PRACTICAL GUIDE FOR CHEMICAL

QUALITY ASSURANCE

NATIONAL DIPLOMA IN ANALYTICAL CHEMISTRY

FACULTY OF APPLIED SCIENCE

CHEMICAL QUALITY ASSURANCE

CQA 200S
Compiled by Dr N Darangwa Bellville Campus
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 TABLE OF CONTENTS

GENERAL COURSE INFORMATION

1. Foreword .................................................................................................................iii

2. Principles for safety in chemical laboratory .......................................................iii

3. Course Policies and Information .........................................................................iii
3.1 Laboratory Notebooks ..................................................................................iii
3.2 Guidelines to be followed ............................................................................iv
3.3 Laboratory Session Attendance ...................................................................iv
3.4 Pre-Lab Tests/Evaluations ..........................................................................iv
3.5 Laboratory Report Policies ..........................................................................iv
4. Criteria for grading of Laboratory Notebook ......................................................v

5. Laboratory Report Format ....................................................................................vi

EXPERIMENTS

1. Experiment 1: Sampling in Analytical Chemistry ...............................................2

1.1 Experiment Objectives .................................................................................2
1.2 Theory .........................................................................................................2
1.3 Experimental ...............................................................................................5
1.3.1 Part 1 Experimental Procedure ............................................................5
1.3.2 Part 2 Experimental Procedure ............................................................6
1.4 Data Analysis ................................................................................................7
1.5 Discussion Questions ..................................................................................8
1.6 References ..................................................................................................8

2. Experiment 2: Quality Control for Analytical Balances ......................................9

2.1 Experiment Objectives .................................................................................9
2.2 Introduction .................................................................................................9
2.3 Experimental ...............................................................................................11
2.3.1 Eccentricity Test ..................................................................................11
2.3.2 Accuracy .............................................................................................12
2.3.3 Reproducibility Test .............................................................................12
2.3.4 Linearity Test .......................................................................................13

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 2.3.5 Linear Error Test ..................................................................................13
2.4 Data Analysis ................................................................................................13
2.5 Discussion Questions ..................................................................................14
2.5 References ..................................................................................................14
2.5 Appendices .................................................................................................15

3. Experiment 3: Use and Calibration of Volumetric Apparatus ............................16

1.1 Experiment Objectives .................................................................................16
1.2 Theory .........................................................................................................16
1.3 Experimental ...............................................................................................18
2.3.1 Using and calibrating a micropipette ....................................................18
2.3.2 Using and calibrating the graduated pipette .........................................19
2.3.3 Using and calibrating the volumetric pipette .........................................20
1.4 Data Analysis ................................................................................................20
1.5 Discussion Questions ..................................................................................21
2.5 Appendices .................................................................................................22

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 GENERAL COURSE INFORMATION
1. FOREWORD

This manual has been prepared for the Chemical Quality Assurance practicals and includes the experiments,
which are related to the topics covered in the Chemical Quality Assurance subject. The main purpose of this
laboratory is to provide the students an appreciation for the potential applications and limitations of the
principles learnt in the subject, and expose the students to general issues quality assurance as applied in
analytical chemistry laboratories . It is also aimed to provide the students an opportunity to develop their
abilities in the laboratory skills required for accurate and precise chemical analyses. Therefore, it is expected
that with an acceptable degree of proficiency the students will grasp and apply the principles of analytical
chemistry and quality assurance to obtain reliable results in the laboratory experiments.

2. PRINCIPLES FOR SAFETY IN THE CHEMICAL LABORATORY

The fundamental assumption made of you as you begin Chemical Quality Assurance practicals is that you are
an adult person with a scientific curiosity ready to learn and expand your knowledge in analytical chemistry. This
may not be a valid assumption in all cases, but if this is not your approach to this course, then try to pretend that
it is. Safe practices in the chemical laboratory are of prime importance. A student should consider it an essential
part of his or her educational experience to develop safe and efficient methods of operation in a lab. To do this,
one must acquire a basic knowledge of properties of materials present in the lab, and one should realize the
types of hazards that exist and the accidents and injuries that can result from ignorance or irresponsibility on the
part of the student or a neighbor.

3. COURSE POLICIES AND INFORMATION

3.1 LABORATORY NOTEBOOKS
A scientific notebook should hold a permanent record of the experimental work. Consequently, the pages should
be securely bound (not a spiral binding) and entries made in ink at the time work is done. The pages are
numbered and the entries are dated. The format of the entries must be such that any scientist who is familiar
with quantitative chemical work could read the book. Readability of the original data is essential.

You should prepare your notebook each week before coming to lab by writing the title of the experiment on a
new numbered page, summarizing relevant critical details from the lab manual, and starting preparations
showing the layout of the work you will do in the lab, etc. Take note of theoretical ideas and special instructions
given by your instructor at the start of each experiment. Your notebook should be a complete record of your
work in lab. You or other chemists should be able to understand the notes in the future, not just during the
current experiment. Good note taking in lab is a valuable skill that you can learn with a little effort and practice.
THE NOTEBOOK MUST BE COMPLETED DURING THE LABORATORY PRACTICAL AND MUST BE
SIGNED BY THE LECTURER BEFORE LEAVING THE LABORATORY. The signed laboratory results MUST

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be readily available when requested and SHOULD NOT BE TORN off from the notebook. PLEASE NOTE
THAT THE LABORATORY WILL BE GRADED FOR THIS MODULE. Please check the grading criteria below.

3.2 GUIDELINES TO BE FOLLOWED:

1) Always bring your notebook with you to lab. You may use your notebook during a lab quiz.
2) Number the pages sequentially and reserve space at the beginning for a table of contents.
3) Take your notebook to the balance room, etc. and record values directly in it - not on loose scraps of
paper.
4) Pencils should never be used for recording in the notebook.
5) Specify each measured quantity by name and include the units.
6) If you make a mistake in your notebook, simply draw a solid line through the error and write the
correction nearby.
7) Tables greatly simplify data entry; they should be set up before coming to lab.
8) Write down all observations such as color and phase changes - don't rely on your memory.
9) Save time by doing trial calculations in your notebook before filling out any report sheets.
10) Save time by making preliminary sketches of graphs on the ruled lines in your notebook.

3.3 LABORATORY SESSION ATTENDENDANCE

Attendance is required and you are expected to attend all scheduled laboratory sessions. If you miss more
than two-lab sessions without a valid reason you will be automatically dropped from lab. Should you be
sick and cannot come to lab, you need to bring a doctor’s excuse from the UNIVERSITY HEALTH CENTER, a
sick note from a private doctor or an official report from a government hospital. Also, if you cannot be
available for an official reason and you know this ahead of time, you should notify the lecturer in advance of the
lab period and make sure you obtain the required documentation. Be on time for all the experiments. Tardiness
will be noted by the teaching assistant and can affect your grade.

3.4 PRE-LAB TEST/EVALUATIONS

An unannounced prelab test/evaluations will be conducted during the start of the laboratory sessions. You must
go through your practical before you come to the laboratory and try to understand the concepts that will be
explored the experiment as highlighted in the objectives section of each practical. The marks for the pre-lab test
will be constitute the overall practical mark.

3.5 LABORATORY REPORT POLICIES

Brief reports of analysis will be submitted for each experiment. The marking guide for each report is provided at
the end of each experiment, to guide you on all the relevant information that must accompany your report. Go
through the marking guide before you do any experiment to make sure that all the required information is in your

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notebook before its signed for that experiment. No information must be added to the NOTEBOOK after leaving
the laboratory. All reports will be submitted in an allocated submissions box that the lecturer will advice you.

The nature of the report required will be specified for each experiment at the end of the section for the
experiment. The reports must be submitted A WEEK after the practical was conducted. The penalty for
late lab reports is 5 % per day late up to a maximum of 50 %. All reports more than 2 weeks late will
automatically receive a grade of 50% if they are reasonably complete regardless of quality. This scale is
designed to reward you for giving a good first effort as soon as you finish the experiment. If you delay, you will
almost always get a lower grade.

There is always a possibility that due to illness or other factors you will need an extension on your lab report.
Extensions will always be granted for any reasonable cause. Extensions will only be granted through an e-
mail request to the lecturer. When you request an extension, you must state your general reason for the
extension and give a specific new due date when you will hand in the report. I can accept the proposed due date
or change it. In any case, you will receive an e-mail reply stating the new due date for the lab. You must copy
that message and include it in your lab report when you hand it in for evaluation. If there is no written
extension document submitted, the full late penalty will be assessed. No open-ended extensions will be
granted.

4. CRITERIA FOR GRADING OF THE LABORATORY NOTEBOOK

Points Deducted

1
First Page of Experiment /2
• Title of experiment, name of partners, date - missing
Objective or Purpose
1
• Wrong, unclear or too long /2
Pre-laboratory Information, Data or Calculations
• Balanced reaction equations, formula weights of reagents to be used, 1
amounts to be used, etc. - incomplete or wrong Literature Reference
Experimental Procedure
1
• Too lengthy /2
• Copied word for word from the practical manual 2
• Incomplete 2
• Reagent source not identified (manufacturer of chemical if available) 1
Data and observations
• No unknown number included, if unknown given 3
• Data not in tabular or organized form 1
• Missing data 1
• Required observations missing 1

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 General Comments
• sloppy or illegible writing or writing in pencil 1
• notebook not signed and dated 1
• significant figures not recorded correctly 1
• units missing (whether 1 or more) 1
• errors blotted out instead of being struck with a single line 1

5. LABORATORY REPORT FORMART

After collecting your raw data you will present your results in a FORMAL REPORT. Ideally, the formal
report should focus on the analytical results obtained, and should be neat and easily understandable.
The actual format of the report will include:
• Sample calculations
• Data in tabular form.
• Graphs where appropriate.
• Statistical analysis of the analytical data.
• Any deviations from the written procedure.
• Justification for data exclusion.

N.B. In thus subject, the format of the report, and any further requirements are states at the end of
each practical.

In a case where propagation of error in the analysis is required, one sample calculation that shows the
propagation of error from start to finish must be shown. YOU MUST CLEARLY LABEL
UNCERTAINTIES OF EACH MEASUREMENT FOR FULL CREDIT.

The formal report should not include:

• Material contained in the written procedure
• Multiple examples of the same calculations.
• Excess wordiness.

TITLE PAGE OF THE LABORATORY REPORT.

Title of the practical

Course Name and Code:

Semester and Year:

Name of Student: Group Members:

Date of Report Submission: Grade:

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EXPERIMENTS

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EXPERIMENT 1 : SAMPLING IN ANALYTICAL CHEMISTRY

1.1 EXPERIMENT OBJECTIVES:

At the end of this experiment, the student should be able to:
1. describe the concepts of sampling, experimental error, precision, accuracy as applied to chemical
analysis.
2. explain the concept uncertainty, identify and describe different contributions to the uncertainty of the
final result in a quantitative chemical analysis experiment.
3. describe the role of sampling and sample preparation on overall uncertainty of and calculate the
combined uncertainty

1.2 THEORY:
When we use an analytical method to solve a problem, there is no guarantee that our results will be
accurate or precise. In general, designing an analytical method involves careful review of potential
sources of determinate error and indeterminate error, and take appropriate steps to minimize their effect.
The use of reagent blanks and calibration of the instruments are examples of such measures to minimize
uncertainty in the final result. Furthermore, laboratory exercises typically focus on analysis of a well-
defined unknown sample. However, real life analytical practice presents additional challenges with so
much uncertainty in the results. The question that arises is why might a carefully designed analytical
method give poor results. One possibility is that we may have failed to account for errors associated with
the sample. If we collect the wrong sample, or if we lose analyte while preparing the sample for analysis,
then we introduce a determinate source of error. If we fail to collect enough samples, or if we collect
samples of the wrong size or type, then our precision may suffer. Under such conditions, even the
choosing and the using of the best laboratory analysis technique for those samples may not be able to
correctly answer the question inspiring the analysis.

Indeed each step of an analysis contributes random error that affects the overall standard deviation of the
result. The overall uncertainty (represented by stotal) is made up of additive contributions from sampling,
sample preparation, and the analytical measurement. For convenience, let’s divide an analysis into these
three steps—sample collection with a standard deviation ssampling, sample preparation with a standard
deviation spreparation, and analyzing of the samples with a corresponding standard deviation smeasurement.
Using a propagation of uncertainty, the relationship between the overall variance, s2total, and the variances
due to sampling, s2sampling, sample preparation, s2preparation, and the analytical method, s2measurement , is as in
the equation below:

s2total = s2sampling + s2preparation + s2measurement 1.1

All the contributions are potentially important. Analyses are typically designed to minimize each.

A sampling error occurs whenever a sample’s composition is not identical to its target population. If the
target population is homogenous, then we can collect individual samples without giving consideration to
where to sample. Unfortunately, in most situations the target population is heterogeneous.
Heterogeneous samples provide the greatest sampling challenges. Collection of representative samples
of air and water is generally easier than for solids, but can also show marked variation with location and
time. Consider analyzing medication that has been dissolved in a glass of water. Due to settling, the
medication available as an oral suspension may have a higher concentration of its active ingredients at
the bottom of the container. Other factors may also complicate sampling of heterogenous samples. The
composition of a clinical sample, such as blood or urine, may depend on when it is collected. A patient’s
blood glucose level, for instance, changes in response to eating and exercise. Other target populations
show both a spatial and a temporal heterogeneity. The concentration of dissolved O2 in a lake is
heterogeneous due both to the changing seasons and to point sources of pollution. The composition
varies markedly over small distances, with depth also another important variable. Sample preparation can
introduce additional variability. For example, acid digestions with heating are often used to dissolve
stubborn solids or to break down organic components. Splattering and resulting loss of material are
notable problems. Of course the measurement process itself also has various measurement uncertainties,

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even when the best technique is used. Both wet chemical and instrumental techniques have
measurement uncertainties, with instrumental methods typically slightly worse than the best wet chemical
analyses.

BACKGROUND DISCUSSION OF SAMPLING:

What are the goals and principles of sampling? Consider the following list, remembering that experience
and common sense are always part of a successful analytical experience.

1. OBTAINING A REPRESENTATIVE SAMPLE. The sample represents a much larger "population", and must
faithfully replicate the population's chemical composition. Choosing appropriate samples can be difficult.
For example, consider a segregated or stratified sample. Should the whole sample be taken, or separate
samples of each layer? Usually, the strata are sampled separately if possible. But very often the layers
cannot be seen by the naked eye and the options include either blind sampling, with full mixing or blindly
stratified samples such as core samples. Do the relative sizes of the layers make a difference? Yes!
Estimates of relative sizes of different layers must be made, and must be included in the calculations, or
the number of samples from each layer must be proportional to the size of the layer. The toughest
sampling problems occur when sampling a smaller number of particles.

2. OBTAINING A HOMOGENEOUS SAMPLE. At some point prior to the actual analysis, the size of the sample
will be reduced to fit the instrument or procedure. Prior to that reduction step, the sample must be
thoroughly mixed (and often ground into tiny pieces, particularly when multiple particle sizes are present).

3. SAMPLING SHOULD INCLUDE RANDOM SAMPLING. Both the initial sample gathering and later sample size
reductions should include randomization. Typically, the sample or sampling area is divided into a number
of real or imaginary zones, from which portions are drawn creating the smaller sample portion. Often the
actual zones which are sampled are chosen by a random number generator. Such combined samples are
called composite samples. Combined zones may be different physical portions of a large sample, or
samples collected at different times (e.g. flowing sewage sampled once every 15 minutes over a 24 hour
period).

4. SAMPLING AND SAMPLE HANDLING SHOULD NOT CHANGE ANALYTE CONCENTRATIONS. Factors such as
adsorption of analyte to container walls, wall catalyzed analyte decomposition, analyte evaporation,
sample contamination during sampling or pretreatment (e.g. contamination by the materials in a
container, a sampling device, or a grinding apparatus), and many others must all be considered. Often,
such considerations result in a requirement of rapid analysis after sampling, within days or even hours.

SAMPLING TECHNIQUES:
1. MIXING: Mixing is combining of components, particles, or layers into a more homogeneous state. The
mixing may be achieved manually or mechanically by shifting the material with stirrers or pumps or by
revolving or shaking the container. The process must not permit segregation of particles of different size
or properties. Homogeneity may be considered to have been achieved in a practical sense when the
sampling error of the processed portion is negligible compared to the total error of the measurement
system.

2. REDUCTION OF SIZE: Decreasing the size of the laboratory sample or individual particles, or both is
achieved by the division of the bulk material. Division of the size of the laboratory sample is generally
accomplished manually by coning and quartering or by riffling or mechanically by rotary dividers.
Reduction of particle size may be accomplished by milling or grinding. Simultaneous division and
reduction may also be achieved with mills having stream diverters.

3. CONING AND QUARTERING: Coning and quartering is one of the methods for preparing representative
sample from the powdered sample by forming a conical heap. The heap is spread out into a circular, flat
cake. The cake is divided radially into quarters and two opposite quarters are combined. The other two
quarters are discarded. The process is repeated as many times as necessary to obtain the quantity
desired for the final use.
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4. RIFFLING: The separation of a free-flowing sample into (usually) equal parts by means of a mechanical
device composed of diverter chutes.

5. MILLING/GRINDING: In milling and grinding mechanical reduction of the particle size of a sample is
achieved by attrition (friction). The required particle size of a sample is related to the size of the test
portion and the number of particles required to ensuring homogeneity among test portions. The reduction
in particle size may sometimes result in particles of different hardness and density, which produces in-
homogeneity during the preparation of the test sample or during the withdrawal of the test portion.

SAMPLE TYPES:
1. RANDOM SAMPLE: The sample so selected that any portion of the population has an equal chance of
being chosen.

2. REPRESENTATIVE SAMPLE: A sampling plan that adequately reflects the characteristics of the population.
The degree of representativeness of the sample may be limited by cost or convenience.

3. SELECTIVE SAMPLE: A sample that is deliberately chosen by using a sampling plan that screens out
materials with certain characteristics and/or selects only material with other relevant characteristics.

4. STRATIFIED SAMPLE: A sample consisting of portions obtained from identified subparts (strata) of the
parent population. Within each stratum, the samples are taken randomly. The objective of taking stratified
samples is to obtain a more representative sample than that which might otherwise be obtained by
random sampling.

5. CONVENIENCE SAMPLE: A sample chosen on the basis of accessibility, expediency, cost, efficiency, or
other reason not directly concerned with sampling parameters.

6. REPLICATE (DUPLICATE) SAMPLE: Multiple (or two) samples taken under comparable conditions. A
duplicate sample is a replicate sample consisting of two portions. The umpire sample is often used to
settle a dispute and the replicate to estimate sample variability.

7. UMPIRE SAMPLE/REFEREE SAMPLE/RESERVE SAMPLE: A sample taken, prepared, and stored in an agreed
manner for the purpose of settling a dispute, if arises.

8. SEQUENTIAL SAMPLE: Units, increments, or samples taken one at a time or in successive predetermined
groups, until the cumulative result of their measurements (typically applied to attributes), as assessed
against predetermined limits, permits a decision to accept or reject the population or to continue
sampling.

9. MULTISTAGE SAMPLING: Samples taken in a series of steps with the sampling portions constituting the
sample (units or increments) at each step being selected from a larger or greater number of portions of
the previous step, or from a primary or composite sample.

SAMPLE PREPARATION, STORAGE AND HANDLING:

In analytical chemistry, sample preparation refers to the ways in which a sample is treated prior to its
analysis. Preparation is a crucial step in most of the analytical techniques, because the techniques are
often not responsive to the analyte in its in-situ form, or the results are distorted by interfering species.
Sample preparation may involve dissolution, reaction with some chemical species, pulverizing, treatment
with a chelating agent (e.g. EDTA), masking, filtering, dilution, sub-sampling or many other techniques.
Sample preservation, storage, and handling must be established in the work plan prior to sample
collection.

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SAMPLE PREPARATION, STORAGE AND HANDLING SHOULD CONSIDER THE FOLLOWING:
Sample volume: Samples should be collected using equipment and procedures appropriate to the matrix,
the parameters to be analyzed, and the sampling objective. The volume of the sample collected must be
sufficient to perform the analysis requested, as well as the quality assurance/quality control requirements

Matrix spike/matrix spike duplicate (MS/MSD) sample: A Matrix Spike and Spike Duplicate (MS/MSD) are
representative but randomly chosen samples that have known concentrations of analytes of interest
added to the samples prior to sample preparation and analysis. They are processed along with the same
un-spiked sample. The purpose of the MS/MSD is to document the accuracy and precision of the method
for that specific sample.

Sample Container: Containers must be compatible with the sample matrix, clean and labeled
appropriately. The exterior of the sample containers must be wiped clean and dry prior to sample
packaging. To prevent leakage of aqueous samples during shipping, sample containers should be no
more than 90 percent full. If air space would affect sample integrity, fill the sample container completely
and place the container in a second container to meet the 90 percent requirement.

Sample Preservation: When a preservative other than cooling is used, the preservative is generally added
after the sample is collected. If necessary, the pH must be adjusted to the appropriate level and checked
with pH paper in a manner, which will not contaminate the sample. The laboratory performing the analysis
should be contacted to confirm the requirements for sample volumes, container types, and preservation
techniques.

PRELAB EXERCISE (2 PTS./EACH):

1. Describe the steps involved in a sampling operation.
2. Discuss the purpose of a sampling step in an analysis.
3. Differentiate between a bulk sample and a laboratory sample
4. Discuss the differences between an aliquot and a laboratory sample
5. What are the factors considered in choosing a representative sample?

1.3 EXPERIMENTAL:
1.3.1 PART 1 EXPERIMENTAL PROCEDURE: Keep careful written experimental notes of both
observations and data. For purposes of this experiment, colored beads (samp and beans) are used to
illustrate different chemicals. The beads (samp and the beans) are all harmless, regardless of what type of
chemical they are illustrating. Assume that all beads have the same size and mass. Thus percent by
number, percent by weight, and percent by volume are all identical. White beads (samp) indicate the
sample matrix, harmless soil. Red beads (beans) indicate a heavy metal metabolic poison which is
harmful at concentrations above a fractional concentration of 0.10.

1. Working in a team of 3 students, obtain a sample of mixed beads (samp and beans). Describe the
sample qualitatively.
1.1 Thoroughly mix the beads up.
1.2 Take a random sample with your containers (200-ml OR 150-ml beaker) provided by your
TA. Fill it to the top, but don't overfill.
1.3 Dump your sample on the bench top. Record the count of each of the two colors of
beads.
1.4 Return the beads to the original container and mix it thoroughly again.
1.5 Repeat steps 1.2 to 1.4 FOUR more times using the same container used above.
1.6 Now repeat steps 1.1 to 1.5 twice more, once with a 50-ml beaker, and once with a 100-
ml beaker. You will have a total of 15 sets of counted samples, five for each sampling
size.
1.7 Be sure all beads are back in the original container, and return the container to the TA.
2. Make a copy of your group's data and give it to the instructor.
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BACKGROUND INFORMATION FOR PART 2
In this experiment, we'll again be looking at a two component mixture. One will be salt, NaCI and the
other is a chemical, potassium dichromate (K2Cr2O7). While the focus remains on sampling, we'll be
making measurements with a spectrophotometer. K2Cr2O7absorbs visible light, and NaCl does not. We
will weigh out samples, dissolve them in water, and use absorbance of light to measure the amount of
potassium dichromate. The absorbance measured is proportional to the concentration of K2Cr2O7
according to equation 1.2 below:

A = ebC (the Beer-Lambert law) 1.2

where A is the measured absorbance, C is the concentration, b is the optical pathlength of the cuvette,
and e is a constant specific to the colored analyte and the wavelength used. For now, all we'll use is the
fact that A and C are proportional.

1.3.2 PART 2 EXPERIMENTAL PROCEDURE: Please wear gloves and goggles when handling these
samples. K2Cr2O7 is chemical that causes redness, pain and skin burns, and also blurred vision and
severe deep burns in the eyes when you are exposed to it. In case of exposure, first rinse with plenty of
water, then remove contaminated clothes and rinse again and seek for medical attention.

1. Obtain a bulk sample of mixed K2Cr2O7 / NaCl from the instructor. Pour your sample onto a large
watch glass, and shape it into a cone using a spatula. Divide the cone into four equal quadrants.
This process is called "piling and quartering" and is designed to separate a sample without
biasing the particle size distribution. Piling and quartering can be repeated if needed to downsize
a really large sample.
2. Weigh out three different 0.25 gram samples of the mixture, from different quadrants of the pile,
recording the mass accurately. Transfer each sample quantitatively into a marked 50 mL
volumetric flask and dilute to the mark with deionized water; then mix by inversion. Be sure all
solid particles have dissolved before final mixing.
3. Dilute the solutions prepared in Step 2 100 times in a 100 mL volumetric flask and measure the
absorbances of these three solutions at 350 nm wavelength. For just the first solution record the
absorbance FIVE times in a row, simply removing the cuvette from the spectrometer and
replacing it between measurements. This additional data will help us measure and discuss the
uncertainty associated with the spectrometer measurement process itself. The second and third
solutions should just be measured once each.

Please Note: Discard the remaining solutions in designated waste bottles.

4. Rinse out your three volumetric flasks and save them for the next stage.
5. Go back to the original bulk sample. Take about a 1-2 gram portion and put it into a glass or
agate mortar. Use a glass or agate pestle to grind the sample really well. It will take several
minutes of real grinding effort (we're seeking to improve the homogeneity).
6. Weigh out three different 0.25 gram samples of the ground mixture, recording the mass
accurately, by difference. Transfer each sample quantitatively into a marked 50 ml volumetric flask
and dilute to the mark, then mix by inversion. Be sure all solid particles have dissolved before final
mixing.
7. As in step 3 above, measure the absorbances of the three solutions at 350 nm, reading each
solution's absorbance just once.
8. Clean all apparatus.

Errors in calculation can lead to significantly inaccurate final answers, and poor grades on
accuracy.

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1.4 DATA ANALYSIS
DATA ANALYSIS FOR PART 1:
1. For each individual sampling, calculate the fractional red bead (beans) concentration (result will be
in the range from 0 to 1). Use these values in all calculations below. (2, 2 X 3)
2. For each sample size (beaker size), combine the five individual measurements of fractional red
bead concentration to calculate the mean, sample standard deviation, relative standard deviation,
% relative standard deviation, and a 95% confidence interval for the fractional red bead
concentration. The needed equations and statistical tables are found in Chapter 4 of the Harris
textbook. (5x3)
3. Theoretically for a two component system like this, the RSD = √[(1-p)/np] where p is the fractional
concentration and n is the number of beads sampled. For each of your three sample sizes,
calculate the theoretical RSD and compare it to your calculated RSD. Use the mean for p, and
calculate the average "number of beads sampled for this beaker size" for n. Does your data agree
with the overall trend predicted for the RSD as a function of sample size? Now compare your
results with those from three other groups. Is the predicted trend clearer when a larger number of
measurements are included? (6)
4. You calculated 95% confidence intervals for the fractional red bead concentration. Look at those
and discuss the answer to the question "Is the concentration of the heavy metal represented by
red beads at a harmful level?" (3)
5. Remember that the meaning of the 95% confidence interval is stated "We are 95% sure that the
true value falls in this interval range." How does that affect the conclusion your draw about
"harmful"? (2)
6. Quantitatively, the value: (% RSD)2 x (# beads in a sample) should be a constant for a specific
analyte (called the Ingamells' sampling constant). Estimate this constant by calculating the
constant for each sampling size, then averaging the three values to get an overall estimate. Then
use this constant to predict the sample size (# beads) which should be used to obtain a %RSD of
1% (use the same equation and solve for the # beads with %RSD set to 1). (6)

DATA ANALYSIS FOR PART 2:

1. For each of the 8 measured absorbances calculate the "absorbance per gram of sample" by
dividing the measured absorbance by the appropriate measured mass. We'll use that as our
uncalibrated measurement of K2Cr2O7 in our samples. Use these values in all calculations below.
(2, 3)
2. Calculate the mean, standard deviation, RSD and %RSD for the one set of values which were
measured FIVE times in a row in the spectrometer. The %RSD here represents the uncertainty of
making the absorbance measurements in our spectrometers. (4)
3. Now calculate the mean, standard deviation, RSD and %RSD for the two different sets of 3
equivalent samples (Set 1 = 3 samples prepared from the bulk sample as obtained, Set 2 = 3
samples prepared after grinding). For the single sample that was measured 5 times, just use one
of the three measurements, ignoring the 2nd and 3rd readings. You now should have statistical
values for the two different samples: "As obtained" and "Ground before analysis". (8)
4. Qualitatively discuss and compare the three different %RSD values you've obtained. (3)
5. To determine if the two sets have different standard deviations, perform an F-test as described in
Quantitative Chemical Analysis by Harris, Chapter 4 (Seventh Edition). Use the F-table on page
63. (3)
6. Now test to see if the effect of grinding is statistically important. Use a t-test as described in the
same textbook as case 2 on pp.60. Look first at your result in step 5 above. If the two sets have
standard deviations that cannot be said to be different, use the t-test as described in equation 4-8
on p. 61, calculating Spooled for use in that equation. If step 5 above found the two standard
deviations to be statistically different, you need to use the t-test in equation 4-8a on p. 61. Use a
95% level of significance. Be sure to state your conclusion after doing the calculations. (3)

7
1.5 DISCUSSION QUESTIONS
1. Our bead sampling was rather simplistic. Identify and discuss two factors that would make
sampling of real samples more complicated. (2)
2. In Part 2, we looked at two different types of uncertainty. One was the uncertainty of measuring
with the spectrometer (smeth) [look at %RSD in data analysis step 2 in part 2]. Another was the
uncertainty with the sampling and sample preparation (ssamp). In this work the effect of both are
evaluated independently. The first process doesn’t include sample preparation, but only weighing
out different powdered samples (look at %RSD in data analysis step 3 for part 2). The other
includes sample preparation step, where the effect of grinding considered. Discuss the relative
sizes of these sources of uncertainty. As you look at grinding, you have tested to see if grinding
changed the measured mean (your t-test in data analysis step 6 for part 2). Grinding may also
improve precision. Compare the %RSD for the two sets of three samples in data analysis step 5
of part 2). Did grinding improve %RSD? Was the difference statistically significant? See your F-
test! (3)
3. Consider sampling a train load of coal for its sulfur content. How would different rail wagons (cars)
be included, how would an individual rail wagon be sampled, and how would you handle the
different particle sizes present in the coal? (3)
4. In Times Beach, Missouri, dioxin contamination of the earth caused the evacuation and eventual
abandonment of the whole town in 1983. Dioxin contaminated oils were originally deposited on
roadways, and migrated into soils. Concentrations of dioxin 1100 times the allowed limit were
found. Discuss the sampling design that you would use in exploring the current dioxin
concentrations in that area, 20-years later. Explain fully. (3)

LAB REPORT
ONLY THE FOLLOWING ITEMS SHOULD BE SUBMITTED AS YOUR LAB REPORT:
1. A photocopy of signed original data. This data could be re-typed as part of the presentation of
your statistical work. N.B. Report with no copy of signed results will not be marked.
2. Statistical values calculated in the data analysis section. Be sure each value is clearly identified.
Sample calculations may be shown, but are not required.
3. Answers to questions posed about statistics. These questions are embedded within the data
analysis section. Be sure you have answered every question asked in this section, and
stated/discussed the conclusion of any statistical tests performed.
4. Submit a one page discussion of your results. It should include among other, comments an
analysis of the standard deviations, mean results, possible sources of error and how they can be
corrected. (5)
5. Answers to the four discussion questions in the "Discussion Question.," section.

1.6 REFERENCES

• Guy R.D., Ramble L. and Wentzell P.D. (1998) An Experiment in the Sampling of Solids for
Chemical Analysis, Journal of Chemical Education, 75, 1028-1033.

• Harvey, D. J. (2002) Two Experiments Illustrating the Importance of Sampling in a Quantitative

Chemical Analysis, Journal of Chemical Education, 79, 360-363.

• Ross M.R., Bacon D.W. and Wolsey W.C. (2000) A Classroom Exercise in Sampling Technique,
Journal of Chemical Education, 77, 1015-1016.

• Vitt J.E. and Engstrom R.C. (1999) Effect of Sample Size on Sampling Error, Journal of Chemical
Education, 76, 99-100.

8
EXPERIMENT 2: QUALITY CONTROL FOR ANALYTICAL BALANCES:
VERIFYING ACCURACY AND PERFORMANCE

EXPERIMENT OBJECTIVES:
At the end of this experiment, the student should be able to:
1. Describe and perform the verification process for the performance of an analytical balance.
2. Calibrate an analytical balance.
3. Compile a validation report.
4. Analyze and interpret experimental data and apply statistical principles on the experimental data to
explain uncertainty in the results.

INTRODUCTION
Accurate analytical measurements are fundamental to the functioning of modern society. It is essential to
establish whether equipment used in the laboratory is operating with the reliability required for QUALITY
results. Inaccurate results can have an enormous social and economic impact. Consider the impact of
inaccurate results on legal cases. It is for this reason that calibration and verification are the most
important actions to ensure the correct indication of measuring instruments and are considered major
requirements for the competence of testing and calibration laboratories according to the International
Standard ISO/IEC 17025. In addition, regular verification and control of measuring equipment is an
essential part of any ISO, GLP or GMP quality management system. Testing assures consistent
performance of the instrument and fulfills current regulatory or normative requirements. Verification is a
process of confirmation, based on evidence (test results) that a certain number of specified requirements
have been fulfilled. For example, the verification of an analytical balance will prove that the performance
of the balance is still in agreement with the calibration certificate.

A well-planned and documented maintenance system forms an essential part of any Quality Assurance /
Good Laboratory Practice / Accreditation process. This maintenance system include documented
evidence of scheduled routine maintenance, any repairs and calibration and verification testing performed
on all equipment. The documented evidence should include records of all original observations,
calculations, results, calibration certificates and test reports for the appropriate period. Also included
should be the identity of personnel involved in testing and re-testing/calibration schedule. This evidence
may be incorporated into a logbook and the status of the equipment should be clearly indicated by
labeling the equipment.

A label stating the equipment is OUT OF CALIBRATION means that it must be calibrated before use.
Generally, equipment that is verified will be labeled as follows:
• Identity of person who performed calibration/verification test
• Date test was performed
• Test period
• Next date of calibration/verification testing.

In this experiment, you will calibrate and verify the performance characteristics of the analytical balance.
In the analytical laboratory, the balance is used on a daily basis. It is therefore important to know whether
it performing to specification. As with any measurement instruments, balances should also be calibrated
regularly. A proper metrologically traceable calibration is the only way to know how accurately balances
are measuring. Before starting the calibration of the balance, there are a few things that’s should be
clarified to get prepared. The technical characteristics of the balance (max weight, d value), the accuracy
requirement (max error allowed and uncertainty) and what to do if the calibration fails (adjustment) must
be known. Typically, the whole measurement range is calibrated and the calibration is performed in the
location where the instrument is being used. The balance must be switched on at least 30 minutes before
the calibration. The temperature of the weights should be stabilized to the same temperature where the
calibration is to be done. The balance should be at a horizontal level (check balance manual on how to
verify this). A few pre-tests by placing weights close to the maximum of the range on the instrument must
be performed to ensure it works normally.
9
The following tests and indicators can be used to evaluate the performance of laboratory balances.

1. MONITORING THE DRIFT: Drift refers to weight readings that do not stabilize, or unstable readings with
no weight applied. All analytical balances show some degree of instability but when the amount of drift
increases substantially then there is a problem with the functioning of the balance. Temperature and
static electricity are two environmental factors that dramatically affect the instrument's stability.

2. TESTING FOR REPRODUCIBILITY: Reproducibility refers to the balance's ability to repeatedly deliver the
same mass reading for a given weight and to return to a zero reading after each weighing cycle. It is
expressed as a standard deviation.

3. CORNERLOAD TESTING: Verifies that the balance delivers the same mass reading regardless of where
the weight is placed on the weighing pan.

Verifies the accuracy of the balance at intermediate readings throughout the weighing range of the
balance. Linearity is a critical parameter since a balance will often be used to weigh amounts much
smaller than the capacity of the balance.

5. HYSTERESIS TESTING: Verifies that there is no error caused by approaching the same weight from
above and below the value

6. CALIBRATION: Refers to the difference between the mass reading on a balance of a given mass
standard, and the actual value of that standard. Calibration involves the use of standard precision weights
with very fine tolerances, usually ASTM Class 1,2 and OIML E2. These weights should be traceable to
national and international standards of measure.

A Calibration Certificate is supplied with each weight when it leaves the factory. The certificate should
include the following information:
• measurement results
• associated uncertainly of measurement and /or
• a statement of compliance with an identified metrological specification.

The procedure used to calibrate and verify the performance of a balance is usually in accordance with the
manufacturer's instructions and is documented in a Standard Operating Procedure (SOP).

Before proceeding with the tests consider the following factors:

1. HANDLING TEST WEIGHTS: The weights used to test laboratory balances are precision tools and should
be handled with great care. Never touch them with bare hands. Wear clean gloves and use tweezers to lift
them up. Avoid sliding the weights across any surface including the weighing pan.

2. MASS READING INTERFERENCES: The accuracy and precision of weighing results are closely associated
with a number factors and the following should be considered
• The temperature of the room should not fluctuate by more than 1 °C
• The power to the balance should be ON continuously
• The weighing pan should be shielded from air drafts. Avoid plastic materials for draft shields.
• Minimize static electricity by removing carpets, by not wearing plastic shoe soles, by not using
plastic draft shields or plastic weighing vessels or by placing the balance on a melamine (Formica)
table top. Low ambient humidity also increases static.

To test for static, place a metal enclosure (coffee tin) over the weighing pan but not touching it. If
the mass readings stabilize then static may be the cause of unstable readings.

10
 • Minimize the effects of vibration or movement on the mass readings by placing the balance on a
sturdy table on a concrete floor.
• Do not place any equipment that might generate electromagnetic radiation or any magnetic
material such as stirring bars near the balance

The following parameters will be evaluated on the Radwag Model AS 220.R2 Balance:
• Accuracy
• Linearity
• Precision (Reproducibility)
• Eccentricity (Corner Loading)
• Linear Error

PRELAB EXERCISE (2 PTS./EACH):

1. What is validation?
2. What is a validation report?
3. What is contained in a validation report?
4. What is the difference between validation and verification?
5. Why is the validation process important in an analytical chemistry laboratory?

EXPERIMENTAL:
1. ECCENTRICITY TEST (CORNER LOADING)
In normal use of a balance the load is not always placed perfectly on the center of the load receptor.
Sometimes the results of a balance can vary slightly depending if the load is placed in different locations
on the balance pan. In order to test how much effect the location of the load has, the eccentricity test is
performed. In the eccentricity test, the test weight is placed in a few different specified locations on the
balance pan. First, the weight is placed in the center of the pan (the weight’s center of gravity) and the
result is observed. Next, the load is placed in four different sectors of the balance pan, as illustrated in the
picture below.

The test weight used in an eccentricity test should be at least one third (1/3) of the max load of the
weighing instrument. The test should preferably be done using just one test weight, if possible. That way
it is easier to be sure that the weight’s center of gravity is in the specified location.

PROCEDURE FOR THE ECCENTRICITY TEST

1. Check the cleanliness and level of the balance.
2. Tare the balance to read all zeros
3. Select a test weight close to the weighing capacity of the balance.
4. Place the test weight on position 1 and record its mass after the reading stabilizes.
5. Remove the test weight from the weighing pan and tare the balance.
6. Place the test weight on position 2 and record its weight.
7. Repeat steps 4 and 5 until you have recorded the masses for each location as indication in the
diagram.

11
 8. Finally, the test load is placed again to position 1 to check that the indication has not drifted from
the earlier indication in position 1.

2. ACCURACY
The accuracy of the balance is checked by weighing at least three different certified weights that cover
the usual weighing range of the balance. It is recommended that the weights have approximately 5%,
50% and 100% of the maximum capacity of the balance (or of the maximum weight used on the balance),
depending on the type of balance. It is recommended that the weighing is repeated at least 5 times for
every weight, particularly, when the results shall also be used in the test for precision.

PROCEDURE FOR THE ACCURACY TEST

1. Select test weights equal to, or nearly equal to 5%, 50% and 100% weighing capacity of the
balance.
2. Tare the balance to read all zeros
3. Place the smallest test weight on the center of the balance pan.
4. Record the mass of the test weight after the reading stabilizes.
5. Remove the weight and tare the balance.
6. Place the weight at exactly the same position and again record the mass of the weight.
7. Repeat steps 5 and 6 until you have 5 replicates for the first weight.
8. Repeat steps 3 and 7 for the other TWO test weights.

3. REPRODUCIBILITY TEST
As any instrument, also balances may suffer from reproducibility issues. This means that when the same
load is measured several times, the result is not always exactly the same. To find out the reproducibility of
the instrument, a repeatability test is done. The repeatability test is performed by replacing the same load
on the same place on load receptor (to avoid any eccentricity error) multiple times. Test should be done in
identical and constant conditions and with identical handling. The load used should be close to the
maximum load of the instrument. Often a repeatability test is done with one load only, but it can be done
also with several different load values separately.

PROCEDURE FOR THE REPRODUCIBILITY TEST

1. Select a test weight equal to, or nearly equal to the weighing capacity of the balance
2. Tare the balance to read all zeros
3. Place the test weight on the center of the balance pan.
4. Record the mass of the test weight after the reading stabilizes.
5. Remove the weight and tare the balance.
6. Place the weight at exactly the same position and again record the mass of the weight.
7. Repeat steps 5 and 6 until you have 10 replicates.

4. LINEARITY TEST
The purpose of the linearity test is to verify the accuracy (calibrate) of the balance to provide consistent
sensitivity throughout the weighing range. The most common practice is the following: start with zeroing
the balance without any load. Set the loads of the first test point, wait for stabilization, and record the
indication. Continue increasing the loads through all the increasing test points. Once the maximum load is
recorded, start decreasing the loads through the decreasing test points. In some cases, the weighing
instrument may be calibrated with increasing loads only or decreasing loads only. Typically, 5 to 10
different loads (test points) are used. The highest load should be close to the maximum of the instrument.
The smallest test load can be 10% of the maximum load, or the smallest weight normally used. Generally,
the test points are selected so that they are equally distributed.

PROCEDURE FOR THE LINEARITY TEST

1. Select 5-6 test weights from the ones provided. They must be of the same type (make sure your
selected weights cover the range of the balance as discussed above).
2. Tare the balance so that its reading is zero
3. Place the smallest weight on the balance and record its mass.

12
 4. Weigh each of the standard weights in ascending order and record the observed weight in a log.
5. Repeat the process by weighing from the largest to the smallest.
6. Use the same weights from the same weight set each time this test is performed.
7. Compare the observed masses of the test weights with previous weightings.
8. The results are judged to be satisfactory if there has been no significant shift of the observed
weights of the weights. If a single weight does not agree, reweigh it and confirm that there has not
been a substitution of weights from a previous calibration verification.

5. LINEARITY ERROR
Linearity error is tested at least 3 times using four weights of defined masses whose aggregate total mass
is approximately equal to half of the maximum capacity, depending on the type of balance.
First, the combined mass of all four weights is weighed and recorded. Then, two sub-sets of weights are
made and their masses are recorded. The linearity error of the balance is an absolute value calculated by
the difference between the combined mass of all four weights and the sum of the masses from the two
sub-sets of weights, divided by 2.

PROCEDURE FOR THE LINEARITY ERROR TEST

1. Select 4 test weights from the ones provided. They must be of the same type (the total mass
should be about half the maximum range for the balance).
2. Tare the balance so that its reading is zero
3. Place the 4 weights on the balance and record the total mass (Tm) of the load.
4. Remove the weights from the pan, and tare the balance.
5. Take two of the weights used in Step 1, place them on the balance and record their mass (TA).
6. Remove the weights from the pan, and tare the balance.
7. Take the remaining two of the weights used in Step 1, place them on the balance and record their
mass (TB).

DATA ANALYSIS

ECCENTRICITY TEST
1. Refer to the reference information given in Table 2 below to verify whether the readings fall within
the typical tolerance specification. Does the balance pass the eccentric test? (2, 2, 3, 3)

ACCURACY TEST
1. Calculate the standard deviation of each set of the recorded data. (3 x 2)
2. Calculated standard deviations larger than allowed by the balance specifications indicate that the
balance is either operating in an unstable environment (warm-up, vibration, air draft, static,
temperature fluctuations, magnetic interferences etc.) or that the balance is in need of repair.
Refer to the reference information given in Table 2.2 below to check whether the readings fall
within the typical tolerance specification. (3)

REPRODUCIBILITY TEST
1. Calculate the standard deviation and the relative standard deviation of the recorded data (3)
2. Calculated standard deviations larger than allowed by the balance specifications indicate that the
balance is either operating in an unstable environment (warm-up, vibration, air draft, static,
temperature fluctuations, magnetic interferences etc.) or that the balance is in need of repair.
Refer to the reference information given in Table 2.2 below to check whether the readings fall
within the typical tolerance specification. (4)

LINEARITY TEST

1. Plot a calibration curve of the measured masses vs. the nominal mass of the test weights and
determine the value of the correlation factor, r. (5)

13
 2. Using criterion in the reference information given in Table 2.2 below, does the balance pass the
linearity test? (3)

LINEAR ERROR
1. Calculate the linearity error (Tm – (TA + TB))/2. The acceptance criteria for the linearity can be
evaluated as follows: linearity error ≤ accuracy of the balances. Does the balance pass the
linearity error test. (5)

DISCUSSION QUESTIONS

1. Why is it important to have 'traceability' of external reference weights? (2)

2. Name two organizations in South Africa that are accredited to certify external reference weights
used in the calibration of balances? (2)
3. If a balance is out of calibration, may the balance still be used? Give reasons for your answer.
(2)
4. Why is it important not to handle reference weights with your hands? (2)
5. What do the terms Type I and II, Grade S, O, P and Q, Class 1 and 2 mean when referring to
balance weights (3)

REFERENCES

1. General Requirements for the competence of calibration and Testing Laboratories SASS 0259 —
1990, ISO/TEC Guide 25:1990
2. Chemical Quality Assurance Theoretical Notes, Faculty of Applied Sciences.
3. Mass Standards Handbook, Troemner
4. vvww.labbalancerepair.com
5. Scorer T, Perkin M, Buckley M, Measurement Good Practice Guide No. 70— Weighing in the
Pharmaceutical Industry http://wvvw.npl.co.uk/mass/quidance/pharmaweiqh.pdf accessed
26/06/07
6. Kenyon A S, Layloff TP Weighing on an Analytical Balance Division of Drug Analysis, FDA
http://www.layloff.net/fda/1994analweioh.pdf accessed 26/06/07
7. Calibration and use of analytical balances
8. http://www.wcaslab.com/ca/sop/2110v11web. PDF accessed 26/06/07
9. OMCL Network of the Council of Europe QUALITY MANAGEMENT DOCUMENT

REQUIREMENTS

Each Member is to hand:

• a report showing results for calculations of the parameters listed above. (Tabulate your
data and only show example calculations)
• Answers to discussion questions
• A photocopy of signed laboratory results. N.B. Report with no copy of signed results
will not be marked.

Please note this is not a FULL REPORT.

• Validation report: should report on parameters investigated, and recommendations

(should be submitted separately with own cover page: marks will also be allocated for
cover page design) (30)

14
APPENDICES

Table 2.1: Technical specifications

Table 2.2: Selected parameters and typical tolerance limits for the qualification of balances. Adapted from
OMCL Network of the Council of Europe QUALITY MANAGEMENT DOCUMENT

Parameter Checked Typical Tolerance Limit

Eccentricity Test Proposed criterion: RSD not more than 0.05%, calculated from all
weighings at different locations on the pan

Accuracy Test Proposed criterion: RSD not more than 0.10% of the test weight value
Reproducibility Test Proposed criterion: SD = max 5*d, where d = (actual) scale interval
(e.g. d=0.1 mg))

Linearity Test Proposed criterion: correlation factor, r = 1±0.0001

Linear Error Proposed criterion: linearity error ≤ accuracy of the balances

15
EXPERIMENT 3 : USING AND THE CALIBRATION OF VOLUMETRIC
APPARATUS USING THE GRAVIMETRIC METHOD:
VOLUMETRIC, GRADUATED AND MICROPIPETTES

EXPERIMENT OBJECTIVES:
At the end of this experiment, the student should be able to:
1. Describe and perform the calibration of a micropipette, graduated pipette and a volumetric pipette.
2. Correctly use the micropipette
3. Compile a standard operating procedure (SOP) for using and calibrating a micropipette.
4. Analyze and interpret experimental data and apply statistical principles on the data to explain
uncertainty in the results.

INTRODUCTION
Volume measurement is an important step in most industrial and analytical measurement operations.
Volume instruments are used in many fields of science. One such instrument is the pipette. Pipettes are
found in each and every chemistry laboratory. Pipettes come in a variety of types and sizes. Among them
are volumetric pipettes, graduated pipettes and the micropipettes. Volumetric pipettes are used to
measure a single volume accurately, typically to 4 significant figures. (volumes typically vary from 0.5 to
100 mL). Graduated pipettes are used to measure various volumes with a single pipette (volumes
delivered depend on the overall size of the pipette). Graduated pipets are not as accurate as volumetric
pipets, due to the fact that each graduation line is not individually calibrated and any imperfection in the
internal diameter will have a greater effect on the volume delivered. On a volumetric pipette, the
specifications indicate:

• How much liquid will be transferred if the liquid is drawn up to the calibration line on the neck
• The temperature at which the calibration was made (remember that the volume of a liquid
changes with temperature!)
• The fact that it is a TD (to deliver) NOT a TC (to contain) measuring device.

The proper use of a volumetric pipette includes: when emptying a volumetric pipettes, the liquid is
allowed to slowly drain out. It is never forced out. After it is emptied, the small amount of liquid, which
remains in the tip, should not be blown out. Because they are designed “to deliver” the correct amount,
pipettes are generally NOT blown out. Micropipettes are used extensively in laboratory to accurately and
precisely deliver small volumes of liquid (volumes typically vary from 5 to 1000 μL). The correct use of
micropipettes is essential to ensure accurate volume delivery.

Laboratories must ensure that results obtained using volumetric instruments are reliable. In order to
reduce and identify possible errors in liquid handling, it is necessary to calibrate volume instruments using
correct methods. At the highest level of traceability chain, the volume can be determined by the primary
method by weighing the contents of a suitable liquid by known temperature and density (gravimetric
method) and is the method most commonly used by laboratories accredited to ISO17025. There are
colorimetric methods in use and these are often used for checking pipettes and for non-accredited
calibration. Gravimetric analysis is preferred due to the simplicity and the traceability to an absolute
standard. Gravimetric methods are also often recognized as a more economical way of calibration.

Gravimetric analysis for pipette calibration entails dispensing samples of distilled water into a receiving
vessel in a precision analytical balance. The density of water is a known constant, the temperature,
barometric pressure and humidity are recorded (the Z-factor used in the final mass calculation) and kept
within certain limits and thus the mass of the dispensed sample provides an accurate indication of the
volume dispensed.

ACCURACY AND PRECISION

Pipettes and micropipettes can deliver accurate and precise volumes of a solution. Our goal is to
determine how accurate and how precise our pipettes and micropipettes are. Accuracy is a measure of

16
how close a measured value is to the accepted or “true” value. It is related to the percent error between
the average volume of solution measured experimentally and the volume that was expected (the accepted
value). Smaller percent error reflects higher accuracy. Percent error can be negative, indicating that the
measured volume was smaller than the expected volume or positive, indicating that the measured volume
was larger than the expected volume. For example, we are attempting to measure two different volumes
of water with our micropipette and two with our graduated pipette. Perfect accuracy would have us
measure the exact volume we desire each time. However, the volume of water that we actually measure
will be close but probably different from these volumes. The farther away from the correct volume, the
lower the accuracy of our pipettes and/or our technique will be.

Precision measures the closeness of a set of values obtained from identical measurements of the same
quantity. It is the measure of reproducibility of a measurement (whether it’s accurate or not). Precision is
related to the standard deviation of a series of measurements of the same thing. For example, if the
micropipette is set to the same volume (300 μL) and four measurements are taken at this volume, a
standard deviation can be taken of these five measurements. The smaller the standard deviation, the
more precise the micropipette is. We will use the standard deviation as a measure of the spread of
potential errors in a given measurement.

USE OF THE MICROPIPETTES

When you push down gently on the plunger of the micropipette, you will feel a “stop” where the
resistance increases. If you push a little harder, the plunger will move even further to a second stop. The
first stop is used to suck up the correct volume. The second stop is used to completely expel the liquid
you are measuring.

Liquid is never drawn into the barrel of the micropipette itself. An appropriate tip should always be placed
firmly on the end. Since the principle by which the micropipette works is the creation of a vacuum in the
tip, causing liquid to be drawn up, it is critical that the tip be on tight enough to make an air-tight seal.
Having said this, do NOT jam the tips on so hard that they are hard to get off. The tips used for the
1000μL pipets are usually blue.

The volume to be taken up is set by turning the plunger on the top of the micropipette and reading the
numerical settings displayed. A setting of 100 μL is equal to 0.100 mL. A setting of 1000 μL is equal to
1.000 mL. Do not set the micropipette below 100 μL or above 1000 μL under any circumstances! Doing
this is essentially the only way that the micropipettes can be broken.

https://inchbyinch.de/pictorial/micropipette/

17
When drawing liquid up into the micropipette, set the dial, and then push the plunger down to the first
stop. While holding it there, put the end of the tip under the surface of the liquid to be measured and
slowly, gently allow the plunger to return to its top position. If you go too fast, you will cause some liquid
to spurt up into the micropipette barrel itself, which is bad for the micropipette, bad for your results and
bad for your chemistry karma, whatever that is. This also lets air in, so the volume of fluid sucked up into
the tip will be lower than the amount that you want. Never let the plunger snap up by itself. Next, place
the end of the tip where you want the liquid to go and push the down the plunger to the second stop to
deliver the exact amount of fluid desired.

Although micropipettes are usually quite accurate when first purchased, they can eventually develop
problems with use. We will spend some time checking the calibration of the micropipettes that we will be
using throughout the semester to ensure that they are delivering a known volume of fluid. We will
measure out different exact volumes of water with the micropipette, and using the density of water at the
temperature of the water, we will determine the volume of fluid delivered based on the fluids mass.

GENERAL GUIDELINES FOR GOOD PIPETTING

• Make sure that you operate the push- button slowly and
smoothly.
• When aspirating, keep the tip at a constant depth below
the surface of the liquid (refer to the Table 3.1 below).
• Change the tip before aspirating a different liquid,
sample, or reagent.
• Change the tip if a droplet remains at the end of the tip
from the previous pipetting operation.
• Each new tip should be pre-rinsed with the liquid to be
pipetted.

Table 3.1: Table - Immersion depth and wait

time for Gilson micropipettes

PRELAB EXERCISE (2 PTS./EACH):

1. What is calibration and how often should it be done on analytical instruments?
2. What is an SOP?
3. Besides the gravimetric method, what other method is used to calibrate pipettes?
4. When calibrating pipettes that are used for small volumes, evaporation rate should be considered.
Why?
5. How is the evaporation rate measured?

EXPERIMENTAL:

PROCEDURE 1: USING AND CALIBRATING A MICROPIPETTE

This section involves the calibration of two micropipettes. The group will split into TWO, a pair will check
the calibration of a 100 μL or 200 μL, and the other pair will check the calibration of a 500 μL or 1000 μL
at the volume settings: nominal, 50% and 20%. Each student must record all the results.

This procedure will use the technique of balance taring (zeroing) prior to each measurement. This
will allow direct measurement of the mass from each use of the micropipette.

1. Collect approximately 50-100 mL of deionized water in a medium sized beaker.

2. Record the temperature to the nearest 0.1 oC.
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 3. Record the atmospheric pressure and humidity of the room.
4. Wear a pair of non-powder disposable gloves to handle the stoppered weighing container
5. Select the pipette and set the volume to read the nominal value (i.e. for the P1000 set the volume
to 1000 μL)
6. Insert the correct pipette TIP onto the micropipette and pre-rinse the tip by aspirating and
dispensing distilled water from the beaker to waste at least five times to prepare it.
7. Place a clean, stoppered container with a capacity larger than ten times the volume of the test
pipette, on a calibrated top-loading balance and tare.
8. Remove the stoppered container from the balance
9. Using the correct pipetting procedure, transfer the set volume of distilled water from the beaker to
the tared container.
10. Place the stoppered weighing container with dispensed water on the balance. Record the reading
11. Tare the balance without removing the container.
12. Remove the stoppered container from the balance. Do not empty the water transferred above.
13. Repeat steps 9-12 for a total of 10 transfers.
14. Obtain measurements for 10 transfers.
15. Change the volume setting to 50 % of the nominal value. Eject the pipette tip used above and
insert a new one, and condition it as highlighted in step 6.
16. Set the volume to read 50 % of the nominal value. Condition a new the pipette tip.
17. Carry out the test using a new clean stoppered weighing vial.
18. Repeat steps 7 to 14 dispensing distilled water at a volume of 50 % of the nominal value using a
new clean weighing vial.
19. Obtain measurements for 10 transfer.
20. Change the volume setting to 20 % of the nominal value.
21. Repeat steps 7 to 14.
22. Tabulate the results obtained from the measurements.

PROCEDURE 2. CALIBRATING THE GRADUATED PIPETTE

This section should also be performed as a group. Each member of the group will practice using a
graduated pipette at three different volumes: 3.00 mL, 5.00 mL and 8.00 mL. (Each member should
perform the actual measurements for one of the three volumes. Each member of the group must record
all data.)

This procedure will use the technique of weighing by difference. All mass values will require the
subtraction of two separate measurements. For this reason, you will get the best results if all
measurements for this portion of the experiment are performed with the same balance. Do not attempt to
use the same technique as was used in the first portion of the experiment. This will require you to tie up
the balance for too long, preventing other students from completing the experiment in a timely fashion.

1. Collect approximately 50-100 mL of deionized water in a medium sized beaker.

2. Record the temperature to the nearest 0.1 oC.
3. Create Table in your notebook to record the results.
4. Tare the 3 balance.
5. Place a clean and dry 50 mL beaker covered with a watch glass on the balance.
6. Measure and record the weight of the beaker and watch glass in your notebook data table.
7. Return to your workstation and add 3.00 mL of deionized water to the beaker with your graduated
pipette.
8. Reweigh the beaker, water, and watch glass. Record its mass in your lab notebook.
9. Repeat steps 7 and 8 until you have 5 recorded measurements at this volume. (You don’t have to
dump the water out of the beaker each time; instead use the ending weight for one measurement
as the beginning measurement for the next.) Both groups must record the data in their laboratory
notebook.

10. Repeat Procedure 2 using a volume of 5.00 mL and 8.00 mL using the same pipette (The other
two to be completed by the other group members). Each member of the group must carry out the
actual experiment and record their results.
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 11. Using the mass of the water dispensed using the pipette, calculate the corresponding volume of
water delivered by the pipette, and calculate the mean and sample deviation for each volume
setting on the graduated pipette.

PROCEDURE 3. USING THE VOLUMETRIC PIPETTE.

1. Clean and rinse 3 pipettes (10.00 or 25.00 mL), one for each member of the group. There is NO
need to rinse the pipettes with acetone. Label the pipettes so that you can tell them apart.

Each member should perform the actual measurements. Each member of the group must record all data.)

2. Measure the mass of a suitably sized beaker (~50 mL) on the analytical balance, and record the
mass in your notebook.
3. Rinse the pipette 3 times with deionized water by filling the pipette to the fill line, and discarding
the rinse. Finally, fill the pipette to the fill line, wipe excess water from the outside of the pipette
and dispense this liquid into the beaker.
4. Measure and record the mass of beaker and water, discard the water, and dry the beaker. Repeat
this procedure until you have 5 measurements made for each pipette.
5. The mass of water dispensed from the pipette is found by subtracting the empty weight of the
beaker from the weight of the beaker and water.
6. Using the mass of the water dispensed using the pipette, calculate the corresponding volume of
water delivered by the pipette, and calculate the mean and sample deviation for each pipette.

DATA ANALYSIS

DATA ANALYSIS FOR PART 1: MICROPIPETTE

For each of the set volumes of the micropipettes, calculate:
1. Average value in grams (3)
2. Standard Deviation in grams (3)
3. Experimental volume (mean volume) dispensed together with the standard deviation
(convert the average and standard deviation from mass to the corresponding volume in microliters
of water. The conversion uses the Z-factor at the measured lab temperature (Table 3.2) and the
equation below:
Mean Volume = Z (mean mass + e).
where e is evaporation rate. Assume e to be 0 since a stoppered container was used this
experiment. (3 x 2)

4. Accuracy (Percent Error)

Calculate a percent error for each micropipette, for all the volume settings verified in this
experiment. Use the volume you set the micropipette to as the “true value” and your average value
as the “experimental value”.

𝑒𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙 𝑣𝑎𝑙𝑢𝑒 − 𝑡𝑟𝑢𝑒 𝑣𝑎𝑙𝑢𝑒

% 𝑒𝑟𝑟𝑜𝑟 = ( 4 𝑥 100
𝑡𝑟𝑢𝑒 𝑣𝑎𝑙𝑢𝑒
(3)
5. Calculate the precision of the pipette (% Relative Standard Deviation). (3)

6. By using the student’s t-Test, at 95 % Confidence, is there evidence of systematic errors for the
two micropipettes you used. (3 x 2, 3)

7. By using the tables on Appendix A, state if the micropipette complies with the manufacture’s
specifications. (3)

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DATA ANALYSIS FOR PART 2: GRADUATED PIPETTE
1. Calculate the mass of water added for each addition. (3)
2. Repeat the same calculations listed in steps 1 through 6 in Part 1 above with the exception that
volumes should be listed in milliliters rather than microliters, and also calculate your volumes using
the density of water at a the measured temperature. Table 3.3 shows values of densities at
different temperature. Use the relationship between density, mass and volume for this calculation.
Show only example calculations in your report. The calculations for the standard deviation may be
done with your calculator and do not need to be expressly shown in your report. The final values
must be summarized in your data tables. (3, 3, 3 x 2, 3, 3, 3 x 2, 3)
3. If the percent error of the pipette is more than 1%, the pipette is out of calibration and must be
repaired or replaced.…. Does your pipette need to be replaced? Does this conclusion agree with
results from the t-Test. (3)

DATA ANALYSIS FOR PART 3: VOLUMETRIC PIPETTE

1. Calculate the mass of water added for each addition.
2. Repeat the same calculations listed in steps 1 through 6 in Part 1 above with the exception that
volumes should be listed in milliliters rather than microliters, and also calculate your volumes using
the density of water at a the measured temperature. Table 3.3 shows values of densities at
different temperature. Use the relationship between density, mass and volume for this calculation.
(3, 3, 3 x 2, 3, 3, 3 x 2, 3)
3. If the percent error of the pipette is more than 1%, the pipette is out of calibration and must be
repaired or replaced.…. Does your pipette need to be replaced? Does this conclusion agree with
results from the t-Test. (3)

DISCUSSIONS

1. Discuss whether the accuracy and precision change as a function of the volume of fluid
measured. Were any trends evident? Explain by referencing your results. (3)
2. Why is it advisable to pre-rinse the tip of a micropipette before pipetting. (3)
3. List recommendations suggested for optimizing one’s pipetting technique using micropipette.
(3)

REQUIREMENTS
Each GROUP is to hand in:
• One complete SOP describing how to use a micropipette. (25)
SOP cover page design will also be evaluated

Each Member is to hand:

• a report showing results for calculations as indicated in Part 1, Part 2 and Part 3.
(Tabulate your data and only show example calculations)
• Answers to discussion questions
• A photocopy of signed laboratory results. N.B. Report with no copy of signed results
will not be marked.

Please note this is not a FULL REPORT.

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APPENDICES

Table 3.2: Values for Z (μL/mg), as a function of temperature and pressure, for distilled water at 1 Atm.

Table 3.3: Values for density, r (g/mL), as a function of temperature and pressure, for distilled water at 1 Atm.

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