CHAPTER 4

1
Student Learning Outcomes:
At the end of this chapter, students should be
able to:

• differentiate between the experimental design

approach and conventional method.
• state the advantage of using experimental
design approach.
• explain the validation parameters.

2
 INTRODUCTION
In general, method development is the setting up of an analytical
procedure that will be appropriate for the analysis of a particular
sample.
(i) Choice of analytical technique: eg. Gas chromatography
(GC), high performance liquid chromatography (HPLC), etc.
- Volatile substances are best separated using GC;
- Provides the best resolution
- Shortest analysis time
- Highest sensitivity
(ii) Choice of phase system (based on the interactive character
of the compound to be analysed, polar/non-polar/ionic)

(iii) Detector selection which provide the required sensitivity,

the necessary linearity and if needed the desired specificity.
3
Method Development

A scientific method consists of the collection of data

through observation and experimentation, the formulation
and testing of hypotheses.

Quality standards often favor the use of standard methods

or collaboratively tested methods whenever possible.

Sometimes a laboratory may have a more suitable

method of its own – “in house method”

4
Method Development

Flow Chart:
Development

Optimization

Pre-Validation

Revalidation Validation

Implementation
5
Method Development

Optimization:

Objectives of optimization:
- improve the efficiency of method
- reduce the cost of chemicals and operations
by reducing the number of experiment.
- environmentally friendly method

6
Method Development: Optimization Method

Traditional approach: one-factor at a time

(COST- Change One Separate factor at a Time)
 Holds all factors but one constant
 Vary the levels of the factor of interest until we get the
“best value”
 Repeat for 2nd, then 3rd variables and so on…

7
Method Development: Optimization
(COST Approach)

Problems associated with the COST approach:

 Does not lead to real optimum.

 Inefficient, unnecessarily many runs.
 Provides no information about what happens when factors
are varied simultaneously (ignores interactions).
 Provides less information about the variability of the
response.
 Isolated, unconnected experiments.
 Slow growth of knowledge as no mapping of experimental
space.

8
Method Development: Optimization
(DoE Approach)

Statistically Designed Approach

(Design of Experiments, DoE)

An experiment that uses statistical tools and methods to

successfully study multiple factors in a single experiment.
Change the levels of more than one factor at a time.

DoE is the methodology of how to conduct and plan

experiments in order to extract the maximum amount of
information in the fewest number of experiment runs.

9
Method Development:

Experimental Design

Design of Experiment involves designing a set of ten to

twenty experiments, in which all relevant factors are
varied systematically.

When the results of these experiments are analyzed,

they help to identify optimal conditions, the factors
that most influence the results, and those that do not,
as well as details such as the existence of interactions
and synergies between factors.

10
Method Development:

Experimental Design: Advantages

11
Method Development: Experimental Design

Phases in the DoE process • What is the purpose?

• What are the objectives?
Planning • Identify factors, factor ranges
Experiment and types of factors (quantitative
or qualitative).
• What is possible, experimentally,
financially, environmentally?
Designing Experiment

Conducting Experiment

Analyzing Results

Confirming Predicted
Results 12
Experimental Design (continued)

GOALS OF EXPERIMENTAL DESIGN:

• TO OPTIMIZE THE COSTS (BY CONDUCTING AS

FEW EXPERIMENTS AS POSSIBLE).
• TO LEARN HOW THE INPUTS (FACTORS) AFFECT
THE OUTPUT (RESPONSES).

13
Experimental Design (continued)

Types of an experimental design:

Choose based on the research objectives
(i) Comparative objective:

We have several factors under investigation, but the

primary goal is to make a conclusion about one a-priori
important factor, and

the question of interest is whether or not that factor is

"significant", (i.e., whether or not there is a significant
change in the response for different levels of that factor),
then we have a comparative problem and we need a
comparative design solution.

14
Experimental Design (continued)

(ii) Screening objective:

To select out main factors which have the largest impact on quality
characteristics. The primary purpose of the experiment is to select or
screen out the few important main effects from the many less important
ones.

(iii) Response Surface Method (RSM)

The experiment is designed to estimate interaction, and therefore

give us an idea of the (local) shape of the response surface we are
investigating. For this reason, they are termed response surface
method (RSM) designs.
• Finding optimal settings of the most important factors
• Finding robust operating conditions where the process is insensitive
to variations in uncontrollable noise factors

15
Experimental Design (continued)

(iv) Optimizing responses when factors are proportions

of a mixture.
have factors that are proportions of a mixture and we want to
know what the "best" proportions of the factors are so as to
maximize (or minimize) a response, then we need a mixture
design.

(v) Optimal fitting of a regression model.

If we want to model a response as a mathematical function
(either known or empirical) of a few continuous factors and
you desire "good" model parameter estimates (i.e., unbiased
and minimum variance), then you need a regression design.

16
Method Development: Requirements
of DoE
Two types of variables: factors and responses

Factors: - input parameters/variables whose levels are

set by the experimenter
Factor: time
Levels: 2 min, 3 min, 10 min.

Responses: should provide useful information

about the quality of the process eg; percent
yield, amount (ppm/ppb), etc.

Selection of factors, levels and ranges:

Based on previous research, provided range (low to high)
17
DoE: Factors and Responses

GROWING TOMATOES ;
Input (factors): Fertilizer; soil pH; Seed hybrid; Water
Output (responses): Yield

CHEMICAL REACTION ;
Input (factors): Pressure; Temperature; Catalyst concentration
Output (responses): percent yield

18
Method Development: Types of DoE

• Full Factorial Designs

• Fractional Factorial Designs
• Central Composite Designs
• Plackett-Burman Design
• Box-Behnken Designs
The choice of an experimental
design depends on the
objectives of the experiment
and the number of factors to
be investigated
19
FULL FACTORIAL DESIGN

A FACTORIAL DESIGN CONSISTS OF CONDUCTION ALL POSSIBLE

COMBINATIONS OF VARIABLES AND LEVELS

EFFECTS OF ALL VARIABLES AND ALL POSSIBLE INTERACTIONS

CAN BE DETERMINED.

NO OF TRIALS INCREASE WITH NO OF VARIABLES AND LEVELS

20
FRACTIONAL FACTORIAL DESIGN

RETAINS SOME ADVANTAGES OF FULL FACTORIAL

PERMIT STUDY OF LARGE NUMBER OF VARIABLES WITH

REDUCED NUMBER OF RUNS

SOME INTERACTION EFFECTS CAN BE CONFOUNDED

CAN BE CONFOUNDED BY ALIAS’

LOSE MULTIFACTOR INTERACTIORS

21
CENTRAL COMPOSITE DESIGN

SPECIALLY SUITED TO FITTING SECOND AND HIGHER ORDER

SURFACES
TOTAL NUMBER OF TREATMENT COMBINATIONS = 2N +2N +1
IF N = 2, TREATMENTS = 9
IF N = 3, TREATMENTS = 15
IF N = 4, TREATMENTS = 27 (COMPARED TO 81 FOR FULL
FACTORIAL
USEFUL FOR BOTH SCREENING AND RESPONSE SURFACE

22
Method Development:

DoE: Analyzing the Design

• Multiple regression
• Analysis of Variance (ANOVA)
• Response Surface Plot

23
Method Development: DoE Application

APPLICATION (Example)
Experimental Design Approach
for the Optimization of Solid
Phase Microextraction (SPME)
for the Extraction of Benzene
and Toulene from Water

24
 Experimental Design Approach for the Optimization of Solid
APPLICATION: Phase Microextraction for the Extraction of Benzene and
Toulene from Water

Identifying the factors which may affect the result of an experiment.

Extraction time, Extraction temperature, Desorption time

Choosing the experimental design

Central composite design

Using statistical analysis to separate and evaluate

the effects of the various factor involved.
ANOVA + RSM

25
APPLICATION: Central Composite Design (Design-Expert Software
Version 6.0.6)

Coded Extraction Extraction Desorption

Level Temperature Time Time
- 27oC 0.5 min 13 sec
-1 30oC 6 min 40 sec
0 34oC 14 min 80 sec
1 38oC 22 min 120 sec
 41oC 27 min 147 sec

26
Sets of experiments generated from Central Composite Design.

Run Order Extraction Time (min) Extraction Temp. (oC) Desorption time (sec)
1 14 34 80
2 14 34 80
3 14 40 80
4 0.5 34 80
5 14 34 13
6 14 34 80
7 27 34 80
8 22 38 40
9 14 34 80
10 22 38 120
11 6 38 40
12 6 30 120
13 22 30 120
14 14 34 147
15 14 34 80
16 14 27 80
17 6 38 120
18 14 34 80
19 22 30 40
27
20 6 30 40
RESPONSE: Amount of Benzene and Toluene extracted from water
Run Order Benzene extracted (GC area count) Toluene extracted (GC area count)
1 61156 139925
2 56382 124331
3 59948 137036
4 12923 22252
5 0 0
6 51757 113327
7 53203 118705
8 50194 109196
9 48598 102567
10 51120 108560
11 53340 98946
12 48690 89840
13 70097 142073
14 63299 126841
15 61831 118660
16 58813 112530
17 51560 89957
18 59205 115688
19 64728 123955 28
20 45250 78363
Results of Significance test for benzene using the Quadratic Model

Variable p value
Et 0.0737
Et =Extraction time. T 0.7029
T=Extraction temperature.
Dt=Desorption time. Dt 0.0546
Et2 0.1845
Significant variable if p value is T2 0.3155
less than 0.05
Dt2 0.1501
EtT 0.2951
EtDt 0.9106
TDt 0.8151

At the 95% confidence level (p is less than 0.05)

Therefore, no variables are significant.
29
 Results of Significance test for toluene using the Quadratic Model

Variable p value
Et 0.0155
Et =Extraction time.
T 0.8934
T=Extraction temperature.
Dt=Desorption time. Dt 0.0406
Et2 0.1009
Significant variable if p value is
T2 0.3853
less than 0.05
Dt2 0.0561
EtT 0.3851
EtDt 0.8474
TDt 0.6168

At the 95% confidence level ( p is less than 0.05) two parameters,

extraction time and desorption time are significant
30
Coffee beans were roasted using a roaster (PROBAT) based on RSM with extraction conditions; temperature of 15-30 minutes and time of 150 °C- 185 °C.

Response surface 3-D plot

132859

113143

93427
Toluene Recovery
73710
X = A: Extraction Time

Toluene Recovery
Y = C: Desorption Time 53994

Actual Factor

B: Extraction Temperature = 34.00

6.0
120
10.0
100
14.0
80
Extraction Time (min) 18.0 60
Desorption Time (sec)
22.0 40
31
 Post-Graduate student’s research

ARABICA COFFEE BEANS ROASTING METHOD:

HIGH IN FLAVOUR COMPOUNDS PYRAZINE AND LOW IN ACRYLAMIDE

Roasting is the important step in the production of coffee

because it enables the development of flavour, aroma and
colour and also at the same time may lead to the formation
of undesirable compounds such as acrylamide.

Method: Coffee beans were roasted using a roaster (PROBAT) based

on RSM with extraction conditions; temperature of 150 °C- 185 °C and
time of 15-30 minutes.
Central composite design arrangement for independent variables A
(Temperature, °C) and B ( Time, minute) and their responses; 2-
Methylpyrazine, 2,3-Dimethylpyrazine, 2,5-Dimethylpyrazine,
2,3,5-Trimethylpyrazine, 2,3,5,6-Tetramethylpyrazine and
Acrylamide.
2,3- 2,5- 2,3,5- 2,3,5,6-
Temperature Time 2-methyl dimethyl dimethy trimethyl tetramethy
Run Block (°C) (minute) pyrazine pyrazine lpyrazine pyrazine lpyrazine Acrylamide

1 Block 1 167.53 22.50 229.00 546.00 138.20 671.80 536.00 976.00

2 Block 1 155.15 17.20 69.00 193.00 78.20 83.00 274.80 155.40
3 Block 1 179.90 27.80 38.00 356.60 133.20 25.00 94.60 245.80
4 Block 1 155.15 27.80 164.60 381.20 137.40 81.80 149.60 798.80
5 Block 1 179.90 17.20 44.60 449.80 121.20 75.20 92.80 1754.40
6 Block 1 167.53 22.50 326.40 443.40 216.60 703.20 485.40 951.60
7 Block 1 167.53 22.50 239.00 477.20 173.20 778.00 468.80 109.00
8 Block 2 185.03 22.50 51.20 414.40 217.40 237.80 166.40 676.40
9 Block 2 167.53 15.00 103.20 289.60 73.00 213.40 124.60 531.00
10 Block 2 167.53 22.50 249.20 585.60 178.60 531.20 390.60 114.00
11 Block 2 167.53 22.50 335.60 598.20 174.80 486.00 369.60 96.00
12 Block 2 167.53 30.00 139.00 92.60 247.20 312.00 103.80 887.20
13 Block 2 150.02 22.50 36.40 54.60 27.20 32.60 145.40 294.60
14 Block 2 167.53 22.50 315.80 543.00 188.80 517.40 350.00 60.40
 The optimized
2-Methylpyrazine condition (time
DESIGN-EXPERT Plot
and temperature)
Sqrt(2 MP)
X = A: Temperature
for roasting of
Y = B: Time
Arabica coffee
16.8527

14.4724
beans in order to
12.0921
produce high
9.71177
quality of coffee
Sqrt(2 MP)

7.33146

beans with high

concentration of
27.80

25.15
179.90 flavour and low
173.71
22.50
167.53
concentration of
B: T ime 19.85

17.20 155.15
161.34
A: T emperature undesirable
acrylamide is by at
temperature
167.68 °C for
22.50 minute.
Method Development: Experimental Design

DoE Software

Complete software package for Multivariate Data

Analysis and Experimental Design

Example:
i. Stat-Ease Design Expert
ii. The Unscrambler
iii. KCS (Kovach Computing Services)

35
 FINAL YEAR QUESTIONS
QUESTION 2 (April 2011)

In the development of a new extraction method using solid

phase microextraction, three important parameters
considered are temperature, extraction time and desorption
time. Compare and contrast the steps involved in optimizing
the method using experimental design (DoE) approach and
one factor at a time approach.
(10 marks)

36
QUESTION 3 (April 2006)

The production of B from A is as shown in Figure 1.

Catalyst X
A B
250 °C
Figure 1

Discuss the steps involved in optimizing the production of B

using Experimental Design (DoE). Details on the justification
for your choice of parameters and experimental values are
required.
(12 marks)

37
 ASSIGNMENT 1
CHM561 (Sept11-Jan12)

A student used Accelerated Solvent Extraction (ASE) to

extract the volatile compounds from coriander seed. Outline
an Experimental Design approach (DoE) that can be
considered in optimizing the oleoresin yield (g/100g) of
coriander seed.

(i) Details on the justification for your choice of parameters

and experimental values are required.
(ii) State the advantages of using DoE approach compare
to one factor at a time approach.

38
ASSIGNMENT:
Journal 1: “An Experimental Design Approach for the Determination of
Polycyclic Aromatic Hydrocarbons from Highly Contaminated Soil using
Accelerated Solvent Extraction” Anal. Chem. 1998, 70, 420-424.Saim et
al (1998) An Experimental approach... Anal.Chem,70,420-424.pdf

Answer the following questions:

1. State the standard method used in this study.
2. What are the sample pretreatment involve in this work?
3. List the ASE experimental parameters considered and state their
levels.
4. Name the software used for DoE.
5. List the responses used in this optimization work.
6. Briefly explain the method used in evaluating the results of this work.
7. “The robustness of ASE for the extraction of PAH in contaminated soil is
demonstrated”. How do the authors arrive with the conclusion?
8. Why the authors used GC-MSD to report the results?
9. If you are required to extract PAHs in sediment sample, what type of
solvent will you select? Give the reason for your answer. 39
ASSIGNMENT:
Journal 2: “An experimental design approach for the extraction of
volatile compounds from tumeric leaves (Curcuma domestica) using
pressurised liquid extraction” LWT-Food Science and Technology.
2009, 42, 233-238.An experimental approach_Zaibunissa et al.,
2009.pdf

Answer the following questions:

1. Name the method use for the extraction of volatile compounds from
tumeric leaves.
2. Explain why the authors used this method instead of using Soxhlet
extraction?
3. List the experimental parameters considered in this study and
state their levels.
4. What are the responses used in this optimization work?
5. Why central composite design (CCD) is chosen in this work?
6. Name the software used for DoE used in this work.
7. State the the most significant factor that may affects the amount of
volatiles extracted according to the authors’ finding. 40
Answer
Journal 1
1. Standard method used is EPA method 3545 based on accelerated solvent extraction (ASE).
2. Sample pretreatment involved in the study: air-dried for 24h and sieving through a 2 mesh sieve.
3. ASE experimental parameters and their level:
Level Pressure (psi) Temp (ºC) Ext Time (min)
- 1000 40 2
-1 1300 70 5
0 1700 120 9
+1 2100 170 13
+ 2400 200 16

4. The software used CSS Stastistica/W.Release 5.0 with industrial unit, Statsoft, Letchworth,
U.K.
5. There are 16 responses used in the optimisation namely naphthalene, acenaphthylene,
acenaphtene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene,
chrysene. Benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, indeno[123-
cd]pyrene, dibenzo[a,b]anthracene, benzo[ghi]perylene.
6. Using the experimental design approach in order to understand the way in which ASE
operating variables affect extraction. Multilinear regression was applied to the results of the
central composite design.

41
7. The robustness is the capacity of a method to remain uneffected by small deliberate variations
in method parameters. The robustness of a method is evaluated by varying method parameters
eg solvent used, temperature, pressure, etc. In this work changes in experimental parameters
not affected the ASE performance.

8. The author used GC-MSD because in complex matrix eg., soil, it was difficult to separate all
16 PAHs using GC-FID for example compounds benzo[b]fluoranthene and
benzo[k]fluoranthene are analysed as a combined concentration as they are not totally
seperated. The sensitivity of GC-MSD can be enhanced by applying SIM (selected ion
monitoring) mode. Thus, it is possible to to separate a very close compounds.

9. Combination solvent for example; n-hexane + acetone give the highest amount of PAHs.

42
Journal 2

1. The method used is pressurised liquid extraction (PLE)

2. PLE method is used instead of using Soxhlet because it is a rapid extraction technique within 5-
30 min, temperature can be optimised to achieved the desired extract and consumed less solvent
compared to Soxhlet extraction, which take long extraction time and consumed large solvent.
3.
Level Temp (ºC) Pressure (kPa) Ext Time (min)

- 60 6895 6
-1 90 8274 10.0
0 130 10.343 17.5
+1 170 12.411 25.0
+ 200 13.790 30.0

4. Responses used are volatile compounds (Terpinolene, -Phellandrene, p-Cymene, 1,8-Cineole) ,

yield (g/100g) and Visual observation.
5. Central Composite Design is chosen in this work because this method is the most useful design
for estimating a multifactor response surface which keeps the number of experiments to a
minimum while simultaneous assessments of variations of all experimented factors studied and
distinguishing the interaction between them.

6. The software used is Design-Expert vertion 6.0.4 (Stat Ease Software). 43

7. The most significant factor that may affect the amount of volatiles extracted.