BIO – STATISTICS

RESOURCE PERSON: DR. MUHAMMAD RASHAD YOUNUS

AKHTAR SAEED COLLEGE OF PHARMACY

ANALYSIS OF VARIANCE (ANOVA)
ANOVA stands for Analysis of Variance, and it is a statistical technique used to analyze the differences
between means of three or more groups. It allows researchers to determine whether there are significant
differences among the means of the groups based on the variation in the data.
ANOVA is particularly useful when dealing with experiments or observational studies involving multiple
groups or factors. It helps researchers understand whether the variation observed in the data is primarily
due to differences between groups or whether it can be attributed to random chance.

The basic idea behind ANOVA is to compare the variance between group means (explained variance) with
the variance within each group (unexplained variance). If the explained variance is significantly larger than
the unexplained variance, it suggests that there are significant differences between the groups being studied.
There are different types of ANOVA depending on the study design:
(i) One-Way ANOVA: Used when there is one independent variable (factor) with two or more levels
(groups).
(ii) Two-Way ANOVA: Used when there are two independent variables (factors) and their interactions
on the dependent variable need to be analyzed.
(iii) N-Way ANOVA: Extends ANOVA to more than two independent variables.
(iv) Repeated Measures ANOVA: Used when the same subjects are measured under different conditions
or at different time points.
ANOVA provides valuable insights into the relationships between variables and helps researchers make
informed decisions about their data. It is commonly used in various fields, including social sciences,
biology, engineering, and market research, among others.
Applications of analysis of variance (ANOVA)
 Comparison of multiple treatment groups: ANOVA is extensively used when comparing means
among three or more treatment groups. It helps determine if there are significant differences in
outcomes across multiple interventions or treatments.
 Experimental designs: ANOVA is employed in various experimental designs, such as completely
randomized designs, randomized block designs, and factorial designs. It enables researchers to analyze
and understand the effects of different factors and their interactions on the outcome of interest.
 Quality control and process improvement: ANOVA is used in industries, including pharmaceuticals,
to analyze and improve manufacturing processes. It helps compare means and identify sources of
variation, contributing to quality control and process optimization.
 Observational studies: ANOVA can be utilized in observational studies to compare means across
different groups or populations. For example, it can be used to assess if there are significant differences
in disease outcomes or health behaviors among different demographic groups.
Multiple Comparison Test
ANOVA (Analysis of Variance) is a statistical technique used to analyze the differences among means of
two or more groups. It is typically employed when there are three or more groups to compare. ANOVA
helps determine whether there are significant differences between the means of these groups and if those
differences are due to random variability or actual group differences.
ANOVA tests the null hypothesis that all group means are equal, against the alternative hypothesis that at
least one group mean is different from the others. If the p-value associated with the ANOVA test is below
a predetermined significance level (e.g., 0.05), it suggests that there is evidence to reject the null hypothesis
and conclude that there are significant differences among the group means.
If the ANOVA test indicates significant differences among the groups, a Multiple Comparison Test (also
known as post hoc test or pairwise comparison test) can be performed to determine which specific groups
differ from each other. Multiple Comparison Tests allow for a more detailed examination of the group
differences after the overall ANOVA result.
There are several types of Multiple Comparison Tests, including:
 In ANOVA, we compare the means of multiple groups to determine if there are significant differences
between them. If the ANOVA test yields a significant result, indicating that at least one group mean is
significantly different from the others, we can then perform post hoc tests to determine which specific
group means are significantly different from each other.
 Tukey's Honestly Significant Difference (Tukey's HSD): This test compares all possible pairs of
means and provides a confidence interval for the difference between each pair. It controls the overall
family-wise error rate, meaning the probability of making a Type I error (rejecting the null hypothesis
when it is true) across all comparisons.
 Bonferroni correction: This test compares each pair of means individually, adjusting the significance
level to account for the increased probability of Type I errors due to multiple comparisons. The
significance level is divided by the number of pairwise comparisons being made.
 Scheffé's test: This test is more conservative than Tukey's HSD and can be used when the sample sizes
are unequal across groups. It controls the family-wise error rate, but at the cost of wider confidence
intervals and less power.
 Dunnett's test: This test compares each group mean with a control mean, rather than performing all
pairwise comparisons. It is useful when there is a control group and the interest lies in comparing other
groups to the control.

The choice of which Multiple Comparison Test to use depends on the specific research question, the nature
of the data, and any assumptions made about the data distribution. It is important to consider the
assumptions and limitations of each test and select the most appropriate one for the given situation.
Least Significance Difference Test (LSD)
The Least Significant Difference (LSD) test, also known as the Fisher's LSD test, is a post-hoc test used in
analysis of variance (ANOVA) to compare the means of different treatment groups. It is applied when a
significant difference is found through ANOVA, indicating that at least one group mean is significantly
different from the others. The LSD test helps identify which specific group means are significantly different
from each other.
Here's how the LSD test works:
 Calculate the LSD value: The LSD value is derived from the residual mean square (MSE) obtained
from the ANOVA. It is computed by multiplying the critical value of the t-statistic (obtained from the
t-distribution table) by the square root of MSE divided by the number of observations per group.
 Perform pairwise comparisons: Each pair of group means is compared by calculating the difference
between them. If the absolute difference between two means is greater than the LSD value, then the
two means are considered significantly different from each other.
 Interpret the results: Based on the comparisons, you can identify which specific groups have
significantly different means. Typically, means that differ by an amount greater than the LSD value are
considered statistically significant.
 The LSD test is straightforward and easy to interpret, but it is conservative and assumes homogeneity
of variances among the treatment groups. If the assumption of homogeneity of variances is violated,
alternative post-hoc tests such as Tukey's Honestly Significant Difference (HSD) or the Bonferroni test
may be more appropriate.
It's important to note that the LSD test is only conducted when the overall ANOVA result indicates a
significant difference among the treatment groups. If the ANOVA result is not significant, there is no need
to proceed with the LSD test as it is not meaningful to compare individual group means.
Procedure of one-way ANOVA
One-way Analysis of Variance (ANOVA) is a statistical method used to compare the means of three or
more groups to determine if there are significant differences between them. The procedure for conducting
a one-way ANOVA can be outlined as follows:

1. Formulate the hypotheses

− Null hypothesis (H0): There is no significant difference between the means of the groups.
− Alternative hypothesis (Ha): There is a significant difference between the means of the groups.
2. Collect Data
− Obtain data for each group you want to compare. Ensure that the data is continuous and
represents independent random samples from each group.
3. Calculate the necessary statistics
− Calculate the mean (average) of each group.
− Calculate the overall mean of all the data points combined.
4. Calculate the Sum of Squares Total (TSS)
− TSS measures the total variability in the data. It is the sum of squared differences between each
data point and the overall mean.
5. Calculate the Sum of Squares Between (BSS)
− BSS measures the variability between group means and the overall mean. It is the sum of squared
differences between each group mean and the overall mean, weighted by the number of
observations in each group.
6. Calculate the Sum of Squares Within (WSS)
− WSS measures the variability within each group. It is the sum of squared differences between
each data point and its respective group mean.
7. Calculate the degrees of freedom (df)
− df Total = Total number of observations – 1
− df Between = Number of groups – 1
− df Within = Total number of observations – Number of groups
8. Calculate the Mean Square Between (MSB)
− MSB = SSB / dfBetween
9. Calculate the Mean Square Within (MSW):
− MSW = SSW / dfWithin
10. Calculate the F-statistic:
− F = MSB / MSW
11. Determine the critical value and p-value:
− Use the F-distribution table or software to find the critical value for a given significance level
(alpha) and degrees of freedom.
− Alternatively, use software to calculate the p-value associated with the F-statistic.
12. Make a decision:
− If the F-statistic is greater than the critical value or the p-value is less than the chosen significance
level (usually 0.05), reject the null hypothesis (H0).
− If the F-statistic is less than the critical value or the p-value is greater than the significance level,
fail to reject the null hypothesis (H0).
13. Post hoc tests (if necessary):
− If the ANOVA results are significant, further investigate pairwise differences between groups
using post hoc tests (e.g., Tukey's HSD, Bonferroni, or Scheffe tests) to identify which groups
differ significantly.

Remember that ANOVA assumes equal variance among groups and normally distributed data. If these
assumptions are violated, alternative non-parametric tests might be more appropriate.
Principles of Experimental Design
Principles of Experimental Design refer to the fundamental guidelines and considerations that researchers
follow when planning and conducting experiments to ensure the validity, reliability, and generalizability of
their findings. These principles are essential for drawing accurate conclusions and making meaningful
inferences from experimental data. Here are the key principles of experimental design:
Randomization
Random assignment of participants or subjects to different groups or conditions helps ensure that each
group is representative and minimizes the effects of confounding variables. This is critical for establishing
causal relationships between variables.
Control
Control over extraneous variables is crucial to isolate the effect of the independent variable. Experimental
design should include measures to minimize or eliminate the influence of variables other than the one being
studied.
Replication
Replicating the experiment with a larger sample size increases the reliability of the results. Repetition of
the experiment helps determine if the findings are consistent and not just due to chance.
Blocking
Blocking involves grouping similar subjects together to create homogeneous blocks. Within each block,
randomization is then used to assign participants to different treatment groups. This helps account for
variations and reduce error in experiments.
Balancing
Balancing ensures that an equal number of participants or subjects are assigned to each treatment group, or
that the assignment is balanced across conditions. This helps prevent bias and enhances the precision of the
results.
Counterbalancing
In experiments with multiple treatment conditions, counterbalancing involves systematically varying the
order in which participants receive the treatments. This helps control for order effects and ensures that the
sequence of treatments does not influence the results.
Blinding
Blinding, or masking, refers to keeping participants, researchers, or assessors unaware of the treatment
conditions during the experiment. This minimizes bias and the potential for placebo effects.
Control Group
In experiments with experimental and control groups, the control group serves as a baseline for comparison.
It receives no treatment or a standard treatment, allowing researchers to measure the effect of the
experimental intervention.
Measurement Validity and Reliability
Careful attention to the selection of measurement instruments and procedures is essential to ensure that the
data collected are valid and reliable, providing accurate representations of the variables under study.
Generalizability
Experimental design should consider the extent to which the findings can be generalized to the broader
population or real-world scenarios. It is crucial to strike a balance between internal validity (control over
extraneous factors) and external validity (generalizability of results).
Ethical Considerations
Researchers must adhere to ethical guidelines when designing and conducting experiments involving
human or animal participants. This includes obtaining informed consent, protecting participants' privacy
and well-being, and minimizing any potential harm.
By following these principles, researchers can enhance the rigor and validity of their experiments, thereby
increasing the reliability of their findings and contributing valuable knowledge to their respective fields of
study.
Completely Randomized Design (CRD)
Completely Randomized Design (CRD) is a basic and commonly used experimental design in which
subjects or experimental units are randomly assigned to different treatment groups. It is suitable when the
main objective is to compare the effects of one factor (independent variable) with multiple levels or
treatments on a dependent variable. CRD is characterized by its simplicity and ability to reduce bias due to
confounding variables, making it a straightforward approach to studying cause-and-effect relationships.
Here are the key features and considerations of Completely Randomized Design:
Randomization
Subjects or experimental units are randomly assigned to different treatment groups. This ensures that any
potential confounding variables are evenly distributed among the groups, reducing the risk of bias and
increasing the internal validity of the experiment.
Single Factor
CRD involves the manipulation of only one independent variable (factor) with two or more levels
(treatments). For example, if testing the effects of different fertilizers on plant growth, the type of fertilizer
would be the single factor with several treatment levels (e.g., no fertilizer, fertilizer A, fertilizer B).
Homogeneous Sample
To increase the validity of the results, the subjects or experimental units should be as similar as possible
(homogeneous) to minimize individual differences that could affect the dependent variable.
Control Group
CRD can include a control group that receives no treatment or a standard treatment, allowing for a baseline
comparison to evaluate the impact of the experimental treatments.
Replication
Replicating the experiment by using a sufficient number of subjects or experimental units in each treatment
group improves the precision and reliability of the results. Replication helps account for natural variability
and increases the ability to detect treatment effects.
Randomization Procedures
The random assignment of subjects to treatment groups can be achieved using various methods, such as
random number tables, random number generators, or statistical software.
Statistical Analysis
After data collection, the analysis of a CRD typically involves performing an analysis of variance
(ANOVA) to determine whether there are significant differences among the treatment groups. Post hoc
tests can then be conducted to identify specific group differences if the overall ANOVA result is significant.
Assumptions
The assumptions of the CRD and ANOVA must be met, such as normality of the data and homogeneity of
variances. Violations of these assumptions may require the use of alternative non-parametric tests.
Limitations
While CRD is a powerful design, it may not be suitable in situations where there are known sources of
variation that should be controlled or accounted for (e.g., blocking or repeated measures). In such cases,
more complex experimental designs like randomized block design or factorial designs may be preferred.

In summary, Completely Randomized Design is a fundamental experimental design that allows researchers
to efficiently investigate the effects of one factor with multiple levels on a dependent variable while
minimizing potential confounding variables. It is widely used in various fields of research due to its
simplicity and ability to provide reliable results.

Randomized Complete Block Design (RCBD)

Randomized Complete Block Design (RCBD) is a more advanced experimental design that incorporates
the principles of randomization and blocking to reduce variability and increase the precision of experiments.
It is particularly useful when there are known sources of variation or extraneous factors that can affect the
outcome, allowing researchers to control these factors more effectively. RCBD is commonly employed in
agricultural, industrial, and social sciences research, as well as in various other fields. Here are the key
features and considerations of Randomized Complete Block Design:
Blocking
RCBD involves dividing subjects or experimental units into homogeneous groups or blocks based on a
known source of variability that could influence the outcome. Each block represents a specific level of the
blocking factor. For example, if testing the effects of different fertilizers on plant growth, different fields
or plots could be considered as blocks.
Randomization within Blocks
Within each block, subjects or experimental units are randomly assigned to different treatment groups. This
randomization ensures that any potential confounding variables within each block are equally distributed
among the treatment groups, reducing the risk of bias and increasing the internal validity of the experiment.
Single Factor
Like Completely Randomized Design (CRD), RCBD typically involves the manipulation of only one
independent variable (factor) with two or more levels (treatments). The factor is applied to all blocks, and
the effect of treatments is evaluated within each block.
Control Group
As in CRD, RCBD can include a control group that receives no treatment or a standard treatment to provide
a baseline for comparison.
Replication
Replication of the experiment is essential in RCBD, just as in CRD. Sufficient replication within each
treatment group and within each block improves the precision and reliability of the results, enabling better
detection of treatment effects and variability.
Analysis of Variance (ANOVA)
After data collection, the analysis in RCBD typically involves performing a two-way ANOVA. The two
factors considered are the treatment factor (the main factor of interest) and the block factor (the blocking
variable). The ANOVA assesses whether there are significant differences among the treatment groups while
also considering the effect of blocking.
Randomization Procedures
Randomization procedures are used to assign subjects or experimental units to different treatment groups
within each block.
Statistical Assumptions
As with CRD, the assumptions of ANOVA should be met, including normality of data and homogeneity of
variances within treatment groups.
Benefit
RCBD allows for more efficient control of known sources of variation, making it a powerful design for
experiments with inherent variability. By isolating the effects of the treatments within each block, it
increases the precision of treatment comparisons.
Limitations
RCBD is most suitable when there are specific blocking factors that are believed to influence the outcome.
In some cases, RCBD might not be the best design if there are no identifiable blocking factors, and a simpler
design like CRD may be more appropriate.
In summary, Randomized Complete Block Design is a robust experimental design that combines
randomization and blocking to increase the validity and precision of experiments. It is well-suited for
situations where there are known sources of variation that can be controlled or accounted for, making it a
valuable tool in various research fields.