When using liquid chromatography for analysis, a component may achieve the desired separation effect through multiple different modes. This provides us with more options in practical applications, allowing us to choose a more suitable analytical method based on the different characteristics and requirements of different samples.

As an example, we will attempt to use three different separation modes, namely “reverse phase chromatography“, “ion exchange chromatography” and “hydrophilic interaction chromatography“, to separate citric acid.

Citric acid (CA), also known as 2-hydroxypropane-1,2,3-tricarboxylic acid or 2-hydroxy-1,2,3-propanetricarboxylic acid, is an important organic acid with a molecular formula of C6H8O7. It is also known as citrate and is easily soluble in water.

Reverse phase chromatography mode

Chromatography column: Welch Organic Acid Analysis Column, Ultimate®OAA (4.6×300mm).

Mobile phase: Gradient elution of phosphate buffer solution and methanol;

Detection wavelength: 210nm;

Column temperature: 30℃;

Flow rate: 0.5 mL/min.

Retention time [min]Peak areaPeak HeightNumber of platesAsymmetryResolution

Ion exchange mode

Chromatography column: Welch Hydrogen Strong Cation Exchange Chromatography Column, Xtimate®Sugar-H (7.8×300mm, 5μm).

Mobile phase: 0.008 mol/L sulfuric acid solution;

Detection wavelength: 210nm;

Column temperature: 30℃;

Flow rate: 0.6 mL/min.

Retention time Peak areaPeak HeightNumber of platesAsymmetryResolution

Hydrophilic mode

Chromatography column: Welch HILIC Column with Zwitterionic Bonded Silica, Ultimate®Amphion-Ⅱ (4.6×150mm, 5μm).

Mobile phase: 0.1 mol/L ammonium acetate solution / acetonitrile = 7/3;

Detection wavelength: 210nm;

Column temperature: 30℃;

Flow rate: 1.0 mL/min.

Retention timeArea HeightArea%Number of theoretical platesTailing Factor


Using Welch Ultisil®OAA (4.6×300mm), Xtimate®Sugar-H (7.8×300mm, 5μm), and Ultisil®Amphion-Ⅱ (4.6×150mm, 5μm) chromatography columns, detection under the corresponding chromatographic conditions can meet the detection requirements. In routine detection or method development, appropriate separation modes can be selected to detect based on the properties of other components to be tested.

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