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, can‘t be fully eluted in an isocratic elution, or can’t achieve satisfactory separation effect in isocratic elution. To sum up, gradient elution can be used in the following situations:

1. The polarity range of the multi-component sample is large, and the sample can’t 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. The separation cannot be achieved by isocratic elution.


After the target peak comes out, it will increase in a short time

Organic phase ratio, elution strongly retains interfering impurities

When detecting samples with complex components, there are often some strongly retained substances. Using isocratic, the strongly retained impurities can’t be eluted. No matter how long one injection is collecting, unknown impurities will interfere with subsequent sample injections. This kind of in some cases, gradient elution can be added to solve the problem by eluting strongly retained components on the gradient.

Isocratic first and second injections, retention time is 19 minutes
There is no interfering peak after the peak disappears

From the third injection, there is interference from unknown impurity peaks,
It should be the strongly retained substance of the first injection that peaks in the third injection

Starting at 30 minutes, increasing gradient elution,
To wash away strong retained impurities, continuous sample injection, no interference

It can be seen from the above three pictures that when this traditional Chinese medicine is eluted with isocratic, due to the strong retention of unknown impurities, the continuous injection will produce peaks in the subsequent injections, which will interfere with the detection. After the component to be tested has peaked out, increase the gradient and increase the proportion of the organic phase, and the strongly retained substances will be eluted in the same needle, and the effect can be completely reproduced by continuous sampling confirmation, and the influence of unknown interference peaks will not appear in the subsequent sampling.


Situations where separation cannot be achieved with isocratic elution

Some substances, especially those with similar structures, cannot be separated under isocratic conditions, but the gradient mode can achieve separation due to the selectivity difference of the components in the organic phase change. In this case, gradient conditions are required for testing. .


  2,3,6-Trichloropyridine 2,3-Dichloropyridine            2,6-Dichloropyridine

Using isocratic elution, three substances had two peaks,
Unable to achieve separation of the three components at the same time

Using gradient elution, the three substances can be completely separated,
Meet testing needs

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