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Latin Square Design

By
H.M. Edi Armanto and Swaditya Rizki
Faculty of Agrotechnology and Food Science, UMT - Malaysia
Presentation Outline
1) Relative Efficiency
2) Latin Square (4 Slides)
3) Advantages of Latin Square
4) Disadvantages of Latin Square
5) Latin Square Layout (3 Slides)
6) Hypothesis
7) The Linear Model
8) ANOVA Procedure
9) Example 1: Fishing Machines
10)Example 2: Protein Sources




Relative Efficiency

1) Relative Efficiency of LS to RCBD (how many
times as many replicates would be needed for
RCRD to have as precise of estimates of
treatment means as LS does):
2) Would need approximately 56 reps per variety
to have as precise of estimates of variety
means if experiment conducted as RCBD

Latin Square (1)
A Latin square is a square array of objects (letters
A, B, C, ) such that each object appears once and
only once in each row and each column.

Example - 4 x 4 Latin Square.

A B C D
B C D A
C D A B
D A B C

A
B C D
A
B C D A
B C D
A
B C D
Latin Square (2)
Variation is already well known in row-blocking and column blocking
The experiment area has some following characters, as follows (at least
one of those is fulfilled):
1) Spatial: pond, row, districts/village, households, etc.
2) Time: week, month or year
3) Age: different age group of animals
4) Height: tree trunk, shelf in the drier etc.
5) Direction of soil fertility gradients
6) Soil heterogeneity, slope, insect migration etc.
7) Predictable directions of insect migration
8) Green house trials and experimental plots in the fields
9) Laboratory trials with replication over time
10) Number of replications is equal to the number of treatments
A Latin Square experiment is assumed to be a three-factor
experiment (rows, columns and treatments).
A Latin Square is to handle two known sources of variation
among experimental units (row-blocking and column blocking)
It is called Latin because we usually specify the treatment by
the Latin letters
Square because it always has the same number of levels (t)
for the row and column nuisance factors
It is assumed that there is no interaction between rows,
columns and treatments.
The degrees of freedom for the interactions is used to estimate
error.
Latin Square (3)
Latin Square (4)
A significant assumption is that the three factors (treatments
and two nuisance factors) do not interact
More restrictive than the RCBD
Each treatment appears once and only once in each row and
column
If you can block on two (perpendicular) sources of variation
(rows x columns) you can reduce experimental error when
compared to the RCBD
This design is used to simultaneously control (or eliminate) two
sources of nuisance variability (confounding variables)


Advantages of Latin Square
1) Allows the experimenter to control two sources of
known variation
2) Columns and rows serve as blocks, and hence the
design minimizes the soil heterogeneity in both
directions
3) Experiments may be laid out in compact square
blocks
4) It is used in many fields, such as agriculture,
industry, medicine etc
Disadvantages of Latin Square
1) Error degree of freedom (df) is small if there are only a few treatments
2) The experiment becomes very large if the number of treatments is
large
3) The statistical analysis is complicated by missing plots and mis-
assigned treatments
4) For large numbers of treatments, the row size increases, which
causes heterogeneity among the plots within the blocks and hence for
large number of treatments, this design is not suitable
5) It can be used only when the number of rows, number of column and
number of treatments are equal
6) Latin square is not useful for fewer than four treatments nor more
than eight

3 x 3 4 x 4
A B C A B C D A B C D A B C D A B C D
B C A B A D C B C D A B D A C B A D C
C A B C D B A C D A B C A D B C D A B
D C A B D A B C D C B A D C B A

5 x 5 6 x 6
A B C D E A B C D E F
B A E C D B F D C A E
C D A E B C D E F B A
D E B A C D A F E C B
E C D B A F E B A D C
Layout of Latin Square Designs (1)

Two blocks (random factors) + 1 treatment (fixed factor)

Canal (Block 1)
Road/
Shade/
Elevation
etc.

(Block 2)



Layout of Latin Square Designs (2)
1) All the treatments should be allocated to each block of both the factors
2) 3 null hypotheses (H
0
): there are no effects of block 1 and block 2 and
the treatments

3) For four treatments (T1, T2, T3 and T4)
4) Randomization?
Canal (Block 1)
Road/
Shade/
Elevation
etc.

(Block 2)



T3 T2 T4 T1

T2 T4 T1 T3

T4 T1 T3 T2
T1 T3 T2 T4

Layout of Latin Square Designs (3)
The Latin square Design
All treats appear once in each row
and each column
Columns
R
o
w
s

1
2
3

t
2
3
t
1
1 3
2
15
soil gradient
field
work
gradient
A Need for Blocking in Two Directions
16
soil gradient
field
work
gradient
B
l
o
c
k

1

B
l
o
c
k

2

B
l
o
c
k

3

B
l
o
c
k

4

Blocks that Vary in Soil Quality
17
soil gradient
field
work
gradient
Block 1
Block 2
Block 3
Block 4
Blocks that Vary Due to Field Work Variables
18
soil gradient
field
work
gradient
Assignment of Treatments to Experimental Units
Using a Latin Square Design
A C B D
A
A
A
B
B
B
D C
D
D
C
C
Latin Square Design (Data table)
Column
Row 1 2 3 4
1 A C D B
2 C B A D
3 B D C A
4 D A B C
Total
Treatment A
Treatment B
Treatment C
Treatment D
Grand total
ANOVA for a Latin Square
Source S.S. d.f. M.S. Fcal Sig
Treat SS
T
t-1 MS
T
MS
T
/MS
E
p< 0.01 *
Rows SS
Row
t-1 MS
Row
p< 0.05 **
Columns SS
Col
t-1 MS
Col
p> 0.05 ns
Error SS
E
(t-1)(t-2)
MS
E
Total SS
T
t
2
- 1
t = number of treatments;
* Significant ** Very significant ns: Not significant
In a Latin square, there are three factors:
Treatments (t) (letters A, B, C, )
Rows (t)
Columns (t)

The number of treatments = the number of rows =
the number of columns = t.
The row-column treatments are represented by cells in
a t x t array.
The treatments are assigned to row-column
combinations using a Latin-square arrangement
The Linier Model
( ) ( ) k ij j i k k ij
y c t + + + + =
i = 1,2,, t j = 1,2,, t
y
ij(k)
= the observation in i
th
row and the j
th
column
receiving the k
th
treatment
= overall mean
t
k
= the effect of the k
th
treatment

i
= the effect of the i
th
row
c
ij(k)
= random error
k = 1,2,, t

j
= the effect of the j
th
column
No interaction between
rows, columns and
treatments
Example 1:
Latin Square Design
Example 1: Fishing Machines
1) A company will test five brands of fishing machines (D,P,F,C
and R) which is used by five fishermen.
2) The brands are all comparable in the purchase prices.
3) The company wants to carry out a study that will enable them
to compare the brands with respect to operating costs.
4) For this purpose they select five fishermen (Rows).
5) In addition the study will be carried out over a five week period
(Columns = weeks)
6) Each week a fisherman is assigned to a machine using
randomization and a Latin Square Design.
7) The average cost per km (RM/km) is recorded at the end of
each week and is tabulated below:

Week
1 2 3 4 5
1 7 4 7 9 8
D P F C R
2 3 8 10 6 8
P D C R F
Drivers 3 7 9 8 12 10
F C R D P
4 6 11 11 12 14
R F D P C
5 12 8 9 11 15
C R P F D

Fishermen
Table 1. The average cost (RM/km) of fishing
machines
Research Characters
1) Rows : Fishermen
2) Columns : Week
3) Experimental Unit : Machine
4) Variable Parameter : cost (RM/km)
5) Treatments : to operate the
1) fishing machines
Data View
Select
Analyze->General Linear Model->Univariate
Select the dependent variable and
the three factors Rows, Cols, Treats
Select Model
Identify a model that has only main effects for
Rows, Cols, Treats
The ANOVA table produced by SPSS
Analysis
From the table is obtained sig of fishermen, week and machine.
Fishermen (Rows) : sig. = 0.000
week (columns) : sig. = 0.007
Machine(treatments) : sig. = 0.001

significance level = 0.05



H
0
: there are no significantly different among any of the fishermen
(1 = 2 = .. = n)
H
1
: at least , one fisherman is significantly different from the
others

If the significance obtained > 0.05 , H
0
is received
If the significance obtained < 0.05, H
0
is rejected

For fishermen: sig = 0.000
That means: 0.000 < 0.05, so H
0
is rejected (H
1
is received).

In other word: at least , one fisherman is significantly different from
the others

1) If the significance (Sig.) obtained > = 0.05,
so H
0
cant be rejected (in other words: H
0
is
received or there are no significant different
among any of the fishermen).
2) If the significance (Sig.) obtained < = 0.05,
so H
0
is rejected (in other words: H
1
is
received or at least, one fisherman is
significantly different from the others).

Decision Making for Fishermen (1)
1) See the value of fisherman (Sig. in Latin
Square Design table) = 0.000, in other words
(Sig.) = 0.000 < = 0.05. It means that H
0
is
rejected (H
1
is received).
2) Therefore, it can be concluded that at least,
one fisherman is significantly different from
the others. Its means that at least, there is
one fisherman which can affect to the cost.

Decision Making for Fishermen (2)

H
0
: there are no significantly different among any of the weeks (1
= 2 = .. = n)
H
1
: at least , one week is significantly different from the others

If the significance obtained > 0.05 , H
0
is received
If the significance obtained < 0.05, H
0
is rejected

For fishermen: sig = 0.007
That means: 0.007 < 0.05, so H
0
is rejected (H
1
is received).

In other word: at least , one week is significantly different from the
others

1) If the significance (Sig.) obtained > = 0.05,
so H
0
cant be rejected (in other words: H
0
is
received or there are no significant different
among any of the weeks).
2) If the significance (Sig.) obtained < = 0.05,
so H
0
is rejected (in other words: H
1
is
received or at least, one week is significantly
different from the others).

Decision Making for Weeks (1)
1) See the value of weeks (Sig. in Latin Square
Design table) = 0.007, in other words (Sig.) =
0.007 < = 0.05. It means that H
0
is rejected
(H
1
is received).
2) Therefore, it can be concluded that at least,
one week is significantly different from the
others. Its means that at least, there is one
week which can affect to the cost.

Decision Making for Weeks (2)

H
0
: there are no significantly different among any of the machines
(1 = 2 = .. = n)
H
1
: at least , one machine is significantly different from the others

If the significance obtained > 0.05 , H
0
is received
If the significance obtained < 0.05, H
0
is rejected

For Machines: sig = 0.001
That means: 0.001 < 0.05, so H
0
is rejected (H
1
is received).

In other word: at least , one machine is significantly different from
the others

1) If the significance (Sig.) obtained > = 0.05,
so H
0
cant be rejected (in other words: H
0
is
received or there are no significant different
among any of the machines).
2) If the significance (Sig.) obtained < = 0.05,
so H
0
is rejected (in other words: H
1
is
received or at least, one machine is
significantly different from the others).

Decision Making for Machines (1)
1) See the value of weeks (Sig. in Latin Square
Design table) = 0.001, in other words (Sig.) =
0.001 < = 0.05. It means that H
0
is rejected
(H
1
is received).
2) Therefore, it can be concluded that at least,
one machine is significantly different from
the others. Its means that at least, there is
one machine which can affect to the cost.

Decision Making for Machines (2)
Post Hoc Test Options Button
Multiple Comparisons
Fishermen
Cost
Tukey HSD
Tuke
(I) Fishermen (J) Fishermen
Mean Difference (I-
J) Std. Error Sig.
95% Confidence Interval
Lower Bound Upper Bound
1 2 .0000 .77526 1.000 -2.4711 2.4711
3 -2.2000 .77526 .090 -4.6711 .2711
4 -3.8000
*
.77526 .003 -6.2711 -1.3289
5 -4.0000
*
.77526 .002 -6.4711 -1.5289
2 1 .0000 .77526 1.000 -2.4711 2.4711
3 -2.2000 .77526 .090 -4.6711 .2711
4 -3.8000
*
.77526 .003 -6.2711 -1.3289
5 -4.0000
*
.77526 .002 -6.4711 -1.5289
3 1 2.2000 .77526 .090 -.2711 4.6711
2 2.2000 .77526 .090 -.2711 4.6711
4 -1.6000 .77526 .295 -4.0711 .8711
5 -1.8000 .77526 .204 -4.2711 .6711
4 1 3.8000
*
.77526 .003 1.3289 6.2711
2 3.8000
*
.77526 .003 1.3289 6.2711
3 1.6000 .77526 .295 -.8711 4.0711
5 -.2000 .77526 .999 -2.6711 2.2711
5 1 4.0000
*
.77526 .002 1.5289 6.4711
2 4.0000
*
.77526 .002 1.5289 6.4711
3 1.8000 .77526 .204 -.6711 4.2711
4 .2000 .77526 .999 -2.2711 2.6711
Based on observed means.
The error term is Mean Square(Error) = 1,503.
*. The mean difference is significant at the ,05 level.
Tukey Test for Fishermen
a) For bold values in Sig column from the output above can be seen
that the values less than 0.05. Its means that the fishermen have the
significant differences. For example: Fisherman 1 compared with
Fisherman 4, has significance value = 0.003. Its means that between
Fisherman 1 and Fisherman 4 have significant differences. As well as
Fisherman 1 compared with Fisherman 5, Fisherman 2 compared
with Fisherman 4, and Fisherman 2 compared with Fisherman 5 have
significant differences.
For light values in Sig column, Fishermen (1 and 2), (1 and 3), (2
and 3), (3 and 4), (3 and 5), and (4 and 5) dont have the significant
differences because the significance values more than 0.05.

Analyses (1)
Tukey HSD
Fishermen N
Subset
1 2
1
5 7.0000
2
5 7.0000
3 5 9.2000 9.2000
4
5 10.8000
5
5 11.0000
Sig.
.090 .204
Means for groups in homogeneous subsets are displayed.
Based on observed means.
The error term is Mean Square(Error) = 1,503.
Tukey Test for Fishermen in other form
1) From output above there are three subsets where subset 1
shows that between fishermen in subset 1 there are no
significant differences (Fisherman 1, Fisherman 2 and Fisherman
3 are no significantly different). As well as subset 2 (Fisherman 3,
Fisherman 4, and Fisherman 5 are no significantly different).
To analyze the difference, we only compare between subset 1
and subset 2. For example: Fisherman 1 and Fisherman 4 is
significantly different. As well as Fisherman (1 and 5), Fisherman
(2 and 4), Fisherman (2 and 5) are significantly different.
Conclusion: Actually, Table 5.3 and 5.4 are the same in terms of
analysis. Its only different from display the output.

Analyses (2)
Multiple Comparisons
Weeks
Cost Tukey HSD
(I) Weeks (J) Weeks Mean Difference (I-J) Std. Error Sig.
95% Confidence Interval
Lower Bound Upper Bound
1 2 -1.0000 .77526 .702 -3.4711 1.4711
3 -2.0000 .77526 .137 -4.4711 .4711
4 -3.0000
*
.77526 .016 -5.4711 -.5289
5 -4.0000
*
.77526 .002 -6.4711 -1.5289
2 1 1.0000 .77526 .702 -1.4711 3.4711
3 -1.0000 .77526 .702 -3.4711 1.4711
4 -2.0000 .77526 .137 -4.4711 .4711
5 -3.0000
*
.77526 .016 -5.4711 -.5289
3 1 2.0000 .77526 .137 -.4711 4.4711
2 1.0000 .77526 .702 -1.4711 3.4711
4 -1.0000 .77526 .702 -3.4711 1.4711
5 -2.0000 .77526 .137 -4.4711 .4711
4 1 3.0000
*
.77526 .016 .5289 5.4711
2 2.0000 .77526 .137 -.4711 4.4711
3 1.0000 .77526 .702 -1.4711 3.4711
5 -1.0000 .77526 .702 -3.4711 1.4711
5 1 4.0000
*
.77526 .002 1.5289 6.4711
2 3.0000
*
.77526 .016 .5289 5.4711
3 2.0000 .77526 .137 -.4711 4.4711
4 1.0000 .77526 .702 -1.4711 3.4711
Based on observed means.
The error term is Mean Square(Error) = 1,503.
*. The mean difference is significant at the ,05 level.
Tukey Test for Weeks
Tukey HSD
Weeks N
Subset
1 2 3
1
5 7.0000
2
5 8.0000 8.0000
3
5 9.0000 9.0000 9.0000
4
5 10.0000 10.0000
5
5 11.0000
Sig.
.137 .137 .137
Means for groups in homogeneous subsets are displayed.
Based on observed means.
The error term is Mean Square(Error) = 1,503.
Tukey Test for Weeks in other form
Multiple Comparisons
Machines
Cost
Tukey HSD
(I) Machines (J) Machines Mean Difference (I-J) Std. Error Sig.
95% Confidence Interval
Lower Bound Upper Bound
C D -.2667 .74225 .996 -2.6325 2.0992
F 1.5333 .74225 .295 -.8325 3.8992
P 2.7333
*
.74225 .021 .3675 5.0992
R 3.3333
*
.79124 .009 .8113 5.8554
D C .2667 .74225 .996 -2.0992 2.6325
F 1.8000 .77526 .204 -.6711 4.2711
P 3.0000
*
.77526 .016 .5289 5.4711
R 3.6000
*
.82229 .007 .9790 6.2210
F C -1.5333 .74225 .295 -3.8992 .8325
D -1.8000 .77526 .204 -4.2711 .6711
P 1.2000 .77526 .554 -1.2711 3.6711
R 1.8000 .82229 .247 -.8210 4.4210
P C -2.7333
*
.74225 .021 -5.0992 -.3675
D -3.0000
*
.77526 .016 -5.4711 -.5289
F -1.2000 .77526 .554 -3.6711 1.2711
R .6000 .82229 .945 -2.0210 3.2210
R C -3.3333
*
.79124 .009 -5.8554 -.8113
D -3.6000
*
.82229 .007 -6.2210 -.9790
F -1.8000 .82229 .247 -4.4210 .8210
P -.6000 .82229 .945 -3.2210 2.0210
Based on observed means.
The error term is Mean Square(Error) = 1,503.
*. The mean difference is significant at the ,05 level.
Tukey HSD
Machines


N


Subset
1 2
R
4 7.0000
P
5 7.6000
F
5 8.8000 8.8000
C
6 10.3333
D
5 10.6000
Sig.
.209 .209
Means for groups in homogeneous subsets are displayed.
Based on observed means.
The error term is Mean Square(Error) = 1,503.
Tukey Test for Machines in other form
Example 2:
Latin Square Design
we are again interested in how weight gain (Y) in rats is
affected by Source of Protein (Beef, Cereal, and Chicken)
and by Level of Protein (High or Low). There are a total of
treatment = 3 X 2 = 6 treatment combinations of the two
factors.
Beef -High Protein
Cereal-High Protein
Chicken-High Protein
Beef -Low Protein
Cereal-Low Protein
Chicken-Low Protein

Example 2: Protein Sources
Latin Square Design
Six Initial Weight categories are identified for the test
animals in addition to Six Appetite categories.
A test animal is then selected from each of the 6 X 6 = 36
combinations of Initial Weight and Appetite categories.
A Latin square is then used to assign the 6 diets to the 36
test animals in the study.
A: represents the high protein-cereal diet
B: represents the high protein-chicken diet
C: represents the low protein-beef diet
D: represents the low protein-cereal diet
E: represents the low protein-chicken diet
F: represents the high protein-beef diet.


The weight gain after a fixed period is
measured for each of the test animals
Appetite Category
1 2 3 4 5 6
1 62.1 84.3 61.5 66.3 73.0 104.7
A B C D E F
2 86.2 91.9 69.2 64.5 80.8 83.9
B F D C A E
Initial 3 63.9 71.1 69.6 90.4 100.7 93.2
Weight C D E F B A
Category 4 68.9 77.2 97.3 72.1 81.7 114.7
D A F E C B
5 73.8 73.3 78.6 101.9 111.5 95.3
E C A B F D
6 101.8 83.8 110.6 87.9 93.5 103.8
F E B A D C

Entering Data to SPSS
Univariate Dialog Box
Latin Square ANOVA
Tests of Between-Subjects Effects
Dependent Variable:weight
Source
Type III Sum of
Squares df Mean Square F Sig.
Model
265702.680
a
16 16606.418 5.221E3 .000
row
1767.083 5 353.417 111.102 .000
column
2195.433 5 439.087 138.034 .000
treatment
4183.913 5 836.783 263.056 .000
Error
63.620 20 3.181
Total
265766.300 36
a. R Squared = 1,000 (Adjusted R Squared = 1,000)
Significance Analysis of Rows
Hypothesis
Assumption:

H
0
= there are no significant different among any of
the rows

H
1
= at least, one row is significantly different from
the others.
n
= = = ...
2 1
1) If the significance (Sig.) obtained > = 0.05,
so H
0
cant be rejected (in other words: H
0
is
received or there are no significant different
among any of the rows).
2) If the significance (Sig.) obtained < = 0.05,
so H
0
is rejected (in other words: H
1
is
received or at least, one row is significantly
different from the others).

Decision Making for Rows (1)
1) See the value of rows (Sig. in Latin Square
Design table) = 0.000, in other words (Sig.) =
0.001 < = 0.05. It means that H
0
is rejected
(H
1
is received).
2) Therefore, it can be concluded that at least,
one row is significantly different from the
others. Its means that at least, there is one
row (initial weight category) which can affect
to the weight gain of animals.

Decision Making for Rows (2)
Significance Analysis of Columns
Hypothesis
Assumption:

H
0
= there are no significant different among any of
the columns

H
1
= at least, one column is significantly different
from the others.
n
= = = ...
2 1
1) If the significance (Sig.) obtained > = 0.05,
so H
0
cant be rejected (in other words: H
0
is
received or there are no significant different
among any of the columns).
2) If the significance (Sig.) obtained < = 0.05,
so H
0
is rejected (in other words: H
1
is
received or at least, one column is
significantly different from the others).

Decision Making for Columns (1)
1) See the value of columns (Sig. in Latin Square
Design table) = 0.000, in other words (Sig.) =
0.000 < = 0.01. It means that H
0
is rejected
(H
1
is received).
2) Therefore, it can be concluded that at least,
one column is significantly different from the
others. Its means that at least, there is one
column (appetite category) which can affect
to the weight gain of animals.

Decision Making for Columns (2)
Significance Analysis of Treatments
Hypothesis
Assumption:

H
0
= there are no significant different among any of
the treatments

H
1
= at least, one treatment is significantly different
from the others.
n
= = = ...
2 1
1) If the significance (Sig.) obtained > = 0.05,
so H
0
cant be rejected (in other words: H
0
is
received or there are no significant different
among any of the treatments).
2) If the significance (Sig.) obtained < = 0.05,
so H
0
is rejected (in other words: H
1
is
received or at least, one treatment is
significantly different from the others).

Decision Making for Treatments (1)
1) See the value of rows (Sig. in Latin Square
Design table) = 0.000, in other words (Sig.) =
0.001 < = 0.05. It means that H
0
is rejected
(H
1
is received).
2) Therefore, it can be concluded that at least,
one treatment is significantly different from
the others. Its means that at least, there is
one treatment (initial weight category) which
can affect to the weight gain of animals.

Decision Making for Treatments (1)
Post Hoc Test
1) To analyze post hoc test, we can follow
the steps as example before. For
Example 2, we can analyze post hoc
test for rows, columns, and treatments.
2) Post hoc tests are conducted when the
factors such rows, columns, or
treatments are significantly different.


Billahi Taufiq Walhidayah,
Wassalamualaikum Wr. Wb

Many thanks for your attention
See you in other occasions

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