Application Note AN 118-01-C
Optimizing the Separation of Polyphenols in Natural Products via OPLC The tailing that is commonly observed when polar natural products such as polyphenols are separated via normal phase chromatography can be dramatically reduced by using Optimum Performance Laminar Chromatography bstract OPLCTM with presaturation of the column. With this technique, development of an analytical scale separation and scale up to a semi-preparative separation are quite rapid; and sufficient sample for structural analysis via MALDI-MS analysis could be obtained in an hour. An additional benefit of OPLC is that the analyst can readily monitor both the eluted compound(s) as well as the non-eluted ones.
A
Introduction
Some commonly used chromatographic techniques, such as Thin Layer Chromatography (TLC) are useful for rapid separations to determine the presence (or absence) of a compound, or can be used to indicate the relative purity of a compound. In most cases, TLC does not provide sufficient resolution to supply a pure compound for further study. In contrast, OPLC provides a considerable improvement in the separation of a complex mixture and is frequently used to isolate sufficient material for further analysis.
TLC of curcumin CHCl3 MeOH 2%
The separation of a polyphenol that is a potential anti-cancer agent extracted from Curcumin longa is an excellent example of this point. Three strongly tailing spots were observed when the extract was separated by TLC (Figure 1). Changing the composition of the mobile phase from 2% CH 3OH to 5% CH3 OH and the addition of various pH modifiers such as triethylamine and acetic acid were of little help in reducing this effect. In this note, we demonstrate that OPLC leads to a rapid and selective separation.
Experimental 10µL of a 5 mg/mL dichloromethane (DCM) solution was deposited on a 5x20cm BSLA1003 HTSorbTM column coated with 11µm particles (Bionisis, SA, Le Plessis Robinson, France) with a Desaga AS30 sampler. The plate was then inserted into an OPLC50 chromatograph (Bionisis). Separation was effected with CHCl3/MeOH (98/2) at a flow rate of 200µL per min. In another experiment, the column was placed in a closed chamber containing 20mL CHCl3/MeOH (95/5) for 30 min at room temperature (the OPLC column did not touch the solvent) for equilibration and the separation was then performed in the same manner as above.
TLC of curcumin CHCl3 MeOH 5%
A preparative scale separation using a 100 µL sample was performed on a larger HTSorb BSLA1011 column (20x20cm Silica 60) with increased capacity and at a flow rate of 800µL/min. 0.3-0.5mL fractions were collected starting after 8 min separation time and continuing for 12 min.
1 µL deposited in all cases 5 mg/mL 0.5 mg/mL
MALDI-MS spectra were collected from each fraction using a 3,4-dihydroxycinnamic acid (DCHA) matrix and an SAI MALDI-TOF Spectrometer (SAI Analytical Instruments, Manchester, UK).
Figure 1: TLC Separation of Polyphenols from Curcuminlonga Upper chromatograms Mobile Phase MeOH: CHCl3 (98:2) Lower chromatograms Mobile Phase MeOH: CHCl3 (95:5) Left chromatograms observation with visible light Right chromatograms Fluorescence using 366 nm hand lamp.
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When a chromatographic separation is used to purify a sample for further study (e.g. for mass spectroscopic analysis), the separation must provide adequate resolution. If the desired level of purity is not obtained, the data obtained from the purified sample will be of questionable value.
Fig 2a: OPLC separation without pre-saturation
Fig 2b: OPLC separation with 30 min pre-saturation with 5% MeOH in CHCl 3
Flow Figure 2:
OPLC Separation of Polyphenols from Curcuminlonga. Upper Chromatogram: Column was not pre-saturated Lower Chromatogram: Column was pre-saturated.
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Results The analytical scale OPLC separation without presaturation of the column led to a significant degree of tailing and diluting the sample did not reduce this effect (Figure 2a). The analytical scale OPLC separation with pre-saturation of the plate is shown in Figure 2b. Tailing is almost completely eliminated when pre-saturation of the column precedes the separation. While not presented here, the use of HTSorb columns without UV254 indicator also contributed to a minor extent in reducing tailing. A preparative run was performed with 500µg applied on a larger 20x20cm HTSorb column and fractions were collected. The column was also removed from the system and observed under 366nm UV light after the prep run. There was no residual fluorescence on the column, suggesting that the components of the sample had eluted completely and that the recovery was likely very high. Each fraction was analyzed for purity by TLC; component 1 was contained in fraction 1 (0.0 to 0.2mL), component 2 in fractions 2-3 (0.2 to 1mL) and component 3 in fractions 7-8 (6.0-7.0.mL). The fractions that were collected were sufficiently pure to obtain structural information via MALDI-MS. After subtraction of matrix peaks, the spectra were simple, showing a clean molecular ion peak MH+ for each fraction: m/z 369 (Fraction 1, compound 1), m/z 339 (Fraction 2, compound 2) and m/z 309 (Fraction 7, compound 3)1. The MS results confirm the structures proposed in a recent article2. We note that the entire sequence of the preparative separation took approximately one hour as follows: • • • •
O
H
O
R1
R2
HO
OH
Fraction 1 Fraction 2 Fraction 7
MH+369 MH+339 MH+319
Compound 1 : R1, R2 = OMe Compound 2 : R1 = H, R2 = OMe Compound 3 : R1, R2 = H
Conclusions Optimum Performance Laminar Chromatography was capable of providing a superb separation of a series of 3 polyphenols from a natural product that could not be well separated by preparative Thin Layer Chromatography. Tailing was minimized by presaturation of the stationary phase with solvent prior to the separation. A preparative separation was performed which provided sufficient material for MALDI-Mass Spectroscopic studies for structure determination. The overall separation took approximately one hour. An important benefit of OPLC is that the column can be monitored. In this instance only a trace quantity of the original sample remained, indicating a very effective separation.
References [1] The slight impurity of compound 1 in fraction 2, observed by chromatography, was not visible in the MS spectra. [2] Kim, JE; Kim, AR; Chung, HY; Han, SY; Kim, BS, Choi, JS Phytotherapy Research 2003, 17, 481.
5 minutes for spotting the samples 30 minutes for pre-saturation of the column 20 minutes to run the separation and collect the fractions, and 10 minutes for the analytical control.
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