Chromatography

Gas Chromatography Liquid Chromatography

Gas-liquid Gas-solid Liquid-liquid Liquid-solid Ion-exchange Exclusion

(GLC) (GSC) (LLC) (LSC) (IEC) (EC)

Bonded-phase Ion-pair
Chromatography (BPC) Chromatography

[Categorized based on the type of phases]

General features.
In Chromatogrphy, two mutually immiscible phases are brought in to contact.
One phase is stationary and the other is mobile.
The stationary phase is kept in a column (distributed through the column) and the
mobile phase is passed through the column.
The sample to be separated is introduced into the mobile phase and is carried
through the column, containing the stationary phase
Different chemical species in the sample undergo repeated interactions with the
stationary phase.
Chemical species in the sample are separated in to bands in the column.
At the other end of the column, separated bands emerge in the order of the
strength of interaction (weakly interacting species first).
These chemical species are detected using a variety of methods.

Sample injection Separation into chemical species  Detection of the chemical

species

Categorization based on the type of interactions

Adsorption chromatography
Stationary phase has a high surface area.
Solute is adsorbed on the surface of the stationary phase.
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Mobile phase is gas or liquid.
Eg. Packed columns containing silica gel, chromasorb (porous silicate material)
Separation is based on the strength of adsorption.

Partition chromatography
Stationary phase is bonded to the wall of the column.
Solute is partitioned between stationary phase and the mobile phase.
Mobile phase is gas or liquid
Eg. Tubular columns with open bore (gas chromatography), Columns with bonded
phases (in High performance liquid chromatography)
Separation is based on partition coefficient.

Ion-exchange chromatography
Stationary phases consists of particles of a resin with exchangeable H+ or OH- ions.
Anion exchange-adsorb anions and release OH-
Cation exchange-adsorb cations and release H+
Mobile phase is liquid.
Separation is based on the strength of complexation with stationary phase.

Molecular exclusion chromatography

(Gel filtration, gel permeation chromatography)
Stationary phase has pores of a certain size.
Small molecules enter the pore structure and delayed.
Large molecules elute first.
Mobile phase is liquid.
Eg. Superdex, Sephadex
Separation is based on the size of molecules.

Affinity chromatography
Stationary phase contains covalently bound ligands specific to biomolecules (Eg.
proteins)
Mobile phase is liquid.
Eg. Antibody bound to the wall interact with a specific protein.
Separation is based on the affinity with the stationary phase.

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Terms used in chromatography

Volume flow rate – cm3 min-1

Linear flow rate – cm min-1

𝑉𝑜𝑙𝑢𝑚𝑒 𝑓𝑙𝑜𝑤 𝑟𝑎𝑡𝑒
𝐿𝑖𝑛𝑒𝑎𝑟 𝑓𝑙𝑜𝑤 𝑟𝑎𝑡𝑒
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑎 𝑢𝑛𝑖𝑡 𝑙𝑒𝑛𝑔𝑡ℎ

Chromatogram: Graph showing detector response vs Time

Retention time (tr ) : Time required to reach the detector from the injector

Retention volume: Volume of the mobile phase required to elute a particular

compound

Adjusted retention time (tr/) = Retention time (tr) – time required by the mobile
phase to reach the detector (tm).
/
𝑡𝑟 = 𝑡𝑟 − 𝑡𝑚

Relative retention (𝜶): Used to compare retention of two compounds.

/
𝑡𝑟2 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑 2
𝛼= /
=
𝑡𝑟1 𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 𝑜𝑓 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑 1
Large 𝛼means better separation.

/
𝑡𝑟 −𝑡𝑚 𝑡𝑟
Capacity factor (𝒌/ ): 𝑘 / = =
𝑡𝑚 𝑡𝑚
Relation between capacity factor and partition coefficient:
𝑡𝑖𝑚𝑒 𝑎 𝑠𝑜𝑙𝑢𝑡𝑒 𝑠𝑝𝑒𝑛𝑑𝑠 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒
𝑘/ =
𝑡𝑖𝑚𝑒 𝑎 𝑠𝑜𝑙𝑢𝑡𝑒 𝑠𝑝𝑒𝑛𝑑𝑠 𝑖𝑛 𝑡ℎ𝑒 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒

𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑖𝑛 𝑠𝑡𝑎𝑡𝑖𝑜𝑛𝑎𝑟𝑦 𝑝ℎ𝑎𝑠𝑒

=
𝑚𝑜𝑙𝑒𝑠 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑒 𝑖𝑛 𝑡ℎ𝑒 𝑚𝑜𝑏𝑖𝑙𝑒 𝑝ℎ𝑎𝑠𝑒
𝐶 𝑉 𝑉
𝑘/ = 𝑠 𝑠 = 𝐾 𝑠 Where K is the partition coefficient.
𝐶𝑚 𝑉𝑚 𝑉𝑚

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 𝑉𝑠
𝑘/ = 𝐾
𝑉𝑚
𝑉
/
𝑡𝑟2
/
𝑘2 𝐾2 𝑠 𝐾2
𝑉𝑚
Therefore relative retention: 𝛼 = / = / = 𝑉 = = ratio of the partition
𝑡𝑟1 𝑘1 𝐾1 𝑠 𝐾1
𝑉𝑚

coefficients.

𝐴𝑥 𝐴𝑠
Response factor (F): =𝐹
[𝑋] [𝑆]
Ax = area of the analyte signal
As = area of the internal standard
[X] =concentration of the analyte
[S] = concentration of the standard

Effect of diffusion on peak width:

Due to diffusion, a chromatographic peak broadens as it moves through the
column. Diffusion occurs due to the presence of a concentration gradient. An
infinitely sharp peak broadens by diffusion to form a Gaussian. Concentration
across the Gaussian profile is given by,
−𝑥 2
𝑚 𝑒 4𝐷𝑡
𝐶=
√4𝜋𝐷𝑡
m = moles /unit cross sectional area (mol m-2)
C = concentration (mol m-3)
D = diffusion coefficient (m2 s-1)
t = time (s)
x = distance measured from the center of the band along the column (m)

𝜎 = √2𝐷𝑡 𝜎 = standard deviation of the band (length scale, m)

𝑥 (𝑚)
𝑡= Ux = linear flow rate (m s-1)
𝑈𝑥 (𝑚𝑠 −1 )

Plate height (H):

𝜎2
OR the height equivalent to a theoretical plate 𝐻 =
𝑥

Number of theoretical plates (N):

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 𝐿
𝑁= L = length of the column (m)
𝐻
16 𝑡𝑟2
𝑁=
∆𝑡 2
∆𝑡= width at base in time scale
𝑡𝑟 = retention time

Number of theoretical plates for an asymmetric peak:

2
𝑡
41.7 ( 𝑟 )
𝑁= 𝐴+𝐵
𝐴
+ 1.25
𝐵
W0.1 = A + B = peak width at 1/10 of peak height (time scale).

∆𝑡𝑟 ∆𝑡𝑟 𝐷𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑖𝑛 𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒𝑠

Resolution ( ): =
𝑊𝑎𝑣 𝑊𝑎𝑣 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑤𝑖𝑑𝑡ℎ 𝑎𝑡 𝑡ℎ𝑒 𝑏𝑎𝑠𝑒 (𝑖𝑛 𝑡𝑖𝑚𝑒 𝑠𝑐𝑎𝑙𝑒)

Width at base of a peak = 𝑊 = 4𝜎 where 𝜎 is the standard deviation.

Half width 𝑊1/2 = 2.35 𝜎

Resolution & theoretical plates:

√𝑁 (𝛾 − 1)
𝑅𝑒𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 =
4
N = number of theoretical plates
𝐿𝑜𝑛𝑔𝑒𝑟 𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒
𝛾=separation factor =
𝑆ℎ𝑜𝑟𝑡𝑒𝑟 𝑟𝑒𝑡𝑒𝑛𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒

Band spreading (broadening):

Broadening can happen at injection, in the column, and at detection.
Variance due to each mechanism can be added.
2
𝜎𝑜𝑏𝑠 = 𝜎12 + 𝜎22 + 𝜎32
(∆𝑡)2
Variance due to injection or detection = 𝜎 2 = (time scale)
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Dependence of theoretical plate height on linear flow rate:

Van Deemter equation
𝐵
𝐻 =𝐴+ + 𝐶𝑈𝑥 Where Ux is the linear flow rate.
𝑈𝑥

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A, B, C are constants for a given column.
Packed column: A, B, C all non-zero.
Open tubular column: A =0
Capillary electrophoresis column: A=0 and C= 0
A = Eddy-diffusion parameter, related to channeling through a non-ideal packing
[m]
B = diffusion coefficient of the eluting molecules in the longitudinal direction,
resulting in dispersion [m2 s−1]
C = Resistance to mass transfer of the analyte between mobile and stationary phase
[s]
Ux = linear flow rate [m s−1]