Anal. Chem. 1994,66, 4359-4365
Detection of Alkylated Cellulose Derivatives in Several Archaeological Linen Textile Samples by Capillary Electrophoresis/Mass Spectrometry Dmitri A. Kouznetsov,* Andrey A. lvanov, and Pave1 R. Veletsky S. A. Sedov Biopolymer Research Laboratories, Inc., 4/9 Grafski Per., Moscow 129626, Russia
Eight different known-age samples of light, nondyed linen textile dated between 1200 B.C. and AD. 1500 were studied by combined capillary electrophoresis/mass spectrometry analysis of the textile cellulose enzymatic hydrolysates. Both field ionization and field desorption variants of mass spectrometry have been used for identification of each electrophoretic fraction. High-resolution patterns were obtained using the 100 mM tetraborate @H 9.0) zone electrophoresis system and the on-column laser-based refractive index detector. It has been shown that all archaeological textiles tested contain alkylated cellulose sequences. Thus, these textile samples differ from each other by alkyl groups (acetyl, carboxy, or methyl) as well as by the structure and abundance of the alkylated glucose residues. A possible origin and meaning of the character of this alkylation is under discussion which includes a special reference to correlations between such a parameters as (1) alkylation peculiarities, (2) geographical area of the textile manufacturing,and (3)the textile calendar age values. There is currently a great deal of interest in the development of new approaches to chemical investigations of archaeological textile relics.1.2 The importance of these studies stems from their potential utility in determining the chemical mechanisms of the textile “aging” and, therefore in for further improving of the accuracy of the ancient textile dating procedures. Besides, detailed chemical studies on archaeological textiles could provide important information about the lost, “archaic” technological processes practiced long ago in different geographical area^.^,^ Finally, any information on the historic textile chemical aging should be important for development of methods for conservation of the relics. The chemical investigation of the old textiles can be performed by a number of methods, including near-infrared/infrared reflectance spectrometry of undisintegrated textile, high-resolution electron microscopy, atomic absorption spectrophotometry for element analysis, and several chromatographic (both gas chromatography and HPLC) and electrophoretic techniques for *All correspondence should be sent to: Dr. D. A. Koumetsov, S.B.R.L., 2544 Menzhinski St., Moscow 129327, Russia. Tel/FAx: 011-70951867409. (1) Cardamone,J. M.; Brown, P. Evaluation of Degradation in MuseumTextiles Property Kinetics. In Historic Textile and Paper Materials: Conservation and Characeterization; Needles, H. L., Zeronian, S. H., Eds.; American Chemical Society Press: Washington, DC, 1986 pp 41-76. (2) Ageyev, D. T. Chemical Methods in Archaeology;Moldova State University Press: Kishinev, 1992 (in Russian). (3) Lee, J. S. Elementary Textiles; F’rince Hall, Inc.: New York, 1953; pp 4759.
0003-2700/94/0366-4359$04.50/0 Q 1994 American Chemical Society
fractionation and quantification of the textile cellulose enzymatic hydrolysate product^.^^^^^ In our opinion, an optimal way to carry out efficient comparison between cellulose sequences of many different museum textile samples is to compare the HPLC or electrophoretic patterns obtained as a result of fractionation of low molecular weight compounds (nonmodified and modified /3-Dglucose, plus cellobiose) of the enzymatically digested cellulose pool isolated from the textiles. Thus, a combination of capillary electrophoresis and mass spectrometry should be very efficient for analysis of textiles: capillary electrophoresis provides significant separation efficiency and high analytical speed for a broad range of substances in solution, while mass spectrometry provides peak identification. Furthermore, the flow rates from the capilllary column are compatible with on-line coupling to a mass spe~trometry.~,~ Capillary zone electrophoresis (CZE), in its numerous variants, is an alternative separation technique to HPLC. Remarkable performance of this analytical tool has been demonstrated by unprecedented separation efficiencies in many studies, including the most recent research on carbohydrate^.^^^ Two significant problems have been identified for the analysis of sugars by CZE. First carbohydrates are not charged species under normal conditions; second, they have poor detectability. Nevertheless, it is easy to reach efficient separation results with the additon of borate ions to the separation medium, promoting the formation of ionic derivatives of sugars. This simple approach permits the formation of negatively charged sugar-borate complexes that can be separated by CZE.7 The low sample volumes injected in CZE make detection of carbohydrate a difficult task. Enhancements in sensitivity by W absorbance (at 195 nm) have been obtained with the formation of the sugar-borate complex. The increase in sensitivity, however, is relatively small (2-20-fold), and the detection of sugars is limited to the nanomole range. At the same time, alternative methods of detection, such as electrochemical (am(4) Yakimov, S. S.; Zheltkov, R T. Field Ionization/Field Desorption Mass Spectrometry in Chemistry and Biochemisby of Carbohydrates. In Appied Chemistry of Cellulose; Samarin, L. IC, Bakhurov, V. L., Eds.; Novosibirsk University Press: Novosibirsk, 1990: pp 126-148 (in Russian). (5) Thompson, T. J.; Foret, F.; Vouros, P.; Karger, B. L. Capillary Electre phoresis/Electrospray Ionization Mass Spectrometry: Improvement of Protein Detection Limits Using On-Column Transient Isotachophoretic Sample Reconcentration. Anal. Chem. 1993,65, 900-906. (6) Bruno, A. E.; kattiger, B.; Maystre, F.; Widmer, M. H. On-Column LaserBased Refractive Index Detector for Capillary Electophoresis. Anal. Chem. 1991,63, 2689-2697. (7) Colon, L A; Dadoo, R; Zare, R N. Determination of Carbohydrates by Capillary Zone Electrophoresis with Amperometric Detection at a Copper Microelectrode. Anal. Chem. 1993,65,476-481.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994 4359
perometric) methods or refractometric methods based on the defatted samples were then submerged into a soluton containing refractive index changes, are able to reach much better sensitivity 7.7% formaldehyde, 7.7% borax, and 0.5 sodium dodecyl sulfate levels in the CZE experiments with mone and 0ligosaccharides.2~~~~ for 3 min and dried in an electric oven at 100 "C for 2 h. Finally, As for the cellulose chains in the ancient textile relics, some all the samples were washed extensively with demineralized of them contain a significant number of chemically modified P-D(deionized chromatographically on Amberlyte resins) water, 120 glucose residues, like hemiacetal derivatives of glucose or its mL/cm2, at room temperature. For this purpose, a textile sample carbonyl (conjugated) derivatives.8~9 was applied as a filter on the water flow. The cleaned and washed Taking into account everything stated above, we have decided textile samples (up to 2.8 cmz each) were air-dried and kept in to carry out a comparative study on the possible chemical sealed, dry flasks for further studies. modification of cellulose from eight archaeological textiles dated Emzymatic Hydrolysis of the Textile Cellulose. Samples between 1200 B.C. and AD. 1500 which were kindly provided by of 2.0-2.8 g of the mechanically desintegrated texiles (crude several European museums (see Acknowledgment). For this fibrous material) were incubated at 37 "C for 6 h in 20 mL of 15 study, we have used capillary zone electrophoresis of the puritied mM Tris-borate @H 6.40) buffer containing 4.5-5.0 units of cellulose enzymatic hydrolysates with mass spectrometric idencellulase per milliliter. These conditions were enough for 98% tification of each peak (see Experimental Section and Figure 3). cellulose depolymerization.' After the incubation, the low molecular weight compounds pool was separated from the hydrolyEXPERIMENTAL SECTION sate by ultrafiltration though the Diaflow YM-1 membranes in Materials and Reagents. Light (nondyed) portions of the an Amicon MMC-10 apparatus (Amicon B.V.) at 2000 psi and then linen textile archaeological samples (75-200 g each) were either concentrated in a rotar evaporator or lyophylized. purchased with a narrow calendar age certification from Russian Capillary Zone Electrophoresis (a). The Elma 2000 CZE and Ukrainian state museums and from one individual owner, Prof. apparatus was used (manufactured by the NPO-Electron InstruMario Moroni of Robbiate, Italy (see the Acknowledgment). ments, Russia, 1992). This apparatus includes a 35 kV highThe following known-age archaeological linen textile samples voltage power supply. Very similar equipment was used by Bruno were purchased: Al, Egyptian relic, 1200-1270 B.C. (from the et aL6 in their CZE experiments. However, we have modified this Russian National Historical Museum, Moscow, Russia, museum apparatus with homemade microcylindrical electrodes. The no. 34118KL9);A2,Coptic relic, AD. 380-450 (from the Russian microcylindrical Cu electrodes were constructed with 2 5 , m National Historial Museum, museum no. 20014AE3); B, Israeli diameter copper wire. One end of a 2-3-cm piece of the fine relic, 100 B.C.-AD. 100 (from the Israel Antiquities Authority, wire was attached to another piece of Cu wire (3-7 cm length, Jerusalem, Israel, courtesy of Prof. M. Moroni, Robbiate, Italy); 380 y m diameter) by means of gold paint, providing electrical C1, Irish relic, A.D. 350-400 (from the Moscow State Institute of connection. A glass capillary (Precision Instruments, Inc., SaraTextile Museum, Moscow, Russia, museum no. S411); C2, sota, FL) was pulled with a vertical pipet puller, resulting in two Southem English relic, AD. 1100-1140 (from the Moscow State capillary pieces with microtip at one end of each. The tip of the Institute of Textile Museum, museum no. R832); D, Middle glass micropipet was gently cut to allow passage of the fine Cu Russian relic, AD. 1460-1520 (from the Museum of Slavic Applied wire. The fine wire was carefully introduced into the glass Art,Vladimir, Russia, museum no. 655E): E, Northern Greek relic, capillary through the end opposite the microtip, exposing 300A.D. 860-930 (from the Crimean State Archaeological Museum, 400 pm of the Cu microwire. Under a microscope, epoxy (Epoxy Simpheropol, Ukraine museum no. TK 4451); and F, Eastem Technology, Inc., Billerica, MA) was applied to the tip of the glass Polish relic, AD. 1280-1330 (from the West Ukrainian Museum capillary to seal the fine Cu wire to it. The electrical wire of Ethnography and Archaeology, Temopol, Ukraine, museum no. protruding at the other end of the glass capillary was also epoxied A8026). to support the electrical connection. The electrode was then Cellulase (1,4 [1,3;1,4]-&glucan4-glucanohydrolase; EC 3.2.1.4.) mounted on a microscope slide and manipulated into the elecwas purchased from Sigma Chemical Co. (St. Louis, MO). One trochemical cell by micromanipulators. Before the microelectrode unit of this enzyme was able to liberate 1.0 mol of glucose from was used in combination with CZE, it was cycled between 0.0 and cellulose in 1 h at pH 5.0 at 37 "C (2 h incubation time). 0.8 V for approximately 4-5 min. Diaflo YM-1 ultrafiltration membranes with an exclusion limit New fused-silica capillaries (see the Materials and Reagents of 1000 Da were purchased from Ainicon B.V. (Holland). section) were treated by flushing ikst with 150 mM NaOH soluton The capillary electrophoresis columns were fused-silica capiland then with a separation electrolyte (100 mM tetraborate buffer, laries with the following parameters: id., 50 ym; o.d., 365 ym, pH 9.0). Before each run, the capillary was flushed with the where 5 mm of the polyamide coating was removed for detection: separation electrolyte. In addition, the electrolyte solution at the capillary length, 70 cm, 55 cm to detector (Polymicro Technoloelectrochemical cell was also replaced before each run. This gies, Inc., Phoenix, AZ). procedure was necessary since the separation current was All chemicals used were of analytical grade (Serva Heidelberg, observed to decrease by approximately 10% during 1 h of GmbH, Germany). continuous running. The capillaries were filled with deionized Cleaning of Textile. The textile samples were defatted with water for overnight storage. an alcohol-benzene (1:2 v/v) mixture for 6 h and air-dried. The The sample was siphon injected by inserting the column into (8) Heller, J. H.: Adler, A D. A Chemical Investigation of the Shroud of Turin. the sample vial and elevating the vial by 20 cm for 10-150 s, Can. SOC. Forensic Sci. J. 1981,14, 81-102. (9) Jumper, E. J.; Adler, A D.; Jackson, J. P.: Pellicomi. S. F.; Heller, J. H.; producing an injection volume of 50-750 nL, corresponding to Druzik, J. R A Comprehensive Examination of the Various Stains and 40.0-500.0 ng of pure glucose. Images of the Shroud of Turin. Archaeological Chemistry, I I t Advances in The separation regime includes the following parameters: Chemistry Series 205; Americam Chemical Society: Washington, DC, 1984; pp 447-475. voltage, 14.0 kV; current, 50 ,uA thermocooler temperature, 27 ~
4360 Analytical Chemisfy, Vol. 66, No. 23, December 1, 1994
14
I
1
5
Figure 1. General scheme of the CZE refractive index detector design. (1) He-Ne laser; (2) optical unit; (3) capillary cell; (4)temperature sensor; (5) mirror; (6) Peltier element; (7)radiator; (8) temperature control unit; (9) motorized translation stage; (10) positron-sensitive diode; (1 1) analog electonics unit; (12) autozero control unit; (13) data conversion acquisition unit; (14) output; (15) CZE pattern recorder; (16) Multiscan DX600 unit for computerizedcomparative analysis of numerous (multiplesimilar) electrophoretic profiles (NPO-Electron Instruments, Inc., Russia).
"C; interference fringe, n - 2; separation time, up to 40 min; separation medium, 100 mM tetraborate, pH 9.0; capillary column geometry, see above (Materials and Reagents); applied electric field, E, 25 V cm-l at the maximal current, 57.0 pA. The oncolumn laser-based refractive index 0 detector has been used designed according to recommendations given by Bruno et ale6A general scheme of the RI detector opticomechanical unit is presented in Figure 1. This RI detector is based on the positron-sensitive diode (PSD) and includes the surrounded liquid (RIMF') cooling system. As for the RI cell itself, the best thermal stability in the cell is achieved when the thermocooler system is set at a temperature slightly higher than ambient. The resulting ca. 1 "C temperature gradient is localized near the cell windows and is directly correlated to an n gradient in the RIMF and fused silica according to their dn/dT thermal coefficients. Small fluctuations in the temperature around the exit air/window/RIMF interface, in contact with the environment, introduce an additional source of noise if a flat exit window is employed. In a cell constructed with two plane parallel windows, the incoming laser beam crosses the entrance air/window/RIMF interface at go", and the n gradient does not alter the lightpropagating path (i.e., no refraction). The capillary tube and the flat entrance window are mounted and sealed with a pair of 0 rings and screws, whereas the cylindrical exit window is epoxied to the cell body. The capillary is coiled into a groove around the external part of the aluminum block before it enters the chamber filled with RIMF after crossing a region in contact with the Peltier element. The Peltier element is in contact with the capillary tube 5 mm before it enters the chamber filled with RIMF. A calibrated thermistor is placed on the opposite side of the thermocooler, in close contact with the capillary tube in a small hole in the aluminum block. Both elements are driven by a thermoelectric system. When no electrical current flows through the buffer, the short-term thermal stability (t = 1 s) of the system is better than 2 x "C and has a typical drift of less than 1 x deg h-l. A good RIMF should have a small dn/dT coefficient, not be photodegrade, and have a good transmittance. We employed a commerical RIMF oil (Nos. 19569 and 19571, R P. Cargille
Laboratories Inc., Cedar Grove, NJ) having n = 1.4571and dn/dt = 3.86 x RIU K-' at 25 "C. In all our RIMF-cooling CZE separation experiments, at the center of the bore, T(r = 0) = 26.10 "C. The RI detector we used measures An originated not only at the capillary bore but also at any point of the optical path from the laser output coupler to the PSD. The laser (Uniphase No. 1103P), optical isolator (constructed with a polarizing filer and a 1/4 plate @rs. Steeg and Reuter, Germany), focusing optics, and cell are mounted on four sliding stainless steel rods (S & H NO. 061216) in contact with each other to prevent air flows in the beam path. The laser beam has a diameter of 600 pm and is focused into the capillary bore with a f = 40 cylindrical lens producing a beam waist of ca. 23 x 600 pm in the capillary bore. The cell volume, defined by the capillary i.d. and the beam waist, for a 5@,pm-i.d. tube is 1.2 nL. An autozero for the instrument is constructed by feeding the output of the PSD into a servo system which drives the PSD to the desired position by means of a motor (NPO-Electron Instruments). The applied RI detection system makes it possible to reach a high level of thermal stability, AT = 2.0 x "C, as described earlier by Bruno et a1.6 This is a result of a highly symmetric design intended to ensure fast thermal response from the thermoelectric system. In this efficient RI detector, the linear dynamic range extends to more than 3 orders of magnitude, with a trpical root-mean-squarenoise level of 3 x lo-* RIU and baseline drifts of 2 x RIU h-l at 1 Hz. Data acquisition was performed with the Nelson software package (Perkin-Elmer,Switzerland) on an IBM AT380 computer. The CZE system was unified (connected) with the mass spectrometer by an on-line coupling link using the capillary electrophoresis/mass spectrometry interface in our modification of the technical approach described by Thompson et al.5 This electrophoresis/mass spectrometry interface diagram is presented on Figure 2; in all our studies, the MK80 field ionizaton (lT)/field desorption (FD) mass spectrometer (NPO-Electron Instruments) was employed. The stainless needle supplied with the instrument was replaced with a polyimide-coated fused-silica capillary used in CE. This change was made to eliminate any junctions that could abe Analytical Chemistry, Vol. 66,No. 23, December I, 1994
4361
LS
I
c;s 1
GST
CE
LiT
HVPS - 1
El + l \ \ El 1N C
I '* r
1
MS
ES
HVPS - 2
Figure 2. General scheme of the capillary electrophoresis/Fl(FD) mass spectrometry interface. S,septum; LST, liquid shealth tube; LS, liquid shealth entry port; GST, gas shealth tube; GS, gas shealth entry port; AR, anode reservoir; Ce, CZE column (capillary); NC, nitrogen curtain drying gas; FU, FI(FD)-MS focusing unit (ion source compartment element); ES, entrance slit to mass analyzer; HVPS-1, high-voltage power supply for CZE; HVPS-2, high-voltage power supply for MS; Ah, height difference between the anode and cathode ends of the CZE column.
detrimental to the separation and to enable the end of the capillary to be located at the electrospray needle tip. The interface utilized a coaxial liquid sheath, as shwon previously, as well as a coaxial gas sheath. The liquid sheath tube was replaced by stainless steel tubing of -0.4 mm i.d. and -0.7 mm o.d., and the gas sheath tip was also replaced with a tip having an orifice of 1.0 mm i.d. The liquid sheath tip was narrowed to -0.5 mm. The fused silica separation capillary terminated 0.5 mm inside the liquid sheath tip. A silicone septum was also added to prevent back flow of sheath liquid along the capillary. Figure 2 shows that the differnce in height (Ah) between the anode reservoir and the tip of the MS needle (the cathode end of CZE column) was -10 cm. A partial vacuum was created due to the flow of the gas sheath at the capillary tip, and, with the ends of the capillary at equal height, a signiscant bulk liquid flow toward the cathode took place. In order to compensate for this pressure drop, the level of the anode reservoir was lowered. The capillary was first filled with the leading electrolyte, and then -150 nL of M Methyl Green dye was siphon injected. The injection end of the capillary was then placed in the terminating electrolyte reservoir, and the current was applied. The current was turned off when the dye had focused into a narrow 2-mm-long band. Movement of the zone could easily be observed through the polyimide coating of the capillary, due to the high concentration of this focused dye. The height of the reservoir was adjusted until no movement of the dye was observed in either the forward or the reverse direction. The MS needle, as shown in Figure 2, was maintained at ground potential, while the sampling orifice was at about -4000 V when the system was operated in the positive ion mode. The drying gas (nitrogen curtain) for the MS was maintained at about 100 "C at a flow rate of 6 L/min, while the sheath gas flow was set at approximately 2 L/min. The liquid sheath consisted of 1% acetic acid in 50%2-propanol/water, flowing at a rate of 4.0 pL/ min. Tuning and calibration of the mass spectrometer were performed using a 5 pmol/pL glucose solution. Mass Spectromery. Due to the functioning of the CZE/MS interface described above (Figure 2), all electrophoretic fractions were transferred into the MK80 mass spectrometer for further analysis of the chemical structure. The pure glycerol was used 4362 Analytical Chemistry, Vol. 66, No. 23, December 7, 1994
as a matrix material. In our observation, the final glucose concentration range in the applied sample was equal to 1.0-30.0 mM, depending on individual CZE fraction, which normally corresponds to 1.0-5.0 pL of the postelectrophoretic solution. Before the sample was introduced into the ion source cell, it was briefly degassed to remove most of the water. Each of the MK80 internal copper probe tips contained of 2 pL of matrix material. The MK80 mass spectrometer (NPO-Electron Instruments) contained two separate independent ion source units specially designed for both field ionizatin and field desorption versions of mass spectrometryP For both FI and FD versions, the anode/cathode potential was equal to 8.0 kV difference, and the potential gradient developed was equal to lo* V cm-l. A conventional version of both FI and FD techniques was used in both cases, ionization occurred when a molecule was subjected to a high potential gradient while close to an anode, which can accept electrones. The positive ions are drawn toward a cathode and then into the mass analyzer. In FI, the sample is evaporated, and molecules come very close to or impinge upon the anode (emitter), where they are ionized. In FD, the sample is coated onto the emitter, and the ions are desorbed from the solid ~ t a t e . ~ , ~ The ion current has been monitored on the rear trapping plate of the analyzer cell and usually is between 1.0 and 10.0 nA. The ion production/injection time is variable and is typically between 5 and 500 ms, although pulses as narrow as 1ms are sufficient to obtain signals. Frequencies ranged between 0.5 and 1.5 MHz. Ions were collected linearly by increasing the length of the injection until saturation (space charge limit) was reached. Saturation was registered normally between 100 and 1000 ms, depending on the strength of the signal. The pumping speed was equal to 170 L/s due tothe turbo pump which operates on the source. Two cryopumps each with a pumping speed of 2000 L/s (for Nz) operate on the ion transport region and on the analyzer region. Pressures in the source during the experiment were normally in the range 10-4-10-5 Torr, while pressures in the analyzer region were maintained at 10-9-10-10 Torr.1oAs it may be seen from above, a conventional MSFI/FD analysis has been used.2
PURIFIED LINEN
FIBERS
CELLULASE HYDROLYSIS
A ULTRAFILTRATION
-ISOLATED POOL O F THE DEPOLYMERIZED CELLULOSE LOW MOLECULAR WEIGHT COMPOUNS
CAPILLARY ELECTROPHORESIS (CE)
SPECTROMETRY (MS)
I
COMPUTERIZED QUANTIFICATION (CE) / IDENTIFICATION (MS) O F GLUCOSE DERWATIVES
Figure 3. General scheme illustrating the consequence of Steps and the final aim of the experimental procedures.
All mass spectra were normalized to the protonated glycerol peak with subsequent computerized interpretation (chemical structure estimate) using a conventional FOK"/PAD algorithm in the IBM AT380 computer connected with a data bank of the Russian National Center for Ecology Studies at Moscow, Russia. For this purpose, the RNCES software for furanose/ pyranose compounds identification was used.4 A series of scans were accumulated for each spectrum. The numbers of accumulated scans vary: for long ion injection time (e.g., 500 ms), only 10 scans were accumulated, while for the short injection time (e.g., 5 ms), up to 100 scans were accumulated. A routine analysis usually lasts several minutes. NOTE: a general scheme of the whole experimental procedure is presented in Figure 3. RESULTS AND DISCUSSION As seen from our data presented in Figures 4 and 5, all eight archaeological textiles we tested contain the alkylated cellulose chains. Thus, this alkylation phenomenon includes formation of such derivatives of the glucose residues as 2-acetyl-&methyl-p-Dglucose and &methyl-B-D-glucose (textiles, Al, A2, and B); 2-carboxy-6methyl-/3-~-glucose(textiles C1 and C2) ; 2-carboxyP-D-glUCOSe (textiles D, E, and F);and 2,&dicarboxy-~-~-glucose (textile E). Furthermore, the presence and abundance of these (10) Carrol, J. A; Ngoka, L.; McCullough, S.; Gard, E.; Jones, D. A; Lebrilla, C B. Quadrupole Fourier Transform Mass Spectrometry of Oligosaccharides. Anal. Chew. 1991,63, 2526-2529.
15
20
25
30
15
20
25
30
RETENTION TIME (R,),min ~~
PEAK
Rt
IDENTIFIED STRUCTURE
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19
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21
p-D-GLUCOSE
-D- GLUCOSE
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22
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9
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6 - METHYL- B -D
METHYL-p -D
- GLUCOSE
- D -GLUCOSE - GLUCOSE
Figure 4. Separation of products of enzymatic digestion of the fibrous cellulose from eight archaeological textile samples by capillary electrophoresis with simultaneous mass spectrometric identification of each fraction. For experiment record codes (A1 -F), and technical details, see the Experimental Section.
clearly identified acetyl-, carboxy-, or methyl-containing glucose residues in the cellulose chains depend on the individual textile sample (Figures 4 and 5, Table 1, see Materials and Reagents). For example, the content of both 2-acetyl-6methyl-~-~-glucose and 6methyl-B-D-ghcose residues in the textile cellulose does increase with the age of the relic (A1 > B > A2,Table 1). It is also interesting to note that all these textiles (Al, A2,and B) were founded and possibly manufactured in one geographical region, Israel and Egypt. As for the other six textiles tested, they had other ages and geographical origins (see Materials and Reagents), and they contain other chemical modifications of cellulose based on permethylation and carboxylation of the latter (Figures 4 and 5). In a group relatively young Western European textiles (C1 and C2) and in the Eastern European medieval textiles 0, E, and F), the same regularity should be noted: cellulose alkylation extent increases with the increasing calendar ages (Figure 4,Table 1). All CZE patterns we obtained were clear and well reproducible. It seems the character of alkyl groups and the the chemical structure of alkylated glucose residues depend on the geographical (regional) origin of the textile (compare Cl/C2 and D/E/F see Figure 4,Table 1). Mass spectromeric identification of each alkylated glucose fraction presented in the CZE pattern is beyond any doubt (Figure 5 ) . Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
4363
181MH'
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,
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m/z Figure 5. Mass spectra of monosaccharide fractions isolated by capillaty electrophoresis of the textile cellulose hydrolysates. Spectra I-Vi, field ionization technique; VII-XII, field desorption technique; I and VII, nonmodified glucose fraction isolated from and textile sample tested (A1 -F); II and VIII, fraction 2 isolated from samples A l , A2, and 8; 111 and IX, fraction 6 isolated from samples C1 and C2; IV and X, fraction 7 isolated from samples D, E, and F; V and XI, fraction 8 isolated from sample E; VI and XII, fraction 9 isolated from samples A l , A2, and 8. Two matrix peaks are labeled corresponding to protonated glycerol (mlz92, GH+) and the protonated dimer (mlz 185, G*H+). The fragment ions at mlz 163, 145, and 127 respresent successive losses of H20 from (MH+). For experiment record codes (A1-F), see the Experimental Section. Table 1. Abundance of Alkylated Qlucose Derivatives in Cellulose Chains from Several Archaeological Textile Samples'
sample*
content of minor glucose derviativesc
A1
2-acetyl-f.+methyl-~-~-glucose
A2
2-acetyl-&methyl-/3-~-glucose
B
2-acetyl-Bmethyl;B-~-glucose
c1
2-carboxy-6-methyl-~-~-glucose 2-carboxy-&methyl-~-~-glucose
&methyl-B-D-glucose &methyl-B-D-glucose
c2 D
E
&methyl-D-D-glucose 2-carboxy-/3-~-glucose 2-carboxy-B-~-glucose 2,Bdicarboxy-~-~-glucose
F
2-carboxy-~-~-glucose
5.40 f 0.08 6.12 f 0.04 2.01 +. 0.007 1.99 f 0.006 4.25 f 0.02 5.12 f 0.02 5.98 f 0.08 2.74 f 0.02 2.88 f 0.03 6.33 f 0.07 3.27 f 0.02 4.18 i 0.02
u'Ikese data were obtained using the refractive index change detection in capillary electrophoresis (see the Experimental Section). * For experiment record codes (A1-F), see the Experimental Section. c Percentage of total cellulose hydrolysate (M f SEM, n = 6).
Summarizing the data on the cellulose alkylation extent (CAE) listed in the Table 1, we have found a crude but clear enough correlation between the calendar age values of the textiles tested and their CAE values. In our opinion, this type of dependence could be a subject for further fruitful investigations leading to the improvement of accuracy of the existing dating methods in archaeology and archaeological chemistry. 4364 Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
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In fact, all modem archaeological textile dating techniques need to be improved and developed for better accuracy and efficiency. It seems that detailed chemical study of cellulose structure in different old textiles is a right way for creation of the efficient additional dating approach. In spite of the existence of the radiocarbon dating method, such additional and sometimes alternative dating techniques as stylistic detailed studies including microscopic r e s e a r ~ h ~ . ~are J ~still J ~ important as elements of interdisciplinary approaches to the dating accuracy problem. It is known that there are a number of unique morphological markers for textiles manufactured in several regions during limited historical periods.3J1,12 Obviously, these markers should be taken into account in each archaeological dating procedure with objects from corresponding sites. It should not be excluded that it is possible to find not only morphological but also molecular markers for ancient textiles of different geographical origins and/or calendar ages. We believe that our data Vable 1,Figures 4 and 5 ) demonstrate the great significance of this kind of research. As for the mechanism of cellulose alkylation in the old textiles, we could note at least two possibilities. First, it is logical to assume that the cellulose alkylation we have found in our work is a result of some unknown (lost, "archaic") technological processes for textile manufacturing. This assumption looks especially logical in the light of our data, which show that the most similar chemical structures of the alkyl derivatives were found in textile samples with common or close geographical origins (compare Al/M/B and Cl/C2; see Figures 4 and 5). Second, it should not be excluded that the cellulose alkylation process is a result of microbial contribution to the chemical moditicaton of textiles. Thus, the carboxylating enzymes of bacterial carboxysomes are active even in bacterial lysates in wet alkali conditions and in the presence of oxygen.13 The carboxylation of polyglucose can be easily promoted at pH 9.0 and in aerobic conditions by the destroyed (lysated) cells of several environmentally common bacteria of the Desulfovibrio genus.'* Finally, the normal autolysis of air and soil microorganisms may lead to significant carboxylation and/or methylation processes with different substrates directed by the released active bacterial e n z y m e ~ . l ~Thus, - ~ alkylation of the old textile cellulose could be the consequence of accumulation of the results of a great number of such a microbial treatments during the very long historic time interval. As for the alkali conditions for bacterial enzyme activity, all common soapfoaming washing procedures create this kind of pH change. Besides, alkalis were used as ingredients in some technological schemes of the past for rewashing of textile after its (11) Cramer, M. Das Christlich-Koptische Agipten Emst und Heute, Eine Orientiemng GmbH: Wiesbaden, Germany, 1959; pp 46-88. (12) Bresee, R R; Chandrashekar, V.; Jones, B. W. Age Determination of Textiles from Single-Fiber Creep Measurements. In Historic Textile and Paper Materials: Conversation and Characterization; Needles, H. L., Zeronian, S. H., Eds.; Americal Chemical Society Press: Washington, DC, 1986; pp 19-40. (13) Price, G. D.; Hewitt, S. M.; Harrison, M.; Badger, M. R Analysis of a Genomic DNA Region from the Cyanobacterium Synechococcus sp. Strain PCC7942 Involved in Carboxysome Assembly and Function. J. Bacterid. 1993,175, 2871-2879. (14) Hensgens, C. M. H.; Vonk, J.; Van Beeumen, J.; Van Bruggen, E. F. J.; Hansen, T. A. (1993) Purification and Characterization of an Oxygen-Labile NAD-Dependent Alcohol Dehydrogenase from Desulfovibrio gigas. J. Bacteriol. 1993,175, 2859-2863. (15) Doyle, R J.; Koch, A L. The Function of Autolysis in the Growth and Division of Bacillus subtilis. Crit. Rev. Microbiol. 1987,15, 169-222.
To the best of our knowledge, the present study is the first report on the discovery of alkylated cellulose sequences in archaeologicaltextiles. Further extensive studies on this subject could help to classtjj~a great number of different archaeological textile relics using such a criterion as the cellulose chemical mod8cation types. The classification mentioned would be an important tool for both dating research in archaeological chemistry and studying ancient textile technology. CONCLUSION The capillary zone electrophoresis equipped with the oncolumn laser (He-Ne)-based RI detector is an efficient approach to investigation of chemical modifications of the cellulose, including the studies in archaeological textile chemistry. ACKNOWLEDGMENT This work was supported by the Guy Berthault Foundation, Meulan, France. We are especially grateful to Prof. Mario Moroni of Robbiate, Italy, for sending us a fragment of linen cloth form a burial at En Gedi site, Israel, historically dated to the Early Roman Period (100 B.C.-AD. 100) by the Israel Antiquities Authority. We would like to express our deep gratitude to Dr. Ivan Kappel, Assistant Keeper at the Russian National Historical Museum
(Moscow, Russia), Dr. Oleg Krutov, Keeper at the Moscow State Institute of Textile Musemm (Moscow, Russia), Mrs. Olga Nenasheva, Deputy Director at the Museum of Slavic Applied Art (Vladimir, Russia), Mr. Sergey Bychkov, Deputy Director at the Crimean State Archaeological Museum (Simpheropol, Ukraine), and Mr. Ignat Tyshko, Keeper at the West Ukrainian Museum of Ethnography and Archaeology (Temopol, Ukraine) for their remarkable courtesy in giving us a set of light, nondyed, knownage samples of archaeological linen textiles. We thank Dr. Alan Adler of the University of Westem Connecticut at Danbury, CT, Prof. Witold Brostow of the University of North Texas at Denton, TX, and Dr. Alexander Volkov of Moscow State University for their critical remarks and fruitful participation in the discussion of our preliminary results. Mr. Tmophey Palevich of the Moscow State University is especially to be thanked for his continued assistance in the preparation of samples for capillary electrophoresis. Received for review March 7, 1994. Accepted August 9, 1994.a e- Abstract published
in Advance ACS Abstracts, September 15, 1994.
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