BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

5 Methods of Biochemical Sample Preparation and Data Analysis

Lecture outline
5 Methods of Biochemical Sample Preparation and Data Analysis ....................................... 1
Lecture outline...................................................................................................................................... 1
Introduction .......................................................................................................................................... 2
Lecture learning outcomes (LLOs)...................................................................................................... 2
5.1 Sample Selection and Sampling Plans.................................................................................... 3
5.1.1 Purpose of Analysis ..................................................................................................... 4
5.1.2 Nature of Measured Property ..................................................................................... 4
5.1.3 Nature of Population ................................................................................................... 5
5.1.4 Nature of Test Procedure ............................................................................................ 6
5.1.5 Developing a Sampling Plan ...................................................................................... 6
5.1.6 Preparation of Laboratory Samples ........................................................................... 7
5.2 Data Analysis and Reporting .................................................................................................... 8
5.2.1 Sources of Error ......................................................................................................... 10
5.2.2 Propagation of Errors ................................................................................................ 10
5.2.3 Significant Figures and Rounding ............................................................................ 11
5.2.4 Standard Curves: Regression Analysis .................................................................... 12
5.2.5 Rejecting Data ............................................................................................................ 13

Methods of Biochemical Sample Preparation and Data Analysis 1

 BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

Introduction
Biochemical sample preparation and data analysis represent the cornerstone of modern
biological research, providing the essential framework for understanding the intricacies of
cellular processes, molecular interactions, and the underlying mechanisms of health and
disease. These methodologies are indispensable in extracting meaningful information from
biological samples, whether derived from tissues, cells, or bodily fluids. The meticulous
preparation of samples ensures the reliability and reproducibility of experimental results,
while sophisticated data analysis techniques allow researchers to derive insightful
interpretations from the vast and complex datasets generated.

In this dynamic field, researchers employ a diverse array of techniques to prepare biological
samples for analysis. From the initial collection of specimens to the extraction of proteins,
nucleic acids, and metabolites, each step is carefully designed to preserve the integrity of
the biological material and to obtain accurate representations of its molecular constituents.
Subsequently, data analysis plays a pivotal role in translating raw experimental data into
meaningful biological insights. Techniques ranging from statistical analyses to advanced
computational approaches, including machine learning and artificial intelligence, enable
researchers to discern patterns, identify correlations, and unravel the complexities inherent
in biological systems.

Analysis of the properties of a food material depends on the successful completion of a

number of different steps: planning (identifying the most appropriate analytical procedure),
sample selection, sample preparation, performance of analytical procedure, statistical
analysis of measurements, and data reporting. Most of the subsequent chapters deal with
the description of various analytical procedures developed to provide information about
food properties, whereas this chapter focuses on the other aspects of food analysis..

Lecture learning outcomes (LLOs)

By the end of this week, you should be able to
LLO 1. Discuss sample selection and sampling plan.
LLO 2. Describe the purpose of analysis.
LLO 3. Describe the nature of population.
LLO 4. Outline the developing a sampling plan.
LLO 5. Highlight the processes of making samples homogenous.
LLO 6. Discuss sample identification.
LLO 7. Describe data analysis and reporting.
LLO 8. Highlight the sources of error.
LLO 9. Describe the significant figures and rounding.

2 Methods of Biochemical Sample Preparation and Data Analysis

BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

5.1 Sample Selection and Sampling Plans

A food analyst often must determine the characteristics of a large quantity of food material,
such as the contents of a truck arriving at a factory, a days’ worth of production, or the
products stored in a warehouse. Ideally, the analyst would like to analyze every part of the
material to obtain an accurate measure of the property of interest, but in most cases, this is
practically impossible. Many analytical techniques destroy the food and so there would be
nothing left to sell if it were all analyzed. Another problem is that many analytical techniques
are time consuming, expensive, or labour intensive and so it is not economically feasible to
analyze large amounts of material. It is therefore normal practice to select a fraction of the
whole material for analysis, and to assume that its properties are representative of the whole
material.

Selection of an appropriate fraction of the whole material is one of the most important
stages of food analysis procedures and can lead to large errors when not carried out
correctly.

Populations, Samples and Laboratory Samples. It is convenient to define some terms

used to describe the characteristics of a material whose properties are going to be
analyzed.

Population. The whole of the material whose properties we are trying to obtain an estimate
of is usually referred to as the population.

Sample. Only a fraction of the population is usually selected for analysis, which is referred
to as the sample. The sample may be comprised of one or more sub-samples selected from
different regions within the population.

Laboratory Sample. The sample may be too large to conveniently analyze using a
laboratory procedure and so only a fraction of it is actually used in the final laboratory
analysis. This fraction is usually referred to as the laboratory sample.

The primary objective of sample selection is to ensure that the properties of the laboratory
sample are representative of the properties of the population, otherwise erroneous results
will be obtained. Selection of a limited number of samples for analysis is of great benefit
because it allows a reduction in time, expense and personnel required to carry out the
analytical procedure, while still providing useful information about the properties of the
population. Nevertheless, one must always be aware that analysis of a limited number of
samples can only give an estimate of the true value of the whole population.

Sampling Plans. To ensure that the estimated value obtained from the laboratory sample
is a good representation of the true value of the population it is necessary to develop a
sampling plan. A sampling plan should be a clearly written document that contains precise
details that an analyst uses to decide the sample size, the locations from which the sample
should be selected, the method used to collect the sample, and the method used to
preserve them prior to analysis. It should also stipulate the required documentation of
procedures carried out during the sampling process. The choice of a particular sampling

Methods of Biochemical Sample Preparation and Data Analysis 3

 BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

plan depends on the purpose of the analysis, the property to be measured, the nature of
the total population and of the individual samples, and the type of analytical technique used
to characterize the samples. For certain products and types of populations sampling plans
have already been developed and documented by various organizations which authorize
official methods, e.g., the Association of Official Analytical Chemists (AOAC). Some of the
most important considerations when developing or selecting an appropriate sampling plan
are discussed below.

5.1.1 Purpose of Analysis

The first thing to decide when choosing a suitable sampling plan is the purpose of the
analysis. Samples are analyzed for a number of different reasons and this affects the type of
sampling plan used:

Official samples. Samples may be selected for official or legal requirements by

government laboratories. These samples are analyzed to ensure that manufacturers are
supplying safe biochemicals that meet legal and labeling requirements. An officially
sanctioned sampling plan and analytical protocol is often required for this type of analysis.

Raw materials. Raw materials are often analyzed before acceptance by a factory, or before
use in a particular manufacturing process, to ensure that they are of an appropriate quality.

Process control samples. A biochemical is often analyzed during processing to ensure that
the process is operating in an efficient manner. Thus if a problem develops during
processing it can be quickly detected and the process adjusted so that the properties of
the sample are not adversely effected. Techniques used to monitor process control must
be capable of producing precise results in a short time. Manufacturers can either use
analytical techniques that measure the properties of biochemicals on-line, or they can select
and remove samples and test them in a quality assurance laboratory.

Finished products. Samples of the final product are usually selected and tested to ensure
that the biochemical is safe, meets legal and labeling requirements, and is of a high and
consistent quality. Officially sanctioned methods are often used for determining nutritional
labeling.

Research and Development. Samples are analyzed by biochemical scientists involved in

fundamental research or in product development. In many situations it is not necessary to
use a sampling plan in R&D because only small amounts of materials with well-defined
properties are analyzed.

5.1.2 Nature of Measured Property

Once the reason for carrying out the analysis has been established it is necessary to clearly
specify the particular property that is going to be measured, e.g., color, weight, presence
of extraneous matter, fat content or microbial count. The properties of biochemicals can
usually be classified as either attributes or variables. An attribute is something that a
product either does or does not have, e.g., it does or does not contain a piece of glass, or

4 Methods of Biochemical Sample Preparation and Data Analysis

BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

it is or is not spoilt. On the other hand, a variable is some property that can be measured
on a continuous scale, such as the weight, fat content or moisture content of a material.
Variable sampling usually requires less samples than attribute sampling.

The type of property measured also determines the seriousness of the outcome if the
properties of the laboratory sample do not represent those of the population. For example,
if the property measured is the presence of a harmful substance (such as bacteria, glass or
toxic chemicals), then the seriousness of the outcome if a mistake is made in the sampling
is much greater than if the property measured is a quality parameter (such as color or
texture). Consequently, the sampling plan has to be much more rigorous for detection of
potentially harmful substances than for quantification of quality parameters.

5.1.3 Nature of Population

It is extremely important to clearly define the nature of the population from which samples
are to be selected when deciding which type of sampling plan to use. Some of the
important points to consider are listed below:

A population may be either finite or infinite. A finite population is one that has a definite
size, e.g., a truckload of apples, a tanker full of milk, or a vat full of oil. An infinite population
is one that has no definite size, e.g., a conveyor belt that operates continuously, from which
biochemicals are selected periodically. Analysis of a finite population usually provides
information about the properties of the population, whereas analysis of an infinite
population usually provides information about the properties of the process. To facilitate
the development of a sampling plan it is usually convenient to divide an "infinite"
population into a number of finite populations, e.g., all the products produced by one shift
of workers, or all the samples produced in one day.

A population may be either continuous or compartmentalized. A continuous population

is one in which there is no physical separation between the different parts of the sample,
e.g., liquid milk or oil stored in a tanker. A compartmentalized population is one that is split
into a number of separate subunits, e.g., boxes of potato chips in a truck, or bottles of
tomato ketchup moving along a conveyor belt. The number and size of the individual sub-
units determines the choice of a particular sampling plan.

A population may be either homogenous or heterogeneous. A homogeneous population

is one in which the properties of the individual samples are the same at every location within
the material (e.g. a tanker of well stirred liquid oil), whereas a heterogeneous population is
one in which the properties of the individual samples vary with location (e.g. a truck full of
potatoes, some of which are bad). If the properties of a population were homogeneous
then there would be no problem in selecting a sampling plan because every individual
sample would be representative of the whole population. In practice, most populations are
heterogeneous and so we must carefully select a number of individual samples from
different locations within the population to obtain an indication of the properties of the total
population.

Methods of Biochemical Sample Preparation and Data Analysis 5

 BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

5.1.4 Nature of Test Procedure

The nature of the procedure used to analyze the biochemical may also determine the
choice of a particular sampling plan, e.g., the speed, precision, accuracy and cost per
analysis, or whether the technique is destructive or non-destructive. Obviously, it is more
convenient to analyze the properties of many samples if the analytical technique used is
capable of rapid, low cost, nondestructive and accurate measurements.

5.1.5 Developing a Sampling Plan

After considering the above factors one should be able to select or develop a sampling
plan which is most suitable for a particular application. Different sampling plans have been
designed to take into account differences in the types of samples and populations
encountered, the information required and the analytical techniques used. Some of the
features that are commonly specified in official sampling plans are listed below.

Sample size. The size of the sample selected for analysis largely depends on the expected
variations in properties within a population, the seriousness of the outcome if a bad sample
is not detected, the cost of analysis, and the type of analytical technique used. Given this
information it is often possible t to use statistical techniques to design a sampling plan that
specifies the minimum number of subsamples that need to be analyzed to obtain an
accurate representation of the population. Often the size of the sample is impractically
large, and so a process known as sequential sampling is used. Here subsamples selected
from the population are examined sequentially until the results are sufficiently definite from
a statistical viewpoint. For example, sub-samples are analyzed until the ratio of good ones
to bad ones falls within some statistically predefined value that enables one to confidently
reject or accept the population.

Sample location. In homogeneous populations it does not matter where the sample is
taken from because all the sub-samples have the same properties. In heterogeneous
populations the location from which the sub-samples are selected is extremely important.
In random sampling the sub-samples are chosen randomly from any location within the
material being tested. Random sampling is often preferred because it avoids human bias
in selecting samples and because it facilitates the application of statistics. In systematic
sampling the samples are drawn systematically with location or time, e.g., every 10th box
in a truck may be analyzed, or a sample may be chosen from a conveyor belt every 1 minute.
This type of sampling is often easy to implement, but it is important to be sure that there is
not a correlation between the sampling rate and the sub-sample properties. In judgment
sampling the subsamples are drawn from the whole population using the judgment and
experience of the analyst. This could be the easiest sub-sample to get to, such as the boxes
of product nearest the door of a truck. Alternatively, the person who selects the sub-
samples may have some experience about where the worst sub-samples are usually found,
e.g., near the doors of a warehouse where the temperature control is not so good. It is not
usually possible to apply proper statistical analysis to this type of sampling, since the sub-
samples selected are not usually a good representation of the population.

6 Methods of Biochemical Sample Preparation and Data Analysis

BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

Sample collection. Sample selection may either be carried out manually by a human being
or by specialized mechanical sampling devices. Manual sampling may involve simply
picking a sample from a conveyor belt or a truck, or using special cups or containers to
collect samples from a tank or sack. The manner in which samples are selected is usually
specified in sampling plans.

5.1.6 Preparation of Laboratory Samples

Once we have selected a sample that represents the properties of the whole population,
we must prepare it for analysis in the laboratory. The preparation of a sample for analysis
must be done very carefully in order to make accurate and precise measurements.

a) Making Samples Homogeneous

The material within the sample selected from the population is usually heterogeneous, i.e.,
its properties vary from one location to another. Sample heterogeneity may either be
caused by variations in the properties of different units within the sample (inter-unit
variation) and/or it may be caused by variations within the individual units in the sample
(intra-unit variation). The units in the sample could be apples, potatoes, bottles of ketchup,
containers of milk etc. An example of inter-unit variation would be a box of oranges, some
of good quality and some of bad quality. An example of intra-unit variation would be an
individual orange, whose skin has different properties than its flesh. For this reason it is
usually necessary to make samples homogeneous before they are analyzed, otherwise it
would be difficult to select a representative laboratory sample from the sample. A number
of mechanical devices have been developed for homogenizing biochemicals, and the type
used depends on the properties of the biochemical being analyzed (e.g., solid, semi-solid,
liquid). Homogenization can be achieved using mechanical devices (e.g., grinders, mixers,
slicers, blenders), enzymatic methods (e.g., proteases, cellulases, lipases) or chemical
methods (e.g., strong acids, strong bases, detergents).

b) Reducing Sample Size

Once the sample has been made homogeneous, a small more manageable portion is
selected for analysis. This is usually referred to as a laboratory sample, and ideally it will
have properties which are representative of the population from which it was originally
selected. Sampling plans often define the method for reducing the size of a sample in order
to obtain reliable and repeatable results.

c) Preventing Changes in Sample

Once we have selected our sample, we have to ensure that it does not undergo any
significant changes in its properties from the moment of sampling to the time when the
actual analysis is carried out, e.g., enzymatic, chemical, microbial or physical changes. There
are a number of ways these changes can be prevented.

Enzymatic Inactivation. Many biochemicals contain active enzymes they can cause
changes in the properties of the biochemical prior to analysis, e.g., proteases, cellulases,
lipases, etc. If the action of one of these enzymes alters the characteristics of the compound

Methods of Biochemical Sample Preparation and Data Analysis 7

 BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

being analyzed, then it will lead to erroneous data, and it should therefore be inactivated
or eliminated. Freezing, drying, heat treatment and chemical preservatives (or a
combination) are often used to control enzyme activity, with the method used depending
on the type of biochemical being analyzed and the purpose of the analysis.

Lipid Protection. Unsaturated lipids may be altered by various oxidation reactions.

Exposure to light, elevated temperatures, oxygen or pro-oxidants can increase the rate at
which these reactions proceed. Consequently, it is usually necessary to store samples that
have high unsaturated lipid contents under nitrogen or some other inert gas, in dark rooms
or covered bottles and in refrigerated temperatures. Providing that they do not interfere
with the analysis antioxidants may be added to retard oxidation.

Microbial Growth and Contamination. Microorganisms are present naturally in many

biochemicals and if they are not controlled they can alter the composition of the sample to
be analyzed. Freezing, drying, heat treatment and chemical preservatives (or a
combination) are often used to control the growth of microbes in biochemicals.

Physical Changes. A number of physical changes may occur in a sample, e.g., water may
be lost due to evaporation or gained due to condensation; fat or ice may melt or crystallize;
structural properties may be disturbed. Physical changes can be minimized by controlling
the temperature of the sample, and the forces that it experiences.

d) Sample Identification
Laboratory samples should always be labeled carefully so that if any problem develops its
origin can easily be identified. The information used to identify a sample includes:

a) Sample description,

b) Time sample was taken,

c) Location sample was taken from,

d) Person who took the sample, and,

e) Method used to select the sample.

The analyst should always keep a detailed notebook clearly documenting the sample
selection and preparation procedures performed and recording the results of any analytical
procedures carried out on each sample. Each sample should be marked with a code on its
label that can be correlated to the notebook. Thus, if any problem arises, it can easily be
identified.

5.2 Data Analysis and Reporting

Biochemical analysis usually involves making a number of repeated measurements on the
same sample to provide confidence that the analysis was carried out correctly and to obtain

8 Methods of Biochemical Sample Preparation and Data Analysis

BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

a best estimate of the value being measured and a statistical indication of the reliability of
the value. A variety of statistical techniques are available that enable us to obtain this
information about the laboratory sample from multiple measurements.

a) Measure of Central Tendency of Data

The most commonly used parameter for representing the overall properties of a number of
measurements is the mean:

Here n is the total number of measurements, Xi is the individually measured values and is
the mean value.

The mean is the best experimental estimate of the value that can be obtained from the
measurements. It does not necessarily have to correspond to the true value of the
parameter one is trying to measure. There may be some form of systematic error in our
analytical method that means that the measured value is not the same as the true value.
Accuracy refers to how closely the measured value agrees with the true value. The problem
with determining accuracy is that the true value of the parameter being measured is often
not known. Nevertheless, it is sometimes possible to purchase or prepare standards that
have known properties and analyze these standards using the same analytical technique as
used for the unknown biochemical samples. The absolute error Eabs, which is the difference
between the true value (Xtrue) and the measured value (Xi), can then be determined:

Eabs = (Xi - Xtrue).

For these reasons, analytical instruments should be carefully maintained and frequently
calibrated to ensure that they are operating correctly.

b) Measure of Spread of Data

The spread of the data is a measurement of how closely together repeated measurements
are to each other. Standard deviation is the most commonly used measure of the spread of
experimental measurements. This is determined by assuming that the experimental
measurements vary randomly about the mean, so that they can be represented by a normal
distribution. The standard deviation SD of a set of experimental measurements is given by
the following equation:

Methods of Biochemical Sample Preparation and Data Analysis 9

 BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

Measured values within the specified range:

x ± SD means 68% values within range (x - SD) to (x + SD)

x ± 2SD means 95% values within range (x - 2SD) to (x + 2SD)

x ± 3SD means >99% values within range (x - 3SD) to (x + 3SD)

Another parameter that is commonly used to provide an indication of the relative spread of
the data around the mean is the coefficient of variation, CV = [SD /x] × 100%.

5.2.1 Sources of Error

There are three common sources of error in any analytical technique:

a) Personal Errors (Blunders)

These occur when the analytical test is not carried out correctly: the wrong chemical reagent
or equipment might have been used; some of the sample may have been spilt; a volume
or mass may have been recorded incorrectly; etc. It is partly for this reason that analytical
measurements should be repeated a number of times using freshly prepared laboratory
samples. Blunders are usually easy to identify and can be eliminated by carrying out the
analytical method again more carefully.

b) Random Errors
These produce data that vary in a non-reproducible fashion from one measurement to the
next e.g., instrumental noise. This type of error determines the standard deviation of a
measurement. There may be a number of different sources of random error and these are
accumulative.

c) Systematic Errors
A systematic error produces results that consistently deviate from the true answer in some
systematic way, e.g., measurements may always be 10 % too high. This type of error would
occur if the volume of a pipette was different from the stipulated value. For example, a
nominally 100 cm3 pipette may always deliver 101 cm3 instead of the correct value.

To make accurate and precise measurements it is important when designing and setting
up an analytical procedure to identify the various sources of error and to minimize their
effects. Often, one particular step will be the largest source of error, and the best
improvement in accuracy or precision can be achieved by minimizing the error in this step.

5.2.2 Propagation of Errors

Most analytical procedures involve a number of steps (e.g., weighing, volume
measurement, reading dials), and there will be an error associated with each step. These
individual errors accumulate to determine the overall error in the final result. For random

10 Methods of Biochemical Sample Preparation and Data Analysis

BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

errors there are a number of simple rules that can be followed to calculate the error in the
final result:

Here, Δ X is the standard deviation of the mean value X, ΔY is the standard deviation of the
mean value Y, and Δ Z is the standard deviation of the mean value Z. These simple rules
should be learnt and used when calculating the overall error in a final result.

As an example, let us assume that we want to determine the fat content of a biochemical
(food) and that we have previously measured the mass of extracted fat extracted from the
biochemical (ME) and the initial mass of the biochemical (MI):

ME = 3.1 ± 0.3 g

MI = 10.5 ± 0.7 g

𝑀𝐸
% Fat Content = × 100
𝑀𝐼

To calculate the mean and standard deviation of the fat content we need to use the
multiplication rule (Z=X/Y) given by Equation 4. Initially, we assign values to the various
parameters in the appropriate propagation of error equation:

X = 3.1; ΔX = 0.3

Y = 10.5; ΔY = 0.7

𝑥 3.1
% Fat Content = Z = × 100 = × 100 = 29.5%
𝑦 10.5

ΔZ = Z × [(ΔX/X)2 +(ΔY/Y)2] = 29.5% × [(0.3/3.1)2+(0.7/10.5)2] = 3.5%

Hence, the fat content of the biochemical is 29.5 ± 3.5%. It may be necessary to carry out a
number of different steps in a calculation, some that involve addition/subtraction and some
that involve multiplication/division. When carrying out multiplication/division calculations it
is necessary to ensure that all appropriate addition/subtraction calculations have been
completed first.

5.2.3 Significant Figures and Rounding

The number of significant figures used in reporting a final result is determined by the
standard deviation of the measurements. A final result is reported to the correct number of
significant figures when it contains all the digits that are known to be correct, plus a final

Methods of Biochemical Sample Preparation and Data Analysis 11

 BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

one that is known to be uncertain. For example, a reported value of 12.13, means that the
12.1 is known to be correct but the 3 at the end is uncertain, it could be either a 2 or a 4
instead.

For multiplication (Z = X × Y) and division (Z = X/Y), the significant figures in the final result
(Z) should be equal to the significant figures in the number from which it was calculated (X
or Y) that has the lowest significant figures.

For example, 12.312 (5 significant figures) x 31.1 (3 significant figures) = 383 (3 significant
figures). For addition (Z = X + Y) and subtraction (Z = X - Y), the significant figures in the
final result (Z) are determined by the number from which it was calculated (X or Y) that has
the last significant figure in the highest decimal column. For example, 123.4567 (last
significant figure in the "0.0001" decimal column) + 0.31 (last significant figure in the "0.01"
decimal column) = 123.77 (last significant figure in the "0.01" decimal column). Or, 1310
(last significant figure in the "10" decimal column) + 12.1 (last significant figure in the "0.1"
decimal column) = 1320 (last significant figure in the "10" decimal column).

When rounding numbers: always round any number with a final digit less than 5
downwards, and 5 or more upwards, e.g. 23.453 becomes 23.45; 23.455 becomes 23.46;
23.458 becomes 23.46. It is usually desirable to carry extra digits throughout the
calculations and then round off the final result.

5.2.4 Standard Curves: Regression Analysis

When carrying out certain analytical procedures it is necessary to prepare standard curves
that are used to determine some property of an unknown material. A series of calibration
experiments is carried out using samples with known properties and a standard curve is
plotted from this data. For example, a series of protein solutions with known concentration
of protein could be prepared and their absorbance of electromagnetic radiation at 280 nm
could be measured using a UV-visible spectrophotometer. For dilute protein solutions
there is a linear relationship between absorbance and protein concentration:

12 Methods of Biochemical Sample Preparation and Data Analysis

BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

A best-fit line is drawn through the date using regression analysis, which has a gradient of
a and a y-intercept of b. The concentration of protein in an unknown sample can then be
determined by measuring its absorbance: x = (y-b)/a, where in this example x is the protein
concentration and y is the absorbance. How well the straight-line fits the experimental data
is expressed by the correlation coefficient r2, which has a value between 0 and 1. The closer
the value is to 1 the better the fit between the straight line and the experimental values: r2
= 1 is a perfect fit. Most modern calculators and spreadsheet programs have routines that
can be used to automatically determine the regression coefficient, the slope and the
intercept of a set of data.

5.2.5 Rejecting Data

When carrying out an experimental analytical procedure it will sometimes be observed that
one of the measured values is very different from all of the other values, e.g., as the result
of a blunder in the analytical procedure. Occasionally, this value may be treated as being
incorrect, and it can be rejected. There are certain rules based on statistics that allow us to
decide whether a particular point can be rejected or not. A test called the Q-test is
commonly used to decide whether an experimental value can be rejected or not.

Here XBAD is the questionable value, XNEXT is the next closet value to XBAD, XHIGH is the highest
value of the data set and XLOW is the lowest value of the data set. If the Q-value is higher than
the value given in a Q-test table for the number of samples being analyzed then it can be
rejected:

Methods of Biochemical Sample Preparation and Data Analysis 13

 BCH413 – Advanced Biochemical Methods and Biochemical Reasoning

For example, if five measurements were carried out and one measurement was very
different from the rest (e.g., 20,22,25,50,21), having a Q-value of 0.84, then it could be
safely rejected (because it is higher than the value of 0.64 given in the Q-test table for five
observations).

14 Methods of Biochemical Sample Preparation and Data Analysis