Anal. Chem. 1997, 69, 1965-1969
Correspondence
Analysis of Ancient Dyed Chinese Papers by High-Performance Liquid Chromatography Peter J. Gibbs† and Kenneth R. Seddon*
School of Chemistry, The Queen’s University of Belfast, Stranmillis Road, Belfast BT9 5AG, Northern Ireland Nadezhda M. Brovenko and Yuri A. Petrosyan
Institute of Oriental Studies, Russian Academy of Sciences, St. Petersburg, Russia Mark Barnard
Oriental and India Office Collections, British Library, Orbit House, 197 Blackfriars Road, London SE1 8NG, England
The paper of the Dunhuang Diamond Sutrasthe world’s oldest, dated, printed booksis dyed yellow with an extract believed to contain the alkaloid berberine as the principal colorant. Unambiguous identification of the dye used on this unique book is essential for its conservation. We have developed here a simple, rapid, high-performance liquid chromatography method that gives excellent separation of the protoberberine alkaloids and allows the unambiguous detection of berberine in dyes extracted from tiny fragments of ancient papers. Examination of 25 ancient papers contemporary with the Dunhuang Diamond Sutra revealed the presence not only of berberine but also of the previously unsuspected yellow compounds, palmatine and jatrorrhizine. The Dunhuang Diamond Sutra is a the earliest extant printed document that can be precisely dated (11 May 868 A.D.), and it is a focus for the conservation of collections of Chinese documents held in London, Paris, and St. Petersburg. The present physical condition of this Sutra is critical, and it is essential that poorquality backing papers, pasted to the document during unfortunate early conservation attempts, are removed. An interesting feature of the Dunhuang Diamond Sutra is that the paper is dyed yellow (probably to protect it from insects):1 the trustees of the Dunhuang Diamond Sutra have agreed that the yellow dye is an intrinsic part of this invaluable document, and that the dye must not be damaged, removed, or affected detrimentally in any way in the name of conservation. As any procedure to wash the pastes that bind the backing papers to the Sutra must not affect the dye, it is vital that the exact nature of the yellow dye is determined before any preservation methods can be developed. The most probable dye, huangbo (or huang bo),1 is an extract from the bark of the † Present address: Chemistry Department, Christopher Ingold Laboratories, University College London, 20 Gordon Street, London WC1H 0AJ, UK. (1) Gibbs, P. J.; Seddon, K. R. In Dunhuang and Turfan: Contents and Conservation of Ancient Documents from Central Asia. Br. Libr. Stud. Conserv. Sci. 1996, 1, 59-69.
S0003-2700(96)00279-X CCC: $14.00
© 1997 American Chemical Society
Amur cork tree (Phellodendron amurense):2 in total there are believed to be at least 40 000 documents in existence dyed yellow in this manner. The high performance liquid chromatography (HPLC) method has been developed to complement a recently reported3-5 mass spectrometric technique for the in situ identification of dyes on ancient papers. This liquid secondary ion mass spectrometric (L-SIMS) method requires a 3 × 1 mm fragment of ancient paper. The HPLC technique was evolved to examine the dye extracted from a similar sized paper fragment, with the intention that, for a valuable document such as the Dunhuang Diamond Sutra, the same fragment could be analyzed by both methods. The analysis of dyes by HPLC is principally applied to the detection of food coloring, e.g., refs 6 and 7, but HPLC has recently been applied to the analysis of the traditional dyestuffs Tyrian purple8 and indigo,9 extracted from ancient textiles. To date, however, there have been no reports of the analysis by HPLC of dyes extracted from ancient papers. Three HPLC separations of berberine and other protoberberine alkaloids have been reported in the literature.10-12 The method of Luo et al.11 gives poor separation of the protoberberinium (2) Bremner, A.-M.; Gibbs, P. J.; Seddon, K. R. In Dunhuang and Turfan: Contents and Conservation of Ancient Documents from Central Asia. Br. Libr. Stud. Conserv. Sci. 1996, 1, 70-82. (3) Abdul-Sada, A. K.; Barnard, M.; Lawson, P.; Brovenko, N. M.; Petrosyan, Y. A.; Gibbs, P. J.; Jordan, J. G.; Seddon, K. R. Eur. Mass. Spectrom. 1995, 1, 217-219. (4) Gibbs, P. J.; Jordan, J. G.; Seddon, K. R.; Cooksey, C. J.; Brovenko, N. M.; Tiomken E. N.; Petrosyan, Y. A. Eur. Mass Spectrom. 1995, 1, 417-421. (5) Gibbs, P. J.; Seddon, K. R. Spectrosc. Eur. 1995, 7, 10-18. (6) Croft, S. N.; Lewis, D. M. Dyes Pigments 1992, 18, 309-317. (7) Gennaro, M. C.; Abrigo, C.; Cipolla, G. J. Chromatogr. A 1994, 674, 281299. (8) Koren, Z. C. Abstracts of Papers, 207th National Meeting of the American Chemical Society, American Chemical Society: Washington, DC, 1994. (9) Wouters, J; Verhecken, A. J. Soc. Dyers Colour. 1991, 107, 266-269. (10) Akaoka, T. Nangano-ken Eisei Kogai Kenkyusho Kenkyu Hokoku 1991, 14, 45; Chem. Abstr. 1992, 116 (16), 159027z. (11) (a) Luo, G.; Wang, Y.; Zhou, G.; Yu, Y. J. Liq. Chromatogr. 1990, 13, 38253832. (b) Wang, Y. M.; Zhao, L. H., Lin, S. L. Dong S. S.; An, D. K. Yaoxue Xuebao. 1989, 24, 275; Chem. Abstr. 1989, 111, 121001v. (12) Misaki, T.; Sagara, K.; Ojima, M.; Kazizawa, S.; Oshima, T.; Yoshizawa, H. Chem. Pharm. Bull. 1982, 30, 354.
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Figure 1. HPLC trace of the dye extract of a 3 × 1 mm sample of modern paper dyed with huangbo. The electronic absorption spectra for the signals collected by the diode array detector are also illustrated.
alkaloids, and it requires partial least-squares analysis, which makes it unsuitable for a routine method of dyed paper analysis aimed at the museum community. The methods by Akaoka10 and Misaki et al.12 were similar in principle, giving good reported separation of the protoberberinium alkaloids; the latter used a surfactant and was selected as a basis for the HPLC method used in this study. The conservation of the Dunhuang Diamond Sutra is the subject of high-level debate, and the trustees have yet to agree to release a fragment of the document for dye analysis. The procedures presented here represent a key component in the microanalytical strategy for the pending identification of that dye and demonstrate the efficacy of the method by examining, inter alia, samples contemporary with the Dunhuang Diamond Sutra, found in the same location. EXPERIMENTAL SECTION Berberinium chloride tetrahydrate and palmatinium chloride hydrate were purchased from Sigma Chemical Co. Ltd., and genuine samples of purified jatrorrhizinium chloride were generously independently donated by Dr. W.-N. Wu and Prof. S.-J. Sheu. Safety Note: Berberine, palmatine, and jatrorrhizine are all extremely toxic. Care should be taken to avoid breathing the dust, and gloves should be worn when handling the pure alkaloids, the huangbo dye, and papers dyed with huangbo. All HPLC analysis was performed using a Shimadzu LC-6A liquid chromatograph connected to a Hewlett-Packard HP 1040A HPLC diode array detector. The HPLC trace was recorded at 345 nm, and the electronic absorption spectrum of each signal recorded in the range 250-500 nm. The data were collected on a PC with Hewlett-Packard Chemstation software. The column used was a 15 cm TSK-GEL ODS-80Tm column supplied by TosoHaas. The flow rate of the mobile phase was 1 cm3 min-1 at room temperature. The reversed-phase ion-pair HPLC method of Misaki et al.12 was found to have a prohibitively high retention time (over 40 1966 Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
min for berberine at a 1 cm3 min-1 flow rate), and the mobile phase was therefore altered to decrease the retention times. The reported mobile phase was sodium lauryl sulfate-0.05 M tartaric acid-methanol-ethanenitrile (0.5:49.5:10:40). The column used was designed principally for separating amino acids, and protoberberine alkaloids are amino acid derived; thus, it was theorized that trifluoroethanoic acid, commonly used as a buffer in amino acid separations, would prove a more effective buffer than the tartaric acid for this system. Using the newly devised sodium lauryl sulfate-0.05 M trifluoroethanoic acid-methanol-ethanenitrile (0.5:49.5:10:40) mobile phase, the retention time for berberine was reduced by over a factor of 3 to about 12.5 min at a flow rate of 1 cm3 min-1. The HPLC method was then optimized for the analysis of dyed paper samples using Japanese papers freshly dyed with huangbo in the traditional manner.13 The dye was extracted from a 3 × 1 mm dyed paper sample by soaking in 0.1 cm3 of the mobile phase for 15 min in a sealed sample tube. The extract was collected by graduated syringe and injected into the chromatograph, and the HPLC trace was recorded. Finally, 25 dyed ancient paper samples, donated by the Oriental Institute in St. Petersburg, were examined. The hues of the paper samples ranged from pale yellow to dark brown. Fragments with dimensions of 3 × 1 mm were removed from the edge of each sample and the dye extracted for each fragment with 0.1 cm3 of the mobile phase by soaking for 15 min in separate sealed sample tubes. RESULTS AND DISCUSSION The trace of the dye extracted from a 3 × 1 mm paper sample dyed with huangbo showed three peaks, with retention times around 7.5, 11.5, and 12.5 min, as well as some material moving rapidly through the column. The diode array detector recorded (13) Tsien, T.-H. In Science and Civilization in China; Needham, J., Ed.; Cambridge University Press: New York, 1987; Vol. 5, pp 74-76.
Figure 2. HPLC traces of (a) berberine, (b) palmatine, and (c) jatrorrhizine. The electronic absorption spectra recorded by the diode array detector are also illustrated.
comparison with the HPLC trace and the electronic absorption spectrum of the commercial sample (see Figure 2a). The marked similarity of the electronic absorption spectra for the other signals, when compared with berberine, suggested that they, too, were protoberberine alkaloids. A survey of the literature14-18 revealed that two other protoberberine alkaloids had been isolated from the bark of P. amurense: palmatine and jatrorrhizine. The latest study18 quantified the average abundance of berberine, palmatine and jatrorrhizine in the bark of P. amurense as 26:13:1.
Figure 3. Graphs illustrating the ratios of (a) palmatine to berberine and (b) jatrorrhizine to berberine in the extracts of different sized fragments of ancient paper sample OR8210.
the electronic absorption spectrum of each signal, and these spectra are also displayed with the trace in Figure 1. The signal around 12.5 min was identified as being due to berberine by
The HPLC traces for both palmatine and jatrorrhizine are displayed in panels b and c of Figure 2, respectively, together with the recorded electronic absorption spectra of the signals (the Analytical Chemistry, Vol. 69, No. 10, May 15, 1997
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Figure 4. Five examples of the HPLC traces of dyes extracted from pre-10th century Chinese papers: B ) berberine, P ) palmatine, and J ) jatrorrhizine.
two independently donated samples of jatrorrhizinium chloride had identical retention times of ∼7.5 min; palmatinium chloride had a retention time of ∼11.5 min). The retention times and electronic absorption spectra confirmed that the two other signals in the chromatogram shown in Figure 1 were due to palmatine and jatrorrhizine. The result also confirmed that the HPLC (14) Liepins, V.; Shreter, A. J. Farmatsiya (Moscow) 1968, 17, 65; Chem. Abstr. 1969, 70 (12), 50440j. (15) Kubota, M.; Asakawa, K. Nagano-ken Eisei Kogai Kenkyusho Kenkyu Hokoku, 1976, 1, 20; Chem. Abstr. 1980, 92, 18841j. (16) Kunitomo, J. J. Pharm. Soc. Jpn. 1962, 82, 611. (17) Kawaguchi, J.; Takahashi, S.; Kozuka, M.; Katsumi, K.; Ishida, M.; Kim B.; Yamazaki, N. JPN. PAT. 01,102,007 (89,102,007), 19th April 1989; Chem. Abstr. 1989, 111, 148935q.
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method used was extremely efficient at separating the protoberberinium alkaloids. The relative retention times for berberine, palmatine, and jatrorrhizine were 1:0.89:0.65. The HPLC method was further investigated to see whether the levels of berberine, palmatine, and jatrorrhizine detected in the dye extracted from a 3 × 1 mm sample of ancient paper could be interpreted quantitatively. A large fragment of dyed pre-10th century Chinese paper (code OR8210; donated by the British Library) was used. The dye was extracted from fragments with areas of 1, 2, 3, 10, 25, 50, 100, 200, 400, and 800 mm2 with the (18) Liu, Y. M.; Sheu, S. J.; Chiou, S. H.; Chang H. C.; Chen, Y. P. Planta Med. 1993, 59, 557.
minimum amount of mobile phase, and the ratios of berberine, palmatine, and jatrorrhizine were calculated from the integrals of the signals of the HPLC traces (these were determined automatically by the Hewlett-Packard Chemstation software). The results are displayed in Figure 3. Figure 3a illustrates that the ratios of palmatine to berberine given by the HPLC trace of a 3 × 1 mm sample of ancient paper could not be interpreted quantitatively for small sample sizes: an extract of a sample with dimension of 100 mm2 or greater is required for quantitative analysis, but this sample size is unrealistic for a unique document such as the Dunhuang Diamond Sutra. Figure 3b shows the ratio of jatrorrhizine to berberine taken from the HPLC traces. The graph shows that the level of jatrorrhizine in the extracted dye was below the limits of detection for sample areas of less than 100 mm2. Even in the dye extracted from fragments with areas greater than 100 mm2, the level of jatrorrhizine varies considerably, indicating that this alkaloid, the least abundant in huangbo, may not be evenly distributed across the paper surface. Sample OR8210 was not a heavily dyed specimen, and it was selected for this study mainly because of its size and historical insignificance (the sample was poor quality with no calligraphy); thus, it is possible that the results given by 3 × 1 mm samples with a stronger dye may be quantitative, but as this could not be confirmed, the study of ancient paper samples was limited to the qualitative detection of berberine, palmatine, and jatrorrhizine in their dyes. The dyes extracted from 3 × 1 mm fragments of twenty five ancient paper samples were analyzed, and typical data are presented in Figure 4. Berberine was unambiguously detected in 21 of the dyes, palmatine in 18 of the extracts where berberine was also found, and jatrorrhizine was detected in the extracts of 5 fragments where both berberine and palmatine were also found. In no instance was palmatine or jatrorrhizine detected when berberine was not also observed. Surprisingly, the HPLC traces of two of the paper samples, both with a dark brown hue, revealed an abnormally high level of jatrorrhizine (see Figure 4, traces d and e). Both samples had a strong level of dye, and the high level of jatrorrhizine can be accepted as real. This result could be simply explained by a random variation in the dye’s contents due either to a different distribution of the alkaloids in the same species of tree within the natural phytogeography, or to a different dyeing procedure. The possibility of significant variation of the alkaloid distribution within trees of the same species was recently studied by Liu et al.18 They studied the alkaloidal composition of 31 different samples of Phellodendron bark, harvested in various locations in China, Taiwan, and Japan, by capillary electrophoresis. The ratio of berberine to palmatine was found to vary between 1.2:1 and 3:1 (the average was 2:1) within the P. amurense samples. However, the abundance of jatrorrhizine was consistently much lower than the other alkaloids in the P. amurense samples: the ratio of berberine to jatrorrhizine ranged between 25:1 and 44:1 (the average was 31: 1), and the ratio of palmatine to jatrorrhizine ranged between 10:1 and 26:1 (the average was 17:1). Although physiological differ-
ences in P. amurense specimens over a millennium ago cannot be ruled out completely, the results of the study of Liu et al.18 do strongly suggest that such a relatively high level of jatrorrhizine is unlikely in this species. As the three alkaloids are all highly soluble in water, it is unlikely that the original dye extraction procedure (the bark was boiled in water)13 would preferentially extricate some of the alkaloids at the expense of others, and the sizes and loads, which would have been added prior to dyeing, are unlikely to affect the dye’s composition even over the time period in question. Due to the similar structures of the three alkaloids, and again their high solubility in polar solvents, it is also unlikely that preferential binding of a particular alkaloid to the paper would affect the composition of the dye extracted from an ancient paper sample for analysis; and it is equally unlikely, again because of the similarity of the structures of the three alkaloids, that selective photodegradation may be the cause of the high level of jatrorrhizine. Finally, there is no extant evidence for the existence of a chemical or biochemical process for the conversion of berberine or palmatine to jatrorrhizine occurring naturally, or in situ on a paper’s surface, or during the dyeing procedure. Therefore, after taking the above factors into consideration, the extremely important observation of unusually high levels of jatrorrhizine in the dyes of some ancient papers does suggest, for the first time, that a plant extract other than huangbo may also have been used to dye ancient Chinese papers. In conclusion, the HPLC method for the analysis of ancient dyed Chinese papers is rapid, effective, and simple, and it is a readily accessible analytical tool for the identification of the components of huangbo on tiny fragments of papers and textiles. The method is ideally suited to the museum community and much more readily available than L-SIMS. However, we would recommend the application of BOTH methods to confirm the presence of the components of huangbo on the surface of the Dunhuang Diamond Sutra. ACKNOWLEDGMENT We are indebted to INTAS (Grant 93-3328) and the Royal Society (Grant 92303862) for funds to establish the co-operation between Russia and the UK, to the EPSRC for a studentship (P.J.G.), to the EPRSC and the Royal Academy of Engineering for the award of a Clean Technology Fellowship (K.R.S.), to Anne-Marie Bremner for her initial investigation, and to Dr. W.-N. Wu (R. W. Johnson Pharmaceutical Research Institute, Washington DC) and Prof. S.-J. Sheu (National Taiwan Normal University) for their generous independent gifts of jatrorrhizinium chloride. Received for review March 21, 1996. Accepted August 5, 1996.X AC960279V X
Abstract published in Advance ACS Abstracts, September 15, 1996.
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