#### When it comes to isolating a compound from a mixture at maximum purity there are many options available through flash column chromatography. In previous posts I have addressed using smaller particle media, higher surface area media, and step gradients to achieve this goal.

#### In this post I will discuss how stacking columns in series may help improve separation quality.

Recently, I was asked if there is any real benefit to stacking columns. Many chemists have the perception that the column length to diameter ratio (L/D ratio) plays a role in separating power.

A while ago I wrote a couple of posts asking the question “Does size matter in flash chromatography” where I discussed both media physical properties in one and flash cartridge length vs. diameter in another.

Indeed, chromatographic theory tells us a longer column contains more theoretical plates and more theoretical plates provide a better separation, Equation 1.

N = 5.54(tr/W_{½})² Equation 1

Where N = plates per column

tr = compound retention

W_{½} = compound peak width at 50% of its height

Plate count, or efficiency as it is also known, increases with column length only if peak width increases at a slower rate than compound retention increases, which is not always the case.

In flash chromatography theoretical plates do matter, of course, but only to the extent that you have sufficient selectivity for the separation and use the correct flow rate for the selected column. That being the case, then the loading amount and separation quality should be more about having the correct sample size per gram of media than column dimensions.

To evaluate this theorem I conducted a test where I compared a 30 gram flash cartridge (Biotage® ZIP 30 gram) with a 3 x 10 gram cartridge stack (Biotage ZIP 10 gram), all with the same silica, Table 1. I compared these at equal flow rate and again at equal linear velocity to see where there is a performance difference, if any.

30 gram cartridge | 10 gram cartridge | 3 x 10 gram stack | 3 x 10 gram stack | |
---|---|---|---|---|

Length (cm) | 11.6 | 6.9 | 20.7 | 20.7 |

Diameter (cm) | 2.7 | 2.0 | 2.0 | 2.0 |

L/D ratio | 4.3 | 3.45 | 10.35 | 10.35 |

Flow rate (mL/min) | 60 | 60 | 34 | |

Linear velocity (cm/min) | 15.5 | 27.6 | 15.6 |

First, I used a 30 gram cartridge and purified a 5-component mix targeting the fourth eluting compound. To determine sample load I used my flash system’s automated TLC to linear gradient capability based on a TLC solvent system of 20% EtOAc in hexane. The system suggested my load should be ~250 mg. In Figure 1 you can see the results of this prediction and the resulting chromatography.

Second, I repeated the same method but replaced the 30 gram column with three 10 gram cartridges in series. I maintained both flow rate and sample size, Figure 2.

Last, I used another series of three 10 gram columns using the same gradient profile and load but running at a flow rate at which I achieved a similar linear velocity to the 30 gram cartridge (~15.5 cm/min), Figure 3.

The results show that there is minor separation enhancement for my target compound (fourth peak) and the last compound when the flow rate was maintained (60 mL/min). The peaks do appear a bit more narrow with the column stack.

When the flow rate was reduced to equalize linear velocity to that used with the 30 gram cartridge the impact of the longer column length is seen with sharper peaks and improved resolution.

So, what does this tell us? It tells us that column stacking really only improves your separation if you adjust the flow linear velocity and that L/D ratio alone is not an important factor in separation performance as borne out in the TLC analysis of peak 4, Figure 4.

Have you tried stacking cartridges to improve your tough separations? If so, did it improve your results?

So at reduced flow rate (to equalise the linear velocity) you saw resolution improvements due to reduced band broadening. But isn’t the opposite true? Slower moving mobile phase causes more band broadening and when it moves faster band broadening is limited? Is there something I am missing here? I would be grateful if you could clarify that. Thanks.

Hi Tomasz,

With chromatography there is a relationship between particle size, column diameter, and flow rate. For and specific particle size there is an optimal linear velocity that holds true regardless of column diameter. Smaller diameter particles allow faster velocities. However, if the linear velocity is too fast or too slow, you will increase diffusion and the separation will diminish – to some degree. This is best demonstrated by the van Deempter curve which is a graph of linear velocity vs. HETP (height equivalent to a theoretical plate) for various particle size media.

In the case of the stacked column study, using a flow rate which achieved a suitable linear velocity on the smaller diameter cartridge proved beneficial versus trying to match the 30 gram cartridge flow rate.

Thank you. I was simply under the impression the faster you go through column the less time for diffusion and band broadening. Thanks again for clarifying that.