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Laboratory Operations

Chromatography

Chromatography is one of the most versatile and powerful separation sciences we have. If electrophoresis is a molecular racetrack, then I want you to think of chromatography as a bustling, crowded shopping mall on a weekend. The goal is to get from the entrance to the exit. Some people (our molecules) are focused and walk straight through without stopping. Others get distracted; they stop to look in a shop window, chat with a friend, or buy a pretzel. Because of these different interactions, a big crowd of people who all entered at the same time will naturally spread out and exit at different times

This is the brilliant principle behind chromatography. It is a technique designed to separate a complex mixture into its individual components by taking advantage of the different ways each component “interacts” with its surroundings. It’s the ultimate tool for untangling a chemical mess, allowing us to isolate and quantify substances with incredible precision

Two Key Players: Stationary and Mobile Phases

Every single chromatographic method, from a simple paper strip to a multi-million dollar instrument, is defined by two key players:

  • Stationary Phase: This is the “shopping mall” itself—the shops, the food court, the benches. It is the part of the system that does not move. It is a solid or a liquid coated onto a solid support, and it is specifically designed to interact with the molecules we want to separate. The chemical nature of the stationary phase determines what kind of “shopping” our molecules will do
  • Mobile Phase: This is the “crowd” that pushes everything forward toward the exit. It is the part of the system that does move. It is a liquid or a gas that flows continuously over or through the stationary phase, carrying our sample mixture along with it

The magic of separation happens in the competition. Each molecule in our sample is constantly choosing between interacting with the stationary phase (stopping to shop) or moving along with the mobile phase (getting pushed by the crowd). Molecules that have a high affinity for the stationary phase will be held back and travel slowly. Molecules that have a low affinity for the stationary phase (or a high affinity for the mobile phase) will spend more time moving and will travel quickly. The result is a beautiful separation, with the fastest molecules exiting first and the slowest molecules exiting last

Major Types of Chromatography in the Clinical Lab

We can mix and match different mobile and stationary phases to create various types of chromatography, each with a specific purpose. For the clinical lab, two major categories dominate

High-Performance Liquid Chromatography (HPLC)

This is the undisputed champion of clinical chromatography. In HPLC, the mobile phase is a liquid, and the stationary phase consists of microscopic, tightly packed particles inside a stainless-steel column. The “High-Performance” part comes from the fact that we use high-pressure pumps to force the liquid mobile phase through this densely packed column. This results in incredibly fast and highly efficient separations

  • How it works: A tiny volume of the patient sample (e.g., blood lysate) is injected into the mobile phase stream just before it enters the column. As the mixture travels through the column, the different molecules separate based on their affinity for the stationary phase. They exit the column at different times, one after another
  • Key Application: Hemoglobin A1c (HbA1c): HPLC is the gold standard for measuring HbA1c. It can beautifully separate the stable, glycated HbA1c from normal adult hemoglobin (HbA) and other common hemoglobin variants, providing a highly accurate picture of a patient’s long-term glucose control
  • Other Uses: Therapeutic Drug Monitoring (TDM), measuring vitamins (like Vitamin D), and analysis of catecholamines

Gas Chromatography (GC)

In Gas Chromatography, the mobile phase is an inert gas (like helium or nitrogen), and the stationary phase is a thin liquid film coated on the inside of a very long, very thin capillary column. Because the mobile phase is a gas, GC has one absolute requirement: the sample we want to measure must be volatile, meaning it can be easily vaporized by heat without decomposing

  • How it works: The sample is injected into a heated port, where it instantly vaporizes. The carrier gas then sweeps this vaporized sample into the column. The column itself sits inside an oven. Separation occurs as the different compounds interact with the liquid stationary phase. Less volatile compounds that “like” the liquid phase more will move slowly, while highly volatile compounds that prefer to stay in the gas phase will move quickly
  • Key Application: Toxicology: GC is the definitive method for separating and quantifying volatile substances. Its most common use is in the measurement of alcohols in serum, including ethanol (drinking alcohol), methanol, and ethylene glycol (antifreeze)

Finish Line: Detectors

Chromatography is a separation technique, not a measurement technique. Once the molecules have been separated and exit the column, we still need a way to “see” them. The column outlet is therefore connected to a detector. The two most important combinations are:

  • LC-MS (Liquid Chromatography-Mass Spectrometry): This is the ultimate dream team. HPLC performs the initial, high-resolution separation, and then a Mass Spectrometer serves as the detector, providing a definitive molecular weight and fingerprint for each peak. This is the gold standard for pain management drug panels and newborn screening
  • GC-MS (Gas Chromatography-Mass Spectrometry): The same principle applies here. GC separates the volatile compounds, and the MS detector provides unambiguous identification, which is critical for legal-level confirmation of drugs of abuse

Key Terms

  • Chromatography: A powerful analytical technique for the separation of a mixture’s components by distributing them between a mobile phase and a stationary phase
  • Mobile Phase: The component of the chromatographic system that moves; it is a liquid (in LC) or a gas (in GC) that carries the sample through the stationary phase
  • Stationary Phase: The non-moving component of the chromatographic system that is responsible for the separation by interacting differently with various sample components
  • Analyte: The specific chemical substance or molecule that is being separated and measured
  • Retention Time: The time it takes for a specific analyte to travel from the point of injection, through the column, to the detector. It is a key characteristic used to identify a compound
  • High-Performance Liquid Chromatography (HPLC): A type of chromatography that uses a liquid mobile phase forced under high pressure through a column packed with a solid stationary phase. It is a cornerstone of TDM and HbA1c analysis
  • Gas Chromatography (GC): A type of chromatography that uses an inert gas as the mobile phase to separate compounds that can be vaporized. It is the definitive method for alcohol analysis