Reverse phase columns are available in many forms, and it isn’t as simple as choosing the length of your alkyl chain length (C18, C8 or other length). The particle size can be different, its shape can be irregular or spherical and have differing pore size distributions (100 Å, 300 Å, or other sizes). Believe it or not, all the silanol groups aren’t completely saturated with C18 chains as they are so bulky, leaving some silanol groups free and able to interact with compounds. For some stationary phases, these silanol groups are left free, but in other cases they can be end-capped. The end cap can range from a methyl end cap to a polar end cap that creates a C18AQ type column that resists phase collapse in highly aqueous mobile phases. All these variations result in differences in retention of different compounds, offering different selectivity between these stationary phases. These variations in selectivity result in different columns having different resolution for a particular sample and affect calculations for creating focused gradients. Teledyne LABS has several different types of reverse phase columns such as the RediSep Silver C18 (40-60 µ irregular particles, no end-capping) and RediSep Gold C18 and C18AQ (20-40 µ spherical particles, end-capped with standard and AQ type end-capping). As an alternative to C18, Teledyne also produces a wide-pore 300 Å RediSep Gold C8 column, that is useful for the purification of large molecules. (Learn more about the importance of pore size in relation to the size of the molecule here: https://www.teledynelabs.com/en-us/resources/Documents/Application-Notes/Effects-of-Reverse-Phase-Chain-Length-on-Peptide-Purification.pdf.)
Focused Gradient
Both the ACCQPrep and Flash Focus Gradient Generators work best using matching column chemistries. One way to ensure matching column chemistry is to use the same column for both the scouting and focused runs. It is possible to use a small column for the scouting run and then calculate the focused gradient for the larger column.
Focused gradients can be determined from scouting gradients run on analytical instruments too. As is true with any scale-up method, the analytical and preparative columns should use matching media. Figure 1 shows variation in retention of seven compounds run on different C18 columns of the same size, using the same scouting gradient. All the columns showed different selectivity for the scouting gradient. The scouting gradients were calibrated to a Teledyne LABS RediSep Prep C18 column. Due to differences in the columns, the calculated gradients are different, with only the Teledyne LABS RediSep C18 analytical column giving results where all columns could be run as a focused gradient.
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Teledyne LABS
Column 1
Column 2
Column 3 |
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Figure 1-Variation in retention from different C18 analytical columns. Each column was run with the same scouting gradient. All columns were 2 mm ID x 50 mm. |
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Calculated focused gradient |
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Compound |
1 |
2 |
3 |
4 |
5 |
6 |
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Teledyne LABS |
6-16 |
11-21 |
31-41 |
46-56 |
80-90 |
92-100 |
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Column 1 |
4-14 |
11-21 |
31-41 |
46-56 |
78-88 |
92-100 |
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Column 2 |
0-10 |
5-15 |
32-42 |
46-56 |
74-84 |
87-97 |
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Column 3 |
0 |
0 |
32-42 |
46-56 |
79-89 |
95-100 |
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Table 1- Calculated gradients for different compounds using different columns calibrated against a RediSep C18 column |
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Flash Chromatography column differences
A major manufacturer of flash columns and instruments claimed that their columns were better than the competition by picking 7 un-named compounds that ran better on their gradient compared to the competition. Such a comparison doesn’t make sense unless those seven unnamed compounds are products of your reaction. We ran Teledyne’s universal test mix (phenacetin and N-benzylbenzamide) on a RediSep Gold column and the competitor column, with the RediSep column showing better resolution. The Focus Gradient Generator was used to calculate the gradient for the first eluting peak. To make the comparison fair, the different column volumes were considered, such that the focused gradient is slightly different for the competitor column. The RediSep Gold column was closest with respect to the type of C18 packing used by the competition but is not the same material.
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Figure 2- RediSep Gold C18 and a competitor C18 column run with Universal Test Mix. For these compounds, the RediSep column had better resolution. |
In this case, the Teledyne LABS column ran better. If a different selectivity were required, Teledyne also manufactures Gold C18AQ, C8, and RediSep Silver C18. Note that the Universal Test Mix compounds wouldn’t normally be found together as a reaction product, either.
The competition further claimed that the column volume listed for a competitor was incorrect based solely on retention time of a retained compound. This would be a useful measurement only if comparing columns packed with the same reverse phase material. The proper way to measure column volume for a reverse phase column is to use a UV absorbing salt such as any nitrate or iodide salt as the anions absorb UV and yet are soluble enough in 10 to 20% methanol in water to give a good measure of the void or column volume as they do not retain on reverse phase columns. The columns in Figure 1 have differing retention for the same compounds, yet all the columns had the same column volume.
Conclusions
The Focus Gradient Generator should be run on the same column chemistry to avoid incorrect results when calculating a focused gradient. As Teledyne manufactures matching columns for analytical UHPLC, preparative HPLC, and flash chromatography, it is easy to get great focused gradient results no matter which instrument you use. Also, competitors will post information that is incorrect- verify the claims of your vendor, and test columns with your own samples!