ASSIGNMENT ONStationary PhaseCourse name: Advanced Pharmaceutical AnalysisCourse code: PHRM 409

SUBMITTED TO:Dr. Shamsun Nahar KhanAssociate ProfessorDepartment of PharmacyEast West University

SUBMITTED BY:Samiya Khondaker RintaID: 2010-3-70-048

Submission date: 9th June, 2014

ChromatographyChromatography 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 PhaseThe 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 silicaii. Octyl bonded, endcapped silica

Figure: Octyl bonded, endcapped silicaCompounds 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 silicaCompounds 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 silicaCompounds 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 silicaCompounds 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 silicaCompounds separated by this: For normal phase extraction of polar compounds.6) Aminopropyl bonded silica

Figure: Diol bonded silicaCompounds 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 counterionCompounds 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 OxideCompounds 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: CelluloseCompounds 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 ApplicationsApart 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 chromatographypentane, propanone and ethanol3:1:1 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 chromatographypentane, propanone and ethanol3:1:1 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 chromatographypentane, propanone and ethanol3:1:1 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 chromatographyinert solid such as silica gel (SiO2.xH2O), alumina (Al2O3 xH2O.) or cellulose supported in a glass columnorganic solventsUsed to analyze metal ions and organic compounds in solutions. test water samples to look for pollution,

ReferencesArbro Pharmaceuticals Ltd. (2012) High Performance Liquid Chromatography: Types of Stationary Phases Available at: http://lab-training.com/landing/free-hplc-training-programme-6/ [Accessed 6th 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 6th 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.