Does anyone in chromatography give a load about sample load?

Analytical and preparative chromatography differ in many ways and how much sample you can load on the column falls into one of these contentious topics. Analytical chromatographers cringe at the idea of sample overload, preparative chromatographers shrug the problem off. Why? Read on to find out.

I am a big tea lover. I drink it all year long and I especially enjoy it on chilly fall and winter evenings. A few nights ago, I was making a cup of loose-leaf tea, but I was in a hurry to get back to the couch and watch an evening TV program. I had a lot of leaves in the strainer, I lost patience and dumped a lot of water in the mug. The leaves were quickly over saturated, and the water spilled out.

So much for quick and efficient. I had to wipe up the hot mess and then patiently repour more water, this time a lot more carefully.

Coincidentally, recently a graph showing the dependence of column efficiency and column loading in chromatography landed on my desk. I had to chuckle, as it made me think of pouring water in my mug of tea. All might seem fine and dandy but if you are not careful and overload, suddenly you can get in big trouble. If efficiency is something you care about.

Let me give you a little background information.

Loading in chromatography refers to the amount of substance introduced into a column, expressed in grams of substance per gram of adsorbent. It is impossible to put a precise number on how much sample load is too much sample load because the maximum possible loading for base line separation differs for each run and depends on the particular separation factor (α) and retention factor (k’).

Still, it is helpful to consider the relationship of plate height and ultimately column efficiency before you power on your chromatography unit. The general relationship is as follows:

Plate height remains constant up to a particular loading amount and then decreases with an increasing sample load.

The efficiency there is similar to pouring water into my tea strainer. The leaves in the sieve can take a certain amount of water at a certain rate, but if I pour too much, the water spills out and the efficiency decreases. This point of no return is called “darn it” in tea pouring and “the point of inflection” in chromatography.

The dependence of the plate height (H) on the loading (B) is displayed in the graph below:

column efficiency, sample loading, sample overloading, plate height, flash chromatography, preparative chromatography, prep HPLC, chromatography, liquid chromatography

Here, the point of inflection is referred to as the linear capacity B0. This point is defined as the load at which a 10% increase of the plate height (H) occurs.

When you overload the column, there are not enough sites on the stationary phase for the quantity of analyte injected. To put it simply, overloaded columns decrease the retention time, reduce the column efficiency and sacrifice resolution. Take a look at an example of chromatograms with a sample load of 2.5 mg/g silica (left) and 25 mg/g of silica (center) and 50 mg/g silica (right) below.

sample load, sample overload, preparative chromatography, flash chromatography, column efficiency, plate height, resolution, prep HPLC

As an increasing amount of sample is loaded, neighboring peaks begin to overlap as the examples above show. Peak overlap results in material loss, but it enables much more sample to be loaded per run, so that the process is faster and resources can be saved.

Now, in analytical chromatography, the actual loading is lower than the capacity of the packing material (B>B0). Preparative chromatography is a different story.

If I really want to fill my mug right to the top and don’t care for the mess or how efficient my tea making is, then I sure would pour the whole pot of water in there and go about my business.

Just like that, preparative chromatography is nearly always performed with the condition of B > B0, meaning that the stationary phase is saturated.

In preparative chromatography, it is impossible to work with very low loading capacities as the process run times would be substantially increased. In practice, 30 mg of sample load is usually a good starting point for successful separations. Often, much higher values ranging between 100 to 300 mg are loaded. As previously said, the sample size strongly depends the sample constituents and how easily the components can be separated from one another.

As a helpful side note, the loading B0 may be further increased by increasing the separation factor (α) through an appropriate change of mobile phase.

I just noticed that my mind seems to have been preoccupied with overloading topics. My last post was all about how to avoid overloading your condenser in freeze drying. But now that I also discussed overloading in chromatography, I can move on to other topics. I won’t be spoiling the surprise though, you just got to keep dropping by to keep up with the discussion.

Till next time,

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