Accuracy and Precision

FSC 455 Food and Nutrition Laboratory

Lab 1-Weighing & Pipetting Accuracy and Precision

Learning Objectives:

The purpose of this exercise is to introduce to you the types of errors in laboratory experiments and how to minimize these errors. You will practice using the balances and pipettes in the lab. At the end of the lab, you should:

  1. Understand the concept of accuracy & precision, and how they are affected by random

& systematic errors.

  1. Know the means to reduce random & systematic errors.
  2. Be familiar with the balances and pipettes in the lab and be able to use them correctly in the subsequent lab sessions.

Lab Report Instructions:

  • Work in groups of 3 or 4.
  • Individual Report, submitted at the end of class. Make sure the TA or the instructor has checked your tables and signed off the pag
  • If you don’t turn in the signed copy of page 6, you will be counted absent for the lab

Introduction:

Accuracy and precision are critical in obtaining consistent and reliable results in experimental research.

Accuracy is having confidence that your measurements are true. For example, if you weigh 5 grams; how do you know it is truly 5 grams (not 5.5 or 4.5 grams)? The accuracy of measurements is often reduced by systematic errors. A systematic error would always be in the same direction and by the same amount. Systematic errors in experimental observation usually come from the instruments:

  1. There is something wrong with the instrument. For example, the instrument is not correctly calibrated.
  2. The instrument is wrongly used. For example, the balance is not set to zero before weig

In order to achieve accuracy, systematic errors need to be minimized by:

  1. Calibration of the instruments – This is done by calibration service companies.
  2. Using the instruments correctly –You are responsible for this part.

The precision of measurements is how close a number of measurements of the same quantity agree with each other. For example, if you measure a 5g standard weight three times, the result may be 4.97g, 5.01g, and 5.02g. Alternatively, it may be 4.9g, 5.0g, and 5.1g. The first set of data is more precise than the second set of data because they are closer to one another. The precision of measurements is affected by random errors. Random errors are unknown and unpredictable changes in the environment or the instruments. Unlike a systematic error, a random error has an equal chance to make the result too high or too low. Random errors can be caused by many things. Examples are:

  1. Temperature fluctuations in the lab.
  2. Electronic noise in the circuit of an electrical instrument.

Random errors can be corrected for by repeating the measurements and averaging the results. An easy way to visualize accuracy and precision is through marks on a target presented in the

following figure.

Figure 1. A “target” illustration to demonstrate accuracy and precision.  a.  The measurements are very close together or precise, but off center (inaccurate). b. The measurements all hit the mark, so some are accurate while others are not as accurate, but all of the marks at least hit the target. The spread of the marks indicates imprecise measurements. Also note that there is an equal chance for a mark to go left, right, up, or down from the center. c. The measurements are accurate and precise, or close together and in the center. The goal of all scientific measurements is to be accurate and precise similar to Figure 1c.

A micropipettor (often called a pipette in general) is used to transfer small amounts (< 1 mL) of liquids. The scales on micropipettors are in microliters (1000µl = 1 mL). They come in three sizes which are capable of pipetting three ranges of volumes (lower and upper limits):

P2 = 0.2-2 µl

P20 = 0.5- 20 µl

P200 = 20-200 µl

P1000 = 200-1000 µl.

They are used in conjunction with disposable (often sterile) plastic tips. The following is an illustration of a micropipettor:

How to use a micropipettor:

Never:

  1. Exceed the upper or lower limits of pipettors
  2. Use w/out the tip in place (this could ruin the precision piston that measures the volume of fluid)
  3. Lay down a micropipettor with a filled tip (fluid could run back into the nose cone (piston))
  4. Let the plunger snap back after withdrawing or ejecting fluid (this could damage the piston)
  5. Immerse the barrel (nose cone) of the micropipettor in fluid

Always:

  1. Set the desired volume by turning the centrally located rings clockwise to increase volume or counterclockwise to decrease volume. Some examples are provided below:
  2. Place a tip on the discharge end of the pipettor.
  3. The plunger will stop at two different positions when it is depressed.
    • 1st Stop: point of initial resistance and is the level of depression that will result in the desired volume of solution being transferred.
    • 2nd Stop: used for the complete discharging of solutions from the plastic tip. You should not reach this second stop when drawing liquid into the pipettor, only when expelling the last drop!
  4. Depress the plunger until you feel the initial resistance, then insert tip into the solution, just barely below the surface of the liquid and not as deep as possible.
  5. Carefully and slowly release plunger. NOTE: If the solution you are pipetting is viscous, allow the pipet tip to fill to final volume before removing it from solution to avoid the presence of bubbles in the plastic tip which will result in an inaccurate volume. If you release the plunger too quickly, it will suck liquid up into the pipettor and damage it.
  6. Discharge the solution into the appropriate container by depressing plunger to the point of initial resistance, wait one second, and then continue pressing the plunger as far as it will go in order to discharge the entire volume of solution.
  7. Remove tip by pressing down on the tip discarder.
  8. When using the balance, make sure the level indicator (water bubble) on the right rear side of the balance is in the center of the circle.

Never put down a pipette with the tip still attached!

Experimental Procedure:

In this lab exercise, you will practice using various pipettes with different volume ranges and determine your accuracy of pipetting using an ANALYTICAL BALANCE.

  1. Find the set of pipettes (1000 μL, 200 μL, and 20 μL) assigned to your group, pipette tips and a beaker of distilled water.
  2. Carefully use each pipette to measure the volumes of distilled water according to the list presented in table 1 and table 2, by pipetting into a paper or aluminum weigh boat. Record the volumes in the table.
  3. Make the same tables in you lab notebook and record the weights of each volume in your lab notebook to complete table 1 and table 2.

Note: The density of water is 1.0 gram/milliliter (1.0 g/mL). Therefore, the weight of 1 mL (1000 μL) should be very close to 1.0 g on the scale. (If you measure 50 μL, it should weigh 0.050 g, etc.)

  1. In your notebook, calculate the % difference for each target volume by using this formula for each data point.
  1. In your notebook, calculate the standard deviation and coefficient of variation of the three scale weights of each designated volume for each data point.
  1. Average the % Difference of all the target volumes for each pipette. Obtain the “Pipetting Mean Accuracy” of each pipette using this formula:
  1. Average together the “Pipetting Mean Accuracy” to obtain your overall “Pipetting

Mean Accuracy”.

  1. Record the accuracy of your pipetting in table 1 and table 2 on the next page.

References:

Department of Biology, Davidson College, Davidson, NC, 28036.2002. How to Use a Micropipettor.  Available from http://www.bio.davidson.edu/courses/Molbio/Protocols/pipette.html. Accessed 2016  May 17.

Thermo Scientific. 2010. Good Laboratory Pipetting Guide. Available from:

https://assets.thermofisher.com/TFS-Assets/LSG/brochures/D16542.pdf. Accessed on 2018 Aug 28.

FSC 455 Lab 1-Weighing & Pipetting Accuracy and Precision        Due at the end of class

Table 1. Determining Student’s Ability to Accurately Pipet Repeatedly (Large Volumes)
Pipette Target Volume Target Weight Scale Weight Difference in Weight % Difference Standard Deviation Coefficient of Variation % Pipetting Mean Accuracy %
1000 µL 750 µL 0.7500 g
750 µL 0.7500 g
750 µL 0.7500 g
200 µL 150 µL 0.1500 g
150 µL 0.1500 g
150 µL 0.1500 g
20 µL 15 µL 0.0150 g
15 µL 0.0150 g
15 µL 0.0150 g
Overall Mean Pipetting Accuracy

Table 2. Determining Student’s Ability to Accurately Pipet Repeatedly (Small Volumes)
Pipette Target Volume Target Weight Scale Weight Difference in Weight % Difference Standard Deviation Coefficient of Variation % Pipetting Mean Accuracy %
1000 µL 100 µL 0.1000 g
100 µL 0.1000 g
100 µL 0.1000 g
200 µL 20 µL 0.0200 g
20 µL 0.0200 g
20 µL 0.0200 g
20 µL 3 µL 0.0030 g
3 µL 0.0030 g
3 µL 0.0030 g
Overall Mean Pipetting Accuracy

My overall ability to pipet large volumes was calculated to be            % accurate on analytical balance #                .

My overall ability to pipet small volumes was calculated to be            % accurate on analytical balance #                 .

Student Name:                                                                      PID       #:                                         

 

Circle your section day and group #.

 

Section:   M    T    W     R     F            Group:   1    2    3     4     5