Isolation purification of hydroxyanthraquinones using the Agilent 1100 Series purification system Application Note
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1. ml min to get simi lar retention times After the adjustment was done the next preparative run was fractionated using time slices The first 30 frac tions were taken starting at 6 min utes with a slice width of 0 2 min utes Another 20 fractions were taken starting at 14 minutes with the same slice width The result of the preparative run and the frac tionation information are shown in figure 4 Mobile Phases 0 1 HOAc in water A 0 1 HOAc in ACN B Gradient 30 B to 50 B in 5 min 50 B to 70 B in 10 min 70 B to 80 Bin 1 min 80 B for 3 min 80 B to 30 Bin 1 min Stop time 20 min Post time 5 min Column Zorbax SB C18 21 2 x 150 mm 5 um Flow 25 ml min Injection 750 pl Column temp ambient UV detector DAD 222 nm 16 reference off Standard cell 3 mm pathlength Analytical column Zorbax SB C18 Preparative column Zorbax SB C18 3 x 150 mm 5 pm V ie 21 2 x 150 mm 5 pm 1 vn Flow 0 6 ml min gt Flow 30 ml min Volume 15 pl injection Volume 750 ul injection X x E RE 2 2 TEX p T X ie CL x max volume column 1 15 yl r radius column 1 1 5mm X max volume column 2 r radius column 2 10 6 mm C ratio lengths of columns 1 Figure 3 Scale up from analytical to preparative column O O O2. 21 29 Injected amount 750 p 12 30 mg Recovered 11 44 mg Recovery 93 Absorbance e mAU 2004 Emodin 1504 ae 50 0 50 n ce ne 0 5 10 15 Time min Figure 5 bi Combined fractions 7 15 Injected amount 750 pl 23 63 mg Recovered 16 30 mg Recovery 69 Absorbance mAU mM a 150 Rhein 100 50 0 50 T T T T 0 4 8 12 16 Time min d Combined fractions 32 39 Injected amount 750 u 5 28 mg Recovered 4 54 mg Recovery 86 Absorbance mAU En M 100 75 Crysophanic 50 1 acid 25 0 25 0 4 8 12 16 Time min Combined fractions of a aloe emodin b rhein c emodin d crysophanic acid Conclusion In this Application Note we demonstrated some features of the Agilent 1100 Series purifica tion system to isolate and purify compounds from crude natural product extracts in preparative scale Four hydroxyathraquinones were isolated from the extract of the rhubarb root Rheum palma tum using fraction collection based on time windows To obtain sufficient amounts of the com pounds the purification was done repetitively and the collected frac tions were pooled automatically As a result at least three of the four compounds were isolated in sufficient amount and purity for NMR and MS spectroscopy for identification and structure eluci dation and for initial activity test ing References 1 D J Roush F D Antia K E G klen Preparative high perfor mance liquid chroma
3. Isolation and purification of hydroxyan thraquinones using the Agilent 1100 Series purification system Application Udo Huber Abstract Semi preparative and preparative HPLC is probably the most common technique for isolation and purification of single compounds from mix tures For example it is applied to isolate synthesis products to purify combinatorial chemistry library compounds or to separate isomers or enantiomers In this Application Note we describe how another typical task for preparative HPLC the isolation of compounds from crude nat ural product extracts can be easily performed using the Agilent 1100 Series purification system s Agilent Technologies f Innovating the HP Way Introduction Today isolation and characteriza tion of single compounds from complex natural product extracts is a standard procedure for a nat ural product chemist Liquid chro matography usually in semi preparative or preparative scale with flow rates over 10 ml min is the tool of choice to separate the compounds 123 The goal is to get the desired compounds in high purity and sufficient amount for structure elucidation usually done by NMR MS and IR spectroscopy With the Agilent 1100 Series purifi cation system Agilent provides a toolset for sample handling and fraction collection at flow rates up to 100 ml min The software allows complete control of the Agilent 220 micro plate sampler and provides an easy to use gra
4. O 2 Absorbance T A Fi A PY en pe A lt i a mau aaa naamaan 2500 2000 1500 7 1000 7 5004 0 T T T T 0 2 4 6 8 10 12 14 16 18 Time min Figure 4 Preparative run with fraction collection Reanalysis of fractions and fraction pooling The 50 fractions were reanalyzed using the analytical method and column to determine the purity of the compounds The results are shown in figure 5 For the com pounds aloe emodin and rhein the recovery is low due to the poor separation of the compounds Therefore fractions containing both compounds were discarded to get the anthraquinones in suffi cient purity At least rhein emodin and crysophanic acid could be iso lated in sufficient purity for fur ther NMR and MS spectrometric analysis For aloe emodin proba bly a second purification step would be necessary For detailed activity tests the iso lated amount would not be suffi cient Therefore 75 11 volumes of sample were injected repetitively ten times and the fractions were pooled This could be done conve niently using the dedicated micro plate sampling software As a result at least 20 mg of each com pound were isolated in good puri ty which would be enough for complete structure elucidation and primary activity tests a Combined fractions 4 5 Injected amount 750 ul 13 68 mg Recovered 8 62 mg Recovery 63 Absorbance mAU o a Aloe emodin Time min c Combined fractions
5. ph ical user interface for study set up and sample fraction tracking The system is complemented with the Agilent 1100 Series diode array detector for detection and spectral information and the Agilent 1100 Series MSD for mass information needed for structure elucidation In this Application Note we describe the isolation of four hydroxyanthraquinones from the extract of rhubarb root Rheum palmatum to demonstrate how a natural product chemist would isolate and purify unknown com pounds from a crude natural prod uct extract Equipment The system included two Agilent 1100 Series preparative pumps an Agilent 1100 Series diode array detector an Agilent 1100 Series column organizer and an Agilent 220 micro plate sampler modified for higher flow rates The system was controlled using the Agilent ChemStation revision A 08 04 and the micro plate sampling soft ware revision A 03 02 Results and Discussion Extraction 37 6 g rhubarb root Rheum palmatum were extracted ultra sonically five times with 100 ml methanol each The combined extracts were evaporated to give 25 ml crude extract Absorbance mAU 4007 3007 2007 1007 Volume overloading on the analytical column For method development the crude extract was first applied to an analytical system The method was optimized regarding selectivi ty of the compounds and run time The chromatogram measured with the optimized method i
6. s shown in figure 1 1 Aloe emodin 3 Emodin 2 Rhein 4 Crysophanic acid Injection 1 ul crude extract Diluted 1 10 with methanol 0 2 4 6 8 10 12 14 16 18 Time min Figure 1 Analytical chromatogram of crude rhubarb extract Absorbance mAU 3500 3000 l 2500 njection meee 2000 volume 1500 Al Sn 10 ul 1000 5 ul s0 N 1l 5 ul 1 10 0 1 pl 1 10 T T T T T T T 0 25 5 7 5 10 12 5 15 17 5 Time min Figure 2 Volume overloading on the analytical column The concentration of the crude extract was not variable because of the extraction process There fore concentration overloading was not possible and volume over loading had to be done The results for volume overloading on the analytical column are shown in figure 2 Mobile Phases 0 1 HOAc in water A 0 1 HOAc in ACN B Gradient 30 B to 50 Bin 5 min 50 B to 70 B in 10 min 70 B to 80 B in 1 min 80 B for 3 min 80 B to 30 B in 1 min Stop time 20 min Post time 5 min Column Zorbax SB C18 3 x 150 mm 5 um Flow 0 6 ml min Injection 5 ul Column temp ambient UV detector DAD 222 nm 16 reference off Standard cell 10 mm pathlength Scale up to preparative scale The scale up from the analytical to the preparative column was calcu lated using the formulae shown in figure 3 After the first run on the prepara tive column the flow rate was adjusted to 25
7. tography of echinocandins J Chromatogr A 827 1998 373 389 2 T Wawrzynowicz M Waksmundzka Hajnos Influence of various chromatographic para meters on the separation of natur al mixtures under column over loading conditions Fresenius J Anal Chem 352 7 8 1995 779 782 3 M Fiorini Preparative high per formance liquid chromatography for the purification of natural anthocyanins J Chromatogr A 692 1 2 1995 213 219 Udo Huber is an application chemist based at Agilent Technologies Waldbronn Germany www agilent com chem Copyright 2000 Agilent Technologies All Rights Reserved Reproduction adaptation or translation without prior written permission is prohibited except as allowed under the copyright laws Printed 10 2000 Publication Number 5988 0637EN lt x Agilent Technologies Innovating the HP Way
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Isolation purification of hydroxyanthraquinones using the Agilent 1100 Series purification system Ap