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Various Mixture Separation Processes

Prepared by Mr. D. Scott, Celina High School

(The material in this outline came from various internet resources including, but not limited to Wikipedia.)

This outline is meant to be an overview or introduction and is not intended to be thoroughly
descriptive or instructional with regard to an particular separation process contained herein.

Adsorption is a process that occurs when a gas or liquid solute accumulates on the
surface of a solid or a liquid (adsorbent), forming a molecular or atomic film (the adsorbate). It is
different from absorption, in which a substance diffuses into a liquid or solid to form a solution. The
term sorption encompasses both processes, while desorption is the reverse process.
Activated carbon adsorption explained:
The best example of activated carbon adsorption explained as a process is in a wastewater
treatment plant where millions of gallons of polluted, unfiltered water enters into a system and must
be treated before being transported to nearby water systems. Activated carbon has an affinity for
various organics and is use for organic contaminant removal from water supplies. There are several
forms of activated carbon: powdered activated carbon (PAC) is ground loose carbon. Adsorption
isotherm tests are run to determine the effectiveness on the substance and its concentration in the
surrounding solution at equilibrium. Granular Activated Carbon (GAC) is typically used in drinking
water systems where liquid flows through stationary beds through the adsorpbent. In generic terms,
the carbon is heavier in weight than water and traps contaminants into its material.
Adsorption is indicative in most natural physical, biological, and chemical systems, and is widely
used in industrial applications such as activated charcoal, synthetic resins and water purification.
Adsorption, ion exchange and chromatography are sorption processes in which certain adsorptives
are selectively transferred from the fluid phase to the surface of insoluble, rigid particles suspended
in a vessel or packed in a column.

Centrifugation is a process that involves the use of the centripetal force for the
separation of mixtures, used in industry and in laboratory settings. More-dense components of the
mixture migrate away from the axis of the centrifuge, while less-dense components of the mixture
migrate towards the axis. In chemistry and biology, increasing the effective gravitational force on a
test tube so as to more rapidly and completely cause the precipitate ("pellet") to gather on the
bottom of the tube. The remaining solution is properly called the "supernate" or "supernatant liquid".
Since "supernatant" is an adjective, its usage alone is technically incorrect, although many
examples can be found in scientific literature. The supernatant liquid is then either quickly decanted
from the tube without disturbing the precipitate, or withdrawn with a Pasteur pipette.
Common Centrifugation Vocabulary and Formulas:

Pellet: hard-packed concentration of particles in a tube or rotor after centrifugation.

Supernatant: The clarified liquid above the pellet.
Adapter: A device used to fit smaller tubes or centrifugal devices in the rotor cavities.
RPM: Revolutions Per Minute (Speed).
Rmax: Maximum radius from the axis of rotation in centimeters.

Rmin: Minimum radius from the axis of rotation in centimeters.

RCF: Relative centrifugal Force. RCF = 11.17 x Rmax (RPM/1000)2
K-factor: Pelleting efficiency of a rotor. Smaller the K-factor, better the pelleting efficiency.

S-value: the sedimentation coefficient is a number that gives information about the molecular
weight and shape of the particle. S-value is expressed in Svedberg units. The larger the Svalue, the faster the particle separates.
Pelleting time: time taken to pellet a given particle. T = K/S where T= pellet time in hours. K
= K-factor of the rotor, and S = sedimentation coefficient.

These diagrams show change over time as the

mixture is spun.
Spinning in a centrifuge causes the effective g-force
(centripetal force) to act like an increase in gravity
that both enables and accelerates the settling
process. Mixtures that wont settle under normal
earth gravity can settle under greater gravitation-like

Chromatography is the collective term for a family of laboratory techniques for

the separation of mixtures. It involves passing a mixture of different dissolved substances in a
"mobile phase" through another material called a stationary phase, which separates the analyte to
be measured from other molecules in the mixture and allows it to be isolated.
Chromatography may be preparative or analytical. Preparative chromatography seeks to separate
the components of a mixture for further use (and is thus a form of purification). Analytical
chromatography normally operates with smaller amounts of material and seeks to measure the
relative proportions of analytes in a mixture. The two are not mutually exclusive.

Chromatography terms

The analyte is the substance that is to be separated during chromatography.

Analytical chromatography is used to determine the existence and possibly also the
concentration of analyte(s) in a sample.
A bonded phase is a stationary phase that is covalently bonded to the support particles or
to the inside wall of the column tubing.

A chromatogram is the visual output of the chromatograph. In the case of an optimal

separation, different peaks or patterns on the chromatogram correspond to different
components of the separated mixture.

Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example
obtained by a spectrophotometer, mass spectrometer or a variety of other detectors)
corresponding to the response created by the analytes exiting the system. In the case of an
optimal system the signal is proportional to the concentration of the specific analyte

A chromatograph is equipment that enables a sophisticated separation e.g. gas

chromatographic or liquid chromatographic separation.
Chromatography is a physical method of separation in which the components to be
separated are distributed between two phases, one of which is stationary (stationary phase)
while the other (the mobile phase) moves in a definite direction.
The effluent is the mobile phase leaving the column.
An immobilized phase is a stationary phase which is immobilized on the support particles,
or on the inner wall of the column tubing.
The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC
and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). A
better definition: The mobile phase consists of the sample being separated/analyzed and the
solvent that moves the sample through the column. In one case of HPLC the solvent consist
of a carbonate/bicarbonate solution and the sample is the anions being separated. The
mobile phase moves through the chromatography column (the stationary phase) where the
sample interacts with the stationary phase and is separated.
Preparative chromatography is used to purify sufficient quantities of a substance for further
use, rather than analysis.
The retention time is the characteristic time it takes for a particular analyte to pass through
the system (from the column inlet to the detector) under set conditions.
The sample is the matter analysed in chromatography. It may consist of a single component
or it may be a mixture of components. When the sample is treated in the course of an
analysis, the phase or the phases containing the analytes of interest is/are referred to as the
sample whereas everything out of interest separated from the sample before or in the course
of the analysis is referred to as waste.
The solute refers to the sample components in partition chromatography.
The solvent refers to any substance capable of solubilizing other substance, and especially
the liquid mobile phase in LC.
The stationary phase is the substance which is fixed in place for the chromatography
procedure. Examples include the silica layer in thin layer chromatography.

Types of Chromatography
Planar Chromatography

Original location
spot of the mixture.

Separated portions of the

mixture appear as bands
or spots on the surface
of the chromatography
substrate. The easiest
part of the mixture for the
solvent to carry is located
the farthest from the
original spot.

Thin layer chromatography is used as shown above to separate components of chlorophyll.

Planar chromatography is a separation technique in which the stationary phase is present as or on
a plane. The plane can be a paper, serving as such or impregnated by a substance as the
stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a
glass plate (thin layer chromatography).
Paper Chromatography
Paper chromatography is a technique that involves placing a small dot of sample solution onto a
strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent
and sealed. As the solvent rises through the paper it meets the sample mixture which starts to
travel up the paper with the solvent. Different compounds in the sample mixture travel different
distances according to how strongly they interact with the paper. This allows the calculation of an Rf
value and can be compared to standard compounds to aid in the identification of an unknown
Thin layer chromatography
Thin layer chromatography (TLC) is a widely-employed laboratory technique and is similar to paper
chromatography. However, instead of using a stationary phase of paper, it involves a stationary
phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate.
Compared to paper, it has the advantage of faster runs, better separations, and the choice between
different adsorbents. Different compounds in the sample mixture travel different distances
according to how strongly they interact with the adsorbent. This allows the calculation of an Rf value
and can be compared to standard compounds to aid in the identification of an unknown substance.

Column chromatography
As the mobile phase (solventlike liquid in this case) passes
downward through the
column, the different parts of
the mixture travel through at
different rates. The different
rates of travel result in a
separation of the mixture.

Column chromatography is a separation technique in which the stationary bed is within a tube. The
particles of the solid stationary phase or the support coated with a liquid stationary phase may fill
the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube
wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open
tubular column). Differences in rates of movement through the medium are calculated to different
retention times of the sample.
In 1978, W. C. Still introduced a modified version of column chromatography called flash column
chromatography (flash). The technique is very similar to the traditional column chromatography,
except for that the solvent is driven through the column by applying positive pressure. This allowed
most separations to be performed in less than 20 minutes, with improved separations compared to
the old method. Modern flash chromatography systems are sold as pre-packed plastic cartridges,
and the solvent is pumped through the cartridge. Systems may also be linked with detectors and
fraction collectors providing automation. The introduction of gradient pumps resulted in quicker
separations and less solvent usage.

Gas chromatography
Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a
separation technique in which the mobile phase is a gas. Gas chromatography is always carried out
in a column, which is typically "packed" or "capillary" .
Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid stationary
phase (often a liquid silicone-based material) and a mobile gas (most often Helium). The stationary
phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix
inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the
high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins
(heat will denature them), frequently encountered in biochemistry, it is well suited for use in the
petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively
in chemistry research.

Liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid
chromatography can be carried out either in a column or a plane. Present day liquid
chromatography that generally utilizes very small packing particles and a relatively high pressure is
referred to as high performance liquid chromatography (HPLC).
In the HPLC technique, the sample is forced through a column that is packed with irregularly or
spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile
phase) at high pressure. HPLC is historically divided into two different sub-classes based on the
polarity of the mobile and stationary phases. Technique in which the stationary phase is more polar
than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called
normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the
mobile phase and C18 = octadecylsilyl as the stationary phase) is called reversed phase liquid
chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is
therefore used considerably more.

Affinity chromatography
Affinity chromatography is based on selective non-covalent interaction between an analyte and
specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the
purification of proteins bound to tags. These fusion proteins are labelled with compounds such as
His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some
of these tags are usually removed and the pure protein is obtained.

Supercritical fluid chromatography

Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid
above and relatively close to its critical temperature and pressure.

Ion exchange chromatography

Ion exchange chromatography utilizes ion exchange mechanism to separate analytes. It is usually
performed in columns but the mechanism can be benefited also in planar mode. Ion exchange
chromatography uses a charged stationary phase to separate charged compounds including amino
acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange
resin that carries charged functional groups which interact with oppositely charged groups of the
compound to be retained. Ion exchange chromatography is commonly used to purify proteins using

Size exclusion chromatography

Size exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC)
or gel filtration chromatography and separates molecules according to their size (or more
accurately according to their hydrodynamic diameter or hydrodynamic volume). Smaller molecules
are able to enter the pores of the media and; therefore, take longer to elute, whereas larger
molecules are excluded from the pores and elute faster. It is generally a low resolution
chromatography technique and thus it is often reserved for the final, "polishing" step of a
purification. It is also useful for determining the structure of purified proteins.

Reversed-phase chromatography
Reversed-phase chromatography is an elution procedure used in liquid chromatography in which
the mobile phase is significantly more polar than the stationary phase.

Two-dimensional chromatography
In some cases, the chemistry within a given column can be insufficient to separate some analytes.
It is possible to direct a series of unresolved peaks onto a second column with different physicochemical (Chemical classification) properties. Since the mechanism of retention on this new solid
support is different from the first dimensional separation, it can be possible to separate compounds
that are indistinguishable by one-dimensional chromatography.

Fast protein liquid chromatography

Fast protein liquid chromatography (FPLC) is a term applied to several chromatography techniques
which are used to purify proteins. Many of these techniques are identical to those carried out under
high performance liquid chromatography.

Countercurrent chromatography
Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the
stationary and mobile phases are liquids. It involves mixing a solution of liquids, allowing them to
settle into layers and then separating the layers.

Chiral chromatography
Chiral Chromatography involves the separation of stereoisomers. In the case of enantiomers, these
have no chemical or physical differences apart from being three dimensional mirror images.
Conventional chromatography or other separation processes are incapable of separating them. To
enable chiral separations to take place, either the mobile phase or the stationary phase must
themselves be made chiral, giving differing affinities between the analytes. Chiral chromatography
HPLC columns (with a chiral stationary phase) in both normal and reversed phase are commercially

Crystallization is the (natural or artificial) process of formation of solid crystals from

a uniform solution. Crystallization is also a chemical solid-liquid separation technique, in which
mass transfer of a solute from the liquid solution to a pure solid crystalline phase occurs.
The crystallization process consists of two major events, nucleation and crystal growth.
Nucleation is the step where the solute molecules dispersed in the solvent start to gather into
clusters, on the nanometer scale (elevating solute concentration in a small region), that becomes
stable under the current operating conditions. These stable clusters constitute the nuclei. However
when the clusters are not stable, they redissolve. Therefore, the clusters need to reach a critical
size in order to become stable nuclei. Such critical size is dictated by the operating conditions
(temperature, supersaturation, etc.). It is at the stage of nucleation that the atoms arrange in a
defined and periodic manner that defines the crystal structure note that "crystal structure" is a
special term that refers to the relative arrangement of the atoms, not the macroscopic properties of
the crystal (size and shape), although those are a result of the internal crystal structure.

The crystal growth is the subsequent growth of the nuclei that succeed in achieving the critical
cluster size. Nucleation and growth continue to occur simultaneously while the supersaturation
exists. Supersaturation is the driving force of the crystallization, hence the rate of nucleation and
growth is driven by the existing supersaturation in the solution. Depending upon the conditions,
either nucleation or growth may be predominant over the other, and as a result, crystals with
different sizes and shapes are obtained (control of crystal size and shape constitutes one of the
main challenges in industrial manufacturing, such as for pharmaceuticals). Once the
supersaturation is exhausted, the solid-liquid system reaches equilibrium and the crystallization is
complete, unless the operating conditions are modified from equilibrium so as to supersaturate the
solution again.

Decantation is a process for the separation of mixtures, carefully pouring a solution

from a container, leaving the precipitate (sediments) in the bottom of the container. Usually a small
amount of solution must be left in the container, and care must be taken to prevent a small amount
of precipitate from flowing with the solution out of the container. It is generally used to separate a
liquid from an insoluble solid, e.g. in red wine, where the wine is decanted from the potassium
bitartrate crystals. Another example is when separating the oil from water after getting them as a
mixed liquid out of olives in order to extract olive oil.

Drying is a mass transfer process resulting in the removal of water moisture or moisture
from another solvent, by evaporation from a solid, semi-solid or liquid (hereafter product) to end in a
solid state. To achieve this, there must be a source of heat, and a sink of the vapor thus produced.
In the most common case, a gas stream, e.g., air, applies the heat by convection and carries away
the vapor as humidity. Other possibilities are vacuum drying, where heat is supplied by contact
conduction or radiation (or microwaves) while the produced vapor is removed by the vacuum
system. Another indirect technique is drum drying, where a heated surface is used to provide the
energy and aspirators draw the vapor outside the room.
Freeze drying or lyophilization is a drying method where the solvent is frozen prior to drying and is
then sublimed, i.e., passed to the gas phase directly from the solid phase, below the melting point
of the solvent. Freeze drying is often carried out under high vacuum to allow drying to proceed at a
reasonable rate. This process avoids collapse of the solid structure, leading to a low density, highly
porous product, able to regain the solvent quickly. In biological materials or foods, freeze drying is
regarded as one of the best if not the best method to retain the initial properties. It was first used
industrially to produce dehydrated vaccines, and to bring dehydrated blood to assist war casualties.
Now freeze drying is increasingly used to preserve some foods, especially for backpackers going to
remote areas. The method may keep protein quality intact, the same as the activity of vitamins and
bioactive compounds.
In turn, the mechanical extraction of the solvent, e.g., water, by centrifugation, is not considered
"drying". The ubiquitous term dehydration may mean drying of water-containing products as foods,
but its meaning is more vague, as it is also applied for water removal by osmotic drive from a salt or
sugar solution. In medicine, dehydration is the situation by which a person loses water by
respiration, sweating and evaporation and does not incorporate, for whatever reason, the "makeup" water required to keep the normal physiological behavior of the body.
Drying may be either a natural or an intentional process. The process of extreme drying is called

Distillation is a technique used to separate mixtures of liquids. It is a method of

separating chemical substances based on differences in their volatilities in a boiling liquid mixture.
Distillation usually forms part of a larger chemical process, and is thus referred to as a unit
Commercially, distillation has a number of uses. It is used to separate crude oil into more fractions
for specific uses such as transport, power generation and heating. Water is distilled to remove
impurities, such as salt from sea water. Air is distilled to separate its components - notably oxygen,
nitrogen and argon - for industrial use. Distillation of fermented solutions has been used since
ancient times to produce distilled beverages with a higher alcohol content.

Laboratory distillation set-up: 1: Heat source 2: Still pot 3: Still head 4: Thermometer/Boiling point
temperature 5: Condenser 6: Cooling water in 7: Cooling water out 8: Distillate/receiving flask 9:
Vacuum/gas inlet 10: Still receiver 11: Heat control 12: Stirrer speed control 13: Stirrer/heat plate
14: Heating (Oil/sand) bath 15: Stirrer bar/anti-bumping granules 16: Cooling bath.

Electrophoresis is a separation technique used with organic molecules, such as

protein. The proteins are placed in a gel. A voltage is applied and the molecules move through
the gel because they are charged. The gel restricts the motion so that different proteins will make
different amounts of progress in any given time.

Evaporation this separation technique removes a volatile liquid solvent from its
dissolved or suspended components.

Extraction Processes
Leaching is the process of extracting a substance from a solid by dissolving it in a liquid.
Brewing of tea and coffee can similarly be considered as leaching process.

Solvent extraction, also known as liquid-liquid extraction and partitioning, is a

method to separate compounds based on their relative solubilities in two different immiscible
liquids, usually water and an organic solvent. It is an extraction of a substance from one liquid
phase into another liquid phase. Liquid-liquid extraction is a basic technique in chemical
laboratories, where it is performed using a separatory funnel. This type of process is commonly
performed after a chemical reaction as part of the work-up.

Solid-phase extraction (SPE) is a separation process that is used to extract

compounds (called analyte) from a mixture of impurities. Analytical laboratories use solid phase
extraction to concentrate and purify samples for analysis. Solid phase extraction can be used to
isolate analytes of interest from a wide variety of matrices, including urine, blood, water samples,
beverages, soil, animal tissue, and consumer products.
The separation ability of solid phase extraction is based on the preferential affinity of desired or
undesired solutes in a liquid, mobile phase for a solid, stationary phase through which the sample is
passed. Impurities in the sample are either washed away while the analyte of interest is retained on
the stationary phase, or vice-versa. Analytes that are retained on the stationary phase can then be
eluted from the solid phase extraction cartridge with the appropriate solvent.

Flocculation is a process where a solute comes out of solution in the form of floc or
"flakes." The term is also used in colloid chemistry to refer to the process by which fine particulates
are caused to clump together into floc. The floc may then float to the top of the liquid, settle to the
bottom of the liquid, or can be readily filtered from the liquid. Flocculation and sedimentation are
widely employed in the purification of drinking water as well as sewage treatment, stormwater
treatment and treatment of other industrial wastewater streams.


Mesh, bag and paper filters are used to remove large particulates suspended in fluids, eg. fly
membrane processes including microfiltration, ultrafiltration, nanofiltration, reverse osmosis,
dialysis (biochemistry) utilising synthetic membranes can separate micrometre-sized or
smaller species.
Oil-water separation - gravimetric separator used to remove suspended oil droplets from
wastewaters in oil refineries, petrochemical and chemical plants, natural gas processing
plants and similar industries.

Some common every-day filters

Technique for folding filter paper:

Precipitation is the formation of a solid in a solution during a chemical reaction. When

the reaction occurs, the solid formed is called the precipitate, and the liquid remaining above the

solid is called the supernate.

Recrystallization is a procedure for purifying compounds. Typically the mixture of

"compound A" and "impurity B" are dissolved in the minimum amount of solvent to fully dissolve the
mixture i.e. a saturated solution. The solution is then allowed to cool. As the solution cools the
solubility of compounds in solution drops. This results in the desired compound dropping
(recrystallizing) from solution. The slower the rate of cooling, the bigger the crystals formed .
The crystallization process requires an initiation step. Once a small crystal has formed, more
crystals can grow from that crystal. Since "Compound A" is in excess this will usually result in these
crystals forming first and thus leaves a greater ratio of impurity in solution. Thus the resulting solid
is more pure than the original mixture.

Sedimentation describes the motion of molecules in solutions or particles in

suspensions in response to an external force such as gravity, centrifugal force or electric force.

Sieving In general, a sieve separates wanted/desired elements from unwanted material

using a tool such as a mesh, net or other filtration or distillation methods. The word "sift" derives
from this term. A strainer is a type of sieve typically associated with separating liquids from solids.

Sublimation is a technique used by chemists to purify compounds. Typically a solid is

placed in a vessel which is then heated under vacuum. Under this reduced pressure the solid
volatilizes and condenses as a purified compound on a cooled surface, leaving the non-volatile
residue impurities behind. This cooled surface often takes the form of a cold finger. Once heating
ceases and the vacuum is released, the purified compound can be collected from the cold surface.
Usually this is done using a sublimation apparatus.

Winnowing Wind winnowing is an agricultural method developed by ancient cultures for

separating grain from chaff. It is also used to remove weevils or other pests from stored grain.
In its simplest form it involves throwing the mixture into the air so that the wind blows away the
lighter chaff, while the heavier grains fall back down for recovery. Techniques included using a
winnowing fan (a shaped basket shaken to raise the chaff) or using a tool (a winnowing fork or
shovel) on a pile of harvested grain.