000 x 100 = 100.3% or .3% error

Preventive maintenance of pipets

Wipe the pipet with alcohol if it gets contaminated with solution

Store properly which is in a stand if possible as you do not want the shaft part of the pipet to get bent from laying on a bench