Simplifying sample prep optimization by

using factorial design

Wan Raihana Wan Aasim

Universiti Sains Malaysia
 Sample prep : Everyone does it!

• Sample prep is a integral part of any analytical

Introduction method
Factorial
design basics • The goal of sample prep is enrichment, cleanup
Analysis of
and signal enhancement
Some commonly used sample prep methods for
factorial
chromatography applications are:
design
Liquid-liquid extraction Solid Phase
7 Steps to Microextraction
factorial
design

Conclusion

Self-
Assessment QuEChERS
Solid Phase Extraction
© 2010 Wan Raihana W.A
 Why optimize sample prep?

• The performance of a sample prep method

Introduction depends on several variables e.g. pH,
temperature, solvent type etc.
Factorial
design basics Optimization of
sample prep is
Analysis of essential to ensure
factorial optimum recovery,
design
selectivity and
7 Steps to sensitivity
factorial
design
Most times, optimization is performed by sequential
Conclusion analysis of experimental variables

Self- A simpler and more efficient alternative is the use of a

Assessment
statistical approach called factorial design
© 2010 Wan Raihana W.A
 Tutorial objectives

• In this tutorial, you will learn :

Introduction
 Why factorial design is an excellent
Factorial tool for sample prep optimization.
design basics
 Basic principles of factorial design.
Analysis of
 Key terminology and concepts
factorial
design  How factorial designs are analyzed
7 Steps to
 How to set up your own factorial design for sample
factorial prep optimization in seven easy steps
design

Conclusion Once you have completed the tutorial, there is

a short quiz to help you assess if you have
Self- mastered the information contained in this
Assessment tutorial
© 2010 Wan Raihana W.A
 How is optimization USUALLY done?

The most common approach to sample prep

Introduction optimization is known as :
Factorial
design basics

Analysis of
OFAT
factorial One-Factor-At-a-Time
design

7 Steps to
 The classic approach to sample prep optimization
factorial  A factor = an experimental variable
design  OFAT involves sequential experiments where one
Conclusion factor is varied and the others are held constant
 This process is repeated until an optimal
Self- combination of factors is found which gives the
Assessment
best results
© 2010 Wan Raihana W.A
 A simple optimization example

• To understand the OFAT approach, consider the

Introduction following sample prep method as an example:
Factorial
design basics • A liquid-liquid extraction (LLE) method for isolation of
amphetamine (AM) from human urine samples
Analysis of •
factorial
design Objective of optimization
• Maximize recovery of the sample prep method
7 Steps to
factorial mentioned above by optimizing the following
design experimental variables at the two levels specified
below:
Variable/Factor to Optimize Levels/values to be tested
Conclusion
Extraction Solvent [ExtSolv] Hexane or ethyl acetate
Self- Urine pH [pH] pH 8 or pH 10
Assessment Extraction time [ExtTime] 30 mins or 60 mins
© 2010 Wan Raihana W.A
 Optimizing with OFAT

• Using an OFAT approach, the following

Introduction experiments would have been performed:
Factorial Factors/Variables Tested
Experiment Highest Recovery
design basics Run
sets ExtSolv pH ExtTime when :

Analysis of ExtSolv 1 Hexane pH 8 30 min ExtSolv = Ethyl

factorial varied 2 EtOAc pH 8 30 min acetate
design 3 EtOAc pH 8 30 min
pH varied pH = 10
4 EtOAc pH 10 30 min
7 Steps to
ExtTime 5 EtOAc pH 10 30 min
factorial ExtTime = 60 min
varied 6 EtOAc pH 10 60 min
design

Conclusion Conclusion : Recovery is highest when :

Extraction solvent : ethyl
Self-
acetate
Assessment
 Urine pH = 10
© 2010 Wan Raihana W.A
 Optimizing with OFAT

Factors/Variables Tested
Experiment Run Highest Recovery
Introduction sets ExtSolv pH ExtTime when

Factorial ExtSolv 1 Hexane pH 8 30 min ExtSolv = Ethyl

design basics Varied 2 EtOAc pH 8 30 min acetate
3 EtOAc pH 8 30 min
Analysis of pH varied pH = 10
4 EtOAc pH 10 30 min
factorial
design ExtTime 5 EtOAc pH 10 30 min
ExtTime = 60 min
varied 6 EtOAc pH 10 60 min
7 Steps to
factorial Disadvantages of approach :
design  Not all combinations of variables are tested
i.e. what about ExtSolv = hexane; pH = 10; ExtTime = 60 min?
Conclusion

Self-  Unable to detect factor interactions

Assessment i.e. what if the extraction time depended on the type of
extraction solvent used?
© 2010 Wan Raihana W.A
 What are factor interactions ?

Factor interactions occur when the

Introduction effect of one factor depends on the
level of another factor
Factorial
design basics

Analysis of A simple example of a factor interaction

factorial
design Baking a chocolate cake
2 factors : oven temperature, oven
7 Steps to
factorial time. Every baker knows that...
design High temperature, low oven time
Conclusion
Low temperature, high oven time

Self-
Assessment
The selection of oven time depends on
the oven temperature used
© 2010 Wan Raihana W.A
 Why use a factorial design approach?

• Using a classic OFAT approach, important

Introduction information regarding potential interactions is left
Factorial
out.
However, by using a factorial design approach, sample
design basics prep methods can be optimized quickly, efficiently and
with greater statistical confidence in the results
Analysis of
factorial
design Advantages of factorial design:
 All factor-level combinations can be tested
7 Steps to
factorial  Factor interactions can be detected
design  Higher statistical power
Conclusion  Easy to implement
 Efficient design – can obtain more information
Self- compared to OFAT
Assessment
© 2010 Wan Raihana W.A
 What is Factorial Design?

• Factorial design is an experimental design which

Introduction simultaneously studies multiple experimental
Factorial
factors/variables at multiple levels
design basics
Pioneering work in this field was by
Analysis of Sir Ronald A. Fisher at Rothamsted
factorial Agricultural Field Research Station,
design
London, England in the early 1920s
7 Steps to
factorial Can be used for
design  Process optimization
 To determine effects of
Conclusion
variables in a process
Self-  Modelling of a process Sir Ronald A. Fisher
(1890 – 1962)
Assessment  Determination of robustness
© 2010 Wan Raihana W.A
 What is Factorial Design?

Introduction
Simultaneous analysis of all possible
factor & level combinations
Factorial
design basics
 Most common designs are 2-level factorial designs
Analysis of where each factor is studied at two discrete levels.
factorial  Represented by the formula 2x where x represents the
design number of factors studied
 The formula also indicates the number of experiments
7 Steps to
factorial required for a specific design
design
Number of Factors Experiments needed
Conclusion 2 22 = 4
3 23 = 8
Self-
Assessment 4 24 = 16

© 2010 Wan Raihana W.A

 But first, understand some terms

• The most important terms to know are :

Introduction Factors
Factorial Levels
design basics Trials/Runs
Analysis of Response
factorial
design
• In order to understand these concepts, the
7 Steps to
factorial
previous LLE sample prep described will be
design used as an example.
A liquid-liquid extraction (LLE) method for
Conclusion isolation of amphetamine (AM) from
human urine samples
Self-
Assessment
© 2010 Wan Raihana W.A
 DoE Lingo : Factors

Factors refer to the experimental variables that are to be

Introduction optimized

Factorial
design basics Factors can either be qualitative or quantitative

Analysis of
factorial
design Variable/Factor to Optimize Levels/values to be tested

7 Steps to Extraction Solvent [ExtSolv] Hexane or ethyl acetate

Qualitative Factor
factorial Urine pH [pH] pH 8 or pH 10
Quantitative Factor
design Extraction time [ExtTime] 30 mins Quantitative
or 60 mins Factor
Conclusion

Self- Factors
Assessment
© 2010 Wan Raihana W.A
 DoE Lingo : Levels

Levels are the specific and discrete values or settings

Introduction assigned to each factor

Factorial
design basics Levels are either qualitative or quantitative depending on
the factor
Analysis of
factorial
design Variable/Factor to Optimize Levels/values to be tested

7 Steps to Extraction Solvent

Qualitative Factor[ExtSolv] Hexane or ethyl acetate
factorial Urine pH [pH]Factor
Quantitative pH 8 or pH 10
design Extraction time [ExtTime] 30 mins or 60 mins
Quantitative Factor
Conclusion

Self- Levels
Assessment
© 2010 Wan Raihana W.A
 DoE Lingo : Runs

Runs refer to the specific number of experiments required

Introduction to perform a factorial design experiment
Runs are also referred to as trials
Factorial
design basics
Number of Factors Experiments needed for
Analysis of a factorial design
factorial
2 4
design
3 8
7 Steps to 4 16
factorial
design

Conclusion
Runs or Trials

Self-
From the above table, it can be seen that the number of
Assessment runs or trials required for a 3 factor, 2-level factorial design
is 8 runs or trials
© 2010 Wan Raihana W.A
 DoE Lingo : Response

Response is the output of the experiment performed.

Introduction

Factorial
For example, in the LLE optimization previously described,
design basics the output of the experiment would be the percentage
recovery of amphetamine from urine
Analysis of
factorial
design A factorial design may have more than one response
For example, if the LLE method was expanded to
7 Steps to
factorial
include another two drugs, the factorial design should
design have three responses to reflect each drug.

Conclusion

Self-
Assessment
© 2010 Wan Raihana W.A
 Standard 2-level Factorial Design Matrix

Standard factorial designs can be obtained from reference

Introduction books and statistical software packages

Factorial Below is a standard 2-level factorial design matrix for three

design basics
factors (number of runs = 8)
Analysis of Experiment Factor A Factor B Factor C
factorial Run 1 -1 -1 -1
design Run 2 1 -1 -1
Run 3 -1 1 -1
7 Steps to Run 4 1 1 -1
factorial Run 5 -1 -1 1
design Run 6 1 -1 1
Run 7 -1 1 1
Conclusion
Run 8 1 1 1
Self-
Assessment
© 2010 Wan Raihana W.A
-1 & 1 are symbols representing different levels in factorial design notation
 From OFAT to Factorial

For our previous Levels

Factor
-1 1
Introduction OFAT example, the
ExtSolv Hexane Ethyl Acetate
equivalent factorial pH pH 8 pH 10
Factorial design would be:
design basics ExtTime 30 min 60 min

Analysis of
factorial Experiment Factor
ExtSolvA Factor
pH B Factor C
ExtTime
design Run 1 -1
Hexane -1 8
pH 30-1
min
Run 2 1
Ethyl Acetate -18
pH 30-1
min
7 Steps to Run 3 -1
Hexane pH110 30-1
min
factorial Run 4 1
Ethyl Acetate pH110 30-1
min
design Run 5 -1
Hexane -18
pH 1
60 min
Run 6 1
Ethyl Acetate -18
pH 60 1min
Conclusion
Run 7 -1
Hexane pH110 1
60 min
Run 8 1
Ethyl Acetate pH110 60 1min
Self-
Assessment
© 2010 Wan Raihana W.A
 Characteristics of a Factorial Design

Experiment ExtSolv pH ExtTime

Run 1 Hexane pH 8 30 min
Introduction
Run 2 Ethyl Acetate pH 8 30 min
Factorial Run 3 Hexane pH 10 30 min
design basics Run 4 Ethyl Acetate pH 10 30 min
Run 5 Hexane pH 8 60 min
Analysis of Run 6 Ethyl Acetate pH 8 60 min
factorial Run 7 Hexane pH 10 60 min
design Run 8 Ethyl Acetate pH 10 60 min

7 Steps to
factorial
design
All possible factor-level combinations are represented in
Conclusion the factorial experiment

Self-
Assessment
© 2010 Wan Raihana W.A
 Running a Factorial Design

Experiment ExtSolv pH ExtTime Response

Introduction Run 1 Hexane pH 8 30 min 65.3
Run 2 Ethyl Acetate pH 8 30 min 81.3
Factorial Run 3 Hexane pH 10 30 min 53.3
design basics Run 4 Ethyl Acetate pH 10 30 min 69.9
Run 5 Hexane pH 8 60 min 61.8
Analysis of Run 6 Ethyl Acetate pH 8 60 min 77.4
factorial
Run 7 Hexane pH 10 60 min 73.9
design
Run 8 Ethyl Acetate pH 10 60 min 89.9

7 Steps to
factorial Each run is performed according to the factor settings
design specified in the design matrix
Conclusion
The response (result) for each run should be recorded
Self-
Assessment
In this example, the response is the percent recovery for
amphetamine (AM)
© 2010 Wan Raihana W.A
 Analyzing Factorial Designs

Most statistical packages can analyze factorial designs

Introduction

Factorial
design basics

Analysis of
factorial In this tutorial, all analyses were
performed with Minitab 15
design
Major analytical tools
7 Steps to
 ANOVA – to identify significant factors and factor
factorial
design interactions
 Normal plot of Effects
Conclusion
 Pareto Chart  Visual approach
 Main Effects Plot  Easiest to
Self-
Assessment understand and
 Interaction Plots
interpret
© 2010 Wan Raihana W.A
 Analysis : Effect Size and ANOVA

Introduction

Factorial
design basics
Effect Size:
Analysis of The measure of the
factorial strength of the effect
design of a variable on the
response
7 Steps to
factorial
design p-values
Values below a specific
chosen significance level
Conclusion Model Fit represent statistically
The value of R-Sq represents the significant factors
Self- represent the proportion of variation in
Assessment the response data explained by the
terms in the model.
© 2010 Wan Raihana W.A
 Analysis : Normal Plot of Effects

Significance
level
Introduction
pH
Factorial
design basics
Extraction Solvent
Analysis of Normal
factorial line & non-
design significant Extraction Time –Solvent
effects Interaction
7 Steps to
factorial  Non-significant factors and interactions fall
design near the normal line (indicated in blue)
Conclusion
 The further away the factor/interaction
Self- A comparison offromthe the normal
relative line, theand
magnitude larger the effect
statistical
Assessment size and significance
significance of factor effects and factor interactions
© 2010 Wan Raihana W.A
 Analysis : Pareto Plot

Non-
Introduction significant

Factorial
pH
design basics
Extraction Time – Solvent Interaction
Analysis of
factorial Extraction Solvent
design
Significance Cut-Off Line
7 Steps to
factorial  Factors and interactions that exceed the
design significance cut-off line are significant at α
Conclusion = 0.05

Self- Another visual  representation

The magnitudeof ofrelative
each effect can be seen
magnitude and
Assessment by the of
length of effects
the representative bar
statistical significance factor and interactions
© 2010 Wan Raihana W.A
 Analysis : Main Effect Plot

Introduction

Factorial %
% Recovery
Recovery
design basics increases
increases when
when pH pH
changes
changes fromfrom the
the low
low
Analysis of level
level (-1,
(-1, pH
pH 8)8) to
to the
the
factorial high
high level
level (1,
(1, pH
pH 10)
10)
design

7 Steps to %
% Recovery
Recovery
%
% Recovery
Recovery does does not
not change
change
factorial increases
increases when
when the
the
when
when extraction
extraction timetime
design extraction
extraction solvent
solvent(-
changes
changes from
from the
the low
low level
level (-
changes
changes from
from hexane
hexane
1,
1, 30
30 min)
min) to to the
the high
high level
level
Conclusion (level
(level -1)
-1) to
to ethyl
ethyl
(1,
(1, 6060 min)
min)
acetate
acetate (level
(level 1)
1)
Self- A main effect plot shows the magnitude and “direction”
Assessment
of the factors being studied
© 2010 Wan Raihana W.A
 Analysis : Interaction Plots

Introduction

Factorial
The interaction plot for
design basics ExtTime vs ExtSolv
indicates that :
Analysis of
factorial  When ExtSolv is
design
Hexane, the optimum
7 Steps to recovery is when
factorial ExtTime is 30 min
design
ExtTime –
 When ExtSolv is Ethyl ExtSolv
Conclusion
Acetate, optimum Interaction
Self- recovery
Factor is whenare detected by crossed lines in
interactions
Assessment ExtTime is 60 min
interaction plots
© 2010 Wan Raihana W.A
 Analysis : Pieces of the Puzzle

What we know so far in our optimization experiment:

Introduction  Statistically significant factors are pH and extraction
solvent
Factorial
design basics  The extraction solvent and extraction time factor
interaction is statistically significant
Analysis of  % Recovery increases when pH changes from pH 8 to
factorial
design 10 and extraction solvent changes from hexane to ethyl
acetate
7 Steps to  A change in extraction time does not increase percent
factorial
design
recovery
 When ExtSolv is Hexane, the optimum recovery is when
Conclusion ExtTime is 30 min
Self-
 When ExtSolv is Ethyl Acetate, optimum recovery is
Assessment when ExtTime is 60 min
© 2010 Wan Raihana W.A
 Analysis :
Putting the Pieces Together

Based on the results from the main effects plot, the

Introduction highest recovery for AM is obtained when
 Urine pH is pH 10
Factorial
design basics  Extraction solvent is ethyl acetate
 Extraction time on its own does not have any effect on
Analysis of
recovery
factorial
design
But taking into account the factor interaction results :
7 Steps to
factorial  When ethyl acetate is used, the highest recovery is
design when extraction time is 60 minutes

Conclusion Optimized factor settings :

Self-
pH 10;
Assessment Extraction solvent : ethyl acetate;
Extraction time : 60 minutes
© 2010 Wan Raihana W.A
 7 steps to experimental design
Setting up your own factorial design optimization
 Step 1 – Identify response

Based on the sample prep to be optimized, identify the

Introduction response that characterizes the process

Factorial
design basics For a single method, multiple responses may be selected,
depending on what needs to be optimized
Analysis of
factorial
design Examples of responses:
Percent recovery, peak areas,
7 Steps to reproducibility, % yield
factorial
design

Conclusion
Factorial designs work best with
Self- quantifiable responses
Assessment
© 2010 Wan Raihana W.A
 Step 2 – Identify factors

Select the factors that need to be optimized

Introduction

Factorial
Select factors that are easily controlled i.e. pH, incubation
design basics temperature rather than factors that are uncontrollable
i.e. ambient temperature.
Analysis of
factorial
design Several factors may influence a sample prep method, but
the number of runs increases greatly with the number of
7 Steps to factors, therefore limit factors to those that are most
factorial
design
important.

Conclusion 3 – 5 factors are usual. More than 5 factors is impractical

Self- as it results in a large number of runs
Assessment
© 2010 Wan Raihana W.A
 Step 3 – Determine levels

Determine the two levels for each of the factors to be

Introduction optimized.

Factorial
design basics For quantitative factors, the levels chosen should span a
logical range
Analysis of
factorial
design The range should
not be too wide
7 Steps to
or too narrow
factorial
design

Conclusion

Self-
Assessment
© 2010 Wan Raihana W.A
 Step 4 – Identify the design

Based on the number of factors to optimize, identify the

Introduction appropriate design matrix

Factorial
design basics Factorial design matrices can be obtained from:
 Statistical software – Minitab has built in designs to
Analysis of
choose from
factorial
design  Design of experiments books

7 Steps to
factorial
design

Conclusion

Self-
Assessment
© 2010 Wan Raihana W.A
 Step 4 – Identify the design

Using the standard design matrix, fill in the variables and

Introduction factor levels in order to obtain the respective factor-level
combinations for each experimental run
Factorial
design basics Filled
Standard
in design
Design
matrix
Matrix
with selected
from statistical
factorsbook
& levels
Analysis of Experiment Factor
ExtSolvA Factor
pH B Factor C
ExtTime
factorial Run 1 Run 1 -1 -1 8
Hexane pH 30-1
min
design
Run 2 Run 2 1
Ethyl Acetate -18
pH 30-1
min
Run 3 -1 1 -1
7 Steps to Run 3 Hexane pH 10 30 min
Run 4 1 1 -1
factorial Run 4 Ethyl Acetate pH 10 30 min
Run 5 -1 -1 1
design Run 5 Run 6 Hexane pH
1 -18 60 min
1
Run 6 Run 7 Ethyl Acetate
-1 pH1 8 60 1min
Conclusion
Run 7 Run 8 1
Hexane pH110 1
60 min
Self- Run 8 Ethyl Acetate pH 10 60 min
Assessment
© 2010 Wan Raihana W.A
 Step 5 – Perform the experiments

Using the design matrix to identify factor-level

Introduction combinations for each run, conduct the experiment

Factorial
design basics Experiments should randomized and replicated

Analysis of
factorial More replicates = more statistical power
design
If replicates are performed, the mean of the replicates for
7 Steps to
factorial each run is taken as the response.
design

Conclusion

Self-
Assessment
© 2010 Wan Raihana W.A
 Step 6 – Analyze the experiments

For each response, analyze using:

Introduction
 ANOVA, to identify model fit and statistical
Factorial
design basics
significance of experiment

Analysis of  Normal plot or pareto chart, to visually identify

factorial
significant factors and interactions
design

7 Steps to  Main effects plot, to identify the optimum levels for

factorial each significant factor
design

Conclusion  Interaction plot, to identify the optimum levels for

each significant factor interaction
Self-
Assessment
© 2010 Wan Raihana W.A
 Step 7: Confirmatory runs & Next Steps

Using the optimized levels for each factor, perform

Introduction confirmation runs in order to check results

Factorial
design basics Non-significant factors need no further optimization and
can be set to any convenient level
Analysis of
factorial
design Next steps:
7 Steps to  If optimized method is acceptable, can proceed to
factorial
design validation etc.
 If further optimization is required, consider other
Conclusion more advanced experimental designs e.g. response
surface methodology or central composite design
Self-
Assessment
© 2010 Wan Raihana W.A
 Conclusion

 Factorial designs provide a

Introduction simple but efficient approach
for optimization of sample
Factorial prep methods
design basics

Analysis of  Factorial designs provide

factorial additional information
design regarding factor interactions
which may have a large impact
7 Steps to
factorial
of sample prep methods
design
 Factorial designs provide
Conclusion higher statistical power and
increase confidence in
Self-
Assessment optimization
© 2010 Wan Raihana W.A
 Recommended reading

1. Bayne, C. K. and I. B. Rubin (1986).

Practical Experimental Designs and
Introduction
Optimization Methods for
Chemists. Weinheim, VCH.
Factorial
design basics 2. Mason, R. L., R. F. Gunst, et al.
(2003). Statistical Design and
Analysis of Analysis of Experiments With
factorial Applications to Engineering and
design Science. Hoboken, New Jersey, John
Wiley & Sons.
7 Steps to 3. Wu, J. C. F. and M. Hamada (2000).
factorial Experiments : Planning, Analysis
design and Parameter Design
Optimization. New York, Wiley
Conclusion 4. Araujo, P. W. and R. G. Brereton
(1996). "Experimental design I.
Self- Screening." TrAC Trends in
Assessment Analytical Chemistry 15(1): 26-31.
© 2010 Wan Raihana W.A
 Self-Assessment

Congratulations! You have reached the end of this tutorial.

Introduction As a final step, use the quiz below to assess if you’ve
mastered the main concepts in this tutorial
Factorial
Use the right
design basics and left
arrows
Analysis of (below the
title bar of
factorial the quiz) to
design navigate the
questions
7 Steps to
factorial
design

Conclusion If you have

finished, click
Self- HERE to end
the tutorial
Assessment
© 2010 Wan Raihana W.A
Simplifying sample prep optimization by
using factorial design

Wan Raihana Wan Aasim

Universiti Sains Malaysia

Thanks for using this tutorial and

good luck with your sample prep optimization!

Copyright © 2010. Wan Raihana Wan Aasim. All rights reserved

Contact the author : wanraihana@gmail.com