B y: Nuriya Khan
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Chromatography is used for the separation,
identification and measurement of the
chemical components in mixtures.
There are a variety of chromotagraphical
techniques.
All of them depend on the components of a
mixture being carried at different rates
through a stationary phase by a mobile
phase.
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Chemisorption
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Physical adsorption
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Desorption
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When a solute is added to a pair of immiscible
liquids it may dissolve in both of them. In this case
the solute will distribute itself between the two
solvents. It may well be more soluble in one
solvent. It may well be more soluble in one solvent
than the other. It is found that the ratio of the two
concentrations is constant:
[concentration of solute in solvent 1]
[concentration of solute in solvent 2]
=k
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In partition chromatography many extraction are
performed in succession in one operation. the
solutes are partitioned between the stationary
phase and the mobile phase.
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The stationary phase stays in place inside the
column or in the fibres of the paper. If the
stationary phase is packed into a column it usually
consists of solid particles or a viscous liquid
coated onto a solid surface.
The mobile phase, which is the solvent, moves
through the column or over the paper and is either
a liquid or a gas. It carries the components of the
analyte.
Picture1:A schematic representation of the process of a
chromatographic separation
Chromatographical Methods
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Liquid Chromatography
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Gas Chromatography
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Ion Exchange
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Gel permeation Chromatography
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The diagram on the following slide illustrates
separation of solutes in a solution by column
chromatography. The stationary phase is an inert
solid.
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A solution of the analyte is poured on to the top of
the column, and the components are adsorbed at
the top of the column. The mobile phase is a
second solvent called the eluant, which carries the
components of the mixture through the stationary
phase. This mixture is permitted to trickle through
the column.
Each solute is partitioned between the adsorbent
and the eluant. The least strongly adsorbed
solutes are desorbed first by the eluant and
carried further down the column before being
readsorbed.When fresh eluant reaches, the
process is repeated carrying it further down the
column.
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A solution of the mixture to be separated is applied
to a strip of chromatography paper. The solvents
used include water, ethanol, butanol.
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As the solvent rises through the paper it meets the
sample and the component bands spread out. The
separation is stopped when the solvent has
travelled nearly the top of the paper. The distance
travelled by the solvent front is measures. Then for
each solute the retardation factor Rf is calculated
by:
Solvent
front
Solute
Rf value=
Starting
Point
Picture 3: The Rf value
x/y
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The stationary phase is the water or other solvent
that is adsorbed as a film on the surface of the
paper. The mobile phase is the second solvent
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Another version of liquid chromatography is the
thin
layer
chromatography.
(TLC).
The
solid
adsorbent, e.g. silica gel or calcium sulphate, is
made into a thick paste with water and spread
evenly over a glass plate.
Picture 4: Apparatus setup of Thin Layer Chromatography
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The particle size of the stationary phase is smaller
in thin layer chromatography than in paper
chromatography. As a result the separations are
much more efficient and more reproducible. Often
separations can be achieved in a few centimetres,
and coated microscope slides are frequently used
for TLC.
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The mobile phase is a gas
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The liquid is spread on the surface of inert solid
particles which pack a long (5-10m) narrow (2-
10mm bore) column
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The injection chamber is 50-100 above the
temperature of the column
Picture 5: Apparatus set-up of Gas Chromatography
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Volatility of compound
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Polarity of compounds
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Column temperature
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Column packing polarity
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Flow rate of the gas through the column
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Length of the column
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In gas chromatography, the mobile phase is a gas.
The liquid which forms the stationary phase is
spread out on the surface of solid particles which
are packed into a column.
Each component is partitioned between the
vapour phase and the liquid phase.
A detector monitors the components as they leave
the column.
Picture 6: Thermal Conductivity Detector
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The ratio of the amount of solute in the stationary
phase, Nsp, compared to the amount of solute in
the mobile phase, Nmp:
k = Nsp/Nmp
The retention factor is most commonly used in
describing GC equilibria and is a strong function
of film thickness (k increases as film thickness
increases).
Picture 7: A graph showing Detector
Signal vs Time
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The thin-layer chromatography parameter that defines
the position of the analyse band on the plate:
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Rf=
(distance of the analyte band from initial spot)
(distance to the solvent front from the initial spot)
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The time required for a solute to travel from
injection to the detection for set instrument
conditions. the value for tr is designated as
occurring at the peak maximum.
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In
thin-layer
chromatography
a
visualization
reagent is used to make an analyte band appear on
a plate. The reagent used can be compound-or
class specific or general( sorbent contains a
fluorescent label that is quenched by the presence
of the analyte).
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Solvent Front is defined as the front line of the
eluent.
Where the eluent is defined as liquid or gas
entering a chromatographic bed and used to effect
a separation by elution.
Picture 8 : A demonstration of
solvent front
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First, each compound leaves the column in the
form of a symmetrical, bell-shaped band or peak.
Second, each band emerges from the column at a
characteristic time that can be used to identity the
compound, just as a melting point can be used for
the qualitative analysis of an organic compound.
This retention tR is measured from the time of
sample injection to the time the band maximum
leaves the column.
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A third characteristic feature is the difference in
retention times between adjacent bands.
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Fourthly, each band is characterized by a band
width tw, as shown for band B in the previous
diagram. Tangent are drawn to each side of the
band and extended to touch the baseline.
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The use of paper as a chromatographic medium is
usually regarded as a typical partition system,
The stationary phase is water, held by adsorption
on cellulose molecules.
Picture 9: Cellulose-Paper form
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Silica Gel is slightly odd in that, although it is
porous, and its pore size certainly influences its
performance as a stationary phase.
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It operates fundamentally as an adsorbent, not as
a molecular sieve.
Picture 10: Picture showing Silica Gel
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Alumina is a powerful adsorbent. It can hydrogen
bond through hydroxyl groups formed on its
surface by hydration, attract by dipole-dipole and
dipole-induced dipole attraction
Picture 11: Picture showing fine
alumina
Chemicals and other materials:
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silica gel 60 (Merck)
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petroleum ether
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acetone
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NaCl
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CaCO3
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Na2SO4
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fresh leaves
Apparatus and glass wares:
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glass chromatography column fitted with a fritted disk at the bottom and a stop cock at the
outlet
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separation funnel 500 mL
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separator funnel 100 mL
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powder funnel
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5 measuring cylinders 25 mL
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beaker 100 mL
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beaker 600 mL
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9 Erlenmeyer flask 100 mL
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volumetric pipette 20 mL
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pipette bulb
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mortar & pestle
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glass rod
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cork ring
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swan-neck lamp
Hazards and safety precautions:
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Petroleum ether is volatile and very flammable.
Petroleum ether presents a high fire risk. The
toxicity of petroleum ether varies according to its
composition. Many of the components are of quite
low toxicity, but some formulations may contain
chemicals that are suspected carcinogens. Avoid
ingestion and inhalation.
Acetone is highly flammable. Irritating to eyes.
Method
Extraction of the leaf pigments:
Using a pestle, fresh leaves are grinded in a mortar containing 22
mL of acetone, 3 mL of petrol ether and a spatula tip-ful of CaCO3.
The pigment extract is filtered. The filtrate is poured into a
separation funnel and is mixed with 20 mL of petrol ether and 20 mL
of 10% aqueous NaCl solution.
The separating funnel is shaken carefully. When the layers have
separated the lower layer is allowed to drain into a beaker. This
phase is thrown away. The upper layer is washed 3-4 times with 5
mL of dest water.
Afterwards the extract is placed in an Erlenmeyer flask and is dried
with about 4 spatula tips of Na2SO4. The liquid is carefully decanted
into a flask.
Picture 12: showing apparatus during experiment
Results and Discussion
The mobile phase slowly flows down through the silica gel
column by gravity leaving behind zones of colour - the
chromatogram. The theory of column chromatography is
analogous to that of thin-layer chromatography. The different
components in the sample mixture pass through the column
at different rates due to differences in their partioning
behaviour between the mobile liquid phase and the
stationary phase.
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Chemicals
Test solution:
a mixture of 7 dyes dissolved in water:
Erythrosine,
Brilliant Black BN, Fast Red E,
Naphthol Red S,
Yellow Orange S,
Ponceau 4R,
Tartrazine.
Reference solutions:
Yellow Orange S Brilliant Black, each dissolved in water.
Developing solvent: 2.5 % sodium citrate solution,
ammonia 25 %,
2-propanol (20 : 5 : 3)
The developing solvent must be freshly prepared.
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Apparatus and materials:
developing chamber (jam glass with a screw cover h = 11 cm, d = 5 cm)
Fertigfolie POLYGRAM® CEL 300 plate (Macherey Nagel)
glass capillaries (1 µL)
Hazards and safety precautions:
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Concentrated ammonia solution is extremely
damaging to eyes. Even contact with dilute
ammonia solution can lead to serious eye damage
Harmful if swallowed or inhaled and in contact
with skin.
2-Propanol is highly flammable.
Safety goggles and protective gloves required. The
developing solvent should be prepared in a
laboratory fume hood!
Method
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Using a soft pencil, a line is drawn approximately 1,5 cm
from the bottom of the plate.
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The spotting points are numbered (1,2,3)
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At the spotting points 1 and 3 the reference solutions are
applied onto the plate, at the spotting point 2 the dye
mixture.
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Using capillaries approx 0.25 µL of the dye solutions are
applied to the TLC plate.
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The capillaries fill themselves quickly when dipped into
organic sample solutions.
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Before emptying the submerged end of the capillary is
rolled horizontally on filter paper.
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The clean upper end of the capillary is placed on the layer vertically
and carefully, vertically so that the capillary empties itself and
carefully to avoid damage to the layer. Easy application of samples
is allowed with a spotting guide.
When the solvent is completely evaporated (approx. 10 min) from
the plate, the loaded TLC plate is carefully placed in the TLC
chamber with the sample line toward the bottom.
The plate whose top is leaned against the jar wall should sit on the
bottom of the chamber and be in contact with the solvent (solvent
surface must be below the extract line)
The TLC chamber is covered.
The TLC plate is allowed to remain undisturbed. When the solvent
front has reached three quarters of the length of the plate, the plate
is removed from the developing chamber and the position of the
solvent front is immediately marked.
The solvent on the plate is allowed to evaporate.
Picture 13: A demonstration of apparatus
during experiment
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Background:
Qualitative analysis of separated components in TLC is based on a
comparison of rates of migration. The retention factor, Rf value, is
used to characterize and compare components of various
samples.
The Rf value is defined as follows:
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In order to get reproducible Rf vakues the atmosphere in the
developing chamber must be saturated with the solvent. The
composition of the mobile phase and the temperature must remain
constant.
Apparatus
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water and rubbing alcohol
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coffee filter (or filter paper?
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water soluble food colors, water-soluble marking pens,
and/or Skittles™ candy and Q-Tips™
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clear glasses or other containers
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Method
Cut the coffee filters into strips about 1 “ wide.
Fill one glass about 1” full of water, the other about 1” full of rubbing
alcohol.
Toward the end of the filter strips, draw a line with a black watercolour
marking pen, or 2 or 3 primary colours of food colouring, or use a Q-Tip™ to
rub off colour from candies and rub onto the filter paper. (May take several
times to get a dense spot of colour.) Let them dry.
Place that end of the papers in the glasses (don’t let the dot of colour touch
the water or alcohol), and watch the water soak in and travel up the papers
(this is called capillary action, which is how water goes up a tree trunk into
the branches). As it does, it will dissolve the colours, which are carried up
the paper. The lighter ones will be carried higher than the others.
After the dots have been completely dissolved, remove the papers from the
liquid and allow them to dry. Observe the results, and compare the
differences between the two liquids.
Try variations, using different kinds of paper, different markers with different
colours, and adding vinegar to the water. Compare results
Picture 14: A demonstration of Paper
Chromatography
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1. What did you see? What colours were actually in the black ink?
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2. Which colours were carried furthest? (the lighter colours) Which
remained lowest? (the darker colours)
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3. Which colour is the lightest in weight? (those lightest in
colour) The heaviest in weight? (the darker colours)
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4. What pattern was there to the change?
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5. What is happening when the colours move up the paper? (the
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molecules of colour are being dissolved by the water and carried
with the water up the paper)
6. What causes the colours to separate? (the different colours have
different affinities for clinging to the paper, and those that cling
hardest to the cellulose in the paper will stop first, and those that
cling the weakest will travel further up the filter paper before
stopping)
7. Predict what might happen with different colours.
Try it again. Do you get the same results?
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These days (GC) is one of the primary analytical
techniques used in every forensic laboratory. GC is
widely used by forensic scientists – from analysis
of body fluids for the presence of illegal
substances, to testing of fibre and blood from a
crime scene, and to detect residue from
explosives. Yet scientists from Ohio University
explored another application of gas
chromatography with differential mobility
spectrometry as a low cost, onsite detection
method for ignitable liquids.
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Reverse Phase- High Performance Liquid Chromatography)
has been increasingly used to provide tens of grams to
kilograms of high purity material in pharmaceutical product
development. However, even with the development of Flash
Chromatography as an alternative, these purification
techniques are struggling to cope with the throughput
demands that the compounds being developed and requiring
purification are causing, primarily due to solubility issues.
By using high performance counter current chromatography
instruments, chemists are achieving high purity (>95%) and
high crude sample masses per injection at low solvent
usage (18 grams of sample injected per litre of solvent
usage). The reason for this is that chemists are able to use a
liquid stationary phase, which offers far superior loading
capacity and the advantage of loading the crude sample in
either the mobile or stationary phases or a mixture of the
two. These options help eliminate many, if not all solubility
issues.
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Residue analysis different environmental samples, such as water,
soil and plant matter, are examined for pesticide residues.
Analytical methods and special analytical equipment are employed
to provide an accurate identification of these organic
environmental pollutants and finally to determine their
concentration in µg dm-3of water or µg kg-1of soil or plant matter.
The substances examined, also known as analyses, have to be
extracted from the sample using an organic solvent or solvent
mixture in the first analytical step. At the same time, these analytes
must be available as authentic standard solutions for comparison
when determining the identity and concentration of the pesticide in
the sample. Since different organic solvents are also used here,
these are indispensable, versatile aids.
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www.chemsoc.org
www.Chemquide.co.uk
www.a-levelchemistry.co.uk
www.demochem..htm
A-Level Chemistry,Cheltenham:Nelson Thorne Limited,2000.
Advanced Chemistry, London: Oxford University Press,2000.
2006 U1 P1 Q8 (All Parts)
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8. a (i) This is the time required for a solute to travel from injection to detection for a set
of instrument conditions. The value for tr is designated as occurring at the peak max.
(ii) The mobile phase, which is the solvent, moves through the column or over the paper,
and is either a liquid or a gas. It carries the components of the analysis. The mobile phase
(solvent) moves through the column or over the paper. It carries the components of the
analysis.
(iii) Stationary phase: Polyethylene glycol acetate (PEG-5). Mobile Phase: Nitrogen gas
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b (i) Recall that :here the solute is the red dye
Sample 1
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Sample 2
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Note that the answer is given to 3 significant figures as the data is given to 3 significant
figures.
(ii) The same red dye is used to make both types of ink.
Reasons are
1. The Rf values is the same for both red spots in the 2 samples.
2. The both sample give spots of the same colours, suggesting that the dyes are the same.
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2003 U1 Q9 P1
a. The general principle is that components of a mixture are carried at different
rates through a stationary phase by a mobile phase.
b.The mobile phase, which is the solvent, moves through the column or over the
paper, and is either a liquid or a gas. It carries the components of the analysis.
(cape exam report 2003)
E.g.. Ethanol can be used as the mobile phase in paper chromatography.
c.The stationary phase stays in place inside the column or in the fibres of the paper.
If packed into a column, it usually consists of solid particles or a viscous liquid
onto a solid surface.
E.g.. Alumina can be used as the stationary phase in adsorption chromatography.
d(i) A is a mixture of 3 components, X, Y and Z where Y and Z are present in
approximately equal amounts and X is present in a much lower amount. X and Y
are easier to separate than Y and Z (using ΔT2) Band width shows that separation is
best for X.
(ii) Y was eluted before Z because it has a lower affinity for the stationary phase,
that is, it is not as strongly held by the stationary phase.