Chromatography lecture notes for 2025, Lecture notes of Biology

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BY 4 5 2

C h r o m a t o g r a p h y

Dr Nigel Brissett

Please read Chapters 44 & 45 ( th Ed): online book via the library. Please read Chapters 50 & 51 ( th Ed): online book via the library.

Please read

Chapters 48

th

Ed): book

in the library.

Remove contaminat es Identify molecule s Estimate concentratio n

Reasons to separate

Chromatography is one of the techniques used in analytical chemistry that can separate a mixture of molecules.

Chroma and graphos come from Greek words meaning colour and to draw, respectively. Different chlorophyll pigments in green leaves

Defined by phases

Chromatography is the process of the separation of molecules between a stationary phase and a moving or mobile phase. Once separated, the molecules can be quantified. This session is about how this process occurs and what factors influence the process.

We place the sample into the mobile phase. As the sample moves with the mobile phase, its components partition themselves between the mobile phase and the stationary phase.

What does it look like?

Planar Chromatography  (^) Can be a layer of paper or gel particles such as silica supported by a glass plate.  (^) Examples: Paper and Thin Layer Chromatography. Column Chromatography  (^) Consists of a tightly packed bead matrix gel.  (^) Examples: Gel Filtration, Ion- Exchange and Affinity. Stationary Phase is fixed and acts as an ADSORBENT

Stationary phase

Stationary Phase is fixed and acts as an ADSORBENT Adsorption process is the interaction of adsorbant molecules with the surface of an adsorbent Adsorbant materials are usually porous. Criteria for adsorbant selection:  (^) Selectivity  (^) Capacity  (^) Chemical and thermal stability  (^) Cost

Stationary phase

Different phases

Chromatography Stationary phase Mobile phase Thin layer Silica, Alumina Solvent Gel-Filtration Sepharose, Sephadex and various polyacrylamide gels Buffers Ion-exchange Gels with charged resins Buffers, salts Affinity Gels with covalently-bound ligands Buffers, salts High performance Silca, C8, C18 Buffer plus solvent Gas-liquid Various greases, gums, resins Gases e.g., helium and nitrogen

Key Facts on Theory

Separation — difference in solute concentration across two the phases and is expressed by its Partition Co- Efficient. Stationary Phase — adsorbent and fixed. Mobile Phase — acts as a carrier for solute.

Beads of stationary phase in column chromatography Hydrophobic Interaction Reversed Phase Multimodal

Gel Filtration Chromatography (GF)

Gel Filtration Chromatography (GF) Exclusion limit is the fixed pore size in a bead of stationary phase.

• (^) Solute molecular weight is greater than the exclusion limit it will elute first.
• (^) Solute molecular weight is less than the exclusion limit it will elute last. Adapted from, SEC Principles and methods, GE Healthcare.

Gel Filtration Chromatography (GF) Large molecule that cannot enter the pores of chromatography beads Target protein that can use a fraction of the pore volume of the beads Salt or other low-molecular- weight substances that can use the entire pore volume of the beads Adapted from, SEC Principles and methods, GE Healthcare.