Gradient elution can be used to deal with samples that cannot be separated by isocratic elution, and the most common factor for using gradients is that multiple components have different retention capabilities, cannot be fully eluted in an isocratic elution, or cannot achieve satisfactory separation effect isocratic in an isocratic manner. To sum up, gradient elution can be used in the following situations:

1. The polarity range of the multi-component sample is wide, and the sample cannot be fully eluted with isocratic;

2. Macromolecular samples;

3. Improve the peak-to-peak shape;

4. After the target peak comes out, increase the proportion of the organic phase in a short period of time to elute strongly retained interfering impurities;

5. When separation cannot be achieved by isocratic elution

#01The polarity range of the multi-component sample is wide, and the sample cannot be fully eluted with isocratic

Multiple components with different retention ranges cannot be completely eluted with the same isocratic. With the same isocratic, there will be some problems that some components cannot be separated without retention, some components with strong retention cannot be eluted, or the elution time of one injection is too long. In this case, gradient elution is selected to make the elution ability of the mobile phase from weak to strong, so that multiple components with different retention value ranges can be separated under the same conditions.

#02 Macromolecular samples

Large molecular weight samples (peptides, proteins, synthetic polymers, etc.) are generally better separated by gradient elution, especially reversed-phase conditions.

#03Improve the peak-to-peak shape;

For some components, when using isocratic elution, the peak shape is not good and can’t meet the detection requirements. In this case, gradient elution can be used to improve the peak shape. Therefore, gradient elution is sometimes used to determine the content of even a single component.

  • Example: Detection of 4-piperidinic acid

Effect diagram of isocratic conditions for 4-piperidinecarboxylic acid, asymmetry 3.84

Retention timePeak areaPeak heightUSPAsymmetryR(USP)

Effect diagram of 4-piperidinecarboxylic acid gradient elution conditions, asymmetry 1.78

Retention timePeak areaPeak heightUSPAsymmetryR(USP)

It can be seen from the comparison chart that the asymmetry of 4-piperidinecarboxylic acid is 3.8 when isocratic elution is used, and the tailing is obvious. After gradient elution, the asymmetry is 1.78, and the tailing appearance is significantly improved.

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