Understanding and characterizing complex compounds is essential for drug discovery and development. Chromatography is a vital technique for separating, identifying, and purifying components in a sample.
What is Chromatography?
The wide range of chromatographic techniques share one common aim: to separate a material into its components. A material, your sample, is dissolved in a solvent, called the mobile phase. This mixture is then passed through a material called the stationary phase. Chromatography works because different components in a sample interact differently with the mobile and stationary phases; it takes longer for some parts of the mixture to pass through than others, enabling their separation.
Types of Chromatography You Should Know
High-Performance Liquid Chromatography (HPLC)
One of the most commonly used chromatography techniques in biopharma is HPLC. It separates compounds in a liquid sample using high-pressure pumps to push the mobile phase through a tightly packed column. Variants include:
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Reverse phase (RP-HPLC) - separates a mixture based on the hydrophobicity of its constituents.
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Ion exchange (IEX) - separates components based on their net surface charge.
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Size exclusion (SEC) - separates molecules based on their size.
HPLC is used as a purification step to prepare samples for further analysis or application, or as an analytical endpoint. HPLC techniques can be coupled with a variety of detection methods, typically based on the absorbance, emittance, and scattering of light.
Affinity Chromatography (AC)
Affinity chromatography purifies proteins based on specific, reversible interactions between a target protein and a ligand attached to the stationary phase. This allows selective protein binding, while other components are washed away.
The protein is then eluted by changing conditions (e.g., pH or adding a competing ligand). AC is highly selective and often used as a first purification step, commonly using tagged proteins, like His- or GST-tagged molecules.
Gas Chromatography (GC)
Gas chromatography separates compounds based on their volatility and affinity for a stationary phase, typically a liquid or solid material fixed within a column. The mobile phase is a gas, normally high-purity helium, nitrogen, or hydrogen. Liquid samples are vaporized at temperatures up to 350 °C before being carried through the stationary phase by the flow of the mobile phase. Components are characterized by a detector following separation, often a flame ionization detector that ionizes molecules as they elute, generating a change in current which can be amplified and translated into a chromatograph.
Thin Layer Chromatography (TLC)
In TLC, the sample is applied near the bottom of a flat plate coated with a thin layer of adsorbent material such as silica gel. The plate is placed in a solvent, which rises by capillary action, carrying the compounds with it. Different compounds travel at different rates, allowing them to be separated and visualized. TLC is relatively quick, simple, and cost-effective.
Why is Chromatography Important in Biopharma?
Purity, potency, and reproducibility are critical in pharmaceutical research and production. Chromatography is used at every stage, from early discovery to quality control in commercial manufacturing.
Some of the ways chromatography supports the drug development pipeline include:
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Protein purification - Essential for isolating therapeutic proteins such as monoclonal antibodies.
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Impurity profiling - Detecting and quantifying trace impurities in drug substances.
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Bioanalytical assays - Measuring drug concentrations in biological fluids during pharmacokinetic studies.
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Process monitoring - Ensuring consistency during the production and purification of biologics
Cytiva is a global supplier of chromatography resins and provides extensive support for scientists at each stage of the drug discovery pipeline. For comprehensive guides to a range of chromatographic techniques download free handbooks from Cytiva.
Watch video: What sets a high-quality chromatography resin apart? From performance to scalability, learn what to look for in chromatography column resin to ensure consistent and high-quality results.
