Types of Liquid Chromatography

strip of paper after liquid chromatography

Photo Credit: Natrij

In high school, did you ever participate in the experiment where you put a dot of black ink on a strip of paper, dip the end in a solvent and watch as the ink moves up the strip of paper, separating into different colors? If you’ve ever completed this popular experiment, you already have an idea of what liquid chromatography is. Liquid chromatography is a materials testing technique that is used to separate a sample into its individual components, just like the ink in the experiment separated into different colors.

The process that inspired this school experiment originated in 1901 when Russian botanist Mikhail Tswett was researching chlorophyll and pigments in plants. Today there are several types of liquid chromatography that are performed in a variety of industries. Used for much more than testing ink samples, liquid chromatography is commonly used for environmental analysis, food analysis, quality control, and cleanliness testing.

Liquid Chromatography Testing Types and Applications

The basic function of chromatography is to separate organic and inorganic samples to the particulate and single cell level for the purpose of measuring and identifying those particulates. The elements of the experiment are:

  • The mobile phase is the catalyst material
  • The stationary phase, which holds the sample and is passed over by the mobile phase. This can be liquid or solid
  • The sample

When you use a piece of paper and pen-ink for chromatography, the ink is the sample, the piece of paper is the stationary phase, and the liquid solvent is the mobile phase which acts as the catalyst for the separation of the dyes. As the ink separates, you can observe the different colors of the dye components within the ink.

The analysis of different types of materials calls for the use of different types of liquid chromatography. The type of analysis to be used is decided based on the physical state and other known characteristics of the sample.

Types of liquid chromatography include:

  • Liquid-solid chromatography
    Describing both normal and reverse phase chromatography, the stationary phase is a column made up of alumina or silica based compound that allows the liquid mobile phase to absorb or pass through it.
  • Normal phase chromatography
    This method is used to measure non-polar samples by using a solid polar stationary phase and non-polar liquid solvent. The polarity difference results in the least polar compounds separating first, and the most polar compounds separating last. The separation occurs using gravity to pull the compounds apart.
  • High-performance liquid chromatography Normal phase chromatography is also used in the liquid-liquid chromatography method called high pressure liquid chromatography, or HPLC, which also has a polar and non-polar stationary and mobile phase. HPLC uses pumps to pass pressurized liquid solvents and samples through columns filled with solid adsorbent materials. HPLC is a popular method for urine testing for performance enhancing drugs, purity testing in pharmaceuticals and cleanliness testing in manufacturing.
  • Reverse phase chromatography
    Opposite of normal phase polarity, reverse phase uses a polar liquid mobile phase and non-polar stationary phase to separate the most polar compounds, followed by compounds with lower polarity. Reverse phase chromatography is also used for HPLC.
  • Flash chromatography
    Instead of relying on gravity to separate the compounds, an inert gas like Nitrogen is used to push the liquid mobile phase through the solid stationary phase at a much faster rate than gravity in normal or reverse phase chromatography. This process uses precise pumps or vacuums and can be more expensive. It is sometimes known as medium pressure chromatography because it uses less pounds per square inch than HPLC.
  • Partition chromatography
    A liquid-liquid method, partition chromatography requires the stationary phase and mobile phase to be immiscible, or to separate into components when they combine like gasoline in water.
  • Ion chromatography
    Also known as ion exchange, ion chromatography separates the ionic compounds within a solution onto a solid stationary phase by their positive or negative ionic exchange.This method is completed using HPLC.
  • Size exclusion chromatography
    Separating compounds according to the size of their molecules, size exclusion chromatography passes a liquid mobile phase through a solid stationary phase. Compounds within the sample separate as they are caught through the precise pore sizes within the silica or polymer column.
  • Affinity chromatography
    Affinity chromatography separates a sample based on its molecular bonds. A mobile phase with a specific metallic bonding property is used to pass the sample through an agarose polymer or porous glass stationary phase, where the bonding molecules, the ligands, are caught and separated from those that do not bond, the analytes.
  • Chiral chromatography
    This method divides non-superimposable mirror image molecules, known as chiral molecules. While symmetrical like a mirror image, chiral molecules are not interchangeable and can be selectively separated from a solution that is racemic, or containing the opposing chiral molecules, based on one of the chiral bonds existent in the stationary or mobile phase. This is used mostly in biochemistry.

These methods take up to several hours to complete at the longest, a much shorter experiment duration than the Russian scientist Tswett would have experienced. Conducting these tests is important for manufacturers who need to ensure that their production methods are not creating any unexpected contaminants. For instance, the Environmental Protection Agency and Motorola collaborated to use liquid chromatography to ensure ionic cleanliness and create circuit production methods that would stand up to safety and cleanliness standards.

Liquid chromatography has many useful applications that extend beyond the high school classroom. Contact Innovatech Labs to learn how liquid chromatography testing can help your business ensure product cleanliness, conduct environmental analysis and more.

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