ECH 3903

MATERIAL ENGINEERING LABORATORY

Equation Chapter 1 Section 1

LABORATORY EXERCISES GUIDE

 TABLE OF CONTENTS

COURSE SYLLABUS
LABORATORY SAFETY AND CODE OF CONDUCT
REPORT WRITING GUIDE AND FORMAT
ERROR ANALYSIS
SOLUTION AND DILUTION

PART I: CHROMATOGRAPHY

HIGH PRESSURE LIQUID CHROMATOGRAPHY

GAS CHROMATOGRAPHY

PART II: SPECTROSCOPY

UV-VIS
AAS
FTIR

PART III: MICROSCOPY

LIGHT MICROSCOPE
SCANNING ELECTRON MICROSCOPY

PART IV: THERMAL ANALYSIS

DSC
TGA

PART V: MECHANICAL ANALYSIS

DMA
RHEOMETER

PART VI: OTHER ANALYSIS

PARTICLE SIZE ANALYSER

REFERENCES
APPENDIX

i
COURSE NAME : MATERIAL ENGINEERING LABORATORY
(Amali Kejuruteraan Bahan)

COURSE CODE : ECH3903

CREDITS : 1(0+1)

TOTAL STUDENT : 44
LEARNING TIME

PREREQUISITE : None

LEARNING : Students are able to:

OUTCOMES (LO)
1. Compare the application and handling of various analytical instruments
(C4)
2. Calibrate parameters by considering the error of measurement device
(P5)
3. Present report in writing and oral as a group (A2, CS, LS)

SYNOPSIS : This course covers the operating methods for analytical equipment that are
used to do material analyses and characterizations. Methods used to
calibrate equipment are also conducted.

(Kursus ini meliputi kaedah pengoperasian peralatan analitikal yang

digunakan untuk pencirian dan analisa bahan. Kaedah menentukur alatan
turut dilaksanakan.)

COURSE :
Contact
CONTENTS
Hours
PRACTICALS 1. Calibrate a chromatography system 3
2. Analyse material using chromatography system 6
3. Calibrate a spectroscopy system 3
4. Analyse material using a spectroscopy method 3
5. Analyse material using microscopic technique 3
6. Classify material using particle size analyser 3
7. Calibrate an elemental analyser unit 3
8. Identify elemental content using elemental analyser 3
9. Test mechanical property of material 3
10. Test rheological property of material 3
11. Test thermal property of material 3
12. Present laboratory report 6

Total 42

EVALUATION : Course Work 100%

2
REFERENCES : 1. Harris, D.C (2010). Quantitative Chemical Analysis (8th Edition).
York: W.H. Freeman
2. Kaufmann, E.N. (2012). Characterization of Materials (2nd
Edition). New York: Wiley-Interscience

3. Leng, Y. (2013). Materials characterization: Introduction to

Microscopic and Spectroscopic Methods (2nd edition).
Singapore: J. Wiley

4. Skoog, D.A., West, D.M., Holler, F.J. & Crouch, S.R. (2014).
Fundamentals of Analytical Chemistry (9th Edition). Thompson:
Brooks Cole

5. Vitalij, K.P. & Peter, Y.Z. (2009).Fundamentals of Powder

Diffraction and Structural Characterization of Materials (2nd
Edition). New York: Springer-Verlag

3
 LABORATORY SAFETY AND CODE OF CONDUCT

This briefing is conducted by the Department’s Safety Officer during Week 1

4
 REPORT WRITING GUIDE AND FORMAT

The purpose of this guideline is to provide ideas on contents that should be included
in your report. Different reporting techniques will provide you with necessary skills
to communicate effectively at different levels. Each report type requires different set
of elements to be presented (Table 1). Late submission will be penalized.

You will also be assessed during a poster/slide presentation session scheduled on

Week 13 and 14. You will present your findings from a mini project in Week 11 and
12.

Element Long Report Journal Report Presentation

●
Title page ●
(as header)
Abstract ● ●
Table of Content ●
List of Figure/Table ●
Introduction ● ●
Theory and Working Equations ●
Refer Table 2
Materials and Method ● ●
Results and Discussion ● ●
(including error analysis)
Conclusion and Recommendation ● ●
Acknowledgement ● ●
References ● ●
Appendices ●
Maximum pages 4 15 slides/
10
(excluding title page) (2-column text) 1 poster
Submission due 2 weeks 1 week Week 13/14

Table 1: Report format and the required elements

5
 Table 2: Assessment criteria for poster/slide presentation

Score
Criteria 1 2 3 4 5

Organization and Poster/slide jumps between Flow of information could be Flow of information could be Logical, smooth flow of Logical, smooth flow of
clarity disconnected topics; main followed but some gaps are followed but some gaps are information in poster; main points information in poster/slide; main
points unclear evident; main points unclear evident; main points stated stated points clearly stated
Content Content patchy, lacks specific Content patchy, lacks specific Content presented/analyzed and Content presented/analyzed and Content thoroughly
important information; little important information; have related to the experiment; key related to the experiment; key presented/analyzed and related
effort to synthesize key points effort to synthesize key points points stated points clearly expressed to the experiment; key points
clearly expressed
Graphics Graphics not properly chosen; Graphics properly chosen; too Graphics properly chosen; able Graphics well-selected, able to Graphics well-selected, make it
too much or not enough detail; much or not enough detail; to understand; ok and understand; neat and presentable easier to understand; neat and
distracting distracting presentable presentable

Mechanics Too many grammatical or Two to five grammatical or Two to five grammatical or One or two grammatical or No grammatical or spelling
spelling errors; format not spelling errors; format not spelling errors; follow the spelling errors; follow the format errors; follow the format
followed followed format
Confidence Presentation seen and heard, Presentation seen and heard, Presentation seen and heard, Presentation clearly seen and Presentation clearly seen and
does not demonstrate does not demonstrate some demonstrate some knowledge heard, demonstrate some heard, demonstrate sufficient
knowledge gain from the knowledge gain from the gain from the experiment; knowledge gain from the knowledge gain from the
experiment; memorization of experiment; memorization of memorization of text script experiment; no memorization of experiment; no memorization of
text script text script text script text script

Addressing Could not answer obvious Questions handled in the best of Questions handled in a Questions handled in a Questions handled with
question questions; speaker struggled to effort with some hesitation; knowledgeable way but with knowledgeable way but with some confidence and in a
link answer to content of demonstrate lack of some hesitation; demonstrate hesitation; demonstrate clear knowledgeable way;
presentation understanding of the subject lack of understanding of the understanding of the subject demonstrate clear understanding
matter. subject matter. matter. of the subject matter.

6
 ERROR ANALYSIS

In general consensus, an error is something that were done wrong. However, in

science, the word “error” means the “uncertainty” which accompanies every
measurement. No measurement of any sort is complete without a consideration of this
inherent error. Unfortunately, errors in experiment could not be eliminated. Hence, the
next best option is to mitigate the magnitude of such error by properly following the
laboratory guideline.

Among the cardinal emphasis in a science education is to learn how to handle and
interpret experimental data and results. This includes the development of
methodologies needed to estimate the inherent error in various types of measurements,
and techniques for data analysis to find out if these error estimates are valid, and the
understanding of the way errors propagate through calculations made. In other words,
perform statistical analysis on the collected data.

Different experiments deal with different aspect of errors. Mastering error analysis
requires extensive practice and will not happen overnight. Consider this document as
a starting resource on how to handle the particular errors in your lab work.

Precision and Accuracy: Two Different Types of Errors

There are two main types of errors associated with an experimental result, namely
precision and accuracy. The precision is usually related to the random error
distribution associated with a particular experiment or even with a particular type of
experiment (for example, in the experiments where the measured parameters have
intrinsically large variations between different samples). The accuracy is related to the
existence of systematic errors, for example, the incorrect calibration.

The object of a good experiment is to improve both precision and accuracy. Usually
in a given experiment one of these two types of errors is dominant, and the scientist
devotes most of his or her efforts towards reducing that one. For example, if you are
measuring the length of a carrot in a sample of carrots to determine an average value
of the length, the natural random variations within the sample of plants are probably
much larger than any possible measurement inaccuracy due to a bad manufacturing of
the ruler that you use. In a physics laboratory, the relative effects of the precision and
accuracy on the final result usually depend on the particular experiment and the
particular apparatus. Some experiments in the introductory physics laboratory have
relatively large random errors that require repeated measurements to increase the
precision. Other experiments are very precise, but the equipment used has built-in
sources of inaccuracy that cannot be eliminated.

7
 Three Major Sources of Errors

a. Reading error

Almost all direct measurements involve reading a scale (ruler, caliper, stopwatch,
analog voltmeter, etc.) or a digital display (e.g., digital multimeter or digital clock).
Sources of uncertainty depend on the equipment we use. One of the unavoidable
sources of errors is a reading error. It refers to the uncertainties caused by the
limitations of our measuring equipment and/or our own limitations at the time of
measurement (for example, our reaction time while starting or stopping a stopwatch).
This does not refer to any mistakes you may make while taking the measurements.
Rather it refers to the uncertainty inherent to the instrument and your own ability to
minimize this uncertainty.

A reading error affects the precision of the experiment. The uncertainty associated
with the reading of the scale and the need to interpolate between scale markings is
relatively easy to estimate. For example, consider the millimeter (mm) markings on a
ruler scale. For a person with a normal vision it is reasonable to say that the length
could be read to the nearest millimeter at best. Therefore, a reasonable estimate of the
uncertainty in this case would be ∆l=±0.5 mm which is half of the smallest division.
A rule of thumb for evaluating the reading error on analogue readout is to use half of
the smallest division (in case of a meter stick with millimeter divisions it is 0.5mm),
but only the observer can ultimately decide what is his/her limitation in error
evaluation. Note that it is wrong to assume that the uncertainty is always half of the
smallest division of the scale. For example, for a person with a poor vision the
uncertainty while using the same ruler might be greater than one millimeter. If the
scale markings are further apart (for example, meter stick with markings 1 cm apart),
one might reasonably decide that the length could be read to one-fifth or one-fourth of
the smallest division.

There are other sources of uncertainty in direct measurements that can be much more
important than uncertainties in the scale or display readings. For example, in
measuring the distance between two points, the main problem may be to decide where
those two points really are. For example, in an optics experiment it is often necessary
to measure the distance between the center of the lens and the position of the focused
image. But even a thin lens is usually several millimeters thick, which makes locating
the center a difficult task. In addition, the image itself may appear to be focused not at
a point, but its location may span a range of several millimeters. Parallax is another
significant source of a reading error, where the reading depends on your line of sight.

In this laboratory course, you will often use instruments that have a digital readout.
For many digital instruments, you may assume that the reading error is ± 1/2 of the
last digit displayed; e.g. if the reading of the timer in the free fall experiment is 682.6

8
ms, the error can be assumed to be ±0.05 ms, and you should quote (682.60±0.05) ms.
Ideally, you must record the specification supplied by the manufacturer of the
equipment used in the experiment.

It is usually difficult or impossible to reduce the inherent reading error in an

instrument. In some cases (usually those in which the reading error of the instrument
approximates a “random error distribution”) it is possible to reduce the reading error
by repeating measurements of exactly the same quantity and averaging them. While
performing the lab, if in doubt how to record the error in a measurement, consult your
lab instructor.

b. Random error

Random error refers to the spread in the values of a physical quantity from one
measurement of the quantity to the next, caused by random fluctuations in the
measured value. For example, in repeating measurements of the time taken for a ball
to fall through a given height, the varying initial conditions, random fluctuations in air
motion, the variation of your reaction time in starting and stopping a watch, etc., will
lead to a significant spread in the times obtained. This type of error also affects the
precision of the experiment.

i. Estimating uncertainties in repeating measurements

For example, while using a stopwatch to measure time intervals, the major uncertainty
is usually not from reading the dial, but from our reaction time in starting and stopping
the watch. The reaction time is unknown and can vary significantly from person to
person and even, for the same person, from measurement to measurement. The
solution is to repeat the measurement several times. Most likely you’ll obtain
somewhat different values for every one of your measurements. The “correct”
measured value lies somewhere between the lowest value and the biggest value (the
interval gives a probable range). We assume that the best estimate of the measured
value is the average value. For example, consider the sequence of four time intervals
2.3, 2.4, 2.5, 2.4 (s). The estimate of time interval is 2.4 s (the arithmetic mean of the
four individual measurements), and the probable range is 2.3 to 2.5 s. Note that the
probable range means that we are reasonably confident (but not necessarily 100%
certain) that the actual quantity lies within this range. Also note that the best estimate,
2.4 s, lies near the midpoint of the estimated range of probable values, 2.3 to 2.5 s.
This is true in most measurements.

ii. Mean and standard deviation

Suppose that we want to measure the value of a quantity, x. Let us imagine that we do
this by making a total number of n measurements of x, the values of each measurement

9
being denoted by xi, where i takes on the values from 1 to n. Either because of
variability in the quantity x or because of inherent and unavoidable random errors in
our measuring procedure, not all values of the individual measurements xi will be the
same. Our best estimate of the “true” value of x is then given by the average or mean
value of x:
1 n
Mean, x   xi (1.1)
n 1

Usually we also require some measure of the spread in the values of xi. This spread is
related to the uncertainty in our estimate of the “correct” value of x. It turns out that
the “best” estimate of the spread is given by a quantity σ, called the standard deviation
of x, given by

n
( xi  x )2
 1 n  1 (1.2)

iii. Standard error, or the error in the mean, σm

σ is associated with the error in each individual measurement, xi. However, what we
often really need to know is the error in our best estimate of x, which is the mean (best)
value. Clearly this error is less than σ, the error in any individual measurement. If it
weren't, we could estimate the “true” value of x as well with one measurement as we
could with many. A standard deviation σ of individual measurements divided by the
square root of the total number of measurements is called the standard deviation of the
mean. It is also called the standard error and is often denoted by σm.

m  (1.3)
n

Thus, our final answer to the question “what is our true measured value of x?” is


x  x m  x  (1.4)
n

Note that we could reduce the standard error by taking more measurements. However
there is no point in taking more than the number required to reduce the standard error
to below the reading error.

Remember that the uncertainty in any measured quantity has the same dimensions as
the measured quantity.

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c. Systematic errors and the role of instrument calibration

Systematic error refers to an error which is present for every measurement of a given
quantity; it may be caused by a bias on the part of the experimenter, a miscalibrated
or even faulty measuring instrument, etc. Systematic errors affect the accuracy of the
experiment.

After evaluating the reading error or the standard error, or both if necessary, we have
to make sure that the scale of our measuring instrument is checked against an
internationally established measuring standard. Such comparison is called calibration.
In the real world, we frequently find that our measuring scale is in slight disagreement
with the standard. For example, if you inspect such simple tools as rulers, you will
find out that no two rulers are exactly the same. It is not uncommon to find a
discrepancy of 1 mm or even more among meter sticks. The correct calibration of
measuring instruments is obviously of great importance. However, in the first year
laboratory, the instruments you will use are usually calibrated by the laboratory staff
and ready to use (unless explicit lab instructions tell you otherwise).

In addition to all the errors discussed above, there can be other sources of error that
may pass unnoticed: variations in temperature, humidity or air pressure, etc. Such
disturbances are more or less constant during our measurements (otherwise they would
appear as random error when the measurement is repeated) and are generally referred
to as the systematic errors. Systematic errors are very difficult to trace since we do not
know where to look for them. It is important to learn to notice all the irregularities that
could become the sources of systematic errors during our experimental work.
Moreover, it is particularly important in data-taking always to record some
information about the surrounding physical conditions. Such information may help us
later on if we discover a serious discrepancy in our experimental results. As a rule, the
place, date and time of measurements, and the type and serial numbers and
specifications of the instruments which were used must be recorded.

Estimate all your reading errors while you take your data and write them down with
your data. Do the same for all manufacturers' error specifications. These usually
cannot be “guessed” later on.

Quoting the Errors: Absolute and Relative Uncertainties

In general, the result of any measurement of physical quantity must include both the
value itself (best value) and its error (uncertainty). The result is usually quoted in the
form
x  xbest  x (1.5)

where xbest is the best estimate of what we believe is a true value of the physical

11
quantity (e.g. mean) and ∆x is the estimate of absolute error/uncertainty (e.g. standard
deviation or standard error). Note that depending on the type of the experiment the
prevailing error could be random or reading error. In case the reading error and random
error are comparable in value, both should be taken into account and treated as two
independent errors. You will learn how to calculate ∆x in this case in the “Propagation
of Errors” section.

The meaning of the uncertainty ∆x is that the true value of x probably lies between
(xbest−∆x) and (xbest+∆x). It is certainly possible that the correct value lies slightly
outside this range. Note that your measurement can be regarded as satisfactory even if
the accepted value lies slightly outside the estimated range of the measured value. ∆x
indicates the reliability of the measurement, but the quality of the measurement also
depends on the value of xbest. For example, an uncertainty of 1 cm in a distance of 1
km would indicate an unusually precise measurement, whereas the same uncertainty
of 1 cm in a distance of 10 cm would result in a crude estimate.

Fractional uncertainty gives us an indication how reliable our experiment is. Fractional
uncertainty is defined as ∆x/xbest. Fractional uncertainty can be also represented in
percentile form (∆x/ xbest)100%. For example, the length l= (0.50 ± 0.01) m has a
fractional uncertainty of 0.01/0.5=0.02 and a percentage uncertainty of 0.02×100=
2%. Note that the fractional uncertainty is a dimensionless quantity. Fractional
uncertainties of about 10% or so are usually characteristic of rather rough
measurements. Fractional uncertainties of 1 or 2% indicate fairly accurate
measurements.

Significant figures

An uncertainty should not be stated with too much precision. The last significant figure
in any stated answer should usually be of the same order of magnitude (in the same
decimal position) as the uncertainty. For example, the answer 92.81s with an
uncertainty of 0.3s should be rounded as (92.8 ± 0.3)s. If the uncertainty is 3s, then
the result is reported as (93 ± 3) s. However, the number of significant figures used in
the calculation of the uncertainty should generally be kept with one more significant
figure than the appropriate number of significant figures in order to reduce the
inaccuracies introduced by rounding off numbers. After the calculations, the final
answer should be rounded off to remove this extra figure.

Practical hints

So far, we have found two different errors that affect the precision of a directly
measured quantity: the reading error and the standard error. Which one is the actual
error of precision in the quantity? For practical purposes you can use the following
criterion. Take one reading of the quantity to be measured, and make your best

12
estimate of the reading error. Then repeat the measurement a few times. If the spread
in the values you obtain is about the same size as the reading error or less, use the
reading error. If the spread in values is greater than the reading error, take three or four
more, and calculate a standard error and use it as the error. In cases where you have
both a reading error and a standard error, choose the larger of the two as “the” error.
Be aware that if the dominant source of error is the reading error, taking multiple
measurements will not improve the precision.

Common mistakes and misconceptions with errors

It is almost always meaningless to specify the error with too many significant digits;
often one is enough. It is a mistake to write: x = (56.7 ± 0.914606) cm, or x = (56.74057
± 0.9) cm. Instead, write: x = (56.7± 0.9) cm. You cannot increase either the accuracy
or precision by extending the number of digits in your mean value beyond the decimal
place occupied by the error. Keep in mind that the error, by its nature, denotes the
uncertainty in the last one or two significant digits of the main number and therefore
any additional digits obtained from multiplication or division should be rounded off
at the meaningful position. So, first calculate your error; round it off to one significant
figure; then quote the value of your measurement to the appropriate number of
significant figures.

When quoting errors in a result do not use the flawed logic that “my result is x, the
handbook gives a value for this quantity as y, thus the error in my result is ±(x - y).”
Your quoted error should be the result of your own analysis of your own experiment
whereas (x - y) relates to a comparison of your work to other people's work. (x – y)
represents the difference between your result and the accepted value. The discrepancy
can be used to characterize the consistency between different sets of measurements,
but has nothing to do with the estimate of error in your own experiment.

If a result you produce differs significantly from the accepted value, you are obligated
to explain what has produced the difference. But in quoting your own result, you must
provide the error of your own experiment.

Propagation of errors

In the majority of experiments the quantity of interest is not measured directly, but
must be calculated from other quantities. Such measurements are called indirect. As
you know by now, the quantities measured directly are not exact and have errors
associated with them. While we calculate the parameter of interest from the directly
measured values, it is said that the errors of the direct measurements propagate. This
section describes how to calculate errors in case of indirect measurements.

As an example, consider the following problem. Suppose we have measured the value

13
of a quantity x with an uncertainty, which we denote ∆x. In order to test a theoretical
formula, suppose that we need to calculate y as function of x, e.g. y = f(x). We want
to know the uncertainty in y due to the uncertainty in the value of x. This is equivalent
to asking what will be the variation in y (call it ∆y) as x varies from x to (x+∆x)?
Mathematically, this variation is given by ∆y = f(x+∆ x) - f(x). The answer comes
from the differential calculus: if y = f(x) and ∆x is small, then

dy df
y  x  x (1.6)
dx dx

This argument can be extended for the calculation of quantities that are functions of
several different measured quantities. All you will need at this point are the results that
you can find below for different types of functions. Note that we neglect the sign in
the differential, since the sign of all errors may take on numerical values which are
either + or -.

a. Propagation of independent errors

Here ∆y and the various ∆x’s are either standard deviations, standard errors or reading
errors, depending on the circumstances.

Rule 1: If two mutually independent quantities are being added or subtracted:

y   x1    x2 
2 2
(1.7)

Rule 2: If two mutually independent quantities are being multiplied or divided:

2 2
y  x   x 
  1   2  (1.8)
y  x1   x2 

Rule 3: If a quantity is raised to a power: y=xn and if ∆x is small:

y x
n (1.9)
y x

Data fitting techniques and graphical analysis

In many experiments, it is very useful to make a plot of the data points as they are
being taken in order to make sure that the experimental data make sense. Carefully
prepared and analyzed graphs are often the best way to extract the values of the

14
unknowns and to confirm or deny the theoretical prediction being tested. They are
essential in presenting your results in a report. Straight line graphs have a special
significance. Their interpretation is often simplest, since all straight lines plotted on
an x−y graph have the form y=ax+b where a is the slope of the line (the tangent of the
angle between the line and the x-axis), while b is the intercept on the y-axis. Usually
x and y depend only on the known or measured quantities while a and b contain the
unknowns which you are trying to find.

Though a plot of your data points in their raw form will not generally lie on a straight
line, you will find that data can usually be made to fit a straight line by manipulating
the equation. If there are two and only two unknowns in your experiment, it is often
possible to put the theoretical relationship into the form of a linear equation.

You also need to show the uncertainty in each point of your graph. This is done by
drawing the so-called error bars. The error bars are the lines corresponding to the size
of the error on either side of the data point. The error bars are drawn vertically for
errors in y values and horizontally for errors in x values.

15
 SOLUTION AND DILUTION

Introduction

There are a number of experimental procedures in this course that requires preparation
of solution and dilution. All solutions must be prepared carefully especially when
measuring weight and volume of the reagents in order to minimize error in the
subsequent experiment. Preparation of solution and dilution are closely related to the
concept of concentration. Concentration of a solution could be expressed in the forms
of weight per unit volume (g/ml), percent composition (%w/w, %w/v, %v/v), parts per
million/billion, molarity, molality, normality, etc.

A simple equation allows the dilution to be calculated readily:

C1V1  C2V2 (1.10)

where C1 is the concentration of the initial/stock solution, V1 is the volume of the

initial/stock solution available to be used for dilution, C2 is the desired final
concentration, and V2 is the desired final volume. In most of the exercises in this
course, the initial concentration and the final concentration are either known/specified
in the lab manual or are chosen by students. The final volume is usually the required
amount for a given experiment. For instance, the experimental procedure
states”…prepare 100ml of 1.0M sodium hydroxide from 3.0M stock solution”, thus,
the initial concentration is 3.0M, the final volume is 100ml, the desired final
concentration is 1.0M, and the volume of initial/stock to be used in the dilution can be
determined from the above equation.

Learning Outcome

At the end of this laboratory, students are expected to comprehend the following:
a. Ability to operate micropipette properly.
b. Understand the concept of concentration and dilution in preparation of
stock/working solution.
c. Ability to perform dilution of concentrated solution in the correct manner.

Equipment and Material

Equipment 1. Analytical balance

Labware 1. Micropipette
2. Micropipette tips
3. Test tube

16
 4. Test tube rack
Material/ chemical 1. Food coloring – yellow, blue, and red.

Experimental Procedure

Part 1: Demonstration on how to use micropipette

The lab instructor will demonstrate the proper way to use a micropipette. Pay close
attention and make some notes on the followings:
i. Do’s and don’ts.
ii. How to prepare micropipette for use – volume adjustment, attach/detach tips
iii. How to withdraw/release liquid

Part 2: Serial dilution of multicomponent solution

1. Select two colours of food colouring. Designate each colour as Colour A and
Colour B.
2. Prepare 100 ml of stock solution of Colour A with percent composition of your
choice.
3. Prepare 100 ml of stock solution of Colour B with weight per unit volume of
your choice.
4. Perform at least two sets of serial dilution of Colour A or Colour B and mixture
of Colour A and B. Use water as diluent (final volume is 5 ml).
Select a range that demonstrates a high contrast between the diluted
solutions.
5. Report the concentration of Colour A and Colour B for all solutions in part per
million (Assume the density of food colouring is similar to water).

Data Analysis and Report

Final
Stock solution Stock solution Water Colour A Colour B
No. volume
Colour A (ml) Colour B (ml) (ml) (ppm) (ppm)
(ml)
1
2
3
…
…

Your report should include the following

1. Discuss how to properly use a micropipette to measure liquid sample and the

17
 consequences of violating such guidelines.
2. Provide several suggestions on how can an experimenter verify that 1 ml of the
liquid that he/she drew using a micropipette is indeed 1 ml?
3. State and justify the dilution series that you have chosen for Part 2. Show the
calculation (dilution step, concentration of individual colour component in the
final solution in parts per million, etc.) for one of the diluted solution.

18
19
PART I
CHROMATOGRAPHY

HPLC
GC

20
PART I – CHROMATOGRAPHY TECHNIQUE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

CALIBRATE A CHROMATOGRAPHY SYSTEM

CALIBRATING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

SYSTEM FOR SUGAR CONTENT ANALYSIS

Introduction

In general, calibration refers to a technique to verify the response provided by any

measuring device. The response is compared against a known quantity, i.e. weight,
volume, concentration, etc., of a standard. In this exercise, student will learn how to
calibrate a HPLC system for sugar analysis.

Learning Outcome

At the end of this laboratory, students are expected to comprehend the following:
a. Ability to calibrate a chromatography system using standard solution
b. Understand the concept of calibration curve

Equipment and Material

Equipment 1. Analytical balance

Labware 1. Micropipette (100 μl – 1000 μl)
2. Micropipette tips
3. Test tube
4. Test tube rack
5. 5 ml syringe
6. Syringe filter
7. Glass vial
Material/ chemical 1. Sugar monomer standard
2. Deionized water

Experimental Procedure

Part 1: Preparing HPLC system for analysis

1. Prepare the suitable mobile phase for the analysis. Selection of mobile phase
depends on the type of column and analytes (compounds of interest).
2. Fill up the mobile phase reservoir to a sufficient level. Make sure the filter for the
mobile phase line is submerged far below the mobile phase level but not touching
the bottom of the container.
3. Turn on the HPLC-Data Acquisition interface module.
4. Turn on the computer. Check the connection status between the computer and the

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PART I – CHROMATOGRAPHY TECHNIQUE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

HPLC system. If there is no connection between the two, restart the computer.
5. Launch the “Instrument Online 1” program from the computer’s desktop
screen/program list. A window with default view of HPLC detector’s signal plot
will be shown.
6. Turn on the pump. At this stage, start with a low flow rate setting.
7. Check for leaking at the connections between HPLC modules.
8. Turn on the column heater. Set the temperature to the value recommended in the
selected test method.
9. Once the temperature has reach the set value, increase the pump’s flow rate setting
to match the recommended value from the selected test method.
10. Monitor the baseline of the signal plot. It should show a stable/flat trend for at
least 15 minutes before conducting the first analysis of the operation cycle/day.
11. The status panel in the main program window should shows a ‘Ready’ status
(green colour).

Part 2: Preparation of standard solution (known concentration) for calibration

1. Weigh enough amount of sugar standard to prepare 20 ml stock solution of 100

mg glucose/ml.
2. Transfer the sugar standard into a clean, dry test tube and add deionized water to
make up the required volume.
3. By using serial dilution technique, prepare two more solutions; each with different
concentration.
4. Report the concentration of all solution prepared in this part of work.

Part 3: Analysis of standard solution in HPLC

1. Check the status panel of the HPLC’s main program window. It should show a
‘Ready’ status (green colour).
2. From “Instrument Online 1” Menu Bar, select ‘Run Control’ from the menu bar,
and select ‘Sample Info’. A new window ‘Sample Info: Instrument 1’ will appear
3. In ‘Sample Info: Instrument 1’ window, provide all the required info.
a. Use a new filename for each sample.
b. Sample name should be the same with filename to avoid confusion.
c. Sample details may be provided in the ‘Comment Box’ (optional).
d. Set the ‘Location’ as ‘1’.
4. Click OK to close the ‘Sample Info: Instrument 1’ window.
5. Using HPLC syringe, draw sample to match the injection volume recommended
by the selected test method. MAKE SURE THERE IS NO AIR BUBBLE
TRAPPED IN THE SYRINGE CAPILLARY
6. Insert the syringe’s needle into the injection port. Be careful not to bend the
delicate needle. DO NOT PRESS THE PLUNGER YET
7. Turn the injection port’s lever upward/ counter-clockwise

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PART I – CHROMATOGRAPHY TECHNIQUE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

8. Press the syringe’s plunger to the max

9. Turn the injection port’s lever downward/ clockwise, all the way to its initial
position. The analysis will commence immediately. The status panel in the main
window will change to blue colour and show ‘Run In Progress: Data Acquisition’
10. Clean the HPLC syringe by withdrawing and dispensing distilled water several
times.
11. Wait for the analysis to complete before running a new sample.

Data Analysis and Report

1. Data

Known
Type of Retention Area under
Peak height Peak width concentration
sugar time (min) the peak
(mg/ml)

From the data,

a. Calculate the area under the peak for each type and concentration of sugar
standard solution.

One method of determining the area under the peak on a chromatogram is

called triangulation. In the triangulation process, the peak is estimated to be a
triangle and the area is calculated as shown in the figure below.

Figure 1: Triangulation method to calculate area under the peak for

HPLC chromatogram

For each sugar type, prepare a calibration curve based on area under the curve
with respect to the known concentration of the standard solution. This
calibration curve will be used to determine the sugars content in samples.

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PART I – CHROMATOGRAPHY TECHNIQUE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

ANALYSE MATERIAL USING CHROMATOGRAPHY SYSTEM

DETERMINATION OF SUGARS CONTENT IN A BEVERAGE

Introduction
High pressure liquid chromatography (HPLC) uses a column filled with a fine
packing, which is coated with a stationary phase. As the sample is moved through this
column by a liquid eluent (mobile phase), the various compounds move at differing
rates, depending on their affinity for the stationary phase. Compounds which have a
stronger attraction to the stationary phase will be retained longer and elute (come out
of the column) later. The sample is detected when it emerges from the column by a
detector that generates signal and send them to the acquisition system/ computer. The
identities of the sample components are found by matching their retention times (how
long it took to pass through the column) with retention times of standard samples that
was analyzed under the same conditions.

Learning Outcomes

At the end of the experiment, you will be able to:

1. Analyze liquid substance and identify its content using HPLC
2. Estimate the concentration of specific compound using HPLC

Equipment and Material

Equipment 1. HPLC system with Refractive Index (RI) detector

2. Rezex™ RCM-Monosaccharide Ca+2 (8%) column
Labware 1. Syringe
2. Syringe filter
3. Beakers
4. Micropipette
5. Micropipette tips
6. Sample vials
7. HPLC syringe
Material/ chemical 1. Beverage sample

Experimental Procedure

Part 1: Preparing sample for analysis

1. Your sample has to be ready prior to characterization process. The preparation

may include size reduction, dilution, digestion, etc.
2. For HPLC analysis, the liquid sample is recommended to be diluted within the

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PART I – CHROMATOGRAPHY TECHNIQUE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

concentration range of the calibration. All samples must be filtered and transferred
to a smaller vial. If the sample is sensitive to light, an amber vial should be used.
If the sample is sensitive to temperature, it should be chilled while waiting for
analysis.
3. Your sample is a liquid sample, e.g. beverage. The unknown samples will be
prepared for you and to be used as it is. Draw several ml of sample into a 5 ml
syringe, attach the filter, and inject into a clean vial.
4. If necessary, prepare a dilution for your sample (final volume 2ml), draw several
ml of the diluted sample into a 5 ml syringe, attach the filter, and inject into a
clean vial. Label all the vials.

Part 2: Preparing HPLC system for analysis

1. Prepare the suitable mobile phase for the analysis. Selection of mobile phase
depends on the type of column and analytes (compounds of interest).
2. Fill in the mobile phase reservoir up to a sufficient level. Make sure the filter for
the mobile phase line is submerged far below the mobile phase level but not
touching the bottom of the container.
3. Turn on the HPLC-Data Acquisition interface module.
4. Turn on the computer. Check the connection status between the computer and the
HPLC system. If there is no connection between the two, restart the computer.
5. Launch the “Instrument Online 1” program from the computer’s desktop
screen/program list. A window with default view of HPLC detector’s signal plot
will be shown.
6. Turn on the pump. At this stage, start with a low flow rate setting.
7. Check for leaking at the connections between HPLC modules.
8. Turn on the column heater. Set the temperature to the value recommended in the
selected test method.
9. Once the temperature has reach the set value, increase the pump’s flow rate setting
to match the recommended value from the selected test method.
10. Monitor the baseline of the signal plot. It should show a stable/flat trend for at
least 15 minutes before conducting the first analysis of the operation cycle/day.
11. The status panel in the main program window should a ‘Ready’ status (green
colour)
12. Prepare to inject sample.

Part 3: Injecting sample into HPLC

1. Check the status panel of the HPLC’s main program window. It should show a
‘Ready’ status (green colour).
2. From “Instrument Online 1” Menu Bar, select ‘Run Control’ from the menu bar,
and select ‘Sample Info’. A new window ‘Sample Info: Instrument 1’ will appear
3. In ‘Sample Info: Instrument 1’ window, provide all the required info.

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PART I – CHROMATOGRAPHY TECHNIQUE
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

a. Use a new filename for each sample.

b. Sample name should be the same with filename to avoid confusion.
c. Sample details may be provided in the ‘Comment Box’ (optional).
d. Set the ‘Location’ as ‘1’.
4. Click OK to close the ‘Sample Info: Instrument 1’ window.
5. Using HPLC syringe, draw sample to match the injection volume recommended
by the selected test method. MAKE SURE THERE IS NO AIR BUBBLE
TRAPPED IN THE SYRINGE CAPILLARY
6. Insert the syringe’s needle into the injection port. Be careful not to bent the
delicate needle. DO NOT PRESS THE PLUNGER YET
7. Turn the injection port’s lever upward/ counter-clockwise
8. Press the syringe’s plunger to the max
9. Turn the injection port’s lever downward/ clockwise, all the way to its initial
position. The analysis will commence immediately. The status panel in the main
window will change to blue colour and show ‘Run In Progress: Data Acquisition’
10. Clean the HPLC syringe by withdrawing and dispensing distilled water several
times.
11. Wait for the analysis to complete before running a new sample.

Data Analysis and Report

Your report should include the following:

2. Prepare a box/block diagram showing primary components of the HPLC system

that you used and their connections.
3. Why the mobile phase needs to be degassed prior to analysis?
4. Report your experimental details (operation parameters); for example, how the
sample was prepared, injection volumes, etc.
2. Print out the chromatogram(s) obtained from the analysis of unknown sample(s).
Using the retention time of the sugar standard(s) in Experiment 2, identify the
sugar peak in your unknown samples and label accordingly.
3. Estimate the concentration for each type of sugar detected in the unknown sample
from the calibration curve prepared in Experiment 2. The dilution factor must be
considered here.

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PART I – CHROMATOGRAPHY TECHNIQUE
GAS CHROMATOGRAPHY

ANALYSE MATERIAL USING CHROMATOGRAPHY SYSTEM

IDENTIFICATION AND QUANTIFICATION OF GAS MIXTURE USING

GAS CHROMATOGRAPHY SYSTEM

Introduction

In chromatography, a sample is separated into its component parts by putting the

mixture into a mobile phase, which flows over or through a stationary phase. The
sample components are distributed between these two phases, depending on their
relative solubility or affinity for each of the phases. If the components differ in this
affinity, they will be separated as they pass through the system. Materials which have
a greater affinity for the stationary phase will take a longer time to pass through the
system, and those which remain more in the mobile phase will exit the system more
rapidly. In a gas chromatograph, the mobile phase is an inert gas, the stationary phase
is a high molecular weight non-volatile liquid or a polymeric solid packed into a
column. The separation depends on the vapor pressure of each component, with those
of highest vapor pressure traveling through the column more rapidly.

At the end of the column, a detector is needed to determine when the components are
exiting the column (eluting) and to record the quantity of each component. In this case
a flame ionization detector (FID) detects organic compounds by passing them into a
small hydrogen-air flame. As they burn, ions are formed in the flame, collected on an
electrode, amplified and recorded. The thermal conductivity detector (TCD) measures
the transfer of heat from a warm filament to the walls of the detector, and indicates the
elution of compounds as the thermal conductivity of the gas stream changes with
concentration. This detector can be used for inorganic gases as well as organics.

Learning outcomes

At the end of the experiment, you will be able to:

1. Learn how to use a gas chromatograph (GC)
2. Learn about some of the practical issues involving separations
3. Perform qualitative and quantitative determination using a GC data

Equipment and Material

Equipment 1. GC system Agilent 6890 Series

Labware 1. GC Syringe

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PART I – CHROMATOGRAPHY TECHNIQUE
GAS CHROMATOGRAPHY

2. Butane lighter
3. Autosampler vials
Material/ chemical 1. Sulfolane
2. Diisopropanolamine
3. Chloform
4. Carbon tetrachloride
5. Dichloromethane
6. Mixture of C5 – C8 hydrocarbons

Experimental Procedure

Part 1: Operating the gas chromatography

A guide to the operating procedure for the Agilent 6890 Series should be referred
to the instructor. Normally, the teaching assistant will have set up the equipment
in advance.

1. Study the GC instrumentation your group is going to use. Draw a neat sketch
of the GC. Specify the type of detector, column and etc. that are being used.
Also report all the operating conditions such as flow rates, temperature etc.
2. The given sample contains sulfolane and diisopropanolamine. Using a
microsyringe, first inject 1 l each of the pure substances provided to you and
report the retention time, t (the time taken for the component peak to appear
after the time of injection of the sample).
This procedure will be done once and copies of the chromatograms will be
given to each student. Make sure that there are no air bubbles in the syringe.
Also inject the syringe into the rubber septum rapidly and release it rapidly.
Prepare accurately a known mixture of the compounds by weighing one gram
of each into a weighing bottle; then inject this sample.
3. Then inject your sample of unknown composition into the GC. Based on
retention time, identify the unknown components. Carry out these procedures
quickly, making sure to cover the weighing bottles because these compounds
are volatile and vaporize rapidly and any evaporation would change the
composition of the mixture, due to different volatilities. Use the peak areas to
estimate the concentration of each component in the given mixture as:
Area of Std./Area of Unknown = mg of standard/mg of Unknown.

4. The typical chromatogram obtained for the separation of diethyl ether, hexane,

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PART I – CHROMATOGRAPHY TECHNIQUE
GAS CHROMATOGRAPHY

toluene and octane will be given to you during your lab session.

Part 2: Optimization of chromatography system

1. First, make sure the instrument in on, the carrier gas is flowing, the flame
ionization detector (FID) is lit, and the computer is responding. Record the
dimensions (L, i.d.), the make and model, and the type and thickness of the
stationary phase for the column installed in the chromatograph. Check with the
lab instructor if any of these items are unclear.
2. Next, using the instructions provided, set the flow rate of the column to 0.80
mL/min, the split ratio to 30:1, and the injector and detector temperatures to
250 °C.
3. In an autosampler vial, deposit 20 μL of carbon tetrachloride (CCl4) using the
micropipette. In a second autosampler vial, deposit 20 μL of chloroform
(CHCl3). In a third autosampler vial, deposit 20 μL of CCl4 and 20 μL of
CHCl3. Finally add sufficient volume of dichloromethane (CH2Cl2) to each
tube to bring the total volumes to 1.000 mL and cap each vial securely.

Part 2a: Determining Dead Time (t0)

1. Each student will make five manual injections of butane to determine the dead
time and to determine the reproducibility of each individual’s injection
technique. Enter a temperature program for the column, in which the oven
temperature is held constant at 50 °C. This is called an isothermal separation
method. Insert the syringe needle into the outlet of a butane lighter and press
the gas release button on the lighter. Do not strike the flint. Withdraw the
plunger on the syringe to between 2 and 4 μL. Prior to injection, keep the
syringe needle pointing down to avoid loss of butane from syringe.
2. Inject the solution and record a chromatogram. A typical manual injection in
split injection mode should take approximately 2.5 seconds (1 second to insert
the needle, 0.5 seconds to inject, and 1 second to withdraw the needle). Be sure
to be consistent in timing the start of the run with the manual injection (insert
syringe inject withdraw syringe  start run).
3. Record the retention time of the peak for each repetition and calculate the
average, the standard deviation, and the relative standard deviation for each
individual five replicates, as well as for the entire group. This is the dead time
for the column under this method.

Part 2b: Effect of Changes in Temperature Program

1. Enter a temperature program for the column, in which the oven temperature is

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PART I – CHROMATOGRAPHY TECHNIQUE
GAS CHROMATOGRAPHY

held constant at 30°C. Inject a 0.5 μL sample of the mixed solution containing
both CCl4 and CHCl3 and record the chromatogram. Print the chromatogram
and the peak analysis report for your records. Make sure all printed
chromatograms are adequately labeled with sample and method information
for future reference. Also, expand the chromatogram on the computer and
record the width at half-height (Wh) for each peak
(Note: width at base (W) may also be recorded, if necessary and if peaks are
adequately resolved).
2. Enter an isothermal temperature program where the oven is set to 50 °C. Inject
a 0.5 μL sample of the mixed solution containing both CCl4 and CHCl3 and
record the chromatogram. Print the chromatogram and the peak analysis
report, and record Wh for each peak.
3. Enter an isothermal temperature program where the oven is set to 90 °C. Inject
a 0.5 μL sample of the mixed solution containing both CCl4 and CHCl3 and
record the chromatogram. Print the chromatogram and the peak analysis
report, and record Wh for each peak.
4. Enter a temperature program in which the oven temperature begins at 50 °C
and increases to 90 °C at a rate of 5 °C/min. This is called a temperature
gradient separation program.
5. Inject a 0.5 μL sample of the mixed solution containing both CCl4 and CHCl3
and record the chromatogram. Print the chromatogram and the peak analysis
report, and record Wh for each peak.
6. Finally, record chromatograms of 0.5 μL injections of each of the two pure
compounds dissolved in CH2Cl2 using the temperature program above. Print
the chromatogram and the peak analysis report for each run, and record Wh
for each peak.

Part 2c: Effect of Changes in Flow Rate

1. Adjust the column flow of the carrier gas to 1.50 mL/min. Set the oven
temperature for an isothermal run at 50 °C, make a 0.5 μL injection of the
mixed solution, and record the chromatogram.
2. Print the chromatogram and the peak analysis report, and record Wh for each
peak.
3. Adjust the column flow of the carrier gas to 3.00 mL/min and repeat the
experiment.
4. Print the chromatogram and the peak analysis report, and record Wh for each
peak.

Part 3: Calibration of gas chromatography system

1. In four (4) autosampler vials, deposit 30, 40, 50, and 60 μL of CCl4,
respectively, using the micropipette.

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PART I – CHROMATOGRAPHY TECHNIQUE
GAS CHROMATOGRAPHY

2. Next, add 20 μL of CHCl3 to each tube.

3. Finally, add sufficient volume of CH2Cl2 to each vial to bring the total
volume to 1.000 mL and cap each vial securely.
4. These are your calibration standard samples. Prepare a sample “blank”, by
filling an autosampler vial with 1 mL of CH2Cl2.
5. In an autosampler vial, add 20 μL of CHCl3 to 500 μL of your unknown.
Add sufficient CH2Cl2 to bring the total volume to 1.000 mL and cap
securely. This is your unknown sample for quantitative determination.
6. Place the sample vials in the autosampler and create a sequence in the
software for the analysis of your samples, using the same temperature
program as previously and a carrier gas flow rate of 0.8 mL/min.
7. Program the autosampler to sequentially inject 0.5 μL of each of the
solutions (calibration standards, blank, unknown).
8. Print the chromatograms and the peak analysis reports for your records.

Part 4: Kovat’s Retention Indices

1. Using the isothermal method with column temperature of 50 °C and flow rate
of 0.8mL/min, record the chromatogram for 0.1 μL injection of a neat mixture
of the C5 – C8 hydrocarbons.
2. Print the chromatogram and the peak analysis report for your records.

Data Analysis and Report

Your report should include the weight percent of the components in the unknown
mixture. Also label the chromatogram to show the individual peaks for all analytes
in addition to any peaks due to air bubbles or impurities in the solvents.

Part 2
Calculate the resolution (R) for the separation of CHCl3 and CCl4 in the 4
chromatograms recorded using the various oven temperature settings (isothermal at
30, 50, and 90 °C; and gradient temperature programming). Also, calculate the
capacity factors (k’) for CHCl3 and CCl4 and the selectivity factor (α) resulting from
each method. Assume t0 for each of the methods is equivalent to that which was
determined initially from the butane injections at 50 °C. How do the calculated values
(R, k’, α ) change for separations using the different oven temperature methods?

Calculate R between CHCl3 and CCl4 at the 1.5 mL/min and the 3.0 mL/min flow
rates.

Calculate the number of theoretical plates (N) and the height equivalent to a
theoretical plate (HETP) for CHCl3 and CCl4 at each of the 3 flow rates (0.8, 1.5, and

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PART I – CHROMATOGRAPHY TECHNIQUE
GAS CHROMATOGRAPHY

3.0 mL/min). Using the HETP values, calculate the A, B, and C constants in the Van
Deemter equation for each of the two compounds (Note: You will need to convert the
flow rates to linear velocities; divide by the area of the cross section of the open portion
of the column).

Finally, take the first derivative of the Van Deemter equation (Equation 1) and
estimate the optimum column flow rate.

Equation 1

Where

▪ A = Eddy-diffusion
▪ B = Longitudinal diffusion
▪ C = mass transfer kinetics of the analyte between mobile and stationary phase
▪ u = Linear Velocity.

To estimate the minimum H, you can differentiate Equation 2:

Equation 2

Part 3
1. Using the chromatograms obtained from your standard solutions perform a
linear regression of the plot of area of the CCl4 peak versus percent CCl4
(external standard method).
2. Determine the slope and the intercept of the best-fit line to the data. Also,
determine the standard deviation of the slope and the intercept.
3. Next, for each of the five chromatograms of the standard samples, divide the
area of the CCl4 peak by the area of the CHCl3 peak and make a second plot
of these values versus percent CCl4 (internal standard method).
4. Determine the slope and the intercept of the best-fit line to the data and
determine the standard deviation of the slope and the intercept. How does the
relative standard deviation of the slope of the second plot compare with the
relative standard deviation of the slope of the first plot?
5. Determine the concentration of the unknown by the external standard method
(using the regression equation from the first plot, determine the concentration
of unknown from the measured area) and the internal standard method (divide
the area of the CCl4 peak by the area of CHCl3 from your unknown
chromatogram and, using the regression equation from the second plot,
determine the concentration of unknown from the normalized area).
6. Estimate the error in these values from the errors in the calibration plot. How
do the absolute values determined and their uncertainty compare? From the

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PART I – CHROMATOGRAPHY TECHNIQUE
GAS CHROMATOGRAPHY

blank injection, estimate the average background signal and standard deviation
of this value. From the slope of the first calibration plot, estimate the limit of
detection (LOD) and the limit of quantification (LOQ) for the CCl4.

Part 4
Make a plot of the log of the adjusted retention times for the C5 to C8 hydrocarbons
versus the number of parafinnic carbon atoms x 100 (e.g. for C5, this equals 500 and
so on). Perform a linear regression and determine the slope and the intercept of the
best-fit line. Use this plot and the adjusted retention times for CHCl3 and CCl4,
calculate the Kovat’s retention index for each compound.

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PART I – CHROMATOGRAPHY TECHNIQUE
GAS CHROMATOGRAPHY

34
PART I – CHROMATOGRAPHY TECHNIQUE
GAS CHROMATOGRAPHY

PART II
SPECTROSCOPY

UV VIS
AAS
FTIR

35
PART II – SPECTROSCOPY
UV-VIS SPECTROPHOTOMETER

CALIBRATE A SPECTROSCOPY SYSTEM

QUALITATIVE AND QUANTITATIVE ANALYSIS USING UV-VIS

SPECTROPHOTOMETER

Introduction

Ultraviolet-Visible (UV-Vis) absorption spectrophotometry can be applied for

quantitative (such as Beer’s Law analysis) and qualitative (identifying compound(s),
checking purity etc.) analysis. This lab will explore the use of the UV-Vis
spectrophotometer to analyze various UV-Vis absorption by organic and inorganic
compounds.

Learning Outcomes

At the end of the experiment, you will be able to:

a. Calibrate UV-Vis for specific quantitative analysis

Equipment and Material

Equipment 1. Analytical balance

2. Hot plate with stirrer
3. UV-1800 Shimadzu UV Spectrophotometer
Labware 1. Plastic cuvette
2. Volumetric flask 100mL (11 pcs)
Material/ chemical 1. Ammonium hydroxide (ammonia solution, NH3, 30%
2. Ammonium chloride,NH4Cl
3. Cobalt salt (i.e. cobalt (II) sulphate heptahydrate) or
standard reference solution
4. Disodium ethylenediaminetetraacetic acid, EDTA
5. Nickel salt (i.e. nickel (II) nitrate hexahydrate) or
standard reference solution
6. Distilled water

Experimental Procedure

Part 1: Preparation of Co and Ni standard solutions

i. From solid metal

1. Accurately weigh enough Co metal to prepare 100 ml of approximately 0.05 M
solution.

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PART II – SPECTROSCOPY
UV-VIS SPECTROPHOTOMETER

2. Place it in a 100 ml volumetric flask and add 15 ml of dilute HNO3 (about 2M)
Heat gently on a hot plate until dissolution is complete. Do not stopper the flasks.
Use the Fume Hood for this procedure.
3. Neutralize with NaOH (about 2M) until the first permanent precipitate of cobalt
hydroxide is visible. Then add a few drops of acetic acid to clear the solution and
dilute to volume.
4. Similarly, prepare 100 ml of a 0.05 M Ni solution.

ii. From metal oxide

1. Accurately weigh enough oxide to prepare 100 ml of approximately 0.05 M metal
solution.
2. Add 8 ml of concentrated HCl, dissolve on a hot plate, neutralize the solution, and
add acetic acid to clear the solution, dilute to volume and mix.

iii. Standard reference solution

1. If ready standard reference solution is used, the dissolution step is not necessary.
Perform dilution if necessary.

iv. Standard solution with unknown concentration

1. If a liquid unknown is used, it should be transferred quantitatively to a 100 mL
volumetric flask and diluted to volume.

v. From metal salt

1. Accurately weigh enough salt to prepare 100 ml of approximately 0.05 M metal
solution and transfer into a volumetric flask.
2. Add deionized water to make up the volume.

Part 2: Preparation of buffer solution

1. Prepare 200 ml of buffer solution which is 1M in NH4Cl and 1M in NH3.

Part 3: Calibration of spectrophotometer with standard solutions

1. Pipet duplicates 40 ml aliquots of Ni and Co standard solution into two different

100 ml volumetric flasks.
2. Add 10 ml of buffer solution to each of the volumetric flasks.
3. Add 1.6g of EDTA disodium salt to each flask containing Ni and Co standards.
Use a powder funnel to add the EDTA. Remove the stopper and warm the flasks
on a hot plate for 20 min to complete formation of the complex. Cool and dilute
to volume.
4. Prepare a blank solution containing 12 ml buffer solution, and 1.6 g. of EDTA in
100 ml of solution.

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PART II – SPECTROSCOPY
UV-VIS SPECTROPHOTOMETER

5. Determine the absorbance of Co and one Ni standard at 20 nm intervals between

350 and 650 nm.
NOTE: Each time the wavelength is changed, the zero and the 100%
adjustments must be made.
6. Plot the absorbance spectra of Co and Ni. From the curves, choose the suitable
wavelengths for analysis of the unknown sample solutions.
7. Proceed to Exercise 5

Data Analysis and Report

1. Sketch of the basic components (block diagram) of a UV-VIS scanning

spectrometer.
2. List the absorbance obtained at each wavelength interval for Co and Ni standard
solutions.
3. Plot from (2) on a same axis and determine the followings:
a. λ1 and λ2

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PART II – SPECTROSCOPY
UV-VIS SPECTROPHOTOMETER

ANALYSE MATERIAL USING A SPECTROSCOPY METHOD

SPECTROPHOTOMETRY OF A TWO-COMPONENT MIXTURE

Introduction

It is possible to analyse a complex mixture containing several species simultaneously

without prior separation. For example, in this experiment the concentration of Cobalt
(Co) and Nickel (Ni) in a mixture will be determined. Both these metals react with
EDTA at a pH of 4 or more. Although the complexes (and therefore the color) are
stable, the rate of reaction with EDTA especially for Co is relatively slow and the
solutions have to be warmed for a long time to ensure complete reaction. This method
is applicable only at high concentrations.

According to Beer’s law, the absorbance (A) of a solution is equal to the molar
absorptivity ( [Lmol-1cm-1]) times the cell length (b [cm]) times the concentration (C
[molL-1]) at a given wavelength or:

A   Bc (1.11)

Since b is usually one cm, the working equation reduces to A = c. If a solution
contains two (or more) absorbing species (chromophores), then the measured
absorbance at a given wavelength will be the sum of all absorbance at that wavelength.
By determining the absorptivity () of each species at each wavelength, we can
theoretically determine the concentration of each component in a mixture by
measuring the total absorbance at each wavelength and solving the resulting equations
simultaneously.

For the binary mixture of this experiment, we have at wavelength λ1

A1  1 Co   2  Ni  (1.12)

and at wavelength λ2

A2   3 Co   4  Ni  (1.13)

where A1 & A2 = absorbance of the mixture at 1 and 2 respectively, 1 - 4 =

absorptivity, [Co] = concentration of cobalt (M),[Ni] = concentration of nickel (M).

If we measure A1 & A2 for our unknown and obtain 1, 2, 3 and 4 from the calibration

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PART II – SPECTROSCOPY
UV-VIS SPECTROPHOTOMETER

standards, we can solve the two equations for the concentrations of Co and Ni. The
highest sensitivity and precision are attained when the measurements are made at
wavelengths at which the absorbance difference is a maximum and the spectral overlap
is minimum.

Figure 2: Spectra of two hypothetical compounds. The best wavelengths are λ1

and λ2, where the difference in absorbance of the compounds is greatest.

Learning Outcomes

At the end of the experiment, you will be able to:

a. Perform quantitative analysis of sample using UV-Vis technique

Equipment and Material

Equipment 1. Analytical balance

2. Hot plate with stirrer
3. UV-1800 Shimadzu UV Spectrophotometer
Labware 1. Plastic cuvette
2. Volumetric flask 100mL (11 pcs)
Material/ chemical 1. Ammonia (NH3) 1M
2. Ammonium chloride (NH4Cl) 1M
3. Cobalt salt/ standard reference solution
4. Deionized water
5. Disodium ethylenediaminetetraacetic acid (EDTA)
6. Nickel salt/ standard reference solution

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PART II – SPECTROSCOPY
UV-VIS SPECTROPHOTOMETER

Experimental Procedure

Part 1: Determination Of Cobalt And Nickel As EDTA Complexes

(continue from Exercise 4)

1. Pipet 40 ml aliquots of the unknown solution into a 100 ml volumetric flasks. Add
3.0g of the EDTA and 10ml of the buffer to the flask. Dilute to volume. Measure
the absorbance of each of the standards and the unknown solution at the two
selected wavelengths.
2. Make at least five sets of readings for the solutions at each of the two selected
wavelengths.

Notes:
1. You will need to solve two equations simultaneously since you have two
unknowns - both metals will be absorbed to some extent at both wavelengths, so
the absorbance you measure will be a sum at that wavelength.
2. Once completed, you may want to try using a couple of other wavelengths. You
should get similar results.
3. If the absorbance is too high, dilute the samples quantitatively until an optimum
absorbance reading can be obtained.

Data Analysis and Report

1. List the absorbance obtained at each wavelength interval the unknown solution.
2. Plot the data in (1) on the same axis and determine the followings:
a. ε1 – ε4.
3. From your result in Exercise 4, estimate the amount (mg) of Co and Ni of the
unknown solution.
4. Discuss your result with regards to Beer’s Law.

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PART II – SPECTROSCOPY
ATOMIC ABSORPTION SPECTROPHOTOMETER

EXERCISE 8: CALIBRATE AN ELEMENTAL ANALYSER UNIT

EXERCISE 9: IDENTIFY ELEMENTAL CONTENT USING ELEMENTAL

ANALYSER

ESTIMATION OF ELEMENTAL HEAVY METALS CONTENT IN WATER

SAMPLES USING ATOMIC ABSORPTION SPECTROSCOPY-FLAME
ATOMIZATION TECHNIQUE

Introduction

This experiment will use atomic absorption spectroscopy to estimate heavy metals
content in water samples using flame atomization technique and prepare statistical
report of the analysis.

Principle

The technique use in atomic absorption spectroscopy (AAS) is that free atoms (gas)
that generated in an atomizer can absorb radiation at specific frequency. It quantifies
the absorption of ground state atoms in the gaseous state. The atoms absorb ultraviolet
or visible light and make transitions to higher electronic energy levels. The analyte
concentration is determined from the amount of absorption. Concentration
measurements are usually determined from a working curve after calibrating the
instrument with standard of known concentration.

Components

The basic requirements for (AAS) are (1) a light source {hollow cathode lamp and
chopper}, (2) a sample cell {flame} and (3) a means of specific light measurement
{monochromator, detector and readout} which is shown as Figure 9.1. Meanwhile the
function of each components are stated in Table 9.1

(Flame)

Monochromator Detector
Hollow Cathode Lamp Readout
Chopper
Cathode Lamp

Fuel Air

Sample

Figure 9.1

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PART II – SPECTROSCOPY
ATOMIC ABSORPTION SPECTROPHOTOMETER

No. Basic Components

Requirements
1. Light source Hollow Cathode Lamp(HCL):
A cathode lamp is a stable light source, which is necessary
to emit the sharp characteristic spectrum of the element to
be determined.
HCL contain cathode, an anode and inert gas (argon or
neon).

Chopper:
To make sure that the detector sees alternating light
intensities.

2.. Sample cell Flames:

All flames require both a fuel and oxidant. Most commonly
used are air-acetylene flames.
A maximum temperature of 2300k is achieved in such a
flame.
Atomization takes place here by exposing the analyte to
high temperatures in a flame.

3. Specific light Monochromator

Measurement To select the specific wavelength of light (absorbed by the
sample) and to exclude other wavelengths.

Detector
The light selected by the monochromator is directed onto
a detector that is typically a photomultiplier tube, whose
function is to convert the light signal into an electrical
signal proportional to the light intensity.

Readout
The electrical current from the photomultiplier is then
amplified and processed by the instrument electronics to
produce a signal which is a measure of the light attenuation
occurring in the sample cell. This signal can be further
processed to produce instrument readout directly in
concentration units.

Table 9.1

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PART II – SPECTROSCOPY
ATOMIC ABSORPTION SPECTROPHOTOMETER

Learning Outcomes

At the end of this experiment, you will be able to:

1. Apply Beer’s Law in atomic absorption spectroscopy to estimate heavy metal.
2. Use atomic absorption spectroscopy in quantitatively determining heavy metal
content in the given sample.
3. Perform repeatability analysis of data obtained.

Equipment and Material

Equipment 1. Hot plate with stirrer

2. Analytical balance

Labware 1. Spatula
2. Volumetric flask (100 mL)
3. Beaker
4. Filter paper (Whatman #1 or equivalent)

Material/ chemical 1. Nitric acid (HNO3) 1M

2. Heavy metal solution (1000 ppm)
3. Certified Reference Material for heavy metal in (2)
4. Deionized water

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PART II – SPECTROSCOPY
ATOMIC ABSORPTION SPECTROPHOTOMETER

Experimental Procedure

Part 1: Calibration of atomic absorption spectroscopy using Beer’s law method

a. Preparation of standard solution for calibration

1. Prepare 1 liter of 1M nitric acid (HNO3) (5%) solution. This solution will be
used in all dilution in this part of experiment.
2. Using the given stock solution having a concentration of 1000 μg metal/mL
(1000ppm), prepare the following Beer’s Law standard solutions:

Stock
1 M HNO3 Final volume
Solution solution
(mL) (mL)
(mL)
a. Blank
b. 1000 ppm
(Stock solution)
c. Dilution of stock solution
i. 100 ppm 25
ii. 5 ppm 100
iii. 3 ppm 100
iv. 1 ppm 100
2
d. QC sample
e. CRM 1
f. CRM 2
g. CRM 3
h. Unknown
Notes:
1. QC = Quality check, CRM = Certified Reference
Material
2. The concentration of QC sample should be between 1
– 3 ppm.

b. Preparing the equipment for analysis

1. See your instructor for an overview of the Thermo Atomic Absorption

Spectrometer (Thermo AAS).
2. Use Section Five of the Thermo AAS Instruction Manual to prepare the
equipment for use.
3. Perform the following steps:
a. Set up gas lines
b. Perform lamp alignment

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PART II – SPECTROSCOPY
ATOMIC ABSORPTION SPECTROPHOTOMETER

c. Perform burner alignment.

d. Perform Nebulizer optimization and Fuel/air ratio optimization
e. Set to Auto Zero mode
f. Check the Absorbance mode to perform Beer’s Law Experiment. For
easy reference use the Cookbook as guide.

c. Preparing standard calibration plots and analyzing samples

1. Prepare a method and sequence for analyzing your standard solutions using flame
atomization technique. Each sample need to be sequentially placed into the
aspirator as prompted by the software. Note and record your method. Justify the
arrangement of sequence that you use.
2. You will conduct the measurement using primary wavelength. Nonetheless, you
have to note the sensitivity of your measurement if other wavelengths are to be
used in determining the absorbance of the given heavy metal components.
3. For the first determination, use a slit width of 0.5nm. Absorbance associated with
the 3 standard CRM solutions, QC solution, and the unknown solution will be
recorded.
4. Measure and record the absorbance associated with the unknown solution. Repeat
this step several times.
5. All data are recorded by SOLAAR software. The lab instructor will provide you
with the data printout.

Data Analysis and Report

1. Sketch and identify the main components of the AAS unit.

2. Prepare a Beer’s Law plot of your standards (Absorbance vs Concentration)
and determine the best fit line or curve for the data.

Absorbance Concentration
Solution
1 2 3 Mean (unit)
Blank
Standard 1
Standard 2
Standard 3
…
…

3. Determine the amount of heavy metals from your calibration curve.

4. Perform a precision study on the measurement of absorbance of the unknown
solution.

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PART II – SPECTROSCOPY
ATOMIC ABSORPTION SPECTROPHOTOMETER

Absorbance Absorbance
Sample Blank True Reading
1 2 3 Mean 1 2 3 Mean
1 1
2 2
3 3
… …

True reading  Samplemean  Blankmean (1.14)

a. Calculate the mean, standard deviation, and relative standard deviation (RSD)
for the true reading
b. Compare your standard deviation and relative standard devitation values with
the APHA Table provided in Appendix 1 and comment.
c. What is the major uncertainty contributor to your measurement?

References:
1. https://www.slideshare.net/sharmasuriti/atomic-absorption-
spectroscopy-15185397
2. http://www.ufjf.br/baccan/files/2011/05/AAS-Perkin.pdf

Link Video:

1. https://www.youtube.com/watch?v=L3OFSJ8ZHog
2. https://www.youtube.com/watch?v=_izbNvw2Tkg

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PART II – SPECTROSCOPY
ATOMIC ABSORPTION SPECTROPHOTOMETER

Appendix 1

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PART II – SPECTROSCOPY
FOURIER TRANSFORM INFRARED SPECTROMETER

FOURIER TRANSFORM INFRARED ABSORPTION

QUALITATIVE ANALYSIS OF ASPIRIN TABLET USING FTIR

TECHNIQUE

Most of the compound containing the covalent bond either it is an organic or inorganic
which will absorbs at certain frequencies of electromagnetic radiation in the infrared
region. This infrared region lies in a wavelength longer than visible light which
approximately from 700 nm to 13,000 nm but shorter than the wavelength in
microwave regions. From this wide range of wavelength, the infrared spectrum is
conveniently divided into near-, mid-, and far-infrared spectrum. Most widely used of
the infrared spectral region is around 4000 nm to 670 nm, which about 2.5 to 15 µm
as illustrated in Figure 1.

Figure 1 : Electromagnetic spectrum

It can be applied to the analysis of organic or inorganic molecules by causing

molecular rotation and/or molecular vibrations (stretching or bending of bonds) in the
molecules. This experiment will measure the absorption of infrared light by salicylic
acid and acetylsalicylic acid. A commercial aspirin tablet containing both of these
molecules will be analyzed and the spectra will be compared.

Learning outcomes
At the end of the experiment, you will be able to:
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PART II – SPECTROSCOPY
FOURIER TRANSFORM INFRARED SPECTROMETER

1. Identify spectrum peaks and relate them to specific structures.

2. Perform qualitative analysis of complex mixture using FTIR.
3. Identify the difference in spectrum between dry and wet samples

Equipment and Materials

Equipment FTIR Spectrum 100

Materials Salicylic Acid
Acetylsalicylic Acid
Commercial Aspirin Tablet
Cooking oil sample / any liquid sample*

Experimental Procedure

1. Measuring the laser energy

2. Background Check
i. Set the parameter for background checking
Resolution : 4.0 cm-1
Range : 4000 cm-1 to 400 cm-1
Unit :%T
Scan number :4
ii. Obtain a background IR spectrum without a sample in the instrument
3. Place the sample on the crystal ensuring good contact
i. Set the parameter for the sample
Resolution : 4.0 cm-1
Range : 4000 cm-1 to 650 cm-1
Unit :%T
Scan number :4
4. Start measuring the samples.

Include in your report:

1. The sketch of the main components of an FTIR spectrophotometer.
2. Explain the ATR principle and compare the advantages of using this ATR

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PART II – SPECTROSCOPY
FOURIER TRANSFORM INFRARED SPECTROMETER

rather than KBr pellet and Liquid cell (Salt Plates)

3. Copies of all spectra acquired during this experiment.
4. Explain why the IR Background spectrum has peaks even though the sample
chamber was empty.
5. Examine the salicylic acid spectrum. Using the structure of salicylic acid
below, identify the major peaks in the spectrum.
6. Examine the acetylsalicylic acid spectrum. Using the structure of
acetylsalicylic acid below, identify the major peaks in the spectrum.
7. Examine the commercial aspirin spectrum and determine spectral
contributions from salicylic acid and acetylsalicylic acid.

Figure 1: Chemical Structures of Salicylic and Acetylsalicylic acid

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PART II – SPECTROSCOPY
FOURIER TRANSFORM INFRARED SPECTROMETER

PART III
MICROSCOPY

LM
SEM

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PART III – MICROSCOPY
LIGHT MICROSCOPE

ANALYSE MATERIAL USING MICROSCOPIC TECHNIQUE

Introduction

A microscope is an optical instrument for viewing objects that are too small to be
observed by unaided eye and the science of investigating small objects using such an
instrument is called microscopy. Microscopes can largely be separated into three
classes namely, optical theory microscopes, electron microscopes, and scanning probe
microscopes. The most common type of microscope available in the laboratory is
optical microscope (e.g. light microscope) which is the simplest and most widely used
type of microscope. This instrument has several lenses, namely optical lens and
objective lens, that produce an enlarged image of an object/specimen placed in the
focal plane of the lens(es). The ocular lens (eyepiece) has a 10x magnification while
for objective lenses, their corresponding magnification value is written on the lens
holder. Common light microscope has three objective lens of different magnification,
namely scanning (4x), low (10x) and high (100x). The total magnification for the
selected lens combination is the ocular lens magnification multiplied by the objective
lens magnification.

Detail information of a sample/specimen such as size, shape, number of feature, etc.,

could be obtained under the microscope but direct quantification is rather difficult due
to the microscopic size of the region of interest and irregular shape of the specimen.
Optionally, the specimen’s image could be captured by using digital camera and then
analysed using image processing software where the specimen’s attributes could be
properly examined. Image processing must be done with caution to preserve the
original image.

Learning Outcomes

At the end of the experiment, you will be able to:

1. To learn the correct method of using light microscope to examine specimen.
2. To use image processing software to extract detailed information of the specimen.

Equipment and Material

Equipment 1. Light microscope equipped with image acquisition
system
Material/ chemical 1. Specimen slide – onion cell
2. Specimen slide – yeast suspension (concentrated)
3. Specimen slide – yeast suspension (diluted)
Computer program 1. ImageJ®

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PART III – MICROSCOPY
LIGHT MICROSCOPE

Experimental Procedures

Total
Magnification Ocular lens
Magnification

Scanning 4x 10x 40x

Low Power 10x 10x 100x

High Power 40x 10x 400x

Figure 1.1: Light microscope and its part

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PART III – MICROSCOPY
LIGHT MICROSCOPE

Part 1: Preparation of specimen for viewing under microscope

a. Plant cell – Specimen: onion cell

1. Cut off the top of an onion and remove the outer skin.
2. Each onion layer is separated by a thin layer of skin. Peel off a section of this thin
layer using tweezers
3. Place this section of skin in a suitable staining agent for about 10 minutes before
transferring onto a clean glass slide. Use diluted staining agent or decolorizing
solution to avoid overstaining the specimen.
4. Cover the specimen using a cover slip.

b. Microbial culture suspension – Specimen: yeast cell

1. Weigh enough dry yeast to prepare the specified concentration of culture

suspension, e.g. 1000mg dry yeast in 50ml water. Add warm distilled water to
volume.
2. Centrifuge the suspension at 5000rpm for 10 minutes and carefully remove the
supernatant (liquid part) without disturbing the accumulated solid at the bottom
of the container.
3. Add staining agent and top up with distilled water to volume. Use vortex mixer
to re-suspend the cells. Wait for at least 10 minutes.
4. Centrifuge the suspension at 5000rpm for 10 minutes and remove the supernatant.
5. Add distilled water to volume. Use vortex mixer to re-suspend the cells.
6. Repeat Step 4 - 5 until a clear supernatant is obtained. Discard the final
supernatant, add distilled water to volume and use vortex mixer to re-suspend the
cells. This is the final suspension is denoted as the stock solution (Suspension A).
7. Prepare another suspension with concentration of choice (Suspension B) by
diluting the stock solution.
8. Prepare a hemocytometer slide and coverslips for use.

A hemocytometer is a thick glass microscope slide with a grid of perpendicular

lines etched in the middle. The grid has specified dimensions so that the area
covered by the lines is known, which makes it possible to count the number of
cells in a specific volume of solution. The most common type of hemocytometer
has an “H” shape engraved in the middle that encloses two separate mirror-like
polished grid surfaces and provides the cover slip mounting area.

9. Ensure that both the hemocytometer and its coverslip are clean by removing any
dust particles with lens paper. Coverslips that are used for mounting on
hemocytometers are specially made to be thicker than the conventional
microscopy coverslips because they must be able to overcome the surface tension

55
PART III – MICROSCOPY
LIGHT MICROSCOPE

of a drop of liquid. Make sure to first place the coverslip over the counting surface
before loading the cell suspension.
10. Then place the pipette tip with your sample into one of the V-shaped wells and
gently expel the sample. The area under the coverslip will be filled by capillary
action. Enough liquid should be introduced so that the mirrored surface is just
covered, usually around 10 µl, but do not overfill the surface. You can load two
samples on one hemocytometer, one into each of the two grids.
11. The loaded hemocytometer is then placed on the microscope stage and the
counting grid is brought into focus at low power. Allow the sample to settle for a
couple of minutes and avoid moving the coverslip as it might introduce air
bubbles and make counting difficult.

Note on counting cell using hemocytometer

1. The full grid on a hemocytometer contains nine squares, each of which is 1 mm2.
The central counting area of the hemocytometer contains 25 large squares and
each large square has 16 smaller squares. The depth of each square is 0.1 mm.
The final volume of each square at that depth is approximately 100nl.

Figure 3: Hemocytometer gridlines

2. When counting, count only those cells on the lines of two sides of the large square
to avoid counting the same cell twice. Suspensions should be dilute enough so
that the cells or other particles do not overlap each other on the grid, and should
be uniformly distributed.

Part 2: Preparing microscope for examining samples

1. Connect the microscope system to an electrical outlet and switch on the

microscope light.
2. Place the provided slide on the stage. Don’t clip the slide yet as the position of the
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PART III – MICROSCOPY
LIGHT MICROSCOPE

slide might require adjustment later.

3. Always start and end with the Scanning Objective. Do not remove slides with the
high power objective into place - this will scratch the lens!
4. Use the Coarse Knob to focus. The image may be small at this magnification, but
this is necessary to ensure that a proper view could be maintained when shifting
to a higher magnification. Move the slide around/use the stage control to get a
sufficient view of the specimen.
5. Once a sufficient view of the specimen is obtained, fix the slide position using the
stage clip. It is expected that all images that will be captured later are from this
position. Be careful not to change the position of the slide until the experiment is
completed.
6. Once a good focus has been achieved on Scanning Objective, switch to Low
Power. Use the Coarse Knob to refocus.
7. Switch to High Power only if necessary. Use the Coarse/Fine Adjustment Knob
to focus.
8. If the viewing field is too light or too dark, try adjusting the diaphragm.
9. The microscope system is equipped with image acquisition fixture. A camera that
is attached to the viewing field allows for image capture.
10. Turn on the computer and locate the image acquisition program (LEICA Q-Win)
on the computer’s desktop screen/program’s list and double-click to open the
program.
11. From the Menu Bar in the main program window, select Window > Image >
Acquisition > Acquire. The program window will now show the viewing field of
the microscope.
12. In the ‘Image Acquire’ window, click ‘Live’ to obtain the real-time view of the
viewing field.
13. Adjust the microscope stage (not the slide) so that the viewed region includes the
pertinent feature of your specimen.
14. In the ‘Image Acquire’ window, click ‘Grab’ to capture the current view of the
viewing field.
15. Save your grabbed image in a new folder in the computer’s hard disk.
16. To return to the ‘Live’ view after using the Grab function, step 11 - 12 has to be
repeated.
17. Copy all images from your experiment into a flash disk.

Part 3: Image processing analysis using ImageJ

ImageJ is a free, open-source software for image processing. The software installer,
documentation, and user guide could be downloaded from http://imagej.nih.gov/ij/

1. Install ImageJ program in your computer.

2. Launch ImageJ

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PART III – MICROSCOPY
LIGHT MICROSCOPE

c. Opening image file and setting the measurement scale

1. From the Menu Bar, select File > Open > Locate your image file > Open. A new
window showing the selected image will appear.
2. Before performing a measurement using ImageJ, a measurement scale need to be
set first. Detailed measurement is not possible if the original image does not
contain any reference to a known scale. Example of this reference includes:
a. A scale bar that accompanies the saved images
b. A ruler/grid that was captured together with the specimen
c. A different image that contains known scale that is opened at the same
time with the image to be analyze (Global setting).
3. By referring to ImageJ User Guide, set a scale for the selected image. Be reminded
that each image requires its own scale.

d. Measuring a feature

1. For this part, use images of onion cell at different magnification obtained in Part
2 and the provided Scanning Electron micrograph.
2. In ImageJ, set the measurement scale using the provided image of hemocytometer
grid/microscope ruler at different magnification. Each grid side has a length of
1mm, while each division of microscope ruler is 0.01mm.
3. Measure the length and width of one onion cell.
4. Obtain at least 20 measurements of each dimension. Perform uncertainty analysis
for these measurements.

e. Particle counting and analysis

1. You will be provided with 2 sets of images (Set A and B) which were acquired
during the analysis of two yeast cell suspensions using hemocytometer; each set
contains 5 images.
2. Open cell suspension image in ImageJ. Start with one image first.
3. For particle count, the scale is not required to be set.
4. Convert the image to greyscale (Image > Type > 8-bit)
5. Threshold the image using the automated routine (Process > Binary > Make
Binary).
6. Analyze Particles (Analyze → Analyze Particles). Enter ’20-Infinity’ as the
particle size, toggle 'Show Outlines', check ‘Display Results’, ‘Summarize’ and
click 'OK'
Selected feature will be counted, numbered and outlined. The data window lists
all the measured data and it could be copied to a spreadsheet.
7. Repeat Step 2 – 6 for all images.

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LIGHT MICROSCOPE

Data Analysis and Report

Your report should include the followings:

Part 2
1. List down main parts of a microscope and briefly explain their respective function
2. Report and discuss on all images from all sections in this experiment
3. Explain some precaution steps that are essential in a) using microscope b)
preparing samples and c) examining sample.

Part 3b
1. Indicate the cell that you selected for this analysis.
2. Tabulate all the measurement for the selected onion cell and perform uncertainity
analysis.
3. Discuss the difference in those measurement based on the (i) uncertainty analysis,
(ii) magnification, and (iii) microscopy technique.

Part 3c
1. Tabulate the cell count in each image for each set.

Set A Set B
Image 1
Image 2
Image 3
Image 4
Image 5
Average
2. The concentration of the corresponding yeast cell suspension for each set could
be estimated from the following large cell formula.

D N
(1.15)
SK

where, D is the dilution factor, N is the average number of cell counter, S is the
number of square counted, and K is the volume of each square.

Based on the result in (1), determine the dilution factor for yeast cell suspension
used in Set B image (Set B is diluted from Set A).

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PART III – MICROSCOPY
SCANNING ELECTRON MICROSCOPY

Scanning Electron Microscopy (SEM)

Morphology and Topography Investigation of Various Solid Materials

Introduction

Scanning Electron Microscopy (SEM) is one of the most versatile methods for analysis
of solid materials. Since its inception in the middle of the 20th Century, the technique
has evolved to allow for imaging of nanoscale features and integration with electron
probe micro analysis (EPMA) techniques. In SEM, an electron beam is moved in a
raster pattern across the surface of a sample. The beam interacts with the sample
surface, producing a number of different signals, which can be analyzed to provide
useful information about topography, composition, crystallography, etc. In many
respects, the optical system in an SEM is analogous to that in an optical microscope,
however, instead of glass lenses used to focus light, electromagnetic lenses are used
to focus and deflect the electron beam. The primary motivation for using SEM instead
of light microscopy is related to the fact that electrons have a much shorter wavelength
than light (higher resolution) and SEM uses a longer focal length (greater depth of
focus).The main classification for different types of SEM instruments is related to the
source of electron illumination. The most common electron guns employ a tungsten
filament which is resistively heated to nearly 3000K until electrons have sufficient
energy to overcome the work-function (Ew) energy barrier (thermionic emission). By
choosing materials with a lower Ew than tungsten, such as LaB6, the brightness of the
electron source can be increased by an order of magnitude. Field emission electron
guns represent another class of electron sources and are generally comprised of
sharpened tip of single crystal tungsten. Field emission guns have two orders of
magnitude greater brightness than is possible with LaB6 thermionic emission, exhibit
a longer lifetime, have a smaller virtual source and a lower energy spread. There are
three main types of field emission electron guns: cold field emission, thermal field
emission and schottky emission. While the enhanced resolution of field emission gun
equipped SEM instrument is useful, even the thermionic gun equipped instrument
used for this lab has a resolution better than 5 nm, however there are several tradeoffs
involved in achieving this performance.

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Learning outcomes:

At the end of the experiment you will be able to:

1. become familiar with the basic operating principles of the Hitachi S3400N
SEM
2. become familiar with the different signals of interest in the SEM and their
utility
3. learn techniques for improving imaging resolution and the compromises you
make in doing so.

Laboratory procedures

Part 1: General Operation of the SEM

Before any imaging or analysis can be performed with the SEM, appropriate sample
preparation is required for the instrument. Fortunately, sample preparation for the
SEM is generally quite straightforward and does not usually require any rigorous and
time-consuming processing. Some general sample requirements are listed below:

Sample size

The sample should be small. A typical sample size prepared is that it can fit on the
standard 15mm diameter aluminum SEM stub. This will allow you to load several
samples at once and avoid unnecessary sample exchanges. Minimize the use of
double-stick tape or non-conductive adhesives for sample mounting to avoid
outgassing. Before loading the sample, measure the sample height using the white
measuring tool. Knowing the sample height will save you plenty of time during the
observation. Make sure the sample height is under the limit.

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Sample type

Powder samples
Disperse a small amount of sample in ethanol and sonicate. Heat the stub using heat
blower
Drop the suspension on the stub. The fast drying of ethanol should spread the
powders fairly well on the surface.
*For sample checking (not for high quality images), you can also disperse your
sample on the black carbon tapes.

Fiber pieces

Carbon or silver paste is preferred to use for fixing samples for better image
(compared with tapes). Completely dry the sample before loading

Sample conductivity

The sample should be electrically conductive. Electrically insulating samples or poor

conductors generally require a conductive coating on the surface, which is grounded
during imaging. This is typically very thin (~20 nm) of a Au/Pd with small grain size
deposited by a physical vapor deposition technique, e.g. sputter coating. Without this
coating, the sample will charge under electron bombardment creating image distortion
and increasing sample damage.

High Vacuum Compatibility

Low vapor pressure materials or materials that can be readily volatilized by the
electron beam should not be loaded in a high vacuum SEM. The S3500N is a variable
pressure SEM, allowing for imaging of some wet samples, but generally speaking your
sample should be dry and relatively stable under electron bombardment.

Surface Information

The SEM obtains information only from near the surface of your sample. Make sure
the surface to be studied is as clean as possible and presents the features of interest.
For example, if you want to look at the internal structure of a polymer, it may be
necessary to cleave it under liquid nitrogen to preserve the morphology. When
possible, avoid cleaning samples with organic solvents as hydrocarbons are readily

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decomposed by the electron beam leaving black ‘contamination’ marks on your

sample. The followings are good guides that should be followed:

1. Always wear gloves when handling your sample and the SEM sample holders.
This helps keep the vacuum system clean and avoids sample contamination.

2. Shut off the high voltage before venting the chamber. If you don’t shut off the
high voltage and bring the chamber up to atmosphere, the filament will rapidly
oxidize and burn out.

3. To change the accelerating voltage, follow this procedure:

a. Shut of the high voltage,

b. Change the voltage level,
c. Turn the voltage back on,
d. Re-saturate the filament. Never change the voltage ‘on the fly’ or you can
destroy the filament.

4. Set the sample height correctly.

Align the lenses and apertures in the instrument so that they are concentric about the
same axis. The alignment of the microscope is affected by many factors, including the
accelerating voltage, condenser lens strength, objective aperture size, working
distance, etc. Every time you change a major parameter, it is necessary to repeat
the alignment procedure in order to obtain the best image.

For step-by-step operation instructions of the S3500N SEM, please refer to the SOP
provided. For this portion of the laboratory, you should capture twenty four (24)
images of the powder and fiber samples (coated and uncoated).

Sample: powder and fiber samples

Accelerating Voltage: range between 3 - 25kV
Beam Current: set between 30-80
Detector: SE Objective
Aperture: 3
Working Distance: 5 – 6 mm
Magnification: Low magnification
High magnification

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Each sample should be measured at two different accelerating voltages, two different
beam currents and at low and high magnifications. Record all parameters that you
varied in your lab report. Record all your observations when changes are done.
What happen to the set working distance value when you fine tune the image?

Part 2: Signals in the SEM

There are a number of useful signals produce by the interaction of the electron beam
with the sample. Among these signals are secondary electrons (SE), backscattered
electrons (BSE), characteristic x-rays, auger electrons and light
(cathodoluminescence). These signals can be utilized to provide information about the
sample topography, composition, electronic structure, crystal structure, magnetic
properties, etc. In this laboratory, you will be primarily concerned with information
provided by the SE and BSE signals. When the electron beam enters the sample, it is
scattered in three-dimensions to produce a so-called interaction volume (IV). The
dimensions of the IV depend on the primary beam energy and sample properties, such
as atomic number and density. The various signals are generated throughout the IV,
but their detection depends on their ability to escape the surface of the sample.

SEM is renowned for its capability to achieve high resolution topographical images
with remarkable depth of focus and the ability to convey this information without any
real understand of how the image is formed. These images are typically generated by
detection of the SE signal. The SE signal is generated via inelastic collisions between
the high energy electrons in the primary beam and valence electrons in the sample.
Once a valence electron has been ejected from its orbit, it may escape the sample
surface provided it has sufficient energy. The energy of SE’s is generally quite low
(<50eV), which results in a short mean free path, hence a shallow escape depth
(~10nm). This makes the information obtained with the SE signal inherently surface
sensitive with high spatial resolution. However, the situation gets a bit more
complicated as the SE signal you detect does not only come from the ‘footprint’ of the
electron probe, which you define as the SE1 signal. There are other SE signals present
as well, including SE2’s, which are generated by backscattered electrons (BSE), and
other SE signals generated by interaction between primary electrons or BSE with
various parts of the SEM (apertures, chamber walls, etc.).The BSE signal is produced
by primary beam electrons that are elastically scattering by the nuclei of atoms in the
sample. As such, backscattered electrons have an energy that is some fraction of the

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primary beam energy. The number of backscattered electrons generated defined by a

backscatter coefficient

(η), which is a function of the atomic number (Z):

η = -0.0254 + 0.016Z – 1.86x10-4Z2+ 8.3x10-7Z3

This atomic number dependence allows for the differentiation of different materials in
a sample based on their average atomic number. The BSE signal, since it is generated
by much higher energy electrons, is an inherently lower resolution signal that the SE1
signal, coming from up to~40% of the maximum penetration depth. The SE2 signal,
created by BSE’s exiting the surface carries the same low resolution information and
can contribute compositional contrast to the SE image. It is a common misconception,
however, that the BSE signal does not contain topographic information. There is both
an angular dependence on the generation of BSE’s and a fixed collection angle, which
provide for topographic information.

For this lab you should capture images of particles using the BSE detectors using the
AV and BC settings from Part I. The SEM Conditions for Part 2:

Sample: powder and fiber (coated & uncoated)

Accelerating Voltage: Based on Part I
Beam Current: Based on Part I
Detector: BSE
Objective Aperture: 3
Working Distance: 5-6 mm
Magnification: Based on Part I

You should also test the coated sample using BSE Detector at low magnification.
Compare and discuss your results with that obtained in Part I.

Part 3: Resolution vs. Signal to Noise

For this portion of the laboratory, you will concentrate on methods for improving
imaging resolution in the SEM and the trade-offs involved. There are two major
aspects to achieving high resolution images: the ability to form small probe of
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electrons and the ability to detect high resolution signals produced by the interaction
with the sample. Let’s take the issue of probe formation first, which starts with the
electron gun. One of the reasons field emission guns can achieve higher resolution is
due to the smaller source size. The tungsten source used in the S3400N SEM has a
size of ~50µm and therefore requires 4 orders of magnitude of demagnification to
reach the 5nm resolution specified by the manufacturer. This demagnification is
achieved through the use of electromagnetic lenses, called condenser lenses, which
introduce a number of issues you need to consider. By increasing the strength of the
condenser lens(es), you can achieve a smaller probe size.

The sample for this portion of the laboratory is a powder and fiber samples (coated).
You will explore the effects of objective aperture size and condenser lens strength on
resolution and signal to noise ratio. You will capture 8 images for this section
comparing at least two different aperture sizes and two different condenser lens
strengths (beam currents).

SEM Conditions for Part 3:

Sample: powder and fiber samples (coated)
Accelerating Voltage: 25kV
Beam Current: 30
Detector: SE Objective
Aperture: 1 and 4
Working Distance: 5 – 6 mm
Magnification: Low magnification
High Magnification

Compare your findings with those obtained in Part I. Discuss the effects of Beam
Currents on the final resolutions of your images.

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Report
Your report should include:

1. Record and explain all methods and procedures performed in the experiment.
Make sure all required parameters are properly recorded and their effects are
observed.
2. Comparison between images obtained under different microscopic
observations – naked eyes and SEM.
3. Describe coating methods that had been done to coat your sample. Discuss
the effects of coating on the images that you obtained and why is it
important.
4. You may have noticed that to get a good BSE image, you need to have weak
condenser lens settings and a large objective aperture. Why do you think this
is the case? Compare the images that you obtained by using SE and BSE
detectors.
5. Why do the smaller features appear darker than the larger ones if they have
the same composition?
6. Note that even though the image on the screen may get noisier with
decreasing aperture size or increasing condenser lens strength, the photo
taken may not show this. Please explain why this occurs. What do you do to
get better image?
7. Since SEM is essentially a surface analysis technique, why doesn’t sputter
coating obscure useful surface information? Can you think of any cases
where the coating might become a problem?
8. Low voltage microscopy is a useful method for minimizing charging effects.
What are some of the other benefits and tradeoffs? Why is cold field
emission well suited for this technique?
9. For all images, describe the overall structure and morphology of the sample.
Properly label all images. Make comparisons based on e.g.
contrast/brightness or resolutions of your images.

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Pre-Lab: Scanning Electron Microscopy

The Pre-lab section should be completed in prior to starting your laboratory

experiment.

Label the following parts:

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1. What is the coating material used to coat your sample?

2. What is the material that you are using as your sample? What type of sample
is it?
3. What are the pre-caution measures that you should implement during the
experiment?

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PART IV
THERMAL ANALYSIS

DSC
TGA

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EXERCISE 12: TEST THERMAL PROPERTY OF MATERIAL

DETERMINATION OF GLASS TRANSITION TEMPERATURE (Tg) USING

DSC TECHNIQUE

Introduction

Differential Thermal Analysis (DTA)/ Differential Scanning Calorimetry (DSC)

curves reflect changes in the energy of the system under investigation - changes that
may be either physical or chemical in origin. DSC measures the heat required to
maintain the same temperature in the sample versus an appropriate reference material
in a furnace. Enthalpy changes due to a change of state of the sample are determined.
DTA differs from DSC in that the temperature difference is determined, rather than
enthalpy differences between the sample and the reference material. A number of
important physical changes in a polymer may be measured by DSC. These include the
glass transition temperature (Tg), the crystallization temperature (Tc), the melt
temperature (Tm), and the degradation or decomposition temperature (TD). Chemical
changes due to polymerization reactions, degradation reactions, and other reactions
affecting the sample can be determined. A typical DSC trace showing these transitions
is shown in Error! Reference source not found..
.

Figure 4: A generic DSC curve depicting several transition stages

There are two types of differential scanning calorimeters: (a) heat flux (AT) and (b)
power compensation (AP). Identify which type will be used in your experiment. In
either type of calorimeter, the measurement is compared to that for a reference material
having a known specific heat. As AT and AP have opposite signs there is some
potential for confusion e.g. at the melting point, Tm, Ts< Tr, and ΔT < 0, whereas Ps >
Pr and ΔP > 0 because latent heat must be supplied (subscripts s and r refer to the
sample and the reference material, respectively).

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Figure 8: a) Power compensation DSC b) heat-flux DSC

Learning outcomes

At the end of the experiment, you will be able to:

1. Describe and discuss theory and techniques used for thermal characterization
of material;
2. Perform data analysis and present report based on collated thermal analysis
data

Equipment and Material

Equipment 1. DSC equipment with data acquisition system

(METTLER TOLEDO DSC823e with STARe software)
2. DSC Cell
3. Cooling system
4. Analytical balance (precision 0.00001 g)

Labware 1. Dewar flask

2. Pointed tip forceps
3. DSC aluminum sample pan and lids
4. Crimper

Material/ chemical 1. Source of pure, low-pressure, dry nitrogen gas

2. Liquid nitrogen
3. Indium (reference material)
4. Three different poly (ethylene terephthalate) samples

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Figure 9: Aluminium: 40µl and 100µl with lid

Figure 10: Crucible Sealing Press/Crimper

Experimental Procedure

Part 1: Preparation for analysis

f. Preparation of DSC instrument

1. One hour in advance of data acquisition, the equipment should be turned on.
2. The polymer samples should be free from impurities; including monomers, water,
and solvent. The polymer sample should be homogeneous and of a large surface
area.
3. Refer the instrument’s operations manual for proper methods of:
a. Temperature and heat calibration verification.
b. Sample pan type and crimping of the sample pan.
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c. Loading sample in pan.

d. Operating temperature controller and setting temperature limits.
e. Setting-up and using data acquisition system.
f. Using the data analysis system.
g. Calibration of DSC equipment

The DSC should be calibrated before analyzing the sample. Calibration should be
verified by using indium (reference material). Temperature and heat values should be
within normal ranges for the instrument or at least within the precision and accuracy
limits allowed.

Part 2: Sample Preparation

1. Use forceps to handle the sample pan and lid. Obtain the tare weight of a sample
pan and lid (the type that can be hermetically sealed is not required).
(Note: Select pans with flat undistorted bottoms so that good contact with the
cell platforms is assured.)
2. Record the mass of the sample pan and lid to five significant figures.
3. Add about 5-10 mg of your sample or calibration standard to the sample pan and
lid and weigh again. A powdered sample provides better thermal contact.
4. Record the mass of the sample to five significant figures.
5. Remove the polymer sample pan and lid carefully from the balance using forceps.
(Note: Skin moisture and oils will be left on the sample pan and lid if they
are picked up by fingers. This extra mass will affect the DSC experiment.)
6. Crimp the samples pan and lid closed.
7. Tare another sample pan and lid. This is the reference pan.
8. Record the weight of the sample to five significant figures.
9. Weigh out an empty reference pan.
10. Crimp the reference pan and lid closed.
11. Purge the cell with nitrogen at a 30 ml/min gas flow rate.

Part 3: Sample Analysis

1. Record the first thermal cycle (used for calibration verification) by heating the
sample at a rate of 20°C/min under nitrogen atmosphere from ambient to 30°C
above the expected melting point or glass transition point or up to a
temperature high enough to erase previous thermal history.
2. Hold the temperature for 1 min.
3. Cool to 50°C below the peak crystallization temperature at a rate of
20°C/min.
4. Repeat heating as soon as possible under nitrogen at a rate of 20°C/min and record
the heating curve.
5. Measure the temperatures for the following: Tf, Tm, Tc, and Te, where Tf is the
extrapolated onset temperature, Tm is the melting peak temperature, Tc is the
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crystallization peak temperature. Report two Tm values if observed.

Data Analysis and Report

Your report should include the followings:

1. Describe the apparatus and experiment in your own words. Explain the
temperature program that you had set.
2. Describe the temperature calibration procedure.
3. Identify the sample atmosphere by pressure, gas flow, etc.
4. Determine K from the result obtained with materials of known heats of fusion.
The following equations may be useful for your calculations:

Equilibrium melting point

H m
Tm  (1.16)
Sm
Enthalpy
Tf
H K
  Tdt (1.17)
m m Ti
Notes: Melting point of indium (156.4oC), Heat of fusion (28.45 KJ/kg)
5.
6. You will be provided with a set of data from DSC analysis.
a. Prepare a thermogram from the provided data.
b. From the thermogram, identify all the available transitions. Tabulate the
onset, endset, peak, inflection temperatures for all transitions (see Figure 8).
c. Determine the extrapolated onset temperature (Tf) for all transitions. At least
one calculation example must be appended in your report.

Figure 5: Different ways of recording transition temperatures

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EXPERIMENT: THERMAL TECHNIQUE BY THERMOGRAVIMETRIC

ANALYSIS (TGA)

Introduction

In this experiment, you will be introduced to the use of thermal methods in performing
material characterization. Thermal gravimetric analysis is a technique that determines
weight changes based on temperature change. Thermal gravimetric analysis is the act
of heating a mixture to a high enough temperature so that one of the components
decomposes into a gas, which dissociates into the air. It is a process that utilizes heat
and stoichiometry ratios to determine the percent by mass ratio of a solute.

Figure 1: Scheme of thermogravimeter

Theory

Based on Figure 2, highly volatile matter is represented by a mass loss measured

between the starting temperature and Temperature X (degraded at 200°C or less);
example as moisture or plasticizer.

Medium volatile matter is represented by the mass loss measured from Temperature
X to Temperature Y (degrade or volatile component in range 200 to 750°C).

Combustible material content is represented by the mass loss measured from

Temperature Y to Temperature Z (example as oxidizable material not volatile at
750°C or some stipulated temperature dependent on material). This region
corresponds to the mass loss as a result of the oxidation of carbon to carbon dioxide.

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Meanwhile, ash is nonvolatile residues in an oxidizing. The use of the recorded first
derivative may be useful in locating the value of X, Y, and Z by examining areas of
the curve where the derivative returns to or approaches the baseline.

Figure 2: Sample Thermogravimetry Curve from ASTM E1131-03

Loss on drying (LOD) is a widely used test method to determine the moisture content
of a sample, although occasionally it may refer to the loss of any volatile matter from
the sample when the sample is dried under specified conditions (temperature and
time).

This test method is applicable to a wide variety of solid or liquid materials, mixtures
or blends where the major component is stable at the test temperature. LOD is useful
for design purposes, service evaluation, regulatory statues, manufacturing control,
quality control, specification acceptance, development and research.

Learning outcomes

At the end of this experiment, you will be able to:

1. Determine compositional analysis of specific material upon heating
2. Determine the loss on drying of specific material

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Material and equipment

1. Gas
– Nitrogen, compressed air.

2. Glasswares, Consumables and Equipment

– TGA Crucible Handling Set: Aluminium oxide crucible 70μL, tweezer,
funnel, crucible holder
– Thermogravimetric analyzer

1. Method for compositional analysis

a) Start up the instrument and open the STARe software
b) Set the method program
c) Choose nitrogen and air at the desired flow rate within range 10 to 100
mL/min as environment in the chamber.
d) Switch the purge gas to nitrogen
e) Zero the instrument and tare the balance
f) Open the furnace to exposed the specimen holder
g) Weighed the sample at the range 10 to 20mg. Carefully place it in the
specimen holder and close it.
h) Record the initial mass
i) Set the program with the desire information:
i. Temperature range (25⁰C to 1000⁰C)
ii. Heating rate (5⁰C to 20⁰C/min)
iii. Type of gas (Nitrogen or Air) and flow rate (10 to 100 mL/min)
j) Initiate the heating program
k) The analysis is complete when the mass plateau is achieved after the
introduction of reactive gas e.g. air
l) Wait until the furnace has cooled completely before begin another sequence

2. Method for Loss on Drying (LOD)

a. Prepare the TGA
b. Cool the specimen test area of the apparatus to ambient temperature. For the
purpose of this test, ambient temperature is 35°C or lower.

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c. Open the furnace to expose the specimen holder. Place sample mass of 5 to
30 mg and close the furnace.
d. Set method to heat specimen at 5°C/min to the isothermal test temperature
and hold for 5 min. Other heating rates may be used and should be indicated
in the report. The time from start to the isothermal portion of the experiment
is taken as ti.
e. Selection isothermal test temperature and test time (tt) can be referred in
Attachment 1, example; polymer at 155°C.
f. Once the test temperature is reached, it is isothermally held there for the
remainder of the experiment.
g. After experiment finished, let the temperature of the furnace reduce at least
70°C.
h. Generate and interpret thermogram using STARe Software. Loss on drying
can be obtained as example LOD: XX% (24 hrs at 155°C for polymer
specimen).
i. Print the result.

Calculation

Highly volatile matter content:

𝑊−𝑅
𝑉= 𝑥 100%
𝑊

Medium volatile matter content:

𝑅−𝑆
𝑂= 𝑥 100%
𝑊

Combustible material content:

𝑆−𝑇
𝐶= 𝑥 100%
𝑊

Ash content:

𝑇
𝐴= 𝑥 100%
𝑊

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where:
C = combustible material content, as received basis (%)
S = mass measured at temperature Y, (mg)
T = mass measured at temperature Z, (mg)
W= original specimen mass (mg)
V = highly volatile matter content, as received basis (%)
R = mass measured at temperature X (mg)
O = medium volatile matter content, as received basis (%)

The percent mass loss value:

(𝑚𝑖 − 𝑚𝑓 ) × 100%
𝑚=
𝑚𝑖

LOD = mean value of m for duplicate determinations

where,

LOD = loss-on-drying (%)

m = percent mass loss (%)
mi = initial mass (mg)
mf = final mass (mg)

The amount of volatile material v, in grams per liter from a liquid sample, was then
calculated using the equation below:

(𝑚𝑖 − 𝑚𝑓 )
𝑣= × 𝜌 × 1000
𝑚𝑓

where,

ρ = density of liquid sample (g/mL)

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Discussion
1. Calculate the high volatile matter content, medium volatile matter content,
combustible material content and ash content.
2. Calculate mass loss on drying of various components of your sample.
3. What is the composition of undegraded components in your sample?
4. Why is the nitrogen gas first used as an atmospheric environment and why is
it switched to air during the experiment?
5. Explain how sample mass, sample shape, and position of the sample on the
sample pan can affect data in a TGA experiment.
6. The sample crucible used in this experiment is made from aluminium oxide
or known as alumina. Why is alumina preferred and why would an
aluminum sample crucible can be inadequate for this experiment?

Attachment

Table 1: Commonly used Isothermal Test Temperatures and Times

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PART V
MECHANICAL
ANALYSIS

DMA
RHEOMETER

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EXPERIMENT 5: DYNAMIC MECHANICAL ANALYSIS (DMA)

TESTING ON MECHANICAL PROPERTIES OF PLASTIC MATERIALS

USING DMA

Introduction

Polymers vary from liquids and soft rubbers to very hard and rigid solids. Many
structural factors determine the nature of the mechanical behavior of such materials.
In considering structure-property relationships, polymers may be classified into one
of several regimes, shown in the volume temperature plot (Error! Reference source
not found.). Dynamic mechanical analysis (DMA) or dynamic mechanical thermal
analysis (DMTA) provides a method for determining elastic and loss moduli of
polymers as a function of temperature, frequency or time, or both. Viscoelasticity
describes the time-dependent mechanical properties of polymers, which in limiting
cases can behave as either elastic solids or viscous liquids (Error! Reference source
not found.). Knowledge of the viscoelastic behavior of polymers and its relation to
molecular structure is essential in the understanding of both processing and end-use
properties. DMA can be applied to a wide range of materials using the different
sample fixture configurations and deformation modes (Error! Reference source not
found.). This procedure can be used to evaluate by comparison to known materials:
(a) degree of phase separation in multicomponent systems; (b) amount type, and
dispersion of filler; (c) degree of polymer crystallinity, (d) effects of certain
pretreatment; and (e) stiffness of polymer composites.

Figure 6: Regime of bulk polymer in terms of volume and temperature

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Figure 7: A plot of the stress relaxation modulus vs time

Dynamic mechanical experiments yield both the elastic modulus of the material and
its mechanical damping, or energy dissipation, characteristics. These properties can
be determined as a function of frequency (time) and temperature. Application of the
time-temperature equivalence principle yields master curves like those in Error!
Reference source not found.. The five regions described in the curve are typical of
polymer viscoelastic behavior.

Table 3: DMA deformation modes for specific applications

Sample Parameter Clamp/deformation mode

Solid polymer Dynamic modulus Flexure
Glass transition temperature Tension
Melting temperature Torsion
Cross-link density Compression
Relaxation behavior Shear
Crystallinity, cure
Film, fiber, coatings Dynamic modulus Flexure
Glass transition temperature Tension
Creep, cure, compliance Shear
Relaxation behavior
Viscous fluids, gels Viscosity Shear
Gelation
Gel-Sol transition
Cure, dynamic modulus

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Learning Outcomes
At the end of the experiment, you will be able to:
1. Describe and discuss theory and technique used to determine cyclic mechanical
properties of polymeric materials using DMA
2. Perform qualitative and quantitative data analysis of sample tested using DMA
technique

Equipment and Material

Equipment 1. DMA Equipment equipped with:

a. Appropriate test fixtures (single/ dual cantilever /
tension clamps)
b. Temperature programmer/controller
c. Sample holder and cooling attachments if needed for
sub ambient testing
d. Data collection and output devices such as X-Y
recorder or chart recorder or computer analysis
system with plotter
Labware 1. Sample preparation tools to size sampler for sample
holder
a. Heatable platen press
b. Cutting tools as needed
Material/ chemical 4. Polymer sample(s)
5. Nitrogen or other gas supply for purging purposes

Experimental Procedure

Part 1: Sample preparation

1. Prepare the sample, if not readily available, in the form of a film approximately 1
mm thick or less for rigid polymers or 2 - 3 mm thick for flexible polymers. Film
preparation methods include casting from solution, milling, or compression
molding. Ask your instructor.
2. Width: Cut a specimen into a 5 - 15 mm wide. Use a single stroke with a sharp
razor blade, and make sure the cut is uniform across the sample to within 0.02
mm.
3. Length: Cut the sample 5 mm longer than the distance between the dual cantilever
supports, so that the sample will lie across the supports without touching the
furnace. This length is approximately 55 to 60 mm for the dual cantilever clamp
orapproximately 30 mm for the single cantilever clamp and tension clamp

Part 2: Preparing instrument for analysis

7. Calibrate the instrument using the procedures outlined by the manufacturer.

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8. Before you start the experiment, ensure that the DMA is connected with the
controller, the sample is loaded, the furnace is closed, and you have entered all
necessary information through the instrument control software.

NOTE: Once the experiment is started, operations are best performed at the computer
keyboard. The DMA is very sensitive to motion and might pick up vibration caused
by touching a key

Part 3: Analyzing sample

1. Use an untested specimen for each measurement.

2. Measure the diameter and height (thickness) of the specimen to the nearest 0.03
mm (0.001 in.) at the center of the specimen. Record the measurement.
3. Mount the test specimen between movable and stationary members.
4. Select the desired frequency (or frequencies) for dynamic linear displacement.
5. Select the linear displacement amplitude.
6. Temperature increases should be controlled to 1 to 2°C/min for linear increases
and 2 to 5°C/min, with a minimum of 1-min thermal soak time, for step increases.
This will allow characterizing of the modulus.
7. The tan δ peak shall coincide with the sudden change in modulus through the glass
transition region.
8. Process collected data using the instrument data analysis system and plot the
calculated values of storage (elastic) modulus (E'), loss (viscous) modulus (E"),
and tan δ versus temperature.

a) b)

Figure x: Clamp type a) Tension Clamp b) Dual Cantilever

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DYNAMIC MECHANICAL ANALYSIS

Figure xx: DMA Cross section

Part 4: Removing samples after analysis

When the experiment has completed, remove the sample from the single/dual
cantilever / tension clamp in the following manner:
a. Wait for the sample to return to room temperature before attempting to remove it.
b. Press the FURNACE key to raise the furnace
c. Press the DRIVE key or STOP to lock the moveable clamp in position.
d. Loose the three clamp center screws that are holding the sample between the
moveable jaws.

Data Analysis and Report

Your report should include the following:

1. Describe the experiment from which the data was acquired. Include a description
of (1) the fixtures used, (2) the sample, the number of samples and its preparation prior
to test, (3) the gaseous atmosphere used during the test, and (4) the frequency or
frequency range used.
2. Prepare a plot of modulus (moduli) and tan δ as a function of dynamic oscillation
(frequency), percent strain, temperature, or time. Discuss your results accordingly.
3. Report the value of the glass transition (Tg) temperature if this region was
investigated.
4. Comment on the significance of tan δ.
5. Comment on the elastic and viscous nature of the material at different
temperatures.

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PART V - MECHANICAL ANALYSIS
DYNAMIC MECHANICAL ANALYSIS

Film Tension Clamp Calibration

https://www.youtube.com/watch?v=Z3sCgEdwtZ0
https://www.youtube.com/watch?v=y4xXCdong5w
https://www.youtube.com/watch?v=r9briIO2p4A

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PART V - MECHANICAL ANALYSIS
DYNAMIC MECHANICAL ANALYSIS

TEST RHEOLOGICAL PROPERTY OF MATERIAL

FLUID RHEOLOGY AND VISCOSITY MEASUREMENT

Introduction

In general, fluid flow patterns are more complex than the relationship between fluid
deformation and stress. Rheology is the discipline of fluid mechanics which studies
this relationship. One goal of rheology is to obtain constitutive equations by which
stresses may be classified into rheological types in reference to the simple shear flow.

Figure 7 shows the rheogram for several types of time-independent fluids. It is a plot
of shear stress versus shear rate for a fluid in simple shear flow. The Newtonian fluids
rheogram is a straight line passing through the origin. The slope of the line is the
viscosity. For a Newtonian fluid, the viscosity is independent of shear rate, and may
depend only on temperature and perhaps pressure. By far, the Newtonian fluid is the
largest class of fluid of engineering importance. Gases and low molecular weight
liquids are generally Newtonian.

Bingham plastic
pseudoplastic

y
Shear stress 

Dilatant Newtonian

Shear rate |du/dy|

Figure 8: Rheogram of time-independent fluid

All fluids for which the viscosity varies with shear rate are non-Newtonian fluids. For
non-Newtonian fluids, the viscosity, defined as the ratio of shear stress to shear rate,
is often called the apparent viscosity to emphasize the distinction from Newtonian
behaviour. Purely viscous, time-independent fluids, for which the apparent viscosity
may be expressed as a function of shear rate, are called generalized Newtonian fluids.
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PART V - MECHANICAL ANALYSIS
DYNAMIC MECHANICAL ANALYSIS

Non-Newtonian fluids include those for which a finite stress y is required before
continuous deformation occurs; these are called yield-stress materials. The Bingham
plastic fluid is the simple yield-stress material; its rheogram has a constant slope,  
called infinite shear viscosity. Highly concentrated suspensions of fine solid particles
frequently exhibit Bingham plastic behaviour.

Shear thinning fluids are those for which the slope of the rheogram decreases with
increasing shear rate. These fluids have also been called pseudoplastic, but this
terminology is outdated and discouraged. Many polymer melts and solutions, as well
as some solids suspensions, are shear-thinning fluids without yield stresses typically
obey a power law model over a range of shear rates. The power law model typically
provides a good fit to data over a range of one to two orders of magnitude in shear
rate; behaviour at very low and very high shear rates is often Newtonian. Shear-
thinning power law fluids with yield stresses are sometimes called Herschel-Bulkley
fluids.

Dilatant, or shear thickening fluids show increasing viscosity with increasing shear
rate. Over a limited range of shear rate, they may be described by the power law model
with n > 1. Dilatancy is rare, observed only in certain concentration ranges in some
fluid.

Learning Outcomes

1. To quantitatively measure the viscosity of Newtonian and Non-Newtonian fluid

using viscometer

Equipment and Materials

1. HAAKE Viscometer 6L with four different spindles: L1 – L4

2. Beaker
3. Syringe
4. Screw driver
5. Newtonian fluid sample – Distilled water
6. Non-Newtonian fluid sample - 30% glucose solution, corn starch suspension,
cooking oil, hair gel, shampoo, tooth paste, sodium alginate solution, etc.

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DYNAMIC MECHANICAL ANALYSIS

Experimental Procedures

Part 1: Setting-up the Viscometer

1. The mains switch is on the rear of the viscometer and can only be activated if the
instrument is connected to the mains.
2. Switch the equipment”ON”. The following message will appear on the viscometer
screen for 2 seconds:
THERMO SCIENTIFIC
VT X-L PLUS VX.X (Version of the Viscotester and the fimware)
GERMAN (Language)
3. During the above presentation message, in order to change the measuring units or
the language, press <START>. Afterwards press <ENTER> (this should be done
within the period of 2 seconds). Once the user has chosen this option, the
equipment is ready to be configured.
4. The equipment is ready to set--up the language. In order to change the language,
the user must press <UP> and <DOWN>, until the desired language appears on
the screen, then press <ENTER> to confirm the selection.
Options to choose: ENGLISH, SPANISH, FRENCH, GERMAN,
ITALIAN, POLISH, JAPANEASE, PORTUGUESE
5. After pressing <ENTER> to confirm the first selection, the user can change the
measuring units. Pressing <UP> and <DOWN> will show the different options
for
Viscosity: ”mPas” or ”cP”
Shear stress: “Nm--2“ or “dyne.cm--2“
Temperature: “Celsius” or “Fahrenheit”
Print mode: “Print” or “Computer”
Press <ENTER> to confirm it.
6. Once the user has selected a language and/or a measuring unit, they will appear
as default when the equipment is used again.

Part 2: Performing auto test for viscometer

1. The following message will appear on the screen:

AUTO TEST?
START-YES STOP-NO
With the <Start> switch an Auto Test is initiated; after pressing the <Stop>
switch, parameters can be entered.
2. With the choice of
START-YES
the following message appears on the screen:
REMOVE SPINDLE
AND PRESS START

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PART V - MECHANICAL ANALYSIS
DYNAMIC MECHANICAL ANALYSIS

3. The spindle has to be removed from the viscometer and on pressing <Start> the
Auto Test will start.
The Auto-Test has always to be performed without spindle.
4. Immediately after pressing <Start>, the following message appears:
AUTOTESTING
SPEED: 10 rpm
5. The speed will increase up to 100 min−1 during the Auto Test.
6. Should there arise any operational problems, they will be displayed on the screen
during this test:
AUTO TEST
O.K.
7. In case of any faults, please refer to the lab’s assistant engineer.

Part 3: Selection of testing parameter

1. On pressing <Start> the parameters can be chosen from the selection tables (see
Table x).
a. Spindle
i. The first parameter is the spindle (SP), the display for this is in the right
top of the screen.
ii. It flashes until the input is completed.
iii. Other spindles (SP) appear on the screen for selection by using the
arrow keys. The selection is made by pressing <Enter>.

b. Speed
i. The second parameter is the speed; the display for this is left top on the
screen.
ii. It flashes until the input is completed.
iii. Other speed stages are displayed for selection by using the arrow keys.
The selection is made by pressing <Enter>.

Part 4: Inserting the spindle

1. The next message to appear on the screen is:

PRESS START
2. After insertion of the correct spindle, <Start> has to be pressed and the instrument
will start operating.
3. If the chosen spindle is of a disk type it should be submerged carefully in the
substance to avoid bubbles forming under its bottom surface.
4. To insert the spindle the shaft is slightly raised holding it firmly with one hand.
With the other hand the spindle is screwed in.
This must be done very carefully to make sure that the spindle is not bent.
5. The spindle and its counterpart with the inner thread should be smooth and clean.
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PART V - MECHANICAL ANALYSIS
DYNAMIC MECHANICAL ANALYSIS

6. Now the spindle can be immersed in the substance up to the immersion point. The
shaft of the instrument should not be knocked against the sides of the container
while the spindle is inserted since this could impair the vertical alignment of the
spindle.
7. The spindles L4 and R7 have to be immersed up to the marked zone (narrow spot).
8. The viscometer is now ready for operation.

Part 5: Performing measurement

1. Press <Start> for measurement.

2. Stable flow conditions is reached quickly and the reading values of the viscometer
can be considered correct within a few seconds (depending on the chosen speed
and the viscosity of the sample).
3. If the message “ERROR” appears on the screen and the alarm signal is activated,
the maximum %-value of scale has been exceeded. In this case either the speed
has to be reduced or a larger spindle should be selected.
4. If the reading is correct and stable the motor of the instrument can be stopped with
the <Stop> -switch. In this case the viscometer will show the last measurement
value on the screen.
5. This Stop-value will change continuously until 0 min−1 is reached. This protects
the more delicate parts of the instrument from unnecessary shaking which could
cause damage.
6. On pressing <Start> again the viscometer will return quickly to stable readings.
7. If the speed or the spindle need to be changed, the viscometer will accept the new
parameters as soon as they are confirmed with “Enter”.
8. Collect enough data to calculate the shear rate and shear stress and produce the
relationship as shown by the rheogram in Figure 7. Classify the materials that you
have tested.
9. Discuss your errors.

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PART VI
OTHER ANALYSIS

PSA

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 EXERCISE 7: CLASSIFY MATERIAL USING PARTICLE SIZE
ANALYSER

Introduction
Laser diffraction measures particle size distributions by measuring the angular
variation in intensity of light scattered as a laser beam passes through a dispersed
particulate sample. Large particles scatter light at small angles relative to the laser
beam and small particles scatter light at large angles. The angular scattering intensity
data is then analyzed to calculate the size of the particles responsible for creating the
scattering pattern, using the Mie theory of light scattering. The particle size is reported
as a volume equivalent sphere diameter. Particle size analysis using wet dispersion is
by far the most widespread method for obtaining reproducible results using laser
diffraction. Wet analysis provides a method of dispersion for samples across a wide
particle size range, ranging from sub-micron pigments through to sands and sediments.
To minimize the effects of particle sedimentation, the viscosity of dispersant plays a
major role in reducing the biasness of the reading. Samples analysed as liquid
dispersions include suspensions, emulsions, and solids dispersed in liquid. These three
general categories of samples have a few unique considerations. Even though the set-
up and the operation of a particle size distribution experiment is simple, few factors
that contribute to realistic measurements should be considered. These include
representative sampling, dispersant selection, measurement settings and dispersion
energy.

The sample to be tested will be provided for you. Determine the suitable refractive
index value based on the dispersant that you use. Remove very large debris (particles)
as the unit is only able to measure particles with size ranging from 0.1 µm to 3000
µm.

Identifying the features

Figure 7.1 is the feature of Mastersizer 2000 attached with Hydro 2000 MU and
the function of features of Mastersizer 2000 attached with Hydro 2000 MU is
shown in Table 7.1. Meanwhile features of a wet cell are shown in Figure 7.2
and the function of features of a wet cell is shown in Table 7.2.

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Figure 7.1: Features of Mastersizer 2000 attached with Hydro 2000 MU

No. Features Function

1. Optical bench To collect the raw data used to calculate the size
of particles in a sample
2. Sample dispersion To prepare the sample then deliver it
units to the optical bench so that it can be measured
3. Computer system To run the Malvern software and to controls the
optical bench and dispersion units.
To analyse the raw data from the optical bench
to determine the size of the particles.
Table 7.1: Function of features of Mastersizer 2000 attached with
Hydro 2000 MU

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 Figure 7.2: Features of a wet cell

No. Features Function

1. Light shroud To stops light entering the cell area
during a measurement and protects the
user from laser emissions.
2. Cell The cell has a pair of windows that
allow the laser to pass through the
sample. The
windows can be removed for
cleaning/replacement
3. “Cell in” and “Cell out” The sample is recirculated through the
connectors cell and back to the dispersion unit.
These
connectors attach the cell to the
dispersion unit. The tubing must be
connected correctly.
4. Locking handle The locking handle is rotated anti-
clockwise to lock the cell in place.
Table 7.2: Function of features of a wet cell

Learning Outcomes

At the end of this experiment, you will be able to:

a. Determine particle size distribution of liquid suspension using a particle sizer
b. Identify effect of measurement settings on the measured particle size

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 Equipment and Material

Equipment 1. Mastersizer 2000 attached with Hydro 2000 MU

Labware 1. 1000ml glass beaker
Material/ chemical 1. Distilled water (or any suitable dispersant)
2. Solid sample (in powder form, not soluble in the
dispersant)
3. Liquid sample containing suspended solids/precipitates
Experimental Procedure

Part 1: Preparing the equipment for analysis

1. Switch on the Hydro 2000 MU unit.
2. Pump distilled water through the system for 5 minutes to ensure the whole system
is cleaned. Set the pump speed to minimum 2000 rpm.
3. Stop the pump and raise the top part of the Hydro 2000 MU unit to drain out the
fluid.
4. Check lens for smudge and particulates. Repeat cleaning (step 2 and 3) with new
batch of clean distilled water if necessary.
5. Discard the cleansing water and pour approximately 500 ml of the dispersant
(distilled water) into the beaker.

Part 2: Analyzing sample

1. Switch on the Mastersizer unit.

2. Switch on the computer and launch the acquisition program (Mastersize 2000)
from the computer’s desktop/program list.
3. Ensure the Mastersizer unit and acquisition program are connected. The
connection status is indicated at the bottom right of the main program window.
4. Switch on ultrasonic to remove large agglomerates/residue in the system.
5. Discard the cleansing water and pour approximately 500 ml of the dispersant
(distilled water) into the beaker.
6. Set the pump speed to your desired value. You will require a high and a low value
to investigate the effect of pumping on measurement accuracy. The advisable
working range is between 500 rpm – 3500 rpm.
7. To begin new measurement, from the Menu Bar in main program window, select
‘File’ > ‘New’ and save using a suitable file name.
8. From the Menu Bar in main program window, select ‘Measure’ > ‘Manual’. A
new window will appear
9. Check the background scatter and make sure the light energy value is less than
20 for each detector number. A value of more than 20 indicates that the lens
requires cleaning (refer Part 1).
10. Make sure that the laser intensity is between 75 – 100%, the laser diffraction is in
OK mode and the laser beam is on OK mode. If there is a humming sound, then
the system needs to be cleaned.
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11. Once the setting is completed, click ‘Start’ and follow the on-screen procedures.
12. The system will measure the background first.
13. Wait for the on-screen instruction to add in your sample. Add your sample slowly
into the beaker until good laser obscuration value is achieved. The acceptable
value is between 10 – 20% (green box).
14. Wait for analysis to complete. Then, record and save your data.
15. Clean the system and using the same sample, re-measure the particle size at a
different pump speed. To investigate the effect of pump speed on the
measurement, increase or decrease the pump speed to a lower or higher value than
that set previously.
16. The measurement is made in triplicate automatically. Make a comparison
between the readings to make sure that the sampling is representative and there is
no destruction occurs on the sample due to the procedures. Justify your basis.
17. Your lab demonstrator will provide you with the analysis result.

Data Analysis and Report

Your report should include the followings:

1. Sketch of the main components of the Particle Sizer unit.

2. Describe in detail the theory used to calculate the mean diameter using laser
diffraction method.
3. State the mean diameter of your sample obtained by D[4,3], D[3,2] techniques.
Explain the differences.
4. For each analysis result, prepare two plots (a) Vol In % vs. particle size, and (b)
Vol In % (cumulative) against particle size. Use logarithmic scale for the x-axis.

From these plots, find the following values (show how you determine the values
from the plot) and provide brief explanation on the significance of these values in
regards to particle size analysis:

1. Mode
2. Median
3. Standard deviation
4. D[v, 0.1] - compare with values obtained by direct calculation
5. D[v, 0.5] - compare with values obtained by direct calculation
6. D[v, 0.9] - compare with values obtained by direct calculation

5. Why is it important to check on the scattering background? What happened if you

have a negative data?
6. Explain the concept of good laser obscuration value and its significance in this
analysis.
7. Elucidate in detail the effect of measurement conditions on your analysis
outcome. You may use the values in 4(a) – 4(f) as discussion points.
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8. Discuss the possible factors that contribute to the uncertainty in your
measurement.

References
1. https://www2.warwick.ac.uk/fac/cross_fac/sciencecity/programmes/internal/the
mes/am2/booking/particlesize/mastersizer_2000_main_manual.pdf

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