CHROMATOGRAPHY III

High Performance Liquid Chromatography

Prof Derick Carboo

Sunday, March 31, 2013

HPLC : High performance liquid chromatography

analytical chemistry to : separate, identify, and quantify compounds based on their polarities and interactions with the stationary phase.
It uses high pressure provided by pumps to force the

liquid eluent and analyte through a closed column.

Sunday, March 31, 2013

HPLC : High performance liquid chromatography

HPLC utilizes a detector that provides a characteristic retention time for the analyte. The detector may also provide other characteristic

information (i.e. UV/Vis spectroscopic data for analyte if so equipped).

Analyte retention time varies depending on :

1. The strength of its interactions with

the stationary phase, 2. the ratio/composition of solvent(s) used, 3. the flow rate of the mobile phase.
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HPLC:
1. 2. 3.

Essential components of HPLC equipment

4.
5. 6.

Solvent reservoir(s) with degasser Solvent delivery system: pump system that can deliver up to 400 atm. pressure Sample injection system Column system (with guard column and oven) Detector to indicate retention time etc. Recorder/computer/display

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An HPLC. From left to right: A pumping device generating a gradient of two different solvents, a steel enforced column and an apparatus for measuring the absorbance.

Sunday, March 31, 2013

HPLC

Schematic representation of an HPLC unit. (1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4) Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position", (6') Switching valve in "load position", (7) Sample injection loop, (8) Precolumn (guard column), (9) Analytical column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Sunday, March 31, 2013Waste or fraction collector.

Solvent
Solvents used for HPLC must be pure and clean (spectroscopic

grade solvent), so as not to damage the column. Solvent must be filtered and degassed. Gas bubbles can interfere with the pump, column or detector. Degassing is achieved by either boiling, purging with helium or sonation using ultrasound ELUOTROPIC SERIES In adsorption chromatography the solvent competes with solute for adsorption sites on the stationary phase. The relative ability of a solvent to displace a given solute from a given stationary phase is independent of the solute but depends on the adsorptive strength of the solvent.

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ELUOTROPIC SERIES The Eluotropic series ranks eluents by their relative abilities to

displace solutes. The eluent strength is the solvents relative ability to displace solutes from a given stationary phase. It is also a measure of adsorption energy In normal phase chromatography, the more polar the solvent, the greater its eluent strength.
By convention n-pentane (C5H12) has been given the ranking

0.00 and all others are relative to it: Example : for alumina
n-Pentane o Cyclohexane o CHCl3 Acetone Ethanol Ethanoic acid = = = = = = 0.00 0.04 0.4 0.56 0.88 large

Sunday, March 31, 2013

Solvent
Gradient elution is the gradual increase of the eluting power (

or polarity) either in discrete steps or continuously during the chromatography. This can be achieved by using more than one solvent, the composition of which changes during the development of the chromatogram. In gradient elution the component solvents in various proportions are mixed in a mixing chamber before getting to the column. Changes in the mixture strength is computer controlled. The polarity can also be influenced by addition of inorganic salts, buffers, acids etc. More than one pump are needed for gradient elution. In Isochratic elution polarity or composition of eluent does NOT change during the chromatography.

Sunday, March 31, 2013

COLUMN SYSTEM

Column length 5 10 cm. Internal diameter 1 5 mm. 4.5mm is standard. Megabore columns have larger internal diameter ( 10mm), have higher capacity but lower sensitivity. Particle size : small ( 10m), spherical and uniform. Small particle size allows for increased rate of mass transfer because distance through which solute diffuses in both phases is reduced. (C-term is reduced). Spherical and uniform particle size reduces

multiple path (A term). Very fine particles create back pressure, hence use of pump to achieve reasonable flow rate
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COLUMN SYSTEM
Particle Size for HPLC column packing refers to the

average diameter of the packing particles. Most HPLC packings contain a narrow range of particle diameters. Particle size affects the back-pressure of the column and the separation efficiency. Column back-pressure is inversely proportional to the square of the particle diameter: when particle size is halved, pressure increases by a factor of four. Example: A well packed column with 3 m packings produces almost twice the separation efficiency of a comparable 5 m column. However, the 3 m column will have about a three-fold higher back-pressure compared to the 5 m column when operated with the same mobile phase and at the same flow rate.
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COLUMN SYSTEM The column length (L) and internal diameter (d)

determine the bed volume (V) of an HPLC column by the following equation: V = L x d2/4 This equation does not account for the reduction in liquid volume due to the packing material. It is an upper limit on the column volume the minimum volume of mobile phase required to elute an unretained analyte from the column. Small column diameters provide higher sensitivity than larger column diameters for the same injected mass because the concentration of the analyte in the mobile phase is greater.
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COLUMN SYSTEM HPLC columns are closed . The tubing are relatively

narrow. This is to prevent voids (i.e. dead volume) that can cause band broadening. Between tubing and column is porous titanium frit to provide uniform flow. Guard column : its role is to collect impurities in the solvent and sample that would otherwise damage the column. Guard column contain same column material and is shorter (1 2 cm long). Must be replaced from time to time.

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COLUMN SYSTEM

Substrate Materials : 1. Micro-porous silica particles (diameter 3 -10m and surface area up to 500m2/g) are the most common substrate material used for HPLC column packings. Silica-based columns can withstand high pressures, are compatible with most organic and aqueous mobilephase solvents, and come in a wide range of bonded phases. Silica based substrate can withstand solvent and sample pH range of 2 to 7.5. The silica surface can be modified as bonded phase.

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COLUMN SYSTEM
2. Polymeric Substrate material : Highly cross-linked styrene-divinylbenzene based packings are compatible with most mobile phase solvents and samples with a pH of 1 to 14. Polymer-based columns have lower mechanical strength and therefore cannot withstand the highest system backpressures. A polymer-based packing is a good alternative, if the sample requires a mobile phase pH outside the normal operating range of standard silica-based columns. Polymer-based columns tend to have lower efficiencies for small molecules compared to silica-based columns due to their smaller average surface area. Polymeric packings are often used for ion-exchange separations, and are also useful in non-aqueous GPC size-exclusion analyses and ion exclusion analyses of organic acids and carbohydrates.
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COLUMN SYSTEM

HPLC Columns allows for all Modes of Separation: 1. Normal-Phase Chromatography : or NP, is the classic form of liquid chromatography using polar stationary phases and non-polar mobile phases. The analyte is retained by the interaction of its polar functional groups with the polar groups on the surface of the packing. NP is useful in the separation of analytes with low to inter-mediate polarity and high solubility in low-polarity solvents. Water-soluble analytes are usually not good candidates for normal-phase chromatography.

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COLUMN SYSTEM

2. Reversed-phase chromatography, or RP, has stationary phase non-polar and the mobile phase polar. The analytes are attracted to the surface by their non-polar functional groups. The most polar analyte elutes from the RP column first followed by other analytes in order of decreasing polarity. RP chromatography is useful for the separation of compounds having high to intermediate polarity.
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COLUMN SYSTEM

3. Ion-exchange chromatography separates analytes by their ionic functionality. Ion-exchange stationary phases are usually comprised of ionic species attached to the surface of the silica substrate or ionic functional groups evenly distributed throughout a polymeric media. Weak ion-exchange phases are usually pH dependent. Strong ion-exchange phases are always charged and therefore independent of typical pH changes. Ion-exchange columns with low capacity are used for ion chromatographic applications where low ionic strength mobile phases are required for conductivity detection.
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COLUMN SYSTEM

4. Affinity chromatography is based on specific interactions in a lock-and-key paradigm between analytes and matrix-bound ligands. Dependent on the secondary structures of biological macromolecules for retention of selected sample components, affinity chromatography is without question the most highly specific, and consequently the most powerful, mode of chromatography.

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COLUMN SYSTEM

Bonded Phase column: A stationary phase which is covalently bonded to support particles or to the inside wall of the column tubing. The most popular support used is microparticulate silica gel. The stationary phase is covalently attached to the silanol group of the silica gel (Si-OH). An organosilane of the type Cl-Si(CH3)2R is used: Example: Cl-Si-(Me)2-C8H17 Cl-Si-(Me)2-CH2-CH2-CH2-CN By using (Cl)2Si(CH3)R there can be cross linking. The R-group is known as the Modifying group:
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Bonded Phase column

non-polar groups

(CH2)3NH2 - (CH2)3CN polar groups - (CH2)2OCH2CH(OH)CH2OH

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 Endcapping:

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 DETECTOR FOR HPLC

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 CHOICE OF MODE OF SEPARATION by HPLC

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