Gradient optimization is a very important step in the development of liquid chromatography. Sometimes, a suitable gradient elution procedure can play a role of “four or two thousand pounds” in the separation of liquid chromatography. However, many small partners mistakenly believe that gradient optimization is very complex because they have done relatively few gradient optimization experiments. Today, Xiaobian will take you to uncover the mystery of reverse phase liquid chromatography gradient program and explore the gradient optimization.

1. Reasons for establishing gradient method.

Why use gradient elution?

Of course, as analysts, if we can achieve our separation goal under an equal condition, we will be happy, but the actual situation is often not as we want. Among the components of mixed samples, there must be components that are easy to separate and difficult to separate, and components that are weak and strong to retain.

The purpose of establishing the gradient elution procedure is to separate the easily separated components in the mobile phase with weak elution ability as soon as possible, while the stronger components stay in the chromatographic column for a long time, interact fully with the surface of the bonded phase, pull apart as far as possible, and finally elute with the mobile phase with strong elution ability to avoid a long elution time.

2.Elution capacity of mobile phase in reversed phase liquid chromatography system.

Since the elution capacity of mobile phase was mentioned earlier, how to judge the elution capacity of mobile phase in reversed-phase chromatography?

We can take a look at the following figure

The above figure should be familiar to all friends. The principle is that in the reverse phase system, as the polarity of the mobile phase continues to increase (the proportion of the aqueous phase continues to increase), the overall elution capacity of the mobile phase gradually decreases, while the retention time gradually increases. Therefore, for the components that are easy to be separated in the previous article, we can try to separate them in the environment of large mobile phase polarity.
At the same time, in order to improve the resolution of the mixed components of the whole sample, a high proportion of water phase is also used as the starting condition of gradient elution as far as possible in the whole gradient program. Therefore, for many friends, when developing a new reverse phase condition method, the most commonly used initial gradient mode is to carry out a linear elution of 95% aqueous phase → 100% organic phase within 0~40min.

3. Central idea of gradient optimization for reversed phase liquid chromatography.

The central idea of gradient optimization of reversed-phase chromatography is to reduce the elution capacity of mobile phase as much as possible, so as to reduce the co elution. The most effective way to weaken the elution capacity of the mobile phase is to replace the solvent with weaker elution strength, or increase or decrease the additives in the mobile phase.
However, in many cases, we carry out experiments according to the established standards, and it is inconvenient to adjust the type of solvent and the amount of additives too much. In such cases, there is another way to adjust the elution intensity of the mobile phase, that is, to slow down the change slope of the mobile phase. Let’s illustrate with the following case:

System suitability solution:
Impurity d+ impurity e
Domperidone + droperidol

From the above figure, we can see that under the condition of gradient (I), domperidone and impurity D are co eluted, and the peaks overlap. So we adjust the gradient program:

System suitability solution:
Mixed standard of 8 substances

In gradient (II), the proportion of acetonitrile is 45% at 10min, while the proportion of acetonitrile is 60% at 10min under gradient (I), and the proportion of acetonitrile does not continue to rise to 100% in the last 7min in gradient (II), which makes the proportion of acetonitrile lower than gradient (I) at every moment in gradient (II), and the elution intensity of mobile phase is weaker than gradient (I) at every moment, as shown in the following figure. In this way, the co elution of impurities is improved, and the result is that as shown in Figure 3, all 8 substances are separated.

4. matters needing attention.

When carrying out gradient optimization of method development, we must pay attention to the compatibility, including the compatibility between mobile phase solvents and samples.
As for the choice of mobile phase solvent, it is better to choose a solvent that can be miscible in any proportion. If the mobile phase solvent is not convenient to replace, it is necessary to slow down the change rate of the mobile phase as much as possible and give the organic phase and the aqueous phase (salt phase) more time to dissolve each other.
For the sample, the properties of the mobile phase must be compatible with the sample solvent to avoid the insoluble and precipitated samples at any time after injection.

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