A Few Essential Tips to Understand for Achieving Liquid-Phase Separation

After receiving a small-scale test result, if you are uncertain about how to scale up or if the preparation results do not meet the expected requirements without understanding the reason, can you really claim that you understand how to prepare liquid phases? In the following discussion, let’s explore some knowledge about preparing liquid phase separation targets together.

As we all know, preparative chromatography is aimed at separation, enrichment, and purification for further use. To achieve effective preparative separation, the following information is usually required to be understood:

Everyone knows that preparative chromatography is aimed at separation, enrichment, and purification for further use.

In order to perform effective preparative separation, the following information is typically required:

1. Composition

How many components are there in the sample;

What are the target components for separation (must be clear);

2. Purity

What is the required purity of the target component.

For preparative separation, there is usually a certain purity requirement for the product. In other words, not all separations require the product purity to reach 100%. The target component only needs to meet the specific purity requirements for our scientific research or production. For example, for trace natural products, enriching them to a content of about 70% is sufficient for preliminary identification; for samples used in NMR and IR testing, a purity of about 95% is sufficient to meet the requirements; for standard substances used in quantitative analysis, the content must be at least 99% or above; and some biologically active substances only need to remove harmful components, and their purity is expressed not in terms of content but in terms of activity. Only by understanding the purity requirements of the product can we establish appropriate methods for preparative separation.

3. Complexity

What are the interfering substances (as much as possible to understand, to facilitate the pretreatment of complex samples);

4. Properties

The physical or chemical properties of the sample (chemical structure, solubility, acidity and alkalinity, detection method for compounds).

The physical or chemical properties of the target component (same as above);

For the target component, if it is an unknown compound, it is necessary to understand what functional groups it roughly has, such as hydroxyl, carboxyl, amino, phenyl, nitrogen-containing heterocyclic, etc. The ultimate goal is to understand the polarity (logP value) and acidity and alkalinity (pKa value) of the compound. These two parameters often determine the types of stationary phase and mobile phase in the development of preparative liquid chromatography methods.

5. Value

Whether the raw material is readily available or expensive;

What is the value of the target substance being separated?

Only by mastering these preliminary information of the sample can we establish a targeted separation method, choose suitable stationary phase and mobile phase, select an analytical column (such as a 4.6mm*250 analytical column with preparative-grade packing of 10μm) for method exploration, and optimize the separation method.

The steps to optimize the separation method can be summarized as follows:

1. Choose the optimal conditions for high performance liquid chromatography separation.
2. Explore preparative separation methods under analytical equipment conditions.
3. Scale up to preparative separation and conduct separation.
4. Collect fractions.
5. Analyze the purity of the product using analytical liquid chromatography, and improve separation conditions if necessary.
6. Merge identical components.
7. Optionally, further purify the product.
8. Collect the target substance.

You may wonder: why not directly explore the conditions on a preparative column? This is because when exploring conditions directly on a preparative column, the scale becomes larger. If the method conditions are not suitable and the desired purity cannot be achieved, it can result in waste of samples and reagents. Over time, it can also cause damage to the preparative column. It should be noted that based on the methods explored on an analytical column, it is best to choose a preparative column with the same brand of packing material as the analytical column to avoid the inability to reproduce the successful method on the preparative column

The following image shows several WelPackerDAC dynamic axial compression column products from Welch Materials for small-scale and medium-scale trials.

Preparative columns with different inner diameters have their own sample loading ranges. If the sample loading is too small, the elution efficiency will be reduced; if the sample loading is too high, the sample may be overloaded in the column and may not be effectively separated from impurities and directly eluted out. It should be noted that most preparative chromatography operates under conditions of moderate overload, and under this condition, the column efficiency decreases exponentially. Therefore, it is not cost-effective to pursue smaller particle size packing materials for preparative chromatography columns, as the cost of 5μm packing materials is much higher than that of 10μm packing materials.

The service life of a preparative chromatography column mainly depends on the quality of the column, the nature of the sample, and the cleanliness of the column. For high-value DACs, extending the column life is important. Generally, DACs need to be equipped with preparative inline filters to prevent particulate matter from blocking the preparative column.

The following figure shows several common preparative inline filters from Welch Materials, the dimensions are 20mm, 47mm, and 100mm, respectively:

Welch Materials Inc. provides professional method development and customized overall preparative chromatography solutions to meet complex exploration and business needs, helping you discover new methods to overcome operational and data challenges.