Measuring Volume Biotech I pg. 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?  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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 m uL or mL uL or mL uL or 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 = g/mL 1 uL would weigh how many mgs?  mgs  Convert the mean water weight to the mean volume weight Mean Volume of water = Mean weight / density

Calibration Procedure Mean of 5 weights / mgs  i.e. if the weights of 2 uL setting were 2.001, 2.001, 2.003, 2.002, mg  What is the mean weight?  What is the mean volume of water? 2.018/ =  What is the accuracy of the pipetor? / 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