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High-Performance Liquid Chromatography is a pivotal technique in analytical chemistry, playing a crucial role in separating, identifying, and quantifying compounds across various scientific disciplines. From pharmaceuticals and food analysis to environmental monitoring and forensics, HPLC stands as a cornerstone technique for researchers and scientists. In this blog, we will delve into the fundamentals of separation techniques in HPLC, shedding light on the key principles and processes that make it a fundamental tool for scientific analysis.

The Fundamental Principles of HPLC Separation

HPLC works on the simple principle of separating compounds within a mixture by their interactions with a stationary phase and a mobile phase. The stationary phase can be a solid adsorbent or a liquid-coated surface, while the mobile phase is typically a liquid, either an aqueous or organic solvent. The sample is injected into the HPLC system, where it is carried through a column filled with the stationary phase. Compounds within the sample interact differently with the stationary phase and mobile phase, leading to their separation based on factors like size, polarity, or charge.

Some common separation techniques in HPLC include:

  1. Reverse-Phase Chromatography: This method separates compounds based on their hydrophobicity. Non-polar compounds elute first, while polar compounds are retained longer.
  2. Normal-Phase Chromatography: In contrast to reverse-phase, this technique separates compounds based on their polarity, with polar compounds eluting first.
  3. Size-Exclusion Chromatography: This technique separates compounds based on their size, with larger molecules eluting earlier than smaller ones.
  4. Ion-Exchange Chromatography: Compounds are separated based on their charge and ionic interactions with the stationary phase.
  5. Affinity Chromatography: This method leverages the specific binding affinity of compounds to separate them selectively.

Fig 1. Example of HPLC Separation

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