Sri Noegrohati, UGM 1

Selectivity and Capacity

Optimization: LLC
Sri Noegrohati
Fac. Of Pharmacy
Gadjah Mada University
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Variables to be Considered
Separation selectivity measured as , is varied by interaction
between the solute and the chromatographic system
(stationary phase and mobile phase).
Major Interaction in Liquid Chromatography
Partitioning
Surface Adsorption
Ion exchange
Size exclusion

Capacity factor, k', can be optimized by changing solvent
composition to provide a larger or smaller k' values for a given
sample
n
S
V
S
k= = K
n
M
V
M
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Partitioning Interaction
Liquid-liquid (LLC) or partition chromatography involves a liquid
stationary phase and moving liquid phase which must be
immiscible
Sample molecules distribute between the mobile and stationary
liquid phases, just as in liquid-liquid extraction within a
separatory funnel.
Separation of solutes are based on the difference of their
relative solubility
Thermodynamic partition coefficient :
K
P
= a
Z,S
/a
Z,m
At low concentration, such as in chromatography:
K
P
= [Z
S
]/[Z
M
]
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Separation Based on Partitioning interaction
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LLC Development
LLC development led to its application to a wide variety of
sample types:
water-soluble
oil-soluble
ionic
Nonionic
Two basic mode of operations:
Normal phase: Polar stationary phase and non-polar
mobile phase
Reverse phase: Non-polar stationary polar phase and
mobile phase
Normal phase was first described but reverse phase is now
more popular
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LC Instrumentation
Useful Liquid Stationary Phases:

Polar phases
Amino R = (CH
2
)
3
NH
2

Cyano R = (CH
2
)
3
CN
Diol R = (CH
2
)
2
OCH
2
CH(OH)CH
2
OH ]

Non-Polar phases
Octadecyl (C
18
) R = (CH
2
)
17
CH
3

Octyl (C
8
) R = (CH
2
)
7
CH
3

Phenyl R = (CH
2
)
3
C
6
H
5

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LC Instrumentation
Liquid-liquid chromatography (LLC)
Stationary phase - viscous liquid coated onto or
chemically bound to exposed silanol groups of
silica (solid support)
Si OH + Cl Si R
CH
3

CH
3
Si O Si R
CH
3

CH
3
Toluene, 110C
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Sri Noegrohati, UGM
LC Instrumentation
Normal-Phase LC
Polar stationary phase
Non-polar mobile phase
Order of elution-non-polar polar
Solvent strength-non-polar (weak), polar (strong)
Reversed-Phase LC (RPLC)
Non-polar stationary phase
Polar mobile phase
Order of elution-polar non-polar
Solvent strength-non-polar (strong), polar (weak)
Most common form of chromatography
Principle: Like-dissolves-like
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Rule of thumb in chromatographic system selection:
Likes dissolved likes
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Interac
tions
The total
interaction of a
solvent
molecule with
an analyte
molecule is the
result of the
four
interactions

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Dispersion and Dipole Interactions
Dispersion interaction or attraction exists between every
pair of adjacent molecules.
X possesses a temporary dipole moment will polarize
the electrons in adjacent molecule S the temporary
excess of electrons on the side of X nearer to S repelling
the electrons in S electrostatic attraction of X and S.
Dipole Interactions
When both the solvent and sample molecule possess
permanent dipole moments strong interaction results by
alignment of the two dipoles in a linear configuration
solvent and analyte molecules having polar functional
groups will selectively interact,
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Hydrogen-Bonding Interactions
between a proton donor and proton acceptor
stronger as the donor is better able to give up a proton and as
the acceptor is better able to accept a proton more acidic
donors and more basic acceptors favor hydrogen bonding
strong donor solvents preferentially interact with and dissolve
strong acceptor analytes, and vice versa.

Compound type Acceptor strength
Aromatic
hydrocarbons
Ethers
Esters Amines
Amides
Sulfoxides
Alcohols
0.0-0.3
0.7-1.2
0.8-1.3
1.5-2.1
2.1-2.4
2.2-2.6
large
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Dielectric Interaction
refers primarily to the interaction of analyte
ions with liquids of high dielectric constant
(e.g., water, the lower alcohols, etc.)
charged sample ion R+ polarizes
molecules of the surrounding solvent,
resulting in electrostatic attraction of R+
and solvent molecules.


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Physico chemical Characteristics of Solvents
commonly used as mobile phase in liquid
chromatography
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Solvent Mixtures
Pure solvents seldom can do the job mixing
Factors to be considered:
Polarity of the mixture
P' =
a
P
a
+
b
P
b
.
Solvent selectivity which
is most readily achieved by
varying the composition of
the mobile phase while
holding solvent strength
constant

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x
d
x
e
x
n
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Example
The greatest change in mobile-phase selectivity results when
the relative importance of the various intermolecular interactions
between solvent and sample molecules is markedly changed.
Correct mobile phase polarity is found to be 20%v
chloroform/hexane having
P' = (0.8 x 0.0) + (0.2 x 4.4)= 0.9
Chloroform belongs to selectivity group VIII
Large change in separation selectivity would be most likely
achieved by groups I and V
Methylene chloride and ethylene chloride belong to group V.
Selecting methylene chloride for its lower volatility and ready
availability, with composition of 26%v CH
2
Cl
2
/hexane giving P'
0.9
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Gradient elution
Sample: Aromatic
Hydrocarbon
Column: ODS 100 x
0.21 cm
Detector: UV 254
Elution:
Isocratic: MeOH :
Water (50:50)
Gradient: MeOH :
Water (40:60) to 100%
MeOH at 8% MeOH


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Additional Methods for RPC
From practical applications, bonded phase
chromatography in Reversed-phase mode (BP-RPC) is
the most popular form of liquid chromatography because
of its broad applicability to neutral compounds of wide
polarity.
In addition:
weak acids and bases can be separated by pH control (ion
suppression chromatography, ISC)
ionic compounds can be separated by using ion pairing with an
additive of opposite charge (ion pair chromatography, IPC);
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ION PAIR
CHROMATOGRAPHY
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ION PAIR CHROMATOGRAPHY
IPC is an effective
chromatographic
technique for separation
of organic ions and
partly ionized organic
analytes.
The ion pair reagent,
usually an alkyl-
sulfonate, an alkyl-
sulfate or an alkyl-
ammonium salt, is
added to the mobile
phase
Since many drugs are basic or acidic, IPC has been applied in pharmaceutical
industry on a routine basis in almost all areas of analytical chemistry where
chromatography is used.
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Example 0f Systems for RP-IPC
Sample Stationary
phase
Mobile phase Counter ion
Amines C
18
H
2
O/CH
3
OH/H
2
SO
4
C
12
H
25
SO
3
2-
Carboxylic acids C
18
pH 7.4 (C
4
H
9
)
4
N
+
Sulfonic acids 1-Pentanol pH 7.4 (C
4
H
9
)
4
N
+

Dyes C
18
pH 2-4 H
2
O/CH
3
OH (C
4
H
9
)
4
N
+

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Supercritical Fluid
Chromatography
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Supercritical Fluid Chromatography
Fluid Critical Temperature
(C)
Critical Pressure
(atm)
CO
2
31.3 72.9
N
2
O 36.5 71.7
NH
3
132.5 112.5
n-Butane 152 37.5
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Examples of LLC applications