With flash chromatography, you either want to yield a large quantity of product or you want it extremely pure. When both are simultaneously true, a large loading capacity is your best friend. It simplifies the purification of all your crude product in a single run, with high yield and high purity. So, what exactly is loading capacity?

WHAT IS LOADING CAPACITY?

In preparative chromatography, the advantages of large sample loads are unignorable: more sample injected means fewer manipulations needed which in turn means savings in time, sorbent, and solvent.

Sample loading is simply the number of grams of sample divided by the number of grams of adsorbent. In other words, it’s how much product you are putting on your column. Subsequently loading capacity is how much of a sample load the column can take while still yielding effective separations. The column itself, the sorbent used as well as the separation difficulty of the mixture will affect loading capacity.

With that in mind, what is your separation goal? Different goals mean different loading capacities. If you are doing analytical chromatography, purity is the most important factor, so decreasing the sample load will make the separation easier to achieve. By contrast, if you are doing preparative chromatography, your objective is most likely to recover enough product for your next step. Then, loading as much as possible on your cartridge is acceptable. In other words, preparative columns have larger loading capacities than analytical columns.

This is not unlike a BBQ with your family and friends. Based on the type of food, amount of food and size of your grill, one often needs to adjust the cooking to ensure a well-cooked meal. If it’s a small party, you may be able to put everybody’s food on the barbecue at once and still manage proper cooking (i.e. small sample load). If you invited the whole neighborhood, and put all the food on at the same time (i.e. large sample load), there is a high chance that it will take longer to cook and result in uneven readiness. In chromatography equivalents, the distance between the peaks may decrease. This is of course unless you have an extra-large barbecue or decided to cook very small and easily cooked items.

Again, it all comes down to what is your objective with this purification.

WHAT INFLUENCES LOADING CAPACITY?

As mentioned earlier loading capacity is dependent on the sorbent used (charcoal, gas or electric?), the sample being tested (burgers or filet mignon?) and the column itself (your great uncle’s crumbling BBQ or the pretentious neighbor’s new shiny grill?). Let’s look at each parameter.

  • Type of Sorbent

    Bare silica tends to have up to 10 times higher loading capacities than a bonded sorbent. Firstly, because bonding uses adsorption sites on the silica, making them inactive to interact with the sample (thus less surface area available on functionalized sorbent). Secondly, separation mechanism affects loading capacity making it lower for bonded sorbents .

  • Size and Shape of Sorbent

    The chemistry of the sorbent is important, but its size and shape also play a role. To get maximum loading, not only choosing bare silica helps but also decreasing the particle size. A smaller particle size offers increased surface area, giving a higher resolution.

    As for the shape, silica comes in both irregular and spherical form. Spherical silica gel allows to reduce the particle size without increasing backpressure, offering more surface area and, as a bonus, making the packing more homogeneous, thus allowing greater sample load .

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    The application note [SS007-0] where SiliaSep PREMIUM spherical cartridges were used with increasing sample load, and [SSP002-0] where spherical and irregular silicas were compared in terms of sample load explored this phenomenon.

  • Sample

    The sample and the compounds of interest have a role to play. If you only cook sausages on the grill, it is easier to load more than if you have a collection of items. Sample complexity. And this refers to the number of compounds in your sample and the complexity of each compound. For example: if the compounds have similar polarities (complexity of the sample) or if they are ionizable, acidic, basic, etc (complexity of the compounds in the sample). In the end the simpler the sample and compounds, the easier the separation, and the more you can load, reducing the number of runs needed thus saving time, sorbent, and solvent and avoiding hungry and impatient guests (nobody likes when Aunt Catherine is hungry).

    Two variables determine how difficult the separation is, which, in turn, determine how much or how little you should load on your cartridge. Selectivity (α) is measured by the retention factor ratio between two similar compounds, and the difference of column volume (ΔCV) is a measure of two analytes separation and resolution. The larger these numbers, the easier the separation. Optimizing the run conditions, finding the right mobile phase, the right gradient, using modifiers, are all possible ways to increase selectivity and number of column volume. The loading technique also has an influence on this. Indeed, solid sample load may improve separation, making larger loading possible. But more on that in another article.

  • Column

    Going back to our definition, if sample loading is the quantity of sample divided by the quantity of sorbent, more sorbent means more sample. Larger columns mean larger loads. But in terms of size, both length and thickness do matter, not only the total quantity of sorbent. We will discuss that in detail shortly.

HOW TO CHOOSE COLUMN SIZE?

To respect loading capacities, you need to select the right column size.

Too small of a column will result in a poorer separation due to overload, too large of a column is a waste of sorbent and solvent, a waste of time spent on a tedious chromatographic run that did not need to be tedious.

First, you need to evaluate the separation difficulty. You can find ΔCV from your TLC method development knowing that the relationship between retention factor (from your TLC) and CV is inversely proportional.

Then, using the load factor for the sorbent type, you can calculate the cartridge size that you need by dividing the amount of crude by the load factor. Load factors which, once again, depend on sample complexity and stationary phase, are shown in the table 1 below.

Table 1. Load Factor
Irregular Bare SilicaSpherical Bare Silica
Very Easy Separation (ΔCV > 6)9-10 %15-20 %
Easy Separation (ΔCV > 3.8)6-9 %8-15 %
Medium Separation (1.9 ≤ ΔCV ≤ 3.8)3-6 %4-8 %
Difficult Separation (ΔCV < 1.9)1-3 %1-4 %

Example: you have 2.75 g of crude, a very easy separation with ΔCV = 6, and you have SiliaSep PREMIUM spherical cartridges on hand.

Calculation: Using the table above with ΔCV = 6 and spherical gel, the load factor is between 15-20 %. Dividing the amount of crude by the found load factor (2.75 g / 15 % to 3.0 g/ 20 %) gives 13.8 to 18.3 g of sorbent needed. The best cartridge available would be the 25 g pre-packed one (see chart below).

In the above example, the lower end would result in a 12 g cartridge however it may not be recommended since the collected fractions may have lower purity. If high purity isn’t the goal, then overloading the 12 g cartridge could be fine and save you time and money.

If packing your own columns, geometry should also be considered even if the mass of adsorbent remains constant. Will a thin and long column behave the same as a thick and short one?

*Tips

A good chromatography technique is to place the sample as a narrow band on the head of the column. Due to diffusion, the band naturally becomes larger as it elutes. If it was wide to begin with, band broadening will lower the separation efficiency.

A longer column with a smaller diameter gives a higher plate count (there are more plates per column) which means better separation efficiency. On the other hand, a shorter column with a larger diameter, increases loading capacity by allowing the sample to be deposited as a narrow band on top of the silica bed.* This means that, when packing your own columns using bulk sorbent, if larger loading capacity is your goal, aim for thick and short columns rather than long and thin.

Now that you understand loading capacity and what affects it, you’ll next have to put in practice to avoid mistakes. We have discussed how the parameters of your column and your separation affect the loading capacity, and how to optimize it to reach your goals. Next, we will discuss loading techniques and how to achieve more with less stress. I must go now, my tofu is about to overcook and that would be a *misteak*.

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