Extraction

• Extraction is the recovery of secondary metabolites from a

biomass (plants, animals, microorganism)

It is the first step to separate the desired natural products from

the raw materials.
Targets could be:

An unknown bioactive compound

A known compound present in an organism

A group of compounds within an organism that are structurally

related. (e.g. alkaloidal extract)

Identification of all secondary metabolites present in an

organism for chemical fingerprinting or metabolomics study
Types of extraction

Solvent

Non-solvent
 Solvent extraction
• the most widely used method.
Process:
(1) the solvent penetrates into the solid matrix;
(2) the solute dissolves in the solvents;
(3) the solute is diffused out of the solid matrix;
(4) the extracted solutes are collected

Any factor enhancing the diffusivity and solubility in the above steps will
facilitate the extraction.
Factors affecting extraction

Temperature, PH, Polarity, Viscosity of the solvent

Pretreatment of raw material – size, moisture content

Solvent-to-solute ratio

Extraction duration
• An ideal solvent:

1. Be highly selective for the compound/s to be extracted

2. Has a high capacity for extraction in terms of coefficient of

saturation of the compound in the medium

3. Does not react with the extracted compound or with other

compounds in the plant material

4. Has a low price

5. Be harmless to man and to the environment

6. Be easily removable from the extract

• Maceration/Percolation

• Sonication

• Decoction Classical methods

• Reflux

• Soxhlet extraction

• Pressurized solvent extraction

• Ultrasound assisted extraction

Modern methods
• Microwave assisted extraction

• Supercritical fluid extraction

• Maceration – is the soaking of plant material with a solvent for
a certain time (mostly 72hrs)

• Percolation - continuous process in which the saturated solvent

is constantly being replaced by fresh solvent.
• Decoction – Boiling of plant material with solvent

• Reflux – Boiling of plant material with a continuous fresh solvent

supply

• Soxhlet - method integrates the advantages of the refux extraction

and percolation, which utilizes the principle of refux and siphoning
to continuously extract the herb with fresh solvent
Disadvantage of classical extraction systems

Require a large volume of solvents and

Long extraction time

Lower yield
• Pressurized solvent extraction – use of high pressure in
extraction
High pressure keeps solvents in a liquid state above their boiling point
resulting in a high solubility and high diffusion rate of lipid solutes in the
solvent, and a high penetration of the solvent in the matrix.

• Ultrasound assisted extraction- uses ultrasonic wave energy in

the extraction
Two major factors that increase the efficiency of using ultrasound waves
are cell disruption and the effective mass trans
• Microwave assisted extraction - Microwaves generate heat by
interacting with polar compounds such as water and some organic
components in the plant matrix following the ionic conduction and
dipole rotation mechanisms.

The transfers of heat and mass are in the same direction in MAE, which
generates a synergistic effect to accelerate extraction and improve
extraction yield.
• Supercritical fluid extraction - uses supercritical fluid (SF) as the
extraction solvent.

SF has similar solubility to liquid and similar diffusivity to gas, and can
dissolve a wide variety of natural products.

• Supercritical carbon dioxide (S-CO2) was widely used in SFE because of its
attractive merits such as low critical temperature (31 °C), selectivity, inertness,
low cost, non-toxicity, and capability to extract thermally labile compounds.

Their solvating properties dramatically changed near their critical points

due to small pressure and temperature changes.
the highest
temperature and
pressure at which a
pure material can
exist in vapor/liquid
equilibrium.
 Non – solvent extraction
Mechanical extraction
• Expression

Distillation - refers to the selective boiling and

subsequent condensation of a component in a
liquid mixture.
• Hydro/steam distillation
Isolation and Separation of
active constituents
Crude extract - complex mixture of unknown components
obtained from the biomass
Separation of the mixture done by the differences in
physical or chemical properties
Classical Separation Methods
• Centrifugation - Centrifugation is a method of separating molecules having
different densities by spinning them in solution around an axis (in a
centrifuge rotor) at high speed.
separation of particles from a solution according to their size, shape,
density, viscosity of the medium and rotor speed.

• Distillation- refers to the selective boiling and subsequent condensation of

a component in a liquid mixture.
Separation based on difference in boiling points
• Crystallization - is the process of atoms or molecules arranging into a
well-defined, rigid crystal lattice in order to minimize their energetic state.
atoms link up in a regular structure.

• Solvent- solvent extraction -based on the relative solubility of the

solute in two different immiscible liquids – Partition coeficient

• Like dissolves like
 Chromatography
Chromatography = color writing

Chromatography basically involves the separation of mixtures due to

differences in the distribution coefficient (equilibrium distribution) of sample
components between two different phases which are immiscible.

One of these phases is a mobile phase and the other is a stationary phase.
• Stationary Phase - in some cases solid support it self form the
stationary phase , while in others , it absorbs liquid phase which
in turn acts as stationary phase. it is solid or liquid.

• Mobile Phase - phase which move over or through stationary

phase and carries sample along with it, thus resulting in the
separation of its component. it is either liquid or gas.
• Distribution Coefficient (Equilibrium Distribution ) is the ratio of
concentration of component in the stationary phase to its concentration in
mobile phase
• Different affinity of components to stationary phase causes the
separation.

• Due to differences in distribution coefficient of various components of a

mixture their mobility in the two phases differ, hence separation
 Types of chromatographic system

• Normal phase: polar stationary phase and a non-polar mobile

phase

• Reverse phase: non-polar stationary phase and a polar mobile

phase
Mechanisms of separation in
chromatography
1. Adsorption
2. Partition
3. Size exclusion
4. Ion-exchange
5. (Bio) affinity
Adsorption
Adsorption chromatography or solid-liquid chromatography is the oldest
form of chromatography.

The stationary phase (the adsorbent) is often a polar solid (almost

always silica or alumina), Mobile phase is a non - polar liquid

separation is based on the differences between the adsorption

affinities of the natural products for the surface of the adsorbents.

The selection of adsorbents (stationary phase) as well as the mobile

phase is crucial to achieve good separation of natural products, maximize
the recovery of target compounds and avoid the irreversible adsorption of
target compounds onto the adsorbents.
Partition
It involves two immiscible liquids, one as the mobile phase and the
other as the stationary phase (mostly fixed on a solid support).
The stationary liquid can either be fixed on the solid by physical
adsorption (LLC), which is easy, but leads to a continuous loss of
stationary phase, or by chemical bonds, which is now the most often
applied method.
separation is based on affinities towards the phases – partition
coeficient
It is suitable for a great number of substances in a wide polarity range.
Size exclusion
Also called gel filtration chromatography

Separation is based on molecular size of constituents

The stationary phase consists of swollen gel particles with a pore size that enables
the small molecules of a sample to penetrate into the pores, while medium size
molecules only partially enter the pores and the greatest molecules are completely
excluded from the pores.

used with a hydrophobic mobile phase, it is then also called gel permeation , and
with a hydrophilic mobile phase, then referred to as gel filtration .
Ion exchange
In this form of chromatography, the sorbent is usually a polymeric
resin, which contains charged groups and mobile counter ions
that may exchange with ions of a component as the mobile
phase migrates through the sorbent.

Separation is achieved by differences in the affinity between

ionic components and the stationary phase.

The technique is limited to mixtures that contain components

carrying a charge
(Bio) affinity
It exploits the unique biological specificity of the protein – ligand interaction

This concept is realized by binding the ligand to an insoluble support just as

in partition chromatography on chemically bonded phases .

The ligands may have low or high molecular weight and consist of nucleic
acids, enzymes or many other compounds.

Using the prepared substance as the column packing, certain proteins with
an appreciable affinity for the ligand will be retained.

These can be eluted by altering the composition or pH of the mobile phase

to favor dissociation and weaken the binding of the ligand.
Chromatographic
Techniques
Planar chromatography

• Paper Chromatography

• Thin layer chromatography

Thin Layer Chromatography (TLC)
• a chromatographic technique used to separate the components of
a mixture using a thin stationary phase supported by an inert
backing.

• TLC functions on the same principle as all chromatography: a

compound will have different affinities for the mobile and
stationary phases, and this affects the speed at which it migrates.
Analytical TLC is a fast, simple, and inexpensive analytical
technique used to determine or monitor:

- The number of components in a mixture.

- The identity of substances.

- The effectiveness of a purification.

- The appropriate conditions and effectiveness for a column

chromatographic separation.

- The progress of a reaction.

Preparative TLC

Sample is applied as a band (not spot)

To obtain fractions….not only to separate

One sample per plate

Stationary Phase

-Adsorbents coated on glass, aluminum, or plastic.

Glass - chemically inert and best withstand reactive stains and heat, but
are brittle and can be difficult to cut.
Aluminum - may not withstand strongly acidic or oxidizing stains
Plastic - does not withstand the high heat required to develop many
stains

• Common adsorbents are silica and alumina

The adsorption strength of compounds increases with increasing
polarity of functional groups, as shown below:

-CH=CH2, -X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH, -CONR2, -CO2H.
(weakly adsorbed) (strongly adsorbed)
(nonpolar) (more polar)

Silica gel is acidic- poor separation of basic molecules and deterioration

of acid-liable molecules
Alumina is basic –Poor separation of acidic molecules

In addition – alumina will not separate sample sizes as large as silica gel
would at a given layer thickness. Also, alumina is more chemically
reactive than silica gel and as a result, would require more care of
compounds and compound classes
Mobile phase
Proper solvent selection is perhaps the most important aspect of TLC
• keep in mind the chemical properties of the analytes.
• determining the best solvent may require a degree of trial and error
• How fast the compounds get carried up the plate depends on two
things:
• How soluble the compound is in the solvent. This will depend on
how much attraction there is between the molecules of the
compound and those of the solvent.
• How much the compound sticks to the stationary phase - the
silica gel, for example. This will depend on how much attraction
there is between the molecules of the compound and the silica
gel.
• Acids, bases, and strongly polar compounds often produce streaks rather
than spots in neutral solvents.
• Streaks make it difficult to calculate an \(R_f\) and may occlude other
spots.
• Adding a few percent of acetic or formic acid to the solvent can
correct streaking with acids.
• Adding a few percent triethylamine or ammonia can improve results
for bases.
• For polar compounds adding a few percent methanol can also
improve results.
• Elution Strength of Mobile Phase (

• Elution strength is generally considered to be equivalent to

polarity. A solvents elution strength depends on Intermolecular
Forces between the solvent and the analytes and between the
solvent and the stationary phase.

• A more polar (or more strongly eluting solvent) will move all of
the analytes to a greater extent, than a less polar, weakly elution
solvent.
• For example, the elution strength of hexane is very low;  = 0.01.
• the elution strength of ethyl acetate is higher;  = 0.45
• the elution strength of ethanol is even higher;  = 0.68
Spotting and Development
Cut the plate to the correct size and using a pencil (never ever use a pen),
gently draw a straight line across the plate approximately 1 cm from the
bottom.
• Do not use excessive forces when writing on a TLC plate as this will
remove the stationary phase.
Using capillary tubes, apply spots of analyte(s) to the line.
Place the plate into the chamber as evenly as possible and lean it against
the side.
• Never allow the bulk solvent to rise above the line you drew.
Allow capillary action to draw the solvent up the plate until it is
approximately 1 cm from the end.
Never allow the solvent to migrate all the way to the end of the plate.
Remove the plate and immediately draw a pencil line across the solvent
front.
Use techniques of visualization and circle the components shown with a
pencil.
Visualization
After plate development compounds could be visualized under UV-Visible light of
certain wavelengths (most commonly 254 and 366 nm).
• If fluorescent plates are used, a number of compounds can be seen by
illuminating the plate with short-wave UV.

Quenching causes dark spots on the surface of the plate.

For compounds which are not UV active, a number of chemical stains can be used.
• Iodine, Permanganate, ceric ammonium molybdate (CAM), and p-
anisaldehyde, Vanillin, ninhydrin….
•
• The Rf value can be used to identify compounds due to their
uniqueness to each compound.

• Rf values and reproducibility can be affected by a number of

different factors such as layer thickness, moisture on the TLC plate,
vessel saturation, temperature, depth of mobile phase, nature of
the TLC plate, sample size, and solvent parameters.
Rf values can be used to aid in the identification of a
substance by comparison to standards.

The Rf value is not a physical constant, and comparison

should be made only between spots on the same sheet,
run at the same time.

Two substances that have the same Rf value may be

identical; those with different Rf values are not identical.
Resolution

The separation between two analytes on a

chromatogram can be expressed as the resolution,
Rs and can be determined using the following equation:

Rs = (distance between center of spots)

(average diameter of spots)

In TLC, if the Rs value is greater than 1.0, the analytes are

considered to be resolved.
Improving Resolution:
For two closely migrating components, optimum resolutions are usually
obtained when the Rf’s of both compounds are between 0.2 and 0.5

* To Improve Rs, change the elution strength of the solvent to optimize Rf’s
change eonet (= in capacity factor), all compounds will be effected similarly.
Alter the composition of the solvent system so that the components affinity
for the mobile phase vs. the solid phase are differentially changed (=
change in selectivity).
Changing the chemical nature of the solvent system, such as changing
a hydrogen bonding solvent to a solvent which cannot hydrogen bond
to the analyte, is often the most effective.

** Improve Rs by decreasing the diameter of the analyte spots. This

can be achieved by applying smaller and less concentrated spots.
Troubleshooting
Overloading: overlapping of other component spots with similar
Rf
Uneven Advance of Solvent Front: no flat bottom, not enough
solvent, plate is not cut evenly
Streaking: spot is too concentrated(overloading), acidic or basic
sample
Spotting: sample spot will be washed off
Column chromatography
• method for separating mixtures of substances in which a liquid or
gaseous solution of the mixture is caused to flow through a tube packed
with a finely divided solid, which may be coated with an adsorbent liquid,
or through a long capillary tube bearing a thin film of adsorbent liquid

Open column chromatography

High performance liquid chromatography (HPLC)

Gas chromatography (GC)

Open column chromatography
Stationary Phase
Dry Vs wet Packing
Column Equilibration

1. Fill the column with the initial mobile phase, taking care to
ensure that the bed is not disturbed.

2. Open the column outlet to allow the solvent to flow naturally

under gravity.

3. Continue the equilibration stage until the stationary phase bed

has a uniform appearance, i.e., no visible dry areas or air pockets.

4. Reduce the solvent level to just above the stationary phase.

Stop the flow by closing the outlet valve.
Mobile phase
The process of washing a compound through a column using a solvent
is known as elution. The solvent is sometimes known as the eluent.

• Sample Elution:

Isocratic elution

Gradient elution
Sample Application

1. Dissolve the sample in the mobile phase. The volume (in milliliters) should
not exceed 0.4D2 (where D is the column diameter in centimeters).

2. Using a Pasteur pipet carefully apply the sample solution, taking care not to
disturb the bed.

3. Open the outlet valve, and allow the sample to flow into the bed. When the
sample has been adsorbed completely, the glass column sides can be washed
carefully with a little amount of the mobile phase and then allowed to flow
again. Care must be taken to ensure the bed is not disturbed and is not
allowed to become dry.

4. This stage can be repeated to ensure complete adsorption of the sample on

the bed.

5. Fill the column with the initial mobile phase required for the separation.
Column Development:
Gravity
Positive pressure (Flash Chromatography, FC))
Vacuum at the bottom (Vacuum Liquid Chromatography,
VLC)
Sample Elution and Fraction Collection

1. Allow the initial mobile phase to flow.

2. Collect the fractions. An automated sample fraction collector can be used or

fractions can be collected manually. For complex separations, small fractions can be
collected (for a 100-mL column, fractions of 5–10mL are ideal). For crude separations,
fractions of larger volumes can be collected for a particular solvent polarity.

3. A different polarity mobile phase is now loaded and samples collected as above.

4. Repeat as required for solvents of different polarity.

For a nonpolar extract, e.g., n-hexane extract, a typical step gradient sequence might be as follows:
initial composition of 100% n-hexane, followed by at least 1 column volume each of 5% EtOAc in n-
hexane, 10–100% EtOAc in n-hexane (increment of 10% in each step), and 5–20% MeOH in EtOAc
(increment of 5% in each step).
High performance liquid chromatography
• High performance liquid Chromatography (HPLC) is a modern
application of liquid chromatography

• guarantees a high sensitivity and reduced time

Gas Chromatography
• In gas chromatography (GC), the sample is vaporized and injected
onto the head of a chromatographic column. Elution is brought
about by the flow of an inert gaseous mobile phase.

• The mobile phase does not interact with molecule of the analyte;
its only function is to transport the analyte through the column.

• Gas-liquid chromatography is based upon the partition of the

analyte between a gaseous mobile phase and a liquid phase
immobilized on the surface of an inert solid.
Stationary phase
Desirable properties for the immobilized liquid phase in a gas-liquid chromatographic column
include:
(1)low volatility (ideally, the boiling point of the liquid should be
at 100 C higher than the maximum operating temperature for
o

the column);
(2)thermal stability;

(3)chemical inertness;

(4) solvent characteristics such that k` and  values for the solutes to be resolved fall within a
suitable range.

The retention time for a solute on a column depends upon its distribution constant which in
turn is related to the chemical nature of the stationary phase.
 Film Thickness
Commercial columns are available having stationary phases that vary
in thickness from 0.1 to 5m. Film thickness primarily affects the
retentive character and the capacity of a column.
Thick films are used with highly volatile analytes because such films
retain solutes for a longer time, thus providing a greater time for
separation to take place.
Thin films are useful for separating species of low volatility in a
reasonable length of time.
For most applications with 0.26- or 0.32-mm columns, a film thickness
of 0.26 m is recommended.
Chromatographic columns vary in length from less than 2 m to 50
m or more. They are constructed of stainless steel, glass, fused
silica, or Teflon.

In order to fit into an oven for thermostating, they are usually

formed as coils having diameters of 10 to 30 cm.

Two general types of columns:

packed and open tubular (capillary).

Packed columns

• Typically have lengths of 2 to 3 m and inside diameters of 2 to 4

mm.

• Tubes are densely packed with a uniform, finely divided packing

material, or solid support, that is coated with a thin layer (0.05 to
m)of the stationary liquid phase.
Open tubular (capillary) columns are of two basic types,namely:
1. wall—coated open tubular (WCOT)
2. support-coated open tubular (SCOT).

Wall-coated columns are simply capillary tubes coated with a thin layer
of the stationary phase.
In support-coated open tubular columns, the inner surface of the
capillary is lined with a thin film of a support material, such as
diatomaceous earth.
SCOT columns hold several times as much stationary phase compared
to WCOT columns and thus have a greater sample capacity.
Instrumentation
Carrier gas supply
Carrier gases, which must be chemically inert: helium, nitrogen,
and hydrogen.
Associated with the gas supply are:
pressure regulators, gauges, and flow meters.

In addition, the carrier gas system often contains a molecular

sieve to remove water or other impurities.
Sample Injection System
Column efficiency requires that the sample be of suitable size and be
introduced as a “plug” of vapor;
• slow injection of oversized samples causes band spreading and poor
resolution.
The most common method of sample injection involves the use of
microsyringe to inject a liquid or gaseous sample through a self-sealing,
silicone-rubber diaphragm or septum into a flash vaporizer port located at the
head of the column (the sample port is ordinarily
about 50 C above the boiling point of the least volatile component of
o

the sample).
For quantitative work, more reproducible sample sizes for both

liquids and gases are obtained by means of a rotary sample valve.

Errors due to sample size can be reduced to 0.5% to 2% relative.

The sampling loop is filled by injection of an excess of sample.

Rotation of the valve by 45 degree then introduces the

reproducible volume into the mobile phase.

Column Ovens
• Column temperature is an important variable that must be controlled to
a few tenths of a degree for precise work.
• Thus, the column is ordinarily housed in a thermostated oven.
• Optimum column temperature depends upon the boiling point of the
sample and the degree of separation required.
• Roughly, a temperature equal to or slightly above the average boiling
point of a sample results in a reasonable elution time (2 to 30 min).
• For samples with a broad boiling range, it is often desirable to employ
temperature programming, whereby the column temperature is increased
either continuously or in steps as the separation proceeds.
Detection Systems
Characteristics of the Ideal Detector: The ideal detector for gas
chromatography has the following characteristics:
1. Adequate sensitivity
2. Good stability and reproducibility.
3. A linear response to solutes that extends over several orders of
magnitude.
4. A temperature range from room temperature to at least 400oC.
5. A short response time that is independent of flow rate.
6. High reliability and ease of use.
7. Similarity in response toward all solutes or a highly selective
response toward one or more classes of solutes.
8. Nondestructive of sample.
Flame Ionization Detectors (FID)
The flame ionization detector is the most widely used and
generally applicable detector for gas chromatography.
• The effluent from the column is mixed with hydrogen and air
and then ignited electrically.
• Most organic compounds, when pyrolyzed at the temperature of
a hydrogen/air flame, produce ions and electrons that can
conduct electricity through the flame.
A potential of a few hundred volts is applied.

The resulting current (~10-12 A) is then measured.

The flame ionization detector exhibits a high sensitivity (~10-13

g/s), large linear response range (~107), and low noise.

A disadvantage of the flame ionization detector is that it is

destructive of sample.
 Thermal Conductivity Detectors(TCD)

A very early detector for gas chromatography, and one that still
finds wide application, is based upon changes in the thermal
conductivity of the gas stream brought about by the presence of
analyte molecules.
• The sensing element of TCD is an electrically heated element
whose temperature at constant electrical power depends upon the
thermal conductivity of the surrounding gas.
• The heated element may be a fine platinum, gold, or tungsten wire
or a semiconducting thermistor.
• The advantage of the thermal conductivity detector is its

simplicity, its large linear dynamic range(~10 ), its general

5

response to both organic and inorganic species, and its nondestructive

character, which permits collection of solutes after detection.

• A limitation of the katharometer is its relatively low sensitivity

(~10 g solute/mL carrier gas).

-8

• Other detectors exceed this sensitivity by factors as large as 10 4

to 10 .
7
Electron-Capture Detectors(ECD)
• The electron-capture detector has become one of the most widely
used detectors for environmental samples because this detector
selectivity detects halogen containing compounds, such as
pesticides and polychlorinated biphenyls.
• The effluent from the column is passed over a  emitter, usually
nickel-63. An electron from the emitter causes ionization of the
carrier gas and the production of a burst of electrons.
• In the absence of organic species, a constant standing current
between a pair of electrodes results from this ionization process.
The current decreases markedly, however, in the presence of those
organic molecules that tend to capture electrons.
• The electron-capture detector is selective in its response being
highly sensitive to molecules containing electronegative
functional groups such as halogens, peroxides, quinones, and
nitro groups.
• It is insensitive to functional groups such as amines, alcohols,
and hydrocarbons.
• An important application of the electron-capture detector has
been for the detection and determination of chlorinated
insecticides.
Thermionic Detectors (TID)
The thermionic detector is selective toward organic compounds
containing phosphorus and nitrogen.
Its response to a phosphorus atom is approximately ten times
greater than to a nitrogen atom and 104 to 106 larger than a
carbon atom.
Compared with the flame ionization detector, the thermionic detector
is approximately 500 times more sensitive to phosphorus-containing
compounds and 50 times more sensitive to nitrogen bearing species.
 Interfacing Gas Chromatography
with Spectroscopic Methods
Gas chromatography is often coupled with the selective
techniques of spectroscopy, thus giving so-called hyphenated
methods that provide the chemist with powerful tools for
identifying the components of complex mixtures.
Gas Chromatography/Mass
Spectrometry (GC/MS)
The flow rate from capillary columns is generally low enough
that the column output can be fed directly into the ionization
chamber of the mass spectrometer. For packed columns and
megabore capillary columns however, a jet separator must be
employed to remove most of the carrier gas from the analyte.
 The Retention Index
The retention index I was first proposed by Kovats for identifying
solutes from chromatograms.
The retention index for any given solute can be derived from a
chromatogram of a mixture of that solute with at least two normal
alkanes having retention times that bracket that of the solute.
That is, normal alkanes are the standards upon which the retention index
scale is based.
• The retention index for a normal alkane is equal to 100 times the
number of carbons in the compound regardless of the column packing,
the temperature, or other chromatographic conditions. Within a
homologous series, a plot of the logarithm of adjusted retention time
t`R (t`R = tR - t`M) versus the number of carbon atoms is linear.
 Qualitative Analysis
Gas chromatograms are widely used as criteria of purity for
organic compounds. Contaminants, if present, are revealed by
the appearance of additional peaks; the areas under these
peaks provide rough estimates of the extent of contamination.
The technique is also useful for evaluating the effectiveness of
purification procedures.
Retention indices should be useful for the identification of
components in mixtures. Gas chromatography provides an
excellent means of confirming the presence or absence of a
suspected compound in a mixture.
 Quantitative Analysis
The detector signal from a gas-liquid chromatographic column
has had wide use for quantitative and semiquantitative analyses.
An accuracy of 1% relative is attainable under carefully controlled
conditions.
Reliability is directly related to the control of variables; the nature
of the sample also plays a part in determining the potential
accuracy.