Calibration of Pipets

A Statistical Point of View

Types of Pipets
Two Main Types
- TC (to contain) - TD (to deliver)

Purpose of Calibration
To determine the random error introduced by human error. Then to determine systematic error introduced due to poor manufacturing tolerances in either the balance or the pipet itself.

Gravimetric Titration
Conventional volumetric calibration procedure using gravimetry i.e. by transferring aliquots of known density from the pipet to a weighing vessel and measuring the masses transferred.

Gravimetric Calibrations
Gravimetric calibration uses an assumed correct and working balance to give a particular amount of a fluid at a known weight using a standard such a H2O. Experimental conditions used in calibration should match as closely as possible the conditions of the experiment itself

Advantage to Gravimetric Titration

Easy to set up Distilled H2O is easy to acquire Cheap Can be done with very few aliquots Calibration of volumetric devices by gravimetry is simple, effective, and wellknown and under ordinary circumstances this procedure and associated data analysis are straight forward

Disadvantages to Gravimetric Calibration

Not an accurate procedure Assumes low error and a precisely calibrated balance The balance MUST be properly calibrated To improve accuracy, many aliquots must be taken Taring can be time consuming depending on vessel

Analysis
Plot large amounts of aliquots and see if they drift aimlessly about the mean, if they dont then some other systematic error must be found and corrected. The smaller the aliquot, the larger and easier errors are to introduce. To counteract this taring is best done performed as well as containment of evaporation along with large numbers of aliquots measured.

EXPERIMENTAL PROCEDURES
Two procedures: tared and untared Same equipment and materials used for both procedures

EXPERIMENT 1 (TARED)
Amount of trials: 40 aliquots

Each aliquot: 20.0% capacity Mean: 0.19069 g Standard deviation: 0.0032 g Standard error: 0.00051 g System Correction: -0.0084 mL The temperature of the water was measured to be 21.7 degrees Celsius +/- 0.2 degrees, and the density of the water was calculated to be 0.9978 g/mL. With a mean of 0.1906 g and a standard error of 0.0005 g, the nominal volume delivered by the micropipette was 0.1911 g.

EXPERIMENT 2 (UNTARED)
Amount of trials: 40 aliquots Each aliquot: 20.0% capacity Mean: 0.19064 g Standard deviation: 0.0034 g Standard error: 0.0006 g System Correction: -0.0094 mL

Values for mean, standard deviation, and standard error were calculated through extrapolation of a residual plot trend.

COMPARISON OF EXPERIMENT 1 AND EXPERIMENT 2

Experiment 1

Experiment 2
Mean: 0.19064 g Standard deviation: 0.0034 g Standard error: 0.0006 g System Correction: 0.0094 mL

Mean: 0.19069 g Standard deviation: 0.0032 g Standard error: 0.0005 g System Correction: -0.0084 mL

Factoring in the standard deviation and standard error, the difference in system correction was 0.0010 mL and the standard error estimate was 0.0008 mL. Evaluating these values with a t test affirms that these values are consistent.

Results

Experiment One vs. Experiment Two vs. Lab1 (Calibration of glassware)

Experiment One
Weighed 40 aliquots , tared immediately before each weighing . Standard deviation- .0032g Volume change of -.0084 +- .0005 The .0005 being rate of water evaporation * time interval.

Experiment One (Cont.)

The independent random component can be found by the residual variable being subtracted from the previous residual.

Experiment One (Cont.)

Volumetric Variance - S= 1.02x10^-5 ml^2 based on 39 degrees of freedom which leads to a volumetric standard deviation of Sv=.0032 mL based on 39 degrees

Experiment Two
Weighed 40 aliquots , Did not tare before each weighing . Standard deviation- .0034 Volume change of-.00094 +- .0006 The .0006 being rate of water evaporation * time interval.

Experiment Two (Cont.)

Experiment Two (Cont.)

Volumetric Variance - S= 1.16x10^-5 ml^2 based on 37 degrees of freedom which leads to a volumetric standard deviation of Sv=.0034 mL based on 37 degrees.

Lab 1 (Calibration of Glassware)

Used measurements of 10ml , 20ml , 30ml , 40ml and 50ml of distilled water. Measured mass of water From mass and density we found the volume .

Lab One ( cont.)

Graphed experimental amount of water vs. the theoretical amount of water.

Discussion
The difference of change in volume between the 2 experiments is 0.0010 mL and the standard error estimate of the difference is .0008 mL. A (t) test for the hypothesis that the difference is within statistical scatter of zero which leads to an acceptance of the hypothesis at any reasonable confidence interval.

Discussion (Cont.)
For the volumetric variances we take the ratio (1.16x10^-5)mL / (1.04x10^-5) mL The ratio of the volumetric variance estimates gives us 1.14 which is less than the tabulated F- statistic for any reasonable confidence interval

Discussion (Cont.)
Thus we may accept the null hypothesis and the two variances are from the statistical population and conclude that the volumetric standard deviations of .0032 and .0034 mL are consistent.

Works Cited
Calibration of pipettes A statistical view (Lowell M. Schwartz)- dept of chemistry , University of Massachusetts , Boston. ( Via ACS.com. Anal. Chem . 1989 61, 1080-1083
Meyer , S. L Data for Analysis for scientist and Engineers ; Willey : New York 1975 chapter 33

Mandel , J.J Am. Statist. Assoc. 1957 , 52 , 552-566

Mandel , J.J the statistical Analysis of Experimental Data ; Willey ; New York, 1964 , Sections 12.7 , 12.8

Resource and Discussion

Statistical uncertainties and accuracies
Matching experimental conditions Properly calibrated weighing machine

The pipet maybe used to contain or deliver the aliquot Two Ways
Calibrated series of vessel (tared) Using single vessel (Untered)

Precision of the Experiment

Highly reliable data
30 times or more replications Use large number of vessel OR Use one vessel to transfer series of aliquots

Tared Vs Untared
Tared Taring vessel is time consuming and troublesome Evaporation of liquid negligible Data analysis is simpler Untared No need to tare the vessel each time Must account evaporation of liquid Data analysis very cumbersome

Two Errors
Systematic error
Denote by v Partly comes from the manufacture tolerance Partly result from the fluctuation of dispensing technique between manufacture and analyst

Random error:
Denote v Result from successive aliquot delivered Magnitude depending on the operation sill of the analyst Taking weighing data of the aliquot

Points of Interest
The density of the liquid be known The liquid must be equilibrated at STP condition When plotting the data, make sure the weighing data scattered randomly about the mean. Balance must be able to operate with minimal mechanical and electrical fluctuation

Comparison with Our 1st Lab

Calibration of Pipet 1. Assumed the balance is calibrated 2. Based on volumetric calculation 3. Uses Single or series of vessel 4. Condition STP 5. Statistical replications 30 times or more Calibration of glassware 1. Same 2. same 3. Used only one vessel 4. Condition STP 5. Statistical replications only five times

Compression Continued
Calibration of pipets 7. Evaporation considered 8. Calibration of Micro pipet (1 micro liters) 9. Data analysis is time consuming and complicated Calibration of glassware 7. Evaporation neglected 8. Calibration of mL glassware's (difference 1000 times) 9. Data analysis is less complicated and straight forward

Conclusion

Error Reduction
Calibration of pipets is a delicate procedure where much care must be taken to assure as little human error as possible is introduced, and all systematic errors are taken into account.

Effectiveness
Gravimetric titration is an easy to perform, lost cost/high value procedure to ensure measurement precision.

Availibility
Almost universally available due to all equipment being easy to procure. IE balance, distilled water, and pipets.

Reliability
Can be very reliable when care is taken and large amounts of aliquots are used to reduce the human error introduced. Reliability is low with less aliquots measured; Also assumes working balance.

Questions