Lecture 8: Chromatography

Learning Outcomes

By the end of this lecture, you should be able to:

✓ Explain Gas/liquid chromatographic methods.
✓ Understand separation mechanisms.
✓ Classify chromatographic methods and their operation
principle.
✓ Know the methods for improvement of column efficiency.

MCEE 5210_13
 Lecture 8: Chromatography

MCEE 5210_14
 Chromatography
Uses the property of molecular
attraction (molecular polarity)
to separate a mixture. Different
molecules have varying
molecular attractions for the
paper (the stationary phase) vs.
the solvent (the mobile phase)

Example: the separation of

plant pigments and dyes

MCEE 5210_15
 What is Chromatography?
Chromatography: separating components
of a mixture that have differing adsorptive
tendencies on a stationary phase as the
mixture is passed over or through the
stationary phase .
Chromatography of
plant pigments
Components:
Mobile phase: a solvent that flows through the supporting
medium
Stationary phase: a layer or coating on the supporting medium that
interacts with the analytes
Supporting medium: a solid surface on which the stationary phase
is bound or coated.
MCEE 5210_16
 History of Chromatography

• Russian Scientist Mikhail Semenovich Tswett is

credited for the discovery of chromatography
(1903)
• German student Fritz Prior is credited for
developing gas chromatography (1947)
• John Porter Martin (UK) is the father of modern
gas chromatography. He developed the first liquid-
gas chromatograph instrument (1950), He won the
Nobel Prize in Chemistry winner (1952)
• Chromatography has application in every branch of
the physical and biological sciences, 12 Nobel
prizes were awarded between 1937 and 1972 alone
for work in which chromatography played a vital
role.

MCEE 5210_17
 Definitions

Term Definition
Mobile liquid phase with no affinity to the stationary phase (i.e.
Solvent
inert towards it) & no effect on solutes.
Any liquid with more affinity to the stationary phase than the
Developer solvent but less than solutes and just capable to move them
through the column.
Effluent Any liquid that passes out of the column.
Any liquid that has lesser affinity to the stationary phase than
Eluent
solutes but is capable to move them out of the column.
Eluate Fraction of eluent containing a required specific substance.
Retention (or retardation volume): Volume of mobile phase that passes out
volume (VR) of the column, before elution of a specific substance.

MCEE 5210_18
 What happens inside a column?

Solutes which interact

more strongly with the
stationary phase take
Strongly Retained
longer to pass through
the column

Weakly Retained
Solutes which only
weakly interact with the
stationary phase or
have no interactions
with it elute very
quickly

MCEE 5210_19
 Chromatography
Chromatogram: graph showing the detector response as a function of
elution time.
Retention time (tr): the time it takes a compound to pass through a column.
Retention volume (Vr): volume of mobile phase needed to push solute
through the column.
Retention time
Non-retained
solute (void
volume)

The strength or degree with which a molecule is retained on the column can
be measured using retention time or retention volume.

MCEE 5210_20
 Efficiency of Separation
o The width of a solute peak is important in determining how well one solute
is separated from another
o One measure of this is the width of the peak at half-height (w½ ) or at its
baseline (wb)

A solute’s retention volume, Vr, is

𝑉𝑟 = 𝑡𝑟 × 𝑢
where u is the mobile phase’s velocity through the column.

MCEE 5210_21
 What happens inside a chromatography column

MCEE 5210_22
 Efficiency of Separation
The separation of two solutes in chromatography depends both on the width of the
peaks and their degree of retention

The separation between the two solutes is given by their Resolution (R)
Δt r 2Δt r
𝑅𝐴𝐵 = =
0.5 WA + WB WA + WB

MCEE 5210_23
 Example 8.1

EXAMPLE 8.1 In a chromatographic analysis of lemon oil a

peak for limonene has a retention time of 8.36 min with a
baseline width of 0.96 min. γ-Terpinene elutes at 9.54 min with a
baseline width of 0.64 min. What is the resolution between the
two peaks?

MCEE 5210_24
 Example 8.1

2Δt r
𝑅𝐴𝐵 =
WA + WB

2 9.54 − 8.36
𝑅𝐴𝐵 = = 1.48
1.64 + 0.96

MCEE 5210_25
 Chromatography
• Ideally, we want sufficient
resolution (Rs of 1.5 or greater
for analyte/solute of interest
peaks)

• We also want the separation

performed in a minimum
amount of time

• Other parameters may also be of importance:

• sufficient quantity if performing preparation scale separation
• sufficient sensitivity for detection (covered more with
instrumentation and quantitation)
• ability to identify unknowns (e.g. with MS detection)

MCEE 5210_26
 Theory of Chromatography
Chromatography is based on a physical equilibrium that results when a solute is
transferred between the mobile and a stationary phase.

𝑆𝑚 ≜ 𝑆𝑠
S
S S 𝑆𝑠
S S 𝐾𝐷 =
𝑆𝑚
S S In the absence of any additional equilibrium
S reactions in the mobile phase or stationary
S phase, KD is equivalent to the distribution
S ratio, D,
S S 𝑆𝑠 𝑚𝑜𝑙 𝑆 𝑠 𝑚𝑜𝑙 𝑆 𝑚
𝐾𝐷 = 𝐷 = = ൘
𝑆𝑚 𝑉𝑠 𝑉𝑚
Cross Section of Equilibrium in a column.
“S” are adsorbed to the stationary phase. where Vs and Vm are the volumes of the
stationary phase and the mobile phase,
“S” are traveling in the mobile phase.
respectively.

MCEE 5210_27
 Theory of Chromatography

𝑚𝑜𝑙 𝑆 𝑡𝑜𝑡𝑎𝑙 = 𝑚𝑜𝑙 𝑆 𝑠 + 𝑚𝑜𝑙 𝑆 𝑚

Thus
𝑆𝑠 𝑚𝑜𝑙 𝑆 𝑡𝑜𝑡𝑎𝑙 − 𝑚𝑜𝑙 𝑆 𝑚 𝑚𝑜𝑙 𝑆 𝑚
𝐷= = ൘
𝑆𝑚 𝑉𝑠 𝑉𝑚
Rearranging this equation and solving for the fraction of solute
in the mobile phase, fm,
𝑚𝑜𝑙 𝑆 𝑚 𝑉𝑚
𝑓𝑚 = =
𝑚𝑜𝑙 𝑆 𝑡𝑜𝑡𝑎𝑙 𝐷𝑉𝑠 + 𝑉𝑚

that is identical to for a liquid–liquid extraction. Because we may not

know the exact volumes of the stationary phase and the mobile phase,

MCEE 5210_28
 Theory of Chromatography
we can simplify equation by dividing both the numerator and
the denominator by Vm; thus
𝑉𝑚
𝑉𝑚 1 𝑉𝑚
𝑓𝑚 = = =
𝐷𝑉𝑠 + 𝑉𝑚 𝐷𝑉𝑠 𝑉 1+𝑘
+ 𝑚
𝑉𝑚 𝑉𝑚
Where
𝑉𝑠 𝑉𝑠
𝑘=𝐷 = 𝑲𝑫
𝑉𝑚 𝑉𝑚
is the solute’s retention factor. Note that the larger the retention factor, the more
the distribution ratio favors the stationary phase, leading to a more strongly
retained solute and a longer retention time.
The retention factor is also called the capacity factor, the capacity ratio, and the
partition ratio, and is sometimes given the symbol k′.

MCEE 5210_29
 Theory of Chromatography
We can determine a solute’s retention factor from a
chromatogram by measuring the column’s void time, tm, 1 − 𝑓𝑚
𝑘=
and the solute’s retention time, tr 𝑓𝑚

Assuming a constant mobile phase velocity, we also can define fm as

𝑡𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 𝑡𝑚
𝑓𝑚 = =
𝑡𝑜𝑡𝑎𝑙 𝑡𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑜𝑛 𝑐𝑜𝑙𝑢𝑚𝑛 𝑡𝑟
𝑡𝑟 − 𝑡𝑚 𝑡𝑟′
rearranging leaves us with 𝑘= =
𝑡𝑚 𝑡𝑚

where t′r is the adjusted retention time.

• The longer a component is retained by the column, the greater the capacity factor
• Capacity factor of a standard can be used to monitor performance of a column

MCEE 5210_30
 Example 8.2

In a chromatographic analysis of low molecular weight acids,

butyric acid elutes with a retention time of 7.63 min. The
column’s void time is 0.31 min. Calculate the retention factor for
butyric acid.

MCEE 5210_31
 Example 8.2

𝑡𝑟 − 𝑡𝑚 7.63 − 0.31
𝑘𝐵𝑈𝑇 = = = 23.6
𝑡𝑚 0.31

MCEE 5210_32
 Example 8.2
Selectivity is a relative measure of the retention of two solutes, which we define using
a selectivity factor, α

𝑘𝐵 𝑡𝑟,𝐵 − 𝑡𝑚
𝛼= =
𝑘𝐴 𝑡𝑟,𝐴 − 𝑡𝑚
When two solutes elute with identical retention time, α = 1.00; for all other
conditions α > 1.00.

MCEE 5210_33
 Theory of Chromatography
Adjusted retention time (tr’): the
t'r = t r − t m
additional time required for a
solute to travel through a where: tm = minimum possible time for
a non-retained solute to pass
column beyond the time
through the column
required for non-retained solute.

Relative Retention (a): ratio of

adjusted retention time between
two solutes.

Greater the relative retention

the greater the separation t'r 2
a= where: tr2’ > tr1’ , so a > 1
between two components. t'r1

MCEE 5210_34
 Example 8.3
EXAMPLE 8.3 An open tubular column has a diameter of
207 µm and the thickness of the stationary phase on the inner
wall is 0.50 µm. Unretained solute passes through in 63s and
a particular solute emerges in 433s. Find the partition
coefficient (K) for this solute and find the fraction of time
spent in the stationary phase.

MCEE 5210_36
 Example 8.3

𝑡𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑖𝑛 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒 𝑡𝑚 63

𝑓𝑚 = = = = 0.145
𝑡𝑜𝑡𝑎𝑙 𝑡𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑜𝑛 𝑐𝑜𝑙𝑢𝑚𝑛 𝑡𝑟 433

1 − 𝑓𝑚 433 – 63
𝑘= = = 5.873
𝑓𝑚 63

MCEE 5210_37
 Example 8.3

𝜋𝑑 2 𝜋 (207 − 𝟐 × 𝟎. 𝟓)𝜇𝑚 2
𝑉𝑚 = 𝐿= 𝐿
4 4

𝑉𝑆 = 𝑉𝑜𝑢𝑡𝑤𝑎𝑙𝑙 − 𝑉𝑖𝑛𝑛𝑒𝑟 = 𝜋 207𝜇𝑚 0.5𝜇𝑚 𝐿

𝑉𝑚
𝐾=𝑘 = 609
𝑉𝑆

MCEE 5210_38
 Measurement of Column Efficiency
Height Equivalent of a Theoretical Plate (H or HETP)
-The distance along the column that corresponds to one “theoretical” separation
step or plate (N) 𝐿
𝑁=
𝐻
where: L = length of column
N = number of theoretical plates

H the height of a theoretical plate

A H decreases, more separation steps per column length are possible

-Results in a narrower peak width and better separation between two neighboring
solutes

MCEE 5210_39
 Measurement of Column Efficiency
If we assume that a chromatographic
peak has a Gaussian profile, then the
extent of band broadening is given by
the peak’s variance or standard
deviation. The height of a theoretical
plate is the variance per unit length of
the column
𝜎2
𝐻=
𝐿
It is more convenient to express the standard deviation
𝜎 𝜎𝑡𝑟
in units of time, τ, by dividing σ by the solute’s average 𝜏= =
linear velocity:
𝒖 𝐿
For a Gaussian peak shape, the width at the baseline, w, is 𝑊 = 4𝜏
four times its standard deviation
𝐿𝑊 2 𝑡𝑟2
Combining equation 𝐻= 𝑁 = 16 2
16𝑡𝑟2 𝑊
MCEE 5210_40
 Example 8.4

A chromatographic analysis for the chlorinated pesticide

Dieldrin gives a peak with a retention time of 8.68 min and a
baseline width of 0.29 min. What is the number of theoretical
plates? Given that the column is 2.0 m long, what is the height
of a theoretical plate in mm?

MCEE 5210_41
 Example 8.3

𝑡𝑟2 8.682
𝑁 = 16 2 = 16 × 2
= 14300 𝑝𝑙𝑎𝑡𝑒𝑠
𝑊 0.29

𝐿 2.0 𝑚 1000 𝑚𝑚
𝐻= = = 0.14 𝑚 𝑚Τ𝑝 𝑙𝑎𝑡𝑒
𝑁 14300 𝑝𝑙𝑎𝑡𝑒𝑠 𝑚

-Similar to number of extractions performed in an extraction separation

-As N increase (number of separating steps) → greater the separation between two
compounds

MCEE 5210_42
 Example 8.3

𝑡𝑟2
𝑁 = 16 2
𝑊

• Column efficiency is used to compare the performance of different

columns. It is expressed as the theoretical plate number, N.
• Columns with high plate numbers are more efficient. A column with a
high N will lead to narrower peak at a given retention time than a
column with a lower N number.
• Parameters influencing column efficiency:
o Column length (increasing colum length increases efficiency)
o Particle size (decreasing particle size increases efficiency)

MCEE 5210_43
 Rate-based theory--Resolution: why bands spread
Remember: Efficiency is dependent on peak width
A band of solute spreads as it travels through the column
-described by a standard deviation (s)

➢Factors include:
-Sample injection
-Longitudinal diffusion
-Finite equilibration between phases
-Multiple flow paths
-others

MCEE 5210_44
 Rate-based theory-- Resolution: why bands spread
➢ Sample injection – sample is injected on the column width a finite width, which
contributes to the overall broadening
- Similar broadening may occur in the detector

➢ Longitudinal diffusion – band slowly broadens

as molecules diffuse from high concentration
in band to regions of lower concentration

2𝛾𝐷𝑚
𝐻𝑑 =
𝑢

Dm is the solute’s diffusion coefficient in the mobile

phase, u is the mobile phase velocity, and γ is a constant
related to the efficiency of column packing.

MCEE 5210_45
 Rate-based theory--Why bands spread: Eddy diffusion
A process that leads to peak (band) broadening due to the
presence of multiple flow paths through a packed column.

❖ Solute 1: longer path ➔ higher retention time broad

❖ Solute 3: shorter path ➔ lower retention time peak
❖ In capillary columns there is no packing ➔ there is no Eddy
diffusion, then peaks are narrower and separation is better

where dp is the average diameter of the particulate packing material,

𝐻𝑝 = 2𝜆𝑑𝑝 and λ is a constant that accounts for the consistency of the packing.

MCEE 5210_46
 Rate-based theory-- Resolution: why bands spread
➢ Finite Equilibration Time Between Phases – a finite time is required to equilibrate between
stationary and mobile phase at each plate
-Some solute is “stuck” in stationary phase as remainder moves forward in mobile phase
-Results in band broadening
Distribution of solute between
mobile and stationary phase

𝑞𝑘𝑑𝑓2
𝐻𝑠 = 2
𝑢
1 + 𝑘 𝐷𝑠

Solute in mobile phase moves

down column → broader peaks

𝑓 𝑘 𝑑𝑃2
𝑯𝒎 = 𝑢
𝐷𝑀
where df is the thickness of the stationary phase, dc is the column’s diameter, Ds and Dm are the
solute’s diffusion coefficient in the stationary phase and the mobile phase, k is the solute’s
retention factor, and q is a constant related to the column packing material.
MCEE 5210_47
 Rate-based theory-- Description of Band Spread
Plate height (H) is proportional to band width
-The smaller the plate height, the narrower the band
-Van Deemter equation

𝐻 ≈ 𝐻𝑝 + 𝐻𝐷 + 𝐻𝑆 + 𝐻𝑚
2𝛾𝐷𝑚 𝑞𝑘𝑑𝑓2 𝑓 𝑘 𝑑𝑃2
= 2𝜆𝑑𝑃 + + 2
𝑢+ 𝑢
𝑢 1 + 𝑘 𝐷𝑠 𝐷𝑀
𝐵
=𝐴+ + 𝐶𝑢𝑥
𝑢𝑥
Multiple paths
equilibration
Longitudinal time
diffusion

where: ux = linear flow rate

A,B,C = constants for a given column and stationary phase

MCEE 5210_48
 Peak capacity
One advantage of improving column efficiency is that we can separate more solutes with
baseline resolution. One estimate of the number of solutes that we can separate is

𝑁 Vmax
𝑛𝑐 = 1 + 𝑙𝑛
4 𝑉𝑚𝑖𝑛
where nc is the column’s peak capacity, and Vmin and Vmax are the smallest and the largest
volumes of mobile phase in which we can elute and detect a solute.

This estimate provides an upper bound on the number of solutes and may help us exclude
from consideration a column that does not have enough theoretical plates to separate a
complex mixture.

MCEE 5210_49
 Peak capacity

mAU
Column: 2.1 x 150 mm, 1.8 µm
60 Back pressure: 402 bar
Peak capacity: 313
40

20

10 20 30 40 50 min
mAU
Column: 2.1 x 300 mm*, 1.8 µm
50
Back pressure: 598 bar
40
Peak capacity: 406
30
20
10
0

20 40 60 80 100 min
*300 mm column by coupling two 150 mm columns

MCEE 5210_50
 Asymmetric Peak

The number of theoretical plates for an asymmetric peak shape is approximately

𝑡𝑟2 𝑡𝑟2
2
𝑊0.1 𝑎+𝑏 2
𝑁 ≈ 41.7 × = 41.7 ×
𝑇 + 1.25 𝑇 + 1.25

where w0.1 is the width at 10% of the peak’s height.

MCEE 5210_51
 Column Efficiency

It is reasonable to assume that their peak widths are nearly identical.

Therefore,
t r,B − t r,A t r,B − t r,A t r,B − t r,A
𝑅𝐴𝐵 = ≈ =
0.5 WA + WB 0.5 2WB WB
N t r,B − t r,A
We get 𝑅𝐴𝐵 = ×
4 t r,B
N 𝑘𝐵 − k A
or 𝑅𝐴𝐵 = ×
4 1 + kB

rearranging, we end up with 𝑅𝐴𝐵 = N × 𝛼 − 1 × 𝑘𝐵

4 α 1 + kB

MCEE 5210_52
 Column Efficiency

N 𝛼−1 𝑘𝐵
𝑅𝐴𝐵 = × ×
4 α 1 + kB
One can improve resolution by improving any of these parameters:
• Selectivity has the highest influence on the resolution. Small changes
in selectivity lead to big hanges in resolutions.
• Retention has only a significant influence at small k-values.
• Efficiency describes the separation power of the column.

MCEE 5210_53
 Column Efficiency
In addition to resolution, another important factor in chromatography is the amount of time
needed to elute a pair of solutes, which we can approximate using the retention time for
solute B.

16R2AB 𝐻 𝛼 2 1 + 𝐤B 3
t r,B = × ×
𝑢 α−1 𝒌2𝐵

• We can vary these terms, to improve resolution and analysis time. The first term, which
is a function of the number of theoretical plates or the height of a theoretical plate,
accounts for the effect of column efficiency.
• The second term is a function of α and accounts for the influence of column selectivity.
• The third term is a function of kB and accounts for the effect of solute B’s retention
factor.

MCEE 5210_54
 Increasing N

1 Lc 1 Lc
For HPLC: 𝑅𝑆 ≈ =
4 𝐻 4 ℎ𝑑𝑝

LC is Column length; dp is Particle size, h, Reduced height of a

theoretical plate
• High plate number (N) provides:
o Sharp and narrow peaks
o Better detection
o Peak capacity to resolve complex samples
• But resolution increases only with the square root of the plate number.
• Plate number increase is limited by experimental conditions
• Analysis time, pressure

MCEE 5210_55
 Increasing a
In HPLC, it is possible to change the mobile phase to affect
solute – solvent interactions and retention.
For example, if molecules A and B are separated by normal phase
HPLC using 15% 2-propanol/85% hexane and are found to co-
elute, solvent changes may resolve.
One might expect that changing solvent to 25% toluene 75%
hexane will increase affinity of compound B for mobile phase
relative to compound A (due to compound B being aromatic)
leading to increase retention of B
H O
12 OH
17
11 13
H H 16
1 9 14
2 10 8 15
H H
3 5 7
4 6
O

compound A compound B

MCEE 5210_56
 Increasing a

The two compounds below are found to give retention

times of 8.91 and 9.02 min. (aniline and
benzaldehyde, respectively) when separated using
HPLC on a C18 column using 60% methanol/40%
water vs. an unretained time of 1.62 min.
There is an easy way to increase a for this separation.
How can the mobile phase be changed to increase a?

NH2
O

MCEE 5210_57
 Increasing α to Optimize Resolution

Window diagram—and
the highest point in each
window gives the
optimum pH within that
range.

MCEE 5210_58
 Increasing selectivity factor

• Same sample, analyzed with same stationary phase and temperature but with
different mobile phases (same gradient).

MCEE 5210_59
 Increasing selectivity factor

• Same sample, analyzed with same stationary and mobile phase, same gradient
but different temperatures.

MCEE 5210_60
 Increasing selectivity factor

• Changes in a with T:
– Example: alkanes and toluene
– In Plot, most alkanes show similar
temp. – retention behavior (similar
slopes – no overlap)
– If two alkanes overlap (e.g. two
branched alkanes), there is not much
chance in increasing a
– If a separation of octane and toluene
had been performed at 150,
coeluting peaks would be observed
– Decreasing T would lead to
improvement because different
slopes lead to a change in a
note: if chromatogram started at 200C, one
would be disappointed by initial change

MCEE 5210_61
 Increasing kB
• To adjust solute B’s retention factor.
increasing kB improves resolution, if
the initial value of kB is small.
• Once kB exceeds a value of
approximately 10, a further increase
produces only a marginal improvement
in resolution.
• May lead to unacceptably long
retention times for other solutes

Parameters influencing retention factor:

• Stationary phase
• Mobile phase
• Gradient slope*
• System dwell volume*

MCEE 5210_62
 Increasing kB

• Same sample, analyzed with different stationary phases but always same
temperature, mobile phase and gradient.

MCEE 5210_63
Classification

According to mobile phase:

1- Liquid chromatography: mobile phase is a liquid.
(LLC, LSC).
2- Gas chromatography : mobile phase is a gas. (GSC,
GLC).
According to the force of separation:
1- Adsorption chromatography.
2- Partition chromatography.
3- Ion exchange chromatography.
4- Gel filtration chromatography.
5- Affinity chromatography.

MCEE 5210_64
 Chromatography

Two key differences between GC and LC:

• No analyte – mobile phase interaction in GC
• Temperature is routinely changed (and always
controlled) in GC

Effects of gases (vs. liquids)

• Much higher diffusivity (larger B term of van Deemter
equation but very small CM term)
• Lower viscosity of gases (backpressure is not as big an
issue)
• Much lower density (capacity of column is a big issue
with liquid samples)
• Gases are compressible

MCEE 5210_65
 Gas Chromatography
• In analytical chemistry, scientists use gas chromatography (GC) to separate
and analyze compounds that can be vaporized without decomposition. They
often use GC to test the purity of a particular substance, or to separate the
components of a mixture to determine the relative amounts of each.
• Scientists use GC for both qualitative and quantitative analysis of volatile
analytes.
• The instrument, called a gas chromatograph, employs a mobile phase and a
stationary phase. That is, a moving gas carries the sample across a stationary
support (a piece of glass or metal tubing called a column) inside the instrument.

MCEE 5210_66
Gas chromatography

Flow meter
Injector Vent
Septum Detector
Pressure Flow GC
regulator controller 
Recorder Chart

Oven

Gas
supply Column

MCEE 5210_67
 Gas chromatography principle
❖Sample is injected then vaporised onto head of a chromatographic column.
❖Elution is produced by the flow of an inert gaseous mobile phase.
❖Separation is based upon the partition of the vaporized analyte between a
gaseous mobile phase and a liquid phase immobilised on the surface of an
inert solid (GLC)
❖Inert carrier gas does not interact with molecules of the analyte.
❖Eluted analytes are detected by a detector and recorded by the data system
❖GC columns are either packed (particles coated with liquid stationary phase)
or capillary (the most used now)

MCEE 5210_68
Gas chromatography
Gas Supply: (usually N2 or He)
Sample Injector: (syringe / septum)
Column: 1/8” or 1/4” x 6-50’ tubing packed with
small uniform size, inert support coated with
thin film of nonvolatile liquid
Detector: TC - thermal conductivity
FID - flame ionization detector

MCEE 5210_69
 Gas chromatography
◼ Packed column
* ~ 3-6mm inner diameter tubing, 1-5 m
long
* used for preparative separations or to
separate gases that are poorly retained
* lower resolution
* small, uniform particle size decreases
Eddy diffusion (requiring higher
pressures)

◼ open tubular (more common):

* 0.1-0.5 mm inner dia., 10-100 m long
* 0.1-5 mm thick sp coated on inner walls
* higher resolution, shorter analysis times,
greater sensitivity compared to packed
columns

MCEE 5210_70
 Gas Chromatography
• Flame Ionization Detector (FID): The
FID consists of a hydrogen / air flame and
a collector plate.
• Atomic Emission Detector (AED):
:Simultaneously determines the atomic
emission of many elements in analyte that
elutes from GC capillary column.
• Chemiluminescence Detector (CD): Uses
quantitative measurements of optical
emission from excited chemical species to
determine analyte concentration (energized
molecules)
• Electron Capture Detector (ECD): Uses a radioactive beta emitter (electrons) to ionize
some of the carrier gas and produces a current between a biased pair of electrodes.Has
application for organic functional groups such as halogens,phosphorus and nitrogen
compounds.
• Flame Photometric Detector (FPD): Used to detect phosphorus and nitrogen containing
compounds. Uses the chemilumiescent reactions of these compounds in a hydrogen / air
flame.

MCEE 5210_71
Gas Chromatography
• Mass Spectrometer (MS) Uses the difference in mass-to-charge ratio (m/e) of ionized
atoms or molecules to separate them from each other.

• Photo Ionization Detector (PID) : Uses ultraviolet light as a means of ionizing an

analyte exiting from a GC column.

• Thermal Conductivity Detector (TCD) Consists of an electrically-heated wire or

thermistor. Changes in thermal conductivity such as when organic molecules displace
some of the carrier gas, cause a temperature rise which is sensed as a change in
resistance. Change in resistance is proportional to the amount of analyte. The TCD is
not as sensitive as the other detectors but it is non-specific and non-destructive.

MCEE 5210_72
Gas Chromatography
Libraries (EI).
i. Over the past forty-fifty
years, since mass
spectrometry has become a
standard tool, libraries of
mass spectra have been
generated.
ii. The newest libraries contain
hundreds of thousands of EI
mass spectra from which an
unknown compound can very
often be identified.

MW 144

MCEE 5210_74
Gas Chromatography
Food Analysis
Analysis of foods is concerned with confirm the presence and determination the
quantities of the analytes (lipids, proteins, carbohydrates, preservatives, flavours, colorants,
and also vitamins, steroids, and pesticide residues).
Drug Analysis
GC is widely applied to identification of the active components, possible impurities as
well as the metabolites.
Environmental Analysis
• The environmental contaminants; e.g. dichlorodiphenyltrichloro-ethane (DDT) and
the polychlorinated biphenyls (PCBs) are present in the environment at very low
concentrations and are found among many of other compounds.
• GC, with its high sensitivity and high separating power, is mostly used in the analysis
of environmental samples.
Forensic Analysis
• In forensic cases, very little sample is available, and the concentration of the sample
components may be very low.
• GC is a useful due to its high sensitivity and separation efficiency.

MCEE 5210_75
Liquid column chromatography
Classification according to the packing of the stationary
phase:
1- Thin layer chromatography (TLC): the stationary
phase is a thin layer supported on glass, plastic or
aluminium plates.
2- Paper chromatography (PC): the stationary phase is a
thin film of liquid supported on an inert support.
3- Column chromatography (CC): stationary phase is
packed in a glass column.

MCEE 5210_76
 Paper Chromatography
o A method of partition
chromatography using filter
paper strips as carrier or inert
support.
o The factor governing separation
of mixtures of solutes on filter
paper is the partition between
two immiscible phases.
o One is usually water adsorbed
on cellulose fibres in the paper
(stationary phase).
o The second is the organic
solvent flows past the sample on
the paper (stationary phase).

MCEE 5210_77
Thin layer chromatography (TLC)
TLC is a method for identifying substances and testing the purity of
compounds.

TLC is a useful technique because it is relatively quick and requires small

quantities of material.

MCEE 5210_78
Thin layer chromatography (TLC)
• Separations in TLC involve distributing a mixture of
two or more substances between a stationary phase
and a mobile phase.
• The stationary phase is a thin layer of adsorbent
(usually silica gel or alumina) coated on a plate.
• The mobile phase is a developing liquid which travels
up the stationary phase, carrying the samples with it.
• Components of the samples will separate on the
stationary phase according to how much they adsorb
on the stationary phase versus how much they dissolve
in the mobile phase.

MCEE 5210_79
Interpreting the Data

• The Rf (retention factor)

value for each spot should be
calculated.
• It is characteristic for any
given compound on the same
stationary phase using the
same mobile phase for
development of the plates.
• Hence, known Rf values can
be compared to those of
unknown substances to aid in
their identifications.

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑓𝑟𝑜𝑚 𝑠𝑡𝑎𝑟t to center of substance spot

𝑅𝒇 =
distance from start to solvent fron𝑡

MCEE 5210_80
Liquid Chromatography
Name of LC Method
Adsorption chromatography
Partition chromatography
Ion-exchange chromatography
Size exclusion chromatography
Affinity chromatography
MCEE 5210_81
Type of Stationary Phase
solid, underivatized support
liquid-coated or derivatized support
support containing fixed charges
porous support
support with immobilized ligand
HPLC columns
 Liquid Column Chromatography

• The instrument used consists of vertical glass tube, where the

adsorbent is packed. A small plug of glass wool or sintered glass
disc at the bottom of the tube supports the column.

• The stationary phase is a solid adsorbent e.g. silica gel, alumina,

Mg oxide. Usually the stationary phase in column
chromatography present in the form of powder or granule with a
particle size 100-200 mesh size (i.e. silica gel used in column
chromatography with a particle size larger than that used in
TLC).

• The mobile phase is liquid. Either single liquid or a mixture of

liquids.

MCEE 5210_82
Three type of elution
A) Simple Elution: one solvent or a mixture of the
solvents is used from the beginning to the end of the
procedure.
B) fractional or step wise Elution: by changing the
mobile phase, during the procedure we can use another
solvent for example more polar that will lead to further
separation of the sample.
C) Gradual Elution: in this method we are not going to
change the solvent completely but we are going to use a
mixture of two solvents e.g. Ethyl acetate +5% Ethanol and
then we can gradually increase the polarity of the mobile
phase.
MCEE 5210_83
 Type of Chromatography
Mode or type Stationary phase Mobile Mechanism
phase
Adsorption Solid that attracts the Liquid Solutes move at different rates
Chromatogra solutes or gas according to the forces of attraction to
phy the stationary phase.
Partition Thin film of liquid Liquid Solutes equilibrate between the 2 phases
Chromatogra formed on the surface or gas according to their partition coefficients
phy of a solid inert
support
Ion Exchange Solid resin that Liquid Solute ions of charge opposite to the
Chromatogra carries fixed ions & contain fixed ions are attracted to the resin by
phy mobile couterions of ing electrostatic forces & replace the mobile
opposite charge electro counterions.
attached by covalent lytes
bonds
Molecular Porous gel with no Liquid Molecules separate according to their
Exclusion attractive action on size:
Chromatogra solute molecules 1.Smaller molecules enter the pores of
phy the gel, and need a larger volume of
eluent.
2.Larger molecules pass through the
column at a faster rate.
Affinity Solid on which Liquid Special kind of solute molecules
Chromatogra specific molecules are or gas interact with those immobilized on the
phy immobilized stationary phase

MCEE 5210_84
Adsorption Column Chromatography

o Solutes are separated based

on their different abilities to
adsorb to the support’s
surface

o Uses an underivatized solid

support (stationary phase =
solid support)
o Oldest type of
chromatography, but not
commonly used

MCEE 5210_85
Adsorption Column Chromatography
Adsorbents:
The most common are Alumina & Silica gel in which the
interactions with solute molecules is due to OH groups
present on their surface.
More polar molecules are adsorbed more strongly & thus, will
elute more slowly
Strength of adsorption of polar groups (solutes) on polar support
is in the following order:
-C=C- < O-CH3 < -COOR < >C = O < -CHO < -NH2 < -
OH < -COOH
Olefins < Ethers < Esters < Lactones < Aldehydes < Amines <
Phenols < Acids.

MCEE 5210_86
Selection of the solid support
The support material should:
• adsorb & retain the mobile stationary phase.
• expose as large surface as possible to the mobile
phase
• be mechanically stable.
• be easy to pack.
• not retard the solvent flow
Examples of solid supports:
Silica gel, diatomaceous earth (as kieselguhr, celite etc.)
& cellulose.

MCEE 5210_87
 Selection of the mobile phase
The liquid stationary & mobile phases should have a
considerable difference between their solvent strength
parameters.

Pure water > Methanol > Ethanol > Propanol >

Acetone > Ethyl acetate> Ether > Chloroform >
Dichloromethane >Benzene > Toluene > Carbon
tetrachloride > Cyclohexane > Hexane > Pentane.

e.g. if the stationary phase is water, pentane would be

the eluent of choice.

MCEE 5210_88
Factors affecting column efficiency
Factor
Particle size of solid stationary
phase (or of support)
Column dimensions
Uniformity of packing
Column temperature
Eluting solvent
Solvent flow rate
Continuity of flow
Condition of adsorbent
Concentration of solutes
MCEE 5210_89
Effect
Decrease of size improves separation (but very small
particles need high pressure).
Efficiency increases as ratio length / width increases.
Non uniform packing results in irregular movement of
solutes through column & less uniform zone formation,
(i.e. band broadning or tailing).
Increase in column temperature results in speed of
elution but does not improve separation (tailing).
Solvents should be of low viscosity (to give efficient
resolution) & h igh volatility (to get rapid recovery of the
substances).
Uniform & low flow rate gives better resolution.
Discontinuous flow disturbs resolution
Deactivation of adsorbent decreases separation.
Substances of high concentration move slowly.
 Partition Column Chromatography
• In this type, the packing consists of a theoretically inert support material
coated with a film of the liquid stationary phase.
• The division into adsorption & partition is only of theoretical significance as
in partition chromatography the adsorption effects of the support can be
felt.
• There are various applications of Paper Chromatography. Some of the
applications are mentioned below:
• To separate and identify amino acids.
• To separate and identify tannins.
• To separate and identify alkaloids.
• To separate and identify carbohydrates.
• To separate and identify glycosides.
• Application of partition chromatography
• Remove the detergent from the protein solution.
• Steroids, bile acids, and mycotoxins are separated.
• Pesticides, phenols, and insecticides are all being removed.
• Concentration of trace metals in aqueous solutions

MCEE 5210_90
Partition Chromatography
Solutes are separated based on their different abilities to partition between the
stationary phase and mobile phase.

-Uses a solid support coated or chemically derivatized with a polar or non-polar

layer
-Most common type of liquid chromatography at present. Good for most organic
compounds
-Reversed Phase: stationary phase is non-polar
-Normal Phase: stationary phase is polar

MCEE 5210_91
Ion exchange Chromatography

-Used to separate ions based on their

different abilities to interact with the
fixed exchange sites.

-Uses a solid support containing fixed charges

(exchange sites) on its surface
-Cation-Exchange: support with negative
groups
-Anion-Exchange: support with positive groups

MCEE 5210_92
Size exclusion Chromatography
-Separates large and small solute based on their different abilities
to enter the pores of the support

-Uses a porous support that does not adsorb solutes

-Commonly used to separate biological molecules or polymers
which differ by size (MW)
MCEE 5210_93
Gel Permeation Chromatography (GPC)
This type is also known as:
• Size Exclusion Chromatography (SEC)
• Molecular Exclusion Chromatography
(MEC)
• Molecular Sieve Chromatography (MSC)
• Gel Filtration Chromatography (GFC)
• Gel Chromatography.

Mobile phase: Mobile phase is a liquid as water

or dilute acohol

Separation mechanism:
Based on difference between the solutes
molecular weights.
Molecules will distribute themselves outside &
inside the pores according to their size.
Larger are excluded, medium sized enter half-
way & smallest permeate all the way.

MCEE 5210_94
 Stationary phase
• Porous polymeric matrix: formed of spongy
particles, with pores completely filled with the
liquid mobile phase (gel).
•
• The gels (polymers) consist of open, three-
dimensional networks formed by cross-
linking of long polymeric chains.
• The pore size varies with the degree of cross-
linking.
• The diameter of the pores is critical as
separation is based on that molecules above
certain size are totally excluded from the
pores because they can not enter the gel.
• The interior of the pores is reached,
partially or wholly, by smaller molecules.

MCEE 5210_95
 Affinity Chromatography
-Separates molecules based on their different abilities to bind to the
affinity ligand

-Uses a support that contains an immobilized biological molecule

(affinity ligand)
-Commonly used to purify and analyze biological molecules
-Most Selective type of Chromatography
MCEE 5210_96
 Ion Exchange Chromatography
Principle
• Process by which ions of an electrolyte solution are
brought into contact with an ion exchange resin.
• The ion exchange resin is an insoluble polymer
consisting of a "matrix" (Lattice or framework) that
carries fixed charges (not exchangeable) and mobile
active ions "counter ions" which are loosely attached to
the matrix.
• In water, the counter-ions move more or less freely in
the framework & can be replaced by ions of the same
sign present in the surrounding solution.
• The "matrix" (framework) of a "cation exchanger" is
considered as a crystalline non-ionized "polyanion" & the
matrix of an "anion exchanger" as a non-ionized
"polycation".

MCEE 5210_97
Cation Exchangers
Active ions (counter ions) are cations.
The polar groups attached to the matrix are acidic (sulphonic acids, carboxylic
acids, phenols, phosphoric acids) e.g. a cation exchanger in the free carboxylic acid
form:
X-COO- H+
X = Frame work (matrix)
-COO- = Fixed charge (anionic), Non-exchangeable
H+ = Counter ion (cation), Exchangeable

Ion exchange process is generally reversible e.g in the following:

2 Na+ + Ca++ 2Cl - → Ca++ + 2 Na+ Cl –
The cation exchanger could be exhausted after exchanging all Na+
for Ca++, the exchanger could be regenerated (loaded again with
Na+) by contacting it with excess Na+ ions e.g. a solution of NaCl.

MCEE 5210_98
Anion Exchangers
Active ions (counter ions) are anions.
The polar groups attached to the matrix are tertiary or quaternary ammonium
groups (basic).
e.g. Anion exchanger in the quaternary ammonium form:
X. NR3+OH –
X = Framework (matrix)
-NR3 + = Fixed charge (cationic) Non exchangeable
-OH– = counter ion (anion), Exchangeable
A- Inorganic ion exchangers
• Common clays, soils, minerals e.g. zeolites used for "softening water".
B. Organic exchangers
• Polycondensation of phenols, sulpho- & carboxy- derivatives with
formaldehyde.
• Polycondensation of aromatic amines with formaldehyde → anionic exchangers.
• These techniques yield products linear in structure & relatively soluble in water
which are now replaced by resin materials based on styrene divinyl benzene
copolymers and polyacrylate.

MCEE 5210_99
Applications of Ion Exchange Chromatography
1- Water softening:
Removal of Ca2+, Mg2+ & other multivalent ions
causing hardness of water by filtration through a layer
of strong cation resin.
2-Water demineralization:
Removal of cations & anions dissolved in water. Usually
carried by the two step technique in which two
columns of strongly acid cation exchanger in [H+]
form & strongly basic anion exchanger in [OH-] form
are used in sequence.
3- Neutralization:
Cationic exchanger in [H+] can be used to neutralize
alkali hydroxide & anionic exchanger in [OH+] form
to neutralize the acidity.

MCEE 5210_100
 High performance liquid chromatography (HPLC)

MCEE 5210_101
 High performance liquid chromatography (HPLC)

MCEE 5210_102
 High performance liquid chromatography (HPLC)

• High Performance Liquid Chromatography (HPLC) is a form of

column chromatography that pumps a sample mixture or analyte
in a solvent (known as the mobile phase) at high pressure through
a column with chromatographic packing material (stationary
phase).
• The sample is carried by a moving carrier gas stream of helium or
nitrogen.
• HPLC has the ability to separate, and identify compounds that are
present in any sample that can be dissolved in a liquid in trace
concentrations as low as parts per trillion.
• Because of this versatility, HPLC is used in a variety of industrial
and scientific applications, such as pharmaceutical,
environmental, forensics, and chemicals.

MCEE 5210_103
 High performance liquid chromatography (HPLC)

Instrumentation:
• Main components in an HPLC system include the solvent
reservoir, or multiple reservoirs, a high-pressure pump, a
column, injector system, the detector and waste.

MCEE 5210_104
 High performance liquid chromatography (HPLC)

• The reservoir holds the solvent, which is referred to

as the mobile phase because it moves. There are
usually a minimum of two reservoirs in a system,
with each holding up to 1000 cc of solvent and
usually fitted with a gas diffuser through which
helium can be bubbled.
• A pump is used to generate a specified flow of the
mobile phase.
• Although manual injection of samples is still
possible, most HPLCs are now fully automated and
controlled by computer.

MCEE 5210_105
 High performance liquid chromatography (HPLC)

• The injector, or auto sampler, introduces the solvent into

a phase stream that carries the sample into the high
pressure (up to 400 bar) column, which contains specific
packing material needed to effect separation. The packing
material is referred to as the stationary phase because it is
held in place by the column hardware.
• A detector is needed to see the separated compound
bands as they elute from the high pressure column.
• The information is sent from the detector to a computer
which generates the chromatogram.
• The mobile phase exits the detector and is either sent to a
waste, or collected, as desired.

MCEE 5210_106
 High performance liquid chromatography (HPLC)

For a given mobile phase, at a given flow rate with a given column,
a known pure standard of acrylamide elutes at 2.85 minutes.
Whenever a real sample is injected that contains acrylamide, you
will see a peak at 2.85 minutes.
MCEE 5210_107
 High performance liquid chromatography (HPLC)

How much is present is measured by the AREA under the peak, which is related to how
much was there. Both samples contain acrylamide, however, Sample B has only 1/10 the
concentration.

MCEE 5210_108
Types of HPLC:

• Normal–phase chromatography.
• Reversed-phase chromatography.

Hydrophilic Interaction
Chromatography (HILIC),
similar to NPLC

MCEE 5210_109
 High performance liquid chromatography (HPLC)

Normal–phase chromatography

• Normal–phase chromatography was one of the first kinds of

HPLC that chemists developed. Also known as normal-phase
HPLC (NP-HPLC) this method separates analytes based on
their affinity for a polar stationary surface such as silica
• NP-HPLC uses a non-polar, non-aqueous mobile phase
(e.g. Chloroform), and works effectively for separating
analytes readily soluble in non-polar solvents. The analyte
associates with and is retained by the polar stationary phase

MCEE 5210_110
 High performance liquid chromatography (HPLC)

Reversed-phase chromatography:

• Reversed phase HPLC (RP-HPLC) has a non-polar

stationary phase , moderately polar mobile phase. One
common stationary phase is a silica which has been
surface-modified with RMe2SiCl, where R is a straight
chain alkyl group such as C18H37 or C8H17. With such
stationary phases, retention time is longer for molecules
which are less polar, while polar molecules elute more
readily (early in the analysis). An investigator can increase
retention times by adding more water to the mobile phase

MCEE 5210_111
Application of HPLC

1. Pharmaceuticals industry
• To control the drug stability
• Quantity of drug determination from pharmaceutical dosage forms, ex.
Paracetamol determination in panadol tablet
• Quantity of drug determination from biological fluids, ex: blood glucose
level

2. Analysis of natural contamination

• Phenol & Mercury from sea water

3. Forensic test
• Determination of steroid in blood, urine & sweat.
4. Clinical test

MCEE 5210_112
 Take home message
• Chromatography is a very versatile separation process, particularly for
– Mixtures of close-boiling compounds that are expensive to separate by
distillation or crystallization (e.g. xylenes, glucose-fructose)
– Thermally sensitive compounds that cannot be distilled or crystallized
(many biological products, natural extracts, flavors, etc.)
• Many process variations have been developed
• High recovery (>99%) and high purity (>99.9%) can be achieved
• Cost depends on process scheme and whether sorbent (stationary phase) and
eluent (mobile phase) can be reused
– For quality control reasons used sorbent and eluent are often discarded in
the pharmaceutical industry

MCEE 5210_114