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AbstractPreparative chromatography occasionally requires solvent that absorbs UV light to allow resolution of compounds from their impurities. One example is the use of toluene in normal phase chromatography to resolve compounds with aromatic character via pi-pi interactions with the solvent. The UV absorption of the solvent blocks detection of the compounds being purified, requiring all fractions to be collected and analyzed. The use of reference spectra allows deconvolution of the UV-absorbing solvent and detection of the desired peak and impurities. Solvent spectra deconvolution using a similarity index allows detection of compounds whose absorbance overlaps commonly used solvents such as ethyl acetate and dichloromethane. Deconvolution of interfering solvent absorbance also allows the use of less expensive solvents, such as acetone replacing either ethyl acetate for normal phase chromatography, or acetonitrile for reverse phase purifications. An example of a synthetic mixture purified using such deconvolution is presented.

BackgroundThere are many solvents that are useful in chromatography from their elutropic properties, yet are rarely used because they adsorb UV light. Some solvents may be less expensive to use. Some examples include the replacement of acetonitrile1 or ethyl acetate2 by acetone, a less expensive solvent. Acetone is also considered a “green” solvent3. Another solvent with unique properties is toluene, which allows solvation and elution of compounds containing aromatic rings. The use of toluene allows a different selectivity compared to a solvent such as dichloromethane, but toluene also strongly absorbs UV light. Many compounds contain chromophores which overlap the absorbance spectrum of ethyl acetate, another commonly used chromatography solvent, rendering detection difficult with UV detection.

The use of UV absorbing solvents can make a baseline which drifts with a gradient, reducing the ability to collect peaks based on the level of UV absorbance. One approach of reducing interference from solvent absorbance is to subtract the solvent spectrum from the baseline, but this doesn’t work well when the solvent absorbance saturates the detector. We find the use of Similarity Indices, also used to determine compound purity4 , work well to resolve compounds from the solvent background. Basically, the solvent spectrum is measured prior to the run. During the run, absorbances which do not have the same adsorption “shape” as the solvent absorbance are deemed to be compounds to be fractionated. Previous methods used to detect compounds over a wavelength range used a “sliding window” which avoided the need to measure the solvent spectrum prior to the run; however peaks wider than the window width would show a sufficiently similar spectrum to that measured at the start of the window as to reset the baseline prior to complete peak elution5.

References1 Funari, C. S.; Carneiro, R. L.; Khandagale, M. M.; Cavalheiro, A. J.; Hilder, E. F. Journal of Separation Science. 2015, 38 (9),

1458–1465.

² Acetone as an Alternative to Ethyl Acetate in Flash Chromatography. www.teledyneisco.com/en-us/liquidChromatography/Chromatography Documents/Application Notes/Acetone as an Alternative to Ethyl Acetate in Flash Chromatography App Note.pdf. Retreived 22 Feb 2018.

³ Shen, Y.; Chen, B.; Beek, T. A. V.  Green Chemistry. 2015, 17 (7), 4073–4081.

4 Silver, J.E. Lewis, R.L. Information rich flash chromatography II: All-wavelength collection and purity measurement. Presented at the 245th ACS meeting general poster session, New Orleans, April 10, 2013, poster 355.

5 Silver, J.E. All-Wavelength Detection: A New Detection Technique for Medium Pressure Liquid Chromatography Suitable for Natural Products. Presented at the American Society for Pharmacognosy meeting, July 2009.

6 Fenwick, O.; Sprafke, J. K.; Binas, J.; Kondratuk, D. V.; Stasio, F. D.; Anderson, H. L.; Cacialli, F. Nano Letters. 2011, 11 (6), 2451–2456.

7 Purification of C60 Fullerene using Non-aqueous Reverse Phase with RediSep Rf Gold® Columns. www.teledyneisco.com/en-us/liquidChromatography/Chromatography Documents/Application Notes/Purification of C60 Fullerene Using Non-aqueous Reverse Phase App Note.pdf. Retreived 22 Feb 2018.

8 Funari, C.S.; Carneiro, R.L.; Khandagale, M.M.; Cavalheiro, A.J.; Hilder, E.F. J. Sep. Sci. 2015, 38 (9).

ConclusionsBaseline correction using a similarity index allow detection and collection of compounds using chromatography solvents that absorb light in the same wavelengths as the compounds being collected. This extends the range of solvents that may be used, and increases the opportunities for a successful purification with the different selectivities from the alternative solvents. In this poster, toluene allowed collection and detection of aromatic compounds using toluene. Toluene, due to interaction with the π-bonds on the compound being purified, allowed a different purification mode than other solvents. The baseline correction allowed the use of a gradient to improve the chromatography or reduce the time needed to purify the compound.

Baseline correction also allows the use of less expensive solvents, solvents which create lower backpressure, or more benign solvents to be used. Acetone was used here for reverse phase, but acetone also makes a useful replacement for ethyl acetate in normal phase as well. Baseline correction also works with traditional solvents, such as ethyl acetate, allowing detection of compounds which are normally hidden due to a limited wavelength absorption range.

Jack E. Silver (Jack.Silver@Teledyne.com), Chester Bailey, Deen Johnson, Steve Paeschke, and Ronald LewisTeledyne ISCO, Lincoln, NE, USA

Improved UV Detection of Peaks During Elution with Absorbing Solvents

ExperimentalSolvents are all ACS grade, purchased from Midland Scientific. The flash system was a modified CombiFlash Rf+ system run with CombiFlash RediSep® Gold columns (Teledyne ISCO, Lincoln, NE, USA).

Other details are provided in each section of the results and discussion.

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Results and DiscussionToluene as a Mobile Phase in Normal PhaseToluene is used in mobile phases to purify compounds containing aromatic rings. This solvent dissolves such compounds via pi-pi interactions between the aromatic groups on the molecule and the toluene. Tetraphenylporphyrin (TPP) is used as a model for other similar compounds. Porphryrins have been used to create diodes and molecular wires for organic electronics6. TPP was synthesized by heating 20 mL propionic acid containing 0.7 mL benzaldehyde to reflux. Pyrrole (0.7 mL) was added dropwise and the mixture refluxed 30 minutes. The mixture was cooled and placed in a separatory funnel containing water to remove the propionic acid; dichloromethane was added to yield a purple solution in the dichloromethane which was evaporated to yield 1.3 g solid. RediSep Rf TLC plates (Teledyne ISCO, PN 69-2203-400) were used to determine the porphryrins eluted at about 50% toluene in hexanes prior to the run. The runs below each used 0.025 g of crude solid which was dissolved in dichloromethane (2 mL) and adsorbed on 1 g silica gel (Teledyne ISCO, PN 60-3874-091). This was run on a 4 g RediSep Rf Gold silica column (Teledyne ISCO, PN 69-2203-344) with a hexane/toluene gradient (45–55% toluene, linear gradient) for 15 column volumes. Peak collection was at 260 nm, and All-Wavelength detection was monitored from 200 to 400 nm. All Wavelength Collection uses the signal from the entire detector within the selected range and averages them to create a composite signal. All-Wavelength detection may be used if the detection wavelength isn’t known for a compound.

Figure 1–Purification of TPP with a toluene gradient with baseline correction (left) and without baseline correction (right).

Toluene in Reverse PhaseAnother example where toluene is used as the mobile phase is the purification of fullerenes using non-aqueous reverse phase7. Soot extract (0.025 g) was dissolved in carbon disulfide and loaded onto a 5 g C18 solid load cartridge (PN 69-3873-237) which acted as a pre-column for the 30 g RediSep Rf Gold C18 column (PN 69-2203-335). A gradient from 20 to 30% toluene in 2-propanol was run using baseline correction at a wavelength of 310 nm. The column was washed with 100% toluene after the gradient to remove other compounds. The first collected peak is C60 fullerene, while the second is mostly C70.

Figure 2–Purification of fullerenes using non-aqueous reverse phase.

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Acetone in Reverse PhaseAcetone has some advantages for reverse phase due to its solvent strength. It is a stronger solvent than acetonitrile, allowing less polar compounds to elute from the column and extends the range of reverse phase chromatography compared to methanol or acetonitrile. Acetone is considered “greener” than acetonitrile because it is less toxic and biodegradable8. The back pressure is lower than that of methanol allowing faster flow rates. It’s boiling point is lower than that of other solvents (Table 1), so it is more easily evaporated from purified fractions. The price of acetone is much lower than acetonitrile, but with similar chromatographic behavior. The use of baseline correction allows the use of acetone even where the absorbance of the compound overlaps that of acetone.

Figure 3–Physical characteristics and costs of common reverse phase solvents.

Figure 4–Purification of crude capsaicin on reverse phase using acetone in the mobile phase with baseline correction (left) and without (right).

For each run, 20.0 mg of crude capsaicin dissolved in DMSO was injected into a 15.5 g RediSep Rf Gold C18 column (PN 69-2203-334). The column was run from 40 to 80% acetone in water. Detection was at 280 nm for both runs. The left chromatogram used baseline correction while the right chromatogram used no baseline correction; the peaks are barely visible within the solvent absorbance.

Solvent Price/ Lt, USD Boiling Point (°C)

Methanol $11.41 65

Acetonitrile $48.60 82

THF $46.61 66

Acetone $17.59 56

Ethyl Acetate $31.33 77

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