CHROMATOGRAPHY

DR. P. M. NGUMO
 Chromatography
• Its an analytical method widely used for the separation, identification &
determination of chemical components in complex mixtures like drugs.
• It is applied in a variety of systems & techniques.
• All these methods have in common the use of;

 Stationary phase (Sp)

 Mobile phase (Mp)

• The components of a mixture are carried through the stationary phase by the
flow of gaseous or liquid mobile phase.
• Separation is based on differences in migration rates among the sample
components. This gives the partition coefficient.
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Chromatography…cont’d
• Chromatography can either be;

a.Preparative
This is meant to separate the components of a mixture for
purification purposes. Thus useful in qualitative analysis.
b.Analytical
Normally done with small amounts of materials & is for measuring
the relative proportions of analytes in a mixture. Thus useful for
quantitative analysis.
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 Classification of
chromatography
• Based on;

a.The physical means by which the stationary phase & the mobile
phase come into contact with each other.
b.Upon whether the mobile phase is a liquid (liquid chromatography)
or a gas (gas chromatography).
c.Mechanisms of separation.

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 Based on physical means
a. Columnar chromatography
The stationary phase is held in a narrow tube (column) and the mobile
phase is forced through the tube under pressure or by gravity or
applied voltage. E.g
-High Performance Liquid Chromatography (HPLC).
-Gas Chromatography (GC).
-Capillary Electrophoresis (CE).

b. Planar chromatography
The Sp is supported on a flat plate or in the pores of a paper. The Mp
moves through the Sp by capillary action or under the influence of
gravity. E.g
- Thin layer chromatography (TLC).
- Paper chromatography (PC). 5
 Upon whether Mp is a liquid or a
gas
• If the Mp is a liquid, its called Liquid Chromatography and if Mp is a gas its
referred to as gas chromatography.
• These are further divided according to the type of equilibrium by which the
solutes distribute themselves between the Mp & the Sp.
• When the Sp is a liquid, a means must be provided to hold it immobilized.
Immobilization is accompanied by;

i. Adsorbing a thin film of the liquid on the surface of a finely divided inert
solid.
ii.The liquid may be retained in the pores or interstices of solid particles.
iii.Adsorption or bonding on the inner walls of a capillary tubing.
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Note
• The solids plays no direct part in the separation, serving only
as a support for the liquid. However, the nature may have an
effect on the separation.

• Liquid chromatography can be performed in columns & on

planar surfaces, but gas chromatography is restricted to
column procedures.

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 Upon whether Mp is a liquid or a
gas…cont’d
General Classification Specific Method Stationary Phase Type of Equilibrium

Liquid chromatography Liquid-liquid/ Partition Liquid adsorbed on a solid Partition between immiscible
(Mp = Liquid) liquids

Liquid-bonded phase Organic species bonded to a Partition /Adsorption

solid surface

Liquid-solid/Adsorption Solid Adsorption

Ion-exchange Ion-exchange resin Ion-exchange

Size-exclusion Liquid in interstices of Partition or Sieving

polymeric solid

Gas Chromatography Gas-liquid Liquid adsorbed on a solid Partition between gas &
(Mp = Gas) liquid

Gas-bonded phase Organic species bonded to a Partition between gas &

solid surface liquid

Gas-solid Solid Adsorption

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Mechanisms of separation
• Partition
• Adsorption
• Ion exchange
• Size exclusion/Molecular
• Reverse phase
• Ion-pair
• Affinity

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 Chromatogram
• Refers to the visual output of the chromatograph.

• If a detector that responds to solute concentration is placed at the

end of the column & its signal is plotted as a function of the time (or
sometimes of volume of added Mp), a series of symmetric peaks is
obtained. This is a chromatogram.

• The positions of the peaks on the time axis can be used to identify
the components of the sample, thus useful in qualitative analysis.

• The areas under the peaks provide a quantitative measure of the

amount of each species hence useful in quantitative analysis.
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 Chromatogram pictorial
. mV
1250 Detector A:210nm

RUF
1000

750

500
DP2
DP3

250
DPI

RRCA

RRCB
0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

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 Chromatography nomenclature
• Base line is any part of the chromatogram where only Mp is emerging from
the column.
• Peak maximum is the highest point of the peak.
• Injection point is that point in time/position when or where the sample is
place.
• Dead point is the position of the peak maximum of an unretained sample. It
can be positive or negative.
• Dead/Void time (to /tm) is the time elapsed between the injection point &
the dead point.
• Dead volume (Vo) is the volume of Mp passed through the column between
the injection point & the dead point.
Vo = Qto where Q = flow rate (mL/min.)
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 Chromatography
nomenclature…cont’d
• Retention time (t ) is the time elapsed between the injection point & the peak
R
maximum. Each solute has a characteristic retention time.
• Retention volume (Vr) is the volume of Mp passed through the column between
the injection point & the peak maximum.
Vr = QtR
• Corrected retention time (tR’) is the time elapsed between the dead point & the
peak maximum.
• Corrected retention volume (Vr’) is the volume of Mp passed through the
column between the dead point & the peak maximum.
'
Vr = Vr - Vo = Q (tR - to)

• Peak height (h) is the distance between the peak maximum & the base line,
geometrically produced beneath the peak. 13
 Chromatography
nomenclature…cont’d
• Peak width (w) is the distance between each of a peak measured at 0.6065 of
the peak height. The peak width at this height is equivalent to two standard
deviations of the Gaussian Curve, thus has significance when dealing with
chromatography theory.
• Peak width at half height (W0.5) is the distance between each side of a peak
measured at half the peak height.
• Peak width at the base (Wb) is the distance between the intersections of the
tangents drawn to the sides of the peak & the peak base geometrically
produced. Wb is equivalent to 4 standard deviations of the Gaussian Curve.

• NB;
W0.5 & h0.5 are useful in determining chromatographic parameters.
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 Retention Time
• Refers to the amount of time elapsed from the injection of a sample into the
chromatographic system to the recording of the peak (band) maximum of the
component in the chromatogram.
• The zero (0) on the time axis corresponds to the instant the sample is injected
onto the column & elution is started.
• The peak at tm is for a species that is not retained by the column. Its rate of
migration is the same as the average rate of motion of the molecules of the
Mp.
• The retention time (tR) for the solute responsible for the second peak is the
time for that peak to reach the detector at the end of the column.
• The average linear rate of solute migration (v̅) is given by;
v= L where L = Length of the column packing
tR
tR = Retention time of the solute
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 Capacity/Retention factor (K’)
• Capacity factor of a solute refers to the ratio of its distribution coefficient to
the phase ratio of the column.
• Its used to describe the migration rates of solutes on columns.
• For solute A, the capacity factor (K’A) is defined as;
K'A = tRA - tm
tm
• tRA & tm are obtained from the chromatogram.
• When the capacity factor for a solute is much less than unity, elution goes on
so rapidly that accurate determination of the retention times is difficult.
• When the capacity factor is very large, elution times become inordinately
long.
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 Capacity factor (K’)….cont’d
• Ideally, separations are performed under conditions in which the capacity
factors for the solutes in a mixture lie in the range between 1 – 5.
• That is, if:
a. K’ < 1, the peaks are very close to each other.
b. K’ > 1, separation takes hours. Thus the peaks take hours to appear.
This is not good for routine analysis of drugs.
• In liquid chromatography, capacity factor can be manipulated by;
Varying the composition of the Mp
Varying the composition of the Sp.
• In gas chromatography, capacity factor can be varied by;
 Changing the temperature
 Changing the column packing.
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 Selectivity/separation factor (α)
• Refers is the ability of the chromatographic system to 'chemically' distinguish
between sample components.
• Usually measured as a ratio of the retention (capacity) factors (k) of the two
peaks in question and can be visualized as the distance between the apices of
the two peaks.
• Calculated as, α = k’B
k’A Where k’B = Capacity factor of solute B.
k’A = Capacity factor of solute A.
• But, k'B = tRB - tm k'A = tRA - tm
tm tm

• Therefore,
α = tRB – tm
tRA - tm 18
Selectivity factor…….cont’d
• Ideally, α > 1.
• If α = 1, peaks for the solutes A & B are together.
• Selectivity factor > 1 means better separation, that is, the peaks
for the solutes are separate.
• Selectivity factor is a measure of separation.
• However, separation does not mean good resolution. Well
separated peaks may not be well resolved due to band
broadening.
• This calls for another chromatographic parameter called
resolution or column resolution (Rs).

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 Factor affecting selectivity
factor
• Choice of the solvent/Mp – whether polar or non-polar.
• Mp pH especially when dealing with samples that contain acidic or
basic compounds.
• Solvent strength.
• Concentration of Mp additives e.g Ion-pairing agents.
• Type of the column.
• Temperature.

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 Resolution (Rs)
• Resolution of a column provides a quantitative measure of its ability to
separate two analytes/solutes.
• In column chromatography, resolution is calculated as;
1.18(tRB - tRA)
Rs =
W0.5A + W0.5B
• For complete separation of two closely eluted peaks, Rs = 1.5. At this Rs, the
overlap is minimal.
• Resolution of a given Sp can be improved by lengthening the column, thus
increasing the number of theoretical plates (N). However, added plates
increase the time required for the resolution.

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 Peak symmetry
• The shape of the peaks produced by the column can be useful in method
development just like the column plate number.
• Columns & experimental conditions that provide symmetrical Gaussian peaks are
always preferred.
• Peak symmetry is am measure of column performance especially in HPLC.
• Symmetrical peak is obtained with low concentration of the analyte & it is a
means of good distribution of the sample between the Mp & the Sp. Diagram
• Peaks with poor symmetry can result in;
 Imprecise quantification
 Degraded resolution & undetected minor bands in the peak tail affecting qualitative
analysis.
Masking smaller peaks which then cannot be quantified.
Problems with retention reproducibility.
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 Peak Tailing
• The degree of asymmetry or peak tailing is measured by the peak Asymmetry
(As) or Tailing factor.
where W0.05 = Width of the peak at 1/20 of the peak height
W
As = 0.05 2d = Distance from the centre line of the peak to the front
2dor tail.
slope

• Good columns produce peaks with As of 0.9 – 1.1.

• Causes of peak asymmetry or tailing
a. Bad column
b. Build up of “garbage” on column inert
c. Sample overload
d. Wrong solvent for a sample
e. Extra-column effects
f. Chemical or secondary retention (silanol) effects
g. Inadequate or inappropriate buffering
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h. Contaminating heavy metals
 Types of peak tailing
a. Back tailing / Adsorption peak
•Occurs in adsorption technique type of chromatography.
•Most common in gas-solid chromatography but may also occur in liquid- solid
chromatography especially if the analyte is basic.
•The peak has a tail at the back while the front is not tailed.
•Most common with drugs.
•As > 1 because b > a Diagram

b. Front tailing / overload peak

 occurs because the solute molecules are interacting more with each other
compared to with the Sp.
 more common in gas-liquid chromatography.
 Peak has a tail at the front.
 As < 1 because b < a Diagram
 Rarely with drugs
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 Partition Ratios in
chromatography
• All chromatographic separations are based upon differences in the extent to
which solutes are partitioned between the Mp & the Sp.
• For solute species A, the equilibrium is given by;
AMp ASp
• The equilibrium constant K for this reaction is called partititon ratio or
partition/distribution coefficient, defined as;
CSwhere
K =
CM
CS = molar analytical concentration of a solute in Sp.
CM = molar analytical concentration of a solute in Mp.

• Chromatography carried out under conditions in which K is more or less

constant is termed as Linear chromatography. 25
 Partition Ratios in
chromatography…cont’d
• The magnitude of K is determined by the relative affinity of the solute for the 2
phases.
• Those solutes interacting more strongly with the Sp will exhibit a larger
distribution coefficient & will be retained longer in the chromatographic
system.
• Molecular interaction results from intermolecular forces (electrical in nature)
that affect the magnitude of K.
• These forces include;
Dispersion forces
Ionic forces
Polar forces

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