How to Optimize TLC to Enhance Purification by Flash Chromatography

In this article I discuss the optimization of solvent ratios to generate ideal Rf (retention factor) values on TLC plates.  Then I show how maximizing efficiency of flash chromatography achieves higher loading with rapid and reliable isolation of compounds, reduced solvent use and improved separation.

In a previous post I explored why choosing flash chromatography solvents based on their separation selectivity is important for effective purification of reaction products.

Here we will look at how to increase loading on a chromatographic column without compromising on effectiveness of purification.

Although a compound is resolved from the other compounds in the mix by TLC, its retention factor (Rf) and mass-transfer kinetics may not be optimal for good purification. That is because compound retention and mass-transfer kinetics are related to separation efficiency and, therefore, loading capacity.

When optimizing flash purification for component retention, TLC again is the most efficient tool for evaluating solvent ratios. Using the chosen solvent (or solvents) from the selectivity study, ratios of those solvents should be adjusted to get the targeted compound to an Rf of between 0.1 and 0.4, especially for isocratic purification. Rf, or retention factor, is the ratio of the distance a compound has moved on TLC divided by the distance the solvent traveled. Rf values range from 0 to 1.

Rf = compound migration distance/solvent migration distance

In flash chromatography, retention is expressed in column volumes (CV). A column volume is the space in a column or cartridge not occupied by the stationary phase.

It just so happens that the number of CV required to elute a compound isocratically using the solvent system used on TLC equals 1/Rf, quite simple! For more on this relationship please see page 24 of the Flash Purification Consumables brochure. With this CV information you can determine the separation quality just by subtracting two adjacent compounds’ 1/Rf or CV values. This generates a value known as delta CV (ΔCV) from which the load amount is determined from a reference table in the same brochure.

ΔCV = 1/Rf2 – 1/Rf1


ΔCV = CV2 – CV1

These four TLC runs highlight the impact of the strong solvent content on compound retention
These four TLC runs highlight the impact of the strong solvent content on compound retention. From left – 10% EtOAC, 20% EtOAC, 30% EtOAc, 40% EtOAc.  Higher EtOAc percentages decrease compound retention resulting in larger Rf values but lower delta CV values.

As an example, a 5-component mixture was separated on TLC using 10%, 20%, 30%, and 40% ethyl acetate in hexane. As seen in the photo above, compound migration distances and Rf values increase with the % of ethyl acetate. Based on the Rf range criteria mentioned previously (Rf 0.1-0.4), only the 10% and 20% ethyl acetate runs show a separation with suitable Rf values.

10% 20% 30% 40%
 Migration distance
1 43 42 44 41
2 20 24 31 41
3 8 16 26 33
4 3 11 20 27
5 2 8 15 22
Solvent Front (SF) 52 50 51 46
Retention Factor (Rf)
1 0.83 0.84 0.86 0.89
2 0.38 0.48 0.61 0.89
3 0.15 0.32 0.51 0.72
4 0.06 0.22 0.39 0.59
5 0.04 0.16 0.29 0.48
Column Volumes (CV)
1 1.21 1.19 1.16 1.12
2 2.60 2.08 1.65 1.12
3 6.50 3.13 1.96 1.39
4 17.33 4.55 2.55 1.70
5 25.00 6.25 3.40 2.09
1-2 1.39 0.89 0.49 0.00
2-3 3.90 1.04 0.32 0.27
3-4 10.83 1.42 0.59 0.31
4-5 8.67 1.70 0.85 0.39

Even having suitable Rf values, and therefore CV values, will not always translate into a good separation unless there is a suitable ΔCV. It is important to understand that the larger the ΔCV, the greater the loading for a particular mixture.

From the data above, we see compound 3 has suitable Rf values of 0.15 and 0.32 in 10% and 20% ethyl acetate, respectively. Though the Rf values convert to CV values of 6.5 and 3.13, we need to look at how well separated compound 3 (our target in this case) is from compounds 2 and 4. What we see from the table above is at 10% ethyl acetate, compound 3 has a ΔCV of 3.90 vs. compound 2 and ΔCV 10.83 vs. compound 4. In 20% ethyl acetate however, the ΔCV values are much smaller (1.04 and 1.42, respectively) which does limit our loading capacity, refer to the flash loading table in the link (page 27). Loading is based on the smaller of the ΔCV values. The table tells us that with a ΔCV of 3.9 we should be able to load between 100 and 500 mg on a 10g silica cartridge but with a ΔCV of 1.04, we are limited to less than a 100 mg load.

In the chromatograms below, isocratic flash runs with 10% and 20% ethyl acetate with a 100 mg loading confirms the load predictions based on ΔCV to be accurate. In 20% ethyl acetate we have poor separation between all of the peaks while in 10% we achieve a complete separation, especially for peak 3.

Flash separation at 10% EtOAc fully resolves each compound at a 100 mg load.
Flash separation using a 10 g Biotage(R) SNAP cartridge at 10% EtOAc fully resolves each compound at a 100 mg load.


Flash separation of 100 mg load at 20% EtOAc shows the cartridge is overloaded.
Flash separation of 100 mg load at 20% EtOAc shows the 10 g cartridge is overloaded due to the low delta CV between compounds.

So, just obtaining a separation on TLC is not enough to take that method to flash; you need to get your targeted compound in the proper Rf range.

Try this and share your results.

Published by

Bob Bickler

Technical Specialist, Biotage

4 thoughts on “How to Optimize TLC to Enhance Purification by Flash Chromatography”

  1. Hello,
    First of all, I want to thank you for your very very very helpful blog. I found the answer to many of my questions here.
    I also have a question. Does the conversion of Rf to CV (TLC to flash column) only apply to flash chromatography or it is also used for simple column chromatography with no pressure on the solvent?

    1. Hello Zary,

      First, thank you for your complement on the blog. I am happy you have found it helpful.

      Your question is a good one. The answer is yes, the same Rf to TLC conversion rules apply whether the chromatography is open column (gravity), automated flash, or even normal-phase HPLC.

  2. Could you be so kind in sending us a sample copy of the article to relation flash column chromatography to TLC.
    Best regards.
    Sincerely your´s:

    Jorge Hernán Prieto
    INTI – Rubber Research Center


    1. Hi Jorge,

      Many thanks for reading the blog, I hope you have found it useful. I believe you can print any of the post just by right-clicking on the article then clicking print.

      If there are other posts you wish to read on the same topic please take a look at the following…

      Invest 10 minutes and save a day of grief
      Using TLC to scout flash chromatography solvents
      How do I choose the right column size

      Please let me know if you need something else.

      These three also explore the TLC/flash column relationship.

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