ASSIGNMENT ON

Stationary Phase
Course name: Advanced Pharmaceutical Analysis
Course code: PHRM 409


SUBMITTED TO:
Dr. Shamsun Nahar Khan
Associate Professor
Department of Pharmacy
East West University


SUBMITTED BY:
Samiya Khondaker Rinta
ID: 2010-3-70-048




Submission date: 9
th
June, 2014


Chromatography
Chromatography is a method by which a mixture is separated by distributing its components
between two phases. It consists of a large group of separatory methods in which the components
of a mixture are separated by the relative attraction of the components for a stationary phase (a
solid or liquid) as a mobile phase (a liquid or gas) passes over the stationary phase (Karger,
2014).
Stationary Phase
The stationary phase is a porous solid (e.g., glass, silica, or alumina) that is packed into a glass or
metal tube or that constitutes the walls of an open-tube capillary. The stationary phase remains
fixed in place while the mobile phase carries the components of the mixture through the medium
being used. The stationary phase acts as a constraint on many of the components in a mixture,
slowing them down to move slower than the mobile phase. The movement of the components in
the mobile phase is controlled by the significance of their interactions with the mobile and/or
stationary phases. Because of the differences in factors such as the solubility of certain
components in the mobile phase and the strength of their affinities for the stationary phase, some
components will move faster than others, thus facilitating the separation of the components
within that mixture (Karger, 2014). Below are discussions of some stationery phase used in
HPLC.
Silica Based Stationery Phase
Some silica based stationery phases are discussed below with their structure and names of the
compounds that they separate.
1) Octadecyl-bonded phases: Amongst the numerous base-deactivated ODS phases obtained by
increasing the bonding density or/and by efficient endcapping treatments, some particular
stationary phases have been developed, to limit the additional interactions of basic compounds
with residual silanols, to work at extreme pH or with rich water mobile phases (Lesellier et al.,
2006).

i. Octadecyl bonded, end capped silica

Figure: Octadecyl bonded, end capped silica
ii. Octyl bonded, endcapped silica

Figure: Octyl bonded, endcapped silica
Compounds separated by these: For reversed phase extraction of nonpolar to moderately polar
compounds, such as end capped silica antibiotics, barbiturates, benzodiazepines, caffeine, drugs,
dyes, essential oils, fat soluble vitamins, fungicides, herbicides, pesticides, hydrocarbons,
parabens, phenols, phthalate esters, steroids, surfactants, theophylline, and water soluble
vitamins (Lesellier et al., 2006)..
2) Butyldimethyl bonded, endcapped silica

Figure: Butyldimethyl bonded, endcapped silica
Compounds separated by this: Less hydrophobic than LC-8 or LC-18 and for extraction of
peptides and proteins.


3) Phenyl bonded silica

Figure: Phenyl bonded silica
Compounds separated by this: Slightly less retention than LC-18 or LC-8 material. For
reversed phase extraction of nonpolar to moderately polar compounds, especially aromatic
compounds.
4) Cyanopropyl bonded, endcapped silica

Figure: Cyanopropyl bonded, endcapped silica
Compounds separated by this: For reversed phase extraction of moderately polar compounds,
normal phase extraction of polar compounds, such as aflatoxins, antibiotics, dyes, herbicides,
pesticides, phenols, steroids. Weak cation exchange for carbohydrates and cationic compounds.
5) Diol bonded silica

Figure: Diol bonded silica
Compounds separated by this: For normal phase extraction of polar compounds.
6) Aminopropyl bonded silica

Figure: Diol bonded silica
Compounds separated by this: For normal phase extraction of polar compounds, weak anion
exchange for carbohydrates, weak anions, and organic acids.

7) Quaternary amine bonded silica with Cl

counterion:

Figure: Quaternary amine bonded silica with Cl counterion
Compounds separated by this: For strong anion exchange for anions, organic acids, nucleic
acids, nucleotides and counterion surfactants. Capacity: 0.2meq/g (Arbro Pharmaceuticals Ltd.,
2012).
Aluminum Oxide as Stationary Phase

Figure: Aluminum Oxide
Compounds separated by this: Columns packed with alumina were combined with common
anion-exchange columns and applied to the ion chromatographic determination of sulphate in
brines and biological fluids and for the trace determination of iodide in mineral waters and fruit
juice samples. Alumina was found to be a highly selective stationary phase for the
preconcentration of sulphate from complex matrices (Buchberger and Winsauer, 1989).
Cellulose as Stationary Phase

Figure: Cellulose
Compounds Separated by this: A hydrophilic interaction liquid chromatography (HILIC)
stationary phase of cellulose-coated silica was synthesized as a novel saccharide separation
material. The material, prepared with a method based on ionic interaction, was demonstrated to
be efficient for immobilization of saccharides on silica supports. The method is more efficient
than traditional immobilized saccharide stationary phase synthesis methods (Sheng et al., 2013).
Ion Exchange Resins


Figure: Ion Exchange Resins

Compounds Separated by this: Synthetic organic resins are normally employed for separation
or water soluble ionizable compounds. Anion exchangers have positive centres on surface and
are used to separate compounds having sulfonate, phosphate or carboxylate groups. Cation
exchangers have negative centers on the surface and are used to separate basic substances such
as amines. Cross-linked styrene divinylbenzene is typical base material with charged groups
linked to phenyl rings. Charges on packing material attract oppositely charged molecules from
mobile phase and release them in inverse order of the attraction forces. Separation of
components can be controlled by control of pH of mobile phase, temperature, ionic composition
and addition of modifiers (Sainio and Tuomo, 2005).
Stationery Phase used in other Chromatographic Applications
Apart from HPLC there are many other types of chromatographic processes. Below are examples
of some stationery phases used in other processes.

Stationary phase Mobile phase Application
Paper
chromatography
pentane, propanone and
ethanol
3:1:1 mixture (by
volume) of butan-
1-ol:ethanol:0.880
ammonia solution.
separating amino
acids and anions,
RNA fingerprinting,
separating and testing
histamines, antibiotics
Paper
chromatography
pentane, propanone and
ethanol
3:1:1 mixture (by
volume) of butan-
1-ol:ethanol:0.880
ammonia solution.
separating amino
acids and anions,
RNA fingerprinting,
separating and testing
histamines, antibiotics
Paper
chromatography
pentane, propanone and
ethanol
3:1:1 mixture (by
volume) of butan-
1-ol:ethanol:0.880
ammonia solution.
separating amino
acids and anions,
RNA fingerprinting,
separating and testing
histamines, antibiotics
Liquid
chromatography
inert solid such as silica
gel (SiO2.xH2O),
alumina (Al2O3 xH2O.)
or cellulose supported in
a glass column
organic solvents Used to analyze metal
ions and organic
compounds in
solutions. test water
samples to look for
pollution,







References
Arbro Pharmaceuticals Ltd. (2012) High Performance Liquid Chromatography: Types of
Stationary Phases Available at: http://lab-training.com/landing/free-hplc-training-
programme-6/ [Accessed 6
th
June, 2014].
Buchberger, W. and Winsauer, K. (1989) Alumina as stationary phase for ion chromatography
and column-coupling techniques. J Chromatogr; 482(2):401-6.
Karger, B. L. (2014) Separation and Purification: Chromatography Available at:
http://www.britannica.com/EBchecked/topic/108875/separation-and-
purification/80490/Chromatography#ref619602 [Accessed 6th June, 2014].
Lesellier, E., West, C. and Tchapla, A. (2006) Classification of special octadecyl-bonded phases
by the carotenoid test Journal of Chromatography A; 1111(2006):6270.
Sainio and Tuomo (2005) Ion-exchange resins as stationary phase in reactive chromatography
Lappeenranta University of Technology. Available at:
http://www.doria.fi/handle/10024/31211 [Accessed 6
th
June, 2014].
Sheng, Q., Ke, Y., Li, K., Yu, D. and Liang, X. (2013) A novel ionic-bonded cellulose
stationary phase for saccharide separation. J Chromatogr A; 1291:56-63.