How to separate very polar substances without destroying your packing material

We like challenges in chromatography and so I’d like to tackle one in this post. If you’ve banged your head trying to figure out how to separate very polar substances without causing irreversible phase collapse, then by all means, read on. I offer you a great stationary phase that can handle high-water content mobile phases needed for separation of polar compounds. I also provide you with practical examples of how to separate very polar vitamins using two different stationary phases.


It happens sometimes that you are at the right place at the right time to witness something tragically hilarious. This happened to me a few days ago. I happened to look out my window to see my neighbor carrying several shopping bags. No big deal, but just then, one of his grocery bags tore apart and his groceries fell all over the sidewalk. I ran out to help him collect a giant cabbage, package of oranges, bananas, kilograms of potatoes and apples. He had overloaded his plastic bag to the point of complete destruction.

But it would be a total tragedy, without any hint of funny, if the same happened to your chromatography performance. What am I talking about? Well, pay close attention, especially if you are trying to isolate very polar substances.

Just like many shoppers use grocery bags from plastic material, many chromatographers use C18 as their preferred column material. Due to its hydrophobic nature, C18 gives very good interactions with non-polar compounds. As polarity of the compounds increases, retention on the C18 material decreases due to reduced interaction with the stationary phase.

To compensate for the reduction in interactions between polar compounds and stationary phase, the content of water in the mobile phase can be increased.

There is a danger to this tactic. When using traditional C18 phases in chromatography , the water content cannot be increased infinitely. This is because water content that is too high will have a negative effect on the material performance. Just like adding more and more potatoes to your plastic bag can damage the material to point of breakage, too much water in the mobile phase can overburden the packing material in your column. In fact, as soon as the water content measures 95% or higher, there is a real risk of phase collapse, as shown in the picture below.

phase collapse, C18, silica, chromatography column, stationary phase

Once the phase has collapsed it is virtually impossible to restore the initial performance of the material.

Even more, when the water content in the mobile phase is too high, there is less interaction possible between polar compounds and the stationary phase in the column. This leads to altered retention times, worse peak shapes and a poor overall performance.

One way my neighbor could have solved his grocery bag problem was to use a bag with different material. A cloth tote would have probably withheld the weight of his fruits and vegetables.

Similarly, chromatographers can try to overcome issues caused by high water content in the mobile phase by choosing a water-stable material for their stationary phase.

For example, C18-AQ, which is made using different bonding techniques, will not cause phase collapse even when the maximal water content (up to 100%) is used in the mobile phase.

I will try to demonstrate the potential of this C18-AQ phase by running a separation of very polar water soluble vitamins like Ascorbic Acid (vit C) and Niacin (vit B3).

molecular structure, vitamin C, vitamin B3, chromatography

If I use traditional C18 columns, I can only use up to 95% water in the mobile phase, which is insufficient for obtaining a full separation of the compounds. In the graph below, you can see that separation of vitamin C and vitamin B3 on such a standard C18 column (95% water and 5% organic solvent in the mobile phase).

C18, column, polar vitamins

Here, you can clearly see that baseline separation cannot be achieved. Increasing the water content to 100% is impossible, as this amount of water will damage the packing material.

If I use a C18-AQ phase instead, I can use 100% water in the mobile phase. With this approach, I can obtain complete separation of the two polar vitamins (see graph below) with the chromatography system.

C18-AQ, polar substances, vitamins, chromatography

Hence, for highly polar compounds, I would recommend using the C18-AQ stationary phase. This will enable you to use 100% water mobile phases and achieve the best separations possible.

I mentioned retention times here and there in this blog post. If you would like to get more details on this particular topic, check out my recent blog post on the retention factor and expand your knowledge. Meanwhile, I hope I can continue to retain your attention as we go on to explore exciting topics in chromatography together. Interested in getting some insights on an issue I haven’t yet touched upon? Leave a comment below and get in touch.

Till next time,

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