Analysis of Cellulose Chemical Modification: a Potentially Promising Technique for Characterizing Cellulose Archaeological Textiles

Journal of Archaeological Science (1996) 23, 23–24

Analysis of Cellulose Chemical Modification: a Potentially Promising Technique for Characterizing Cellulose Archaeological Textiles Dmitri A. Kouznetsov, Andrey A. Ivanov and Pavel R. Veletsky S. A. Sedov Biopolymer Research Laboratories, Inc., 4/9 Grafski Per., Moscow 129626, Russia (Received 14 March 1994, revised manuscript accepted 16 August 1994) Fifteen different known age samples of light non-dyed linen textile dated within the range of  1200– 1500 years 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. A high resolution pattern was obtained using the 100 m tetraborate (pH 9·0) capillary 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 sequences of different 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 discussed, including special reference to correlations between such parameters as (1) alkylation peculiarities, (2) geographical area of the textile manufacturing, and (3) the textile calendar age values. ? 1996 Academic Press Limited Keywords: CAPILLARY ZONE ELECTROPHORESIS (CZE), REFRACTIVE INDEX DETECTION, ARCHAEOLOGICAL TEXTILE CELLULOSE MODIFICATION.

In our opinion, the best 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 (non-modified and modified â--glucose, plus cellobiose) of the enzymatically digested cellulose pool isolated from the textiles compared. Thus, a combination of capillary electrophoresis and mass spectrometry should be very efficient for the 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 capillary column are compatible with on-line coupling to a mass spectrometer (Yakimov & Zheltkov, 1990; Thompson et al., 1993). Capillary zone electrophoresis (CZE), in its numerous variants, is an alternative separation technique to HPLC. The remarkable performance of this analytical tool has been demonstrated by unprecedented separation efficiencies in many studies including the most recent research on carbohydrates (Bruno et al., 1991; Colon, Dadoo & Zare, 1993). Two significant problems have been identified in the analysis of sugars by CZE. First, carbohydrates are not charged species under normal conditions; secondly,

Introduction here is currently a great deal of interest in the development of new approaches to chemical investigations of archaeological textile relics (Cardamone & Brown, 1986; Ageyev, 1992). The importance of these studies stems from their potential to illuminate the chemical mechanisms of textile ‘‘ageing’’ and, therefore, be used for further improvement of the accuracy of the ancient textile dating procedures. In addition detailed chemical studies on archaeological textiles could provide important information about lost ‘‘archaic’’ technological processes practised long ago in different geographical areas (Lee, 1953; Ageyev, 1992). Finally, any information on historic textile chemical ‘‘ageing’’ should be important for development of methods for conservation of the relics. Chemical investigation of old textiles can be performed by a number of methods such as near-infrared/ infrared reflectance spectrometry of undisintegrated textile; high resolution electron microscopy; atomic absorption spectrophotometry for element analysis; several chromatographic (e.g. gas chromatography and HPLC) and electrophoretic techniques for fractionation and quantification of textile cellulose enzymatic hydrolysate products (Cardamone & Brown, 1986; Yakimov & Zheltkov, 1990; Ageyev, 1992).

T

23 0305-4403/96/010023+12 $12.00/0

? 1996 Academic Press Limited

24

D. A. Kouznetsov, A. A. Ivanov and P. R. Veletsky

Table 1. Calendar age and origin characteristics of the linen textile samples studied by capillary electrophoresis/mass spectrometry analysis of the cellulose enzymatic hydrolysates Calendar age (years)

Museum number

Experiment record code

34118KL9

A1

Coptic Linen, Bagauate site

†  1200–1270 †  380–450

20014AE3

A2

Israel Antiquities Authority (Jerusalem, Israel)*

Palestine, El Gedi site

 100– 100

Moscow State Institute of Textile Museum (Moscow, Russia)

Ireland, Limerick site

S411

C1

Southern England

†  350–400 †  1100–1140

R832

C2

Museum of Slavic Applied Art (Vladimir, Russia)

Middle Russia

 1460–1520

655E

D

Crimean State Archaeological Museum (Simpheropol, Ukraine)

Northern Greece

 860–930

TK4451

E

West Ukrainean Museum of Ethnography and Archaeology (Ternopol, Ukraine)

Eastern Poland, Gniezno site

 1280–1330

A8026

F

Museum Russian National Historical Museum (Moscow, Russia)

Area of origin Egypt, Alexandria site

B

*Courtesy of Professor Mario Moroni and Mrs Tamar Schick. †These calendar ages were estimated by radiocarbon methods at the Russian Academy of Sciences Radiochemical Centre, Moscow, Russia. All other samples listed in this table were dated using historical evidence and stylistic details only.

they have poor detectability. Nevertheless, it is easy to reach an efficient separation by adding borate ions to the separation medium to promote 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 (Colon et al., 1993). The low sample volumes injected in CZE make detection of carbohydrates a difficult task. Enhancements in sensitivity by UV-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 nmol range. At the same time, alternative methods of detection such as electrochemical (amperometric) methods or refractometric methods based on the refractive index changes registration can reach higher sensitivity in the CZE experiments with mono- and oligosaccharides (Bruno et al., 1991; Ageyev, 1992; Colon et al., 1993). Some of the cellulose chains in ancient textile relics contain a significant number of chemically modified â--glucose residues like hemiacetal-derivatives of glucose or its carbonyl- (conjugated) derivatives (Heller & Adler, 1981; Jumper et al., 1984). Taking into account everything stated above, we have decided to carry out a comparative study on the possible chemical modification of cellulose from 15 different archaeological textiles dated within the range of  1200– 1500 years which were kindly provided

by several European museums (see Table 1 and Figure 6). For this study, we have used CZE of the purified cellulose enzymatic hydrolysates with a mass spectrometric identification of each peak (see ‘‘Experimental Procedures’’ and Figure 3).

Experimental Procedures Materials and reagents Light (non-dyed), clean-looking, small portions of the linen textile archaeological samples (75–200 g each) were purchased with calendar age certification from Russian and Ukrainian state museums and from one individual owner, Professor Mario Moroni of Robbiate, Italy. Cellulase (1,4-[1,3;1,4]-â--Glucan 4-glucanohydrolase; E.C. 3.2.1.4) was purchased from Sigma Chemical Co. (U.S.A.). One unit of this enzyme was able to liberate 1·0 mol of glucose from cellulose in 1 hour at pH 5·0 at +37)C (2 h incubation time). Diaflo YM-1 ultrafiltration membranes with an exclusion limit of 1000 Da were purchased from Amicon B.V. (Holland). The capillary electrophoresis columns were fused silica capillaries with the following parameters: i.d.=50 ìm, o.d.=365 ìm where 5 mm of the polyamide coating were removed for detection; capillary length 70 cm, 55 to detector (Polymicro Technologies, Inc., U.S.A.). All chemicals used were of analytical grade (Serva Heidelberg, GmbH, Germany).

Analysis of Cellulose Chemical Modification 25

Cleaning of textiles The textile samples were defatted with an alcohol– benzene (1:2, v/v) mixture for 6 h and air dried. Then, the defatted samples were submerged in a solution containing 7·7% formaldehyde, 7·7% borax, and 0·5% sodium dodecylsulphate for 3 min, and dried in an electric oven at 100)C for 2 h. Finally, all the samples were washed extensively with demineralized (deionized chromatographically on Amberlyte resins) water, 120 ml/cm "2, at room temperature. For this purpose, a textile sample has been applied as a filter in a water flow. The cleaned and washed textile samples (up to 2·8 cm2 each) were air dried and kept in sealed dry flasks for further studies. Enzymatic hydrolysis of the textile cellulose Mechanically disintegrated textile samples (2·0–2·8 g) (crude fibrous material) were incubated at +37)C for 6 h in 20 ml of 15 m, Tris-borate (pH 6·40) buffer containing 4·5–5·0 units of cellulase per ml. These conditions are enough for 98% cellulose depolymerization (Ageyev, 1992). After incubation, the low molecular weight compounds pool was separated from the hydrolysate by ultrafiltration through the Diaflo YM-1 membranes in Amicon MMC-10 apparatus (Amicon B.V., Holland) at 2000 p.s.i. and then concentrated in a rotor evaporator or lyophylized. Capillary zone electrophoresis (CZE) An Elma 2000 CZE apparatus was used (manufactured by the NPO-Electron Instruments, Russia, in 1992). This apparatus includes a 35 kV high-voltage power supply. Very similar equipment has been used by Bruno et al. (1991) in their CZE experiments. However, we have modified this apparatus with our own microcylindrical electrodes. Thus, the microcylindrical Cu electrodes were constructed with 25 ìm-diameter copper wire. One end of a 2–3 cm piece of the fine wire was attached to another piece of Cu wire (3–7 cm length, 380 ìm diameter) by means of gold paint, providing an electrical connection. A glass capillary (Precision Instruments, Inc., Sarasota, FL, U.S.A.) was pulled with a vertical pipette puller, resulting in two capillary pieces with a microtip at one end of each. The tip of the glass micropipette was cut gently to allow passage of the fine Cu wire. The fine wire was introduced carefully into the glass capillary through the end opposite the microtip, exposing 300–400 ìm of the Cu microwire. Using a microscope, epoxy (Epoxy Technology, Inc., Billerica, MA, U.S.A.) was applied to the tip of the glass capillary to seal the fine Cu wire to it. The electrical wire protruding from the other end of the glass capillary was also epoxied to support the electrical connection. The electrode was then mounted on a microscope slide and manipulated into the electrochemical cell by micromanipulators. Before using

the microelectrode in combination with CZE, it was cycled between 0·0 and 0·8 V for approximately 4–5 min. New fused silica capillaries (see ‘‘Materials and Reagents’’) were treated first by flushing with 150 m NaOH solution and then with a separation electrolyte (100 m tetraborate buffer, pH 9·0). Before each run, the capillary was flushed with the separation electrolyte. In addition, the electrolyte solution in the electrochemical cell was also replaced before each run. This procedure was necessary since the separation current was observed to decrease by approximately 10% during 1 h of continuous running. The capillaries were filled with deionized water for overnight storage. The sample was siphon-injected by inserting the column into the sample vial and elevating the vial by 20 cm for 10–150 s, producing an injection volume of 50–750 nl corresponding to 40·0–500·0 ng of pure glucose. The separation regime includes the following parameters: voltage, 14·0 kV; current, 50 ìA, thermocooler temperature, 27)C; interference fringe, n—2; separation time, up to 40 min; separation medium, 100 m tetraborate, pH 9·0; capillary column geometry, see above; applied electric field, E= 25 V cm "1 at a maximal current of 57·0 ìA. The on-column-laser-based refractive index (RI) detector was designed according to recommendations given by Bruno et al. (1991). A general scheme of the RI detector optico–mechanical unit is presented in Figure 1. This RI detector is based on a positronsensitive diode (PSD) and includes the surrounded liquid (RIMF)-cooling system. In 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 c. 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 the 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 plan parallel windows, the incoming laser beam crosses the entrance air/ window/RIMF interface at 90), and the n gradient does not alter the light propagating path (i.e. no refraction). The capillary tube and the flat entrance window are mounted and sealed with a pair of ‘‘O’’ 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 aluminium 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 field with RIMF. A calibrated thermistor is placed on the opposite side the thermocooler, in close contact with the capillary tube in a small hole in the aluminium block. Both elements

26

D. A. Kouznetsov, A. A. Ivanov and P. R. Veletsky

Figure 1. A 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; (7) radiator; (8) temperature control unit; (9) motorized translation stage; (10) positron sensitive diode; (11) analogue electronics unit; (12) autozero control unit; (13) data conversion/acquisition unit; (14) output; (15) CZE pattern recorder; (16) MultiScan DX600 unit for computerized comparative analysis of numerous (multiple similar) electrophoretic profiles.

are driven by a thermoelectric system. When no electrical current flows through the buffer, the short-term thermal stability (ô=1 s) of the system is better than 2#10 "4)C and has a typical drift of less than 1#10 "2)C h "1. A good RIMF should have a small dn/dT coefficient, not be photodegradable, and have good transmittance. We employed a commercial RIMF oil (no.s 19569 and 19571, R.P. Cargille Laboratories Inc., Cedar Grove, NJ, U.S.A.) having n=1·4571 and dn/dT=3·86#10 "4 RIU K "1 at 25)C. In all our RIMF-cooling CZE separation experiments, T(r=0)=26·10)C at the centre of bore. The RI detector we have used measures Än originating not only at the capillary bore but 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 filter and a ë/4 plate (Dr. Steeg & Reuter, Germany), focusing optics, and cell are therefore 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 ìm and is focused into the capillary bore with a f=40 cylindrical lens producing a beam waist of c. 23#600ìm in the capillary bore. The cell volume, defined by the capillary i.d. and the beam waist, for a 50 ìm 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, Russia). The RI-detection system makes it possible to reach a high level of thermal stability, ÄT=2·0#10 "4)C, as described earlier by Bruno et al. (1991). This is a result of a highly symmetric design aimed to ensure fast thermal response from the thermoelectric system. In

this efficient RI-detector, the linear dynamic range extends to more than three orders of magnitude with a typical RMS noise level of 3#10 "8 RIU and baseline drifts of 2#10 "8 RIU h "1 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) to the mass spectrometer by the on-line coupling link using the capillary electrophoresis/mass spectrometry interface in our modification of the technical approach described by Thompson et al. (1993). This electrophoresis/mass spectrometry interface diagram is presented in Figure 2; in all our studies, the MK80 Field Ionization (FI)/Field Desorption (FD) Mass Spectrometer (NPO Electron Instruments, Russia) was employed. The stainless steel 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 be 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 shown previously, as well as a coaxial gas sheath. The liquid sheath tube was replaced by stainless steel tubing of 20·4 mm i.d. and 20·7 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 20·5 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 difference in height (Äh) between the anode reservoir and the tip of the MS needle (the cathode end of CZE column) was

Analysis of Cellulose Chemical Modification 27

Figure 2. A general scheme of the capillary electrophoresis/FI(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 analyser; HVPS-1: high voltage power supply for CZE; HVPS-2: high voltage power supply for MS; Äh: the height difference between the anode and cathode ends of the CZE column.

210 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 significant 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 2150 nl of 10 "3  methyl green dye was siphoninjected. The injection end of the capillary was then placed in the terminating electrolyte reservoir, and 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 operating 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 "1, while the sheath gas flow was set at approximately 2 l min "1. The liquid sheath consisted of 1% acetic acid in 50% 2-propanol/water, flowing at a rate of 4·0 ìl min "1. Tuning and calibration of the mass spectrometer were performed using a 5 pmol ìl "1 glucose solution. Mass spectrometry 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. Pure glycerol has been used as a matrix material. In our observation, the final glucose concentration range in the applied sample was equal to 1·0–30·0 m depending on the individual CZE fraction which normally corresponds to 1·0–5·0 ìl of the post-electrophoretic solution. Before the sample is introduced into the ion source cell, it is briefly degassed to remove most of the water. Each MK80 internal Cu probe tip contains 2 ìl of matrix material. The MK80 mass spectrometer (NPO Electron Instruments, Russia) contains two separate independent ion source units especially designed for both FI and FD versions of mass spectrometry (Yakimov & Zheltkov, 1990). For both FI and FD versions, the anode/cathode potential difference was equal to 8·0 kV, a potential gradient developed was equal to 108 V cm "1. A conventional version of both FI and FD techniques has been used: in both cases, ionization occurred when a molecule was subjected to a high potential gradient while close to an anode, which can accept electrons. The positive ions are drawn towards a cathode and then into the mass analyser. 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 state (Yakimov & Zheltkov, 1990; Ageyev, 1992). The ion current has been monitored on the rear trapping plate of the analyser cell and is usually between 1·0 and 10·0 nA. The ion production/injection time is variable and is typically between 5 and 500 ms

28

D. A. Kouznetsov, A. A. Ivanov and P. R. Veletsky

although pulses as narrow as 1 ms are sufficient to obtain signals; frequencies range 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 "1 due to the turbo pump which operates on the source. Two cryopumps, each with a pumping speed of 2000 l s "1 (for N2), operate on the ion transport region and on the analyser region. Pressures in the source during the experiment were normally in the range 10 "4– 10 "5 Torr, while pressures in the analyser region were maintained at 10 "9–10 "10 Torr (Carrol et al., 1991). As seen from above, a conventional MS-FI/FD analysis has been used (Ageyev, 1992). All mass spectra were normalized to the protonated glycerol peak followed by computerized interpretation (chemical structure estimate) using a conventional FORTRAN/PAD algorithm in the IBM AT380 computer connected with a data bank of the Russian National Centre for Ecology Studies at Moscow, Russia. For this purpose, the RNCES software for furanose/pyranose compounds identification has been used (Yakimov & Zheltkov, 1990). A series of scans are accumulated for each spectrum. The number of accumulated scans varies, for long ion injection time (e.g. 500 ms) only 10 scans are accumulated, while for the short injection time (e.g. 5 ms) up to 100 scans are accumulated. A routine analysis usually lasts several minutes.*

Results and Discussion As seen from the data presented in Figures 4 and 5, all eight archaeological textiles tested (Table 1) contain alkylated cellulose chains. Thus, this alkylation phenomenon includes formation of such derivatives of the glucose residues as 2-acetyl-6-methyl-â--glucose and 6-methyl-â--glucose (textiles A1, A2, and B); 2-carboxy-6-methyl-â--glucose (textiles C1 and C2); 2-carboxy-â--glucose (textiles D, E, and F); and 2,6dicarboxy-â--glucose (textile E). Furthermore, the presence and abundance of these clearly identified acetyl-, carboxy-, or methyl-containing glucose residues in the cellulose chains depends on the individual textile sample (Figures 4, 5 and 8, Table 2). For example, the content of both 2-acetyl-6-methylâ--glucose and 6-methyl-â--glucose residues in the textile cellulose does increase with the age of relic (A1>B>A2, Table 2); it is also interesting to note that all these textiles (A1, A2, B) were found and possibly manufactured in one geographical region, Israel and Egypt. The other six textiles tested (Table 1) are of *A general scheme of the whole experimental procedure is presented in Figure 3.

Purified linen fibres

Cellulase hydrolysis

Ultrafiltration

Isolated pool of the depolymerized cellulose low molecular weight compounds

Capillary electrophoresis (CE)

Mass spectrometry (MS)

Computerized quantification (CE)/ identification (MS) of glucose derivatives Figure 3. General scheme illustrating the consequence of steps and the final aim of the experimental procedures.

different ages and geographical origins and contain other chemical modifications of cellulose based on permethylation and carboxylation of the latter (Figures 4 and 5). In a group of relatively young western European textiles (C1 and C2) and in the eastern European mediaeval textiles (D, E, F) the same regularity should be noted: cellulose alkylation increases with calendar age (Figure 4, Table 2). All CZE-patterns we obtained were clear and easily reproduced. It appears that the character of alkyl-groups and the chemical structure of alkylated glucose residues depends on the geographic (regional) origin of the textile (compare C1/C2 and D/E/F—see Figure 4, Tables 1 and 2). It would therefore be interesting to reveal any correlations between the parameters estimated in our study for further discussion. The mass spectrometric identification of each alkylated glucose fraction presented in the CZE-pattern is beyond doubt (Figure 5). Summarizing the data on cellulose alkylation extent (CAE) listed in Table 2, we have found a crude but clear correlation between the calendar ages of the textiles tested and their CAE values (Figure 6). On the

Analysis of Cellulose Chemical Modification 29 A1

3 1

C2

3 1

2 9 4

5 4

5

A2

3 1

Refractive index change

D

3 1

4

2

5

5

B

E

7

3 1

2 4

9

4

5

6

8

5

C1

F

3

3 1

1

7

5 4

15

7

4

9

3 1

6

20

4

5

25 30 15 20 Retention time (Rt) (min)

25

Peak

Rt

2

16

Buffer

2

19

2-acetyl-6-methyl-β-D-glucose

3

21

β-D-glucose

4

22

Cellobiose

5

23

Impurity (non-pyranoses/furanoses pool)

6

24

2-carboxy-6-methyl-β-D-glucose

7

26

2-carboxy-β-D-glucose

8

29

2,6-dicarboxy-β-D-glucose

9

30

6-methyl-β-D-glucose

30

Identified structure

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.

30

D. A. Kouznetsov, A. A. Ivanov and P. R. Veletsky 100 I 80 60 40 20

Relative abundance

100 80 60 40 20

+

181MH 121 +

92GH

163

+ +

+

O OH

185G2H

185G2H

127 145

CH2OH

181MH

VII

92GH

OH

+

HO OH

+

II

+

VII

253MH 163

+

92GH

O

CH2

253MH

+

185G2H

+

O OH

185G2H

OH

+

92GH

127 145

CH3

O

HO

O

C CH3

100 III 80 60 40 20

+

+

IX

181MH +

239MH

185G2H

185G2H

163

+

92GH

CH3

O OH

+

OH

92GH

127 145

O

CH2

+

O

HO

C

O

OH

100 IV 80 60 40 20

100 V 80 60 40 20

+

225MH + 185G2H 163

+

X

CH2OH

225MH +

O OH

185G2H

+

92GH

OH

+ 127 145 92GH

O HO

O

C OH

+

+

+

+

145

CH2

+

185G2H

185G2H 92GH

O

269MH

XI

269MH

C

OH

O OH

+

163

O

92GH

OH

127 O

HO

O

C OH

100 80 60 40 20

+

VI

+

XII

195MH

CH2

195MH

+

+

185G2H + 163 92GH 127 145

185G2H +

92GH

120

160

200

240

80

280

CH3

OH HO

80

O

O OH

120

160

200

240

OH

280

m/z Figure 5. Mass spectra of monosaccharide fractions isolated by capillary electrophoresis of the textile cellulose hydrolysates. I–VI: field ionization technique; VII–XII: field desorption technique; I and VII: non-modified glucose fraction isolated from any textile sample tested (A1–F); II and VIII: fraction 2 isolated from samples A1, A2, and B; III 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 A1, A2, and B. Two matrix peaks are labelled corresponding to protonated glycerol (m/z 92, GH + ) and the protonated dimer (m/z 185, G2H + ). The fragment ions at m/z 163, 145 and 127 represent successive losses of H2O from (MH + ).

other hand, textile E does not contribute to a linear relationship between the CAE and textile ages at all, which is hard to explain. Perhaps more data on cellulose chemical modifications in the textiles of a single region will help to clarify this relationship. It would be logical to use the advanced statistical methods to clarify the crude CAE/AGE relationship illustrated in Figure 6. For this purpose, we have used a bivariate statistical approach which has been developed specifically for

comparison of two or more biopolymer primary structures on the basis of monomer content (abundance) data obtained by chromatographic or electrophoretic techniques (Sokal & Rohlf, 1981). Originally, this statistical approach has been designed for analysis of proteins and nucleic acids, i.e. for more heterogeneous macromolecules than polysaccharides. Nevertheless, this approach can be easily generalized for analysis of intramolecular heterogeneity of each class of biopolymers including the alkylated polysaccharides.

Analysis of Cellulose Chemical Modification 31 Table 2. The abundance of alkylated glucose derivatives in cellulose chains from several archaeological textile samples Sample studied (experiment record code)* A1

^

B C1 C2 D E F

(5·40&0·08) (6·12&0·04) (2·0&0·007) (1·99&0·006) (4·25&0·02) (5·12&0·02) (5·98&0·08) (2·74&0·02) (2·88&0·03) (6·33&0·07) (3·27&0·02) (4·18&0·02)

These data were obtained using Refractive Index Change detection in capillary electrophoresis. Analogous results were obtained also by computerized square peak analysis of electrophoregrams registered using optical density detection at ë=190 nm (Ultrascan RS80 Detector/Analyser, LOMO Instruments, Russia). *For experiment record codes (A1–F), see Table 1.

12.0 A1 E

B 9.0 CAE (%)*

R=

Content of minor glucose derivatives % of total cellulose hydrolysate (M&SEM, n=6) 2-acetyl-6-methyl-â--glucose 6-methyl-â--glucose 2-acetyl-6-methyl-â--glucose 6-methyl-â--glucose 2-acetyl-6-methyl-â--glucose 6-methyl-â--glucose 2-carboxy-6-methyl-â--glucose 2-carboxy-6-methyl-â--glucose 2-carboxy-â--glucose 2-carboxy-â--glucose 2,6-decarboxy-â--glucose 2-carboxy-â--glucose

A2

correlation coefficient is given in vector notation below:

C1 6.0 F

A2 C2

3.0

1000

1000 BC

D

2000

AD

Archaeological time (years)

Figure 6. The relationship between textile cellulose alkylation extent and calendar age in the eight linen samples tested. *Cellulose alkylation extent, i.e. total relative abundance of all alkylated glucose residues in the textile cellulose calculated as a total contribution of all alkyl-residues to the textile cellulose pool (%).

In general, the correlation coefficient (actually, the Product–Moment Correlation Coefficient) is a measure of the ‘‘goodness of fit’’ between a computed (standard) line and a set of experimental points (Sokal & Rohlf, 1981). A more generalized form for the

^

a^ · b . ^ ^ {( a^ · a^ )( b · b )}Y

The vector, a, is represented by the ordered monomer (pure non-modified fibrous ^cellulose) composition of the standard and the vector, b, is represented by the ordered monomer composition of the test polymer (alkyl–cellulose in archaeological textile). For this work, we ordered the alkyl–glucose derivatives compositions alphabetically for simplicity, although any order would be acceptable so long as the same system is used for both the standard pure non-modified cellulose assay and the test alkylated cellulose. The correlation coefficient is given as the vector dot product of the monomer composition of the standard and the test polymer is divided by the square root of the vector dot product of the standard to itself and the vector dot product of the sample to itself. In simple terms, the dot product of two vectors is the sum of the product of each of the corresponding elements of each vector. If the elements of one vector are designated pi, and those corresponding elements of a second vector as qi, then the dot product of the two vectors is Ópiqi. But the vectors may need to be normalized, and the elements of each vector must be ordered in the same fashion. In our research, the monomer assay data are normalized to 100% by virtue of the analysis procedure, and the elements have been ordered by listing the assay results in alphabetical order of the individual alkyl–glucose compounds. The result is that the regression correlation coefficient (R) calculated using the statistical approach described above should be sensitive to the level of difference between the absolutely homogeneous fibrous non-modified cellulose (standard) primary structure and the primary structure of heterogeneous alkylated textile cellulose. So the greater the difference between the test R value and R=1·0 (standard), the greater the difference between primary structures of alkylated cellulose in the textile tested and the pure non-alkylated cellulose (standard). In other words, R is a convenient criterion for quantitative evaluation of the heterogeneity of alkylated cellulose by comparison of its composition and the composition of 100% homogeneous pure cellulose: the highest level of heterogeneity corresponds to the smallest value of R (Sokal & Rohlf, 1981). Taking into account the above, we have calculated the R values for the cellulose pools of each of the eight archaeological textiles listed in Table 1 and the results are presented in Figure 7. These results show a nearlinear dependence of the R values on the calendar ages of the textiles. In our opinion, this type of relationship should be investigated to improve the accuracy of the existing dating methods.

32

D. A. Kouznetsov, A. A. Ivanov and P. R. Veletsky

1.00

A1

B A2 E

F D

R*

C1 C2

0.90

0.80

1000

1000 BC

2000

AD

Archaeological time (years)

Figure 7. The relationship between textile calendar age and differences in the primary structures of the compared homogeneous cellulose chain (standard) and the alkylated textile cellulose tested. R*-Regression Correlation Coefficient characterizing the extent of difference between primary structures of two compared polymers, standard (unmodified cellulose) and tested ones (alkylated cellulose), calculated according to bivariate statistics as the product–moment correlation coefficient (Sokal & Rohlf, 1981).

In fact, all modern archaeological textile dating techniques need to be improved and developed for greater accuracy and efficiency. It seems that detailed chemical studies on cellulose structure in different old textiles is a good way to create an efficient additional dating approach. In spite of the existence of radiocarbon dating methods, such additional and sometimes alternative dating techniques as stylistic studies, including microscopic research (Lee, 1953; Cramer, 1959; Bresee, Chandrashekar & Jones, 1986; Cardamone & Brown, 1986), are still important in interdisciplinary approaches to the problem of dating accuracy. It is known that there are a number of unique morphological markers for textiles manufactured in several regions during limited historic periods (Lee, 1953; Cramer, 1959; Bresee et al., 1986). Obviously, these markers should be taken into account in each archaeological dating procedure with objects from corresponding sites. It should not be forgotten that we can find not only morphological but also molecular markers for ancient textiles of different geographical origins and/or calendar ages. We believe that our data (Table 2, Figures 4, 6, 7) make it possible to demonstrate the potential of this kind of research. There are at least two possibilities for the mechanism of cellulose alkylation in the old textiles.

First, it is logical to assume that the cellulose alkylation we have found in our work is a result of unknown (lost ‘‘archaic’’) technological processes for textile manufacturing. Secondly, it should be remembered that the cellulose alkylation results from a microbial contribution to the chemical modification of textile. Thus, the carboxylating enzymes of bacterial carboxysomes are active even in bacterial lysates in wet alkali conditions and in the presence of oxygen (Price et al., 1993). The carboxylization 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 genus Desulfovibrio (Hensgens et al., 1993). Finally, normal autolysis of air and soil microorganisms may produce significant carboxylation and/or methylation, with different substrates directed by released active bacterial enzymes (Doyle & Koch, 1987; Price et al., 1993; Hensgens et al., 1993). Thus, the alkylation of old textile cellulose could be a consequence of the accumulation of a great number of such microbial treatments over a very long interval. All common soap-foaming washing procedures create exactly the pH change resulting in alkaline conditions from bacterial enzymes activity. Alkalis were also ingredients in some technological methods used for rewashing textiles after dyeing (Lee, 1953; Cramer, 1959). It is quite clear that if the second hypothesis is true, we could have a new efficient dating technique based on quantitative evaluation of textile cellulose alkylation estimated in different unknown and known-age archaeological textile samples. This idea requires further extensive experimental study. In a separate series of experiments, we have carefully investigated a set of seven known-age burial linen textiles excavated from the same site at Bukhara, Uzbekistan. For this purpose, our electrophoretic approach has been applied. As seen from our data, all these textiles contain carboxylated cellulose and moreover the degree of cellulose carboxylation is a function of calendar age (Figure 8). Thus, it is logical to assume that textile cellulose chemical modification analysis could provide an additional method of dating textiles in screening-studies of numerous samples from a common geographical origin. In all our studies, clean-looking non-dyed (‘‘white’’, i.e. light grey) textile samples were tested. Any possible contamination from stained areas was completely removed by our cleaning procedure. The data presented in Figures 4 and 5 confirm this. We have therefore reached complete separation/identification of carbohydrate fractions without encountering such compounds as heterocyclic, aromatic, steroid and other ‘‘stain-related’’ substances. We have found one unidentified peak in the CZE patterns (Figure 4) but a good separation of all fractions prevents any impact of stain-related compounds on our data and conclusions (compare Figures 4 and 5).

Content of minor glucose derivatives in a total cellulose hydrolysate (%)

Analysis of Cellulose Chemical Modification 33

20.0 BK9716 BK8112

r = 0.81

BK7407 BK7016 10.0 BK6884 BK6631

r = 0.86

BK5999

200

400

600

800 1000 Calendar age (years AD)

1200

1400

1600

Figure 8. Cellulose carboxylation as a function of calendar age in the case of seven non-dyed, known-age burial linen textiles excavated in 1928–1987 in Bukhara, Uzbek Republic. (All samples tested were purchased from the Middle-Asian Museum of Ethnography and Anthropology at Samarkand, Uzbekistan.) All data presented were obtained by our CZE technique with Refractive Index Detection for identification and quantitative evaluation (relative content estimation) of the glucose minor alkyl-derivatives using the textile cellulose enzymatic hydrolysates for electrophoretic tests. Museum curation codes presented in this figure correspond to the following samples: BK9716:  210–280, radiocarbon-dated by the Soviet Committee on Asian Studies in 1989; BK8112:  420–490, radiocarbon-dated by the Soviet Committee on Asian Studies in 1989; BK7407:  710–780, dated by historical evidence and stylistic details; BK7016:  860–940, dated by historical evidence and stylistic details; BK6884:  1080–1170, radiocarbon-dated by the USSR Academy of Sciences Institute of Experimental Physics in 1979; BK6631:  1300–1360, dated by historical evidence and stylistic details; BK5999:  1560–1640, dated by historical evidence and stylistic details. The historical/stylistic dating was performed at the Middle-Asian Museum of Ethnography and Anthropology at Samarkand. X–X: 2-carboxy-â--glucose; ,–,: 2,6-dicarboxy-â--glucose.

The ecological effect on the chemical structure of archaeological objects is a still undeveloped but very important area of research (Westbroek et al., 1993). We believe that a further search for correlations between chemical parameters characterizing both old textiles and biological materials (fossils, grain seeds, wood, etc.) excavated in a single site, could lead to interesting conclusions concerning the dependence of textile structure on environmental factors. In any case, the correlations between textile cellulose chemical modification, calendar age values, and geographic (regional) origin of textiles (Table 2, Figures 4, 6–8) look interesting and non-random. To our knowledge, our study is the first report of alkylated cellulose sequences in archaeological textiles. Further extensive studies on this subject could help to classify a great number of different archaeological textile relics using criteria like cellulose chemical modification. This classification would be an additional tool both for dating research in archaeological chemistry and for ancient textile technology studies.

Acknowledgements This work was supported by Guy Berthault Foundation, Meulan, France. We are especially grateful to

Professor Mario Moroni of Robbiate, Italy, for sending us a fragment of linen cloth from a burial at the En Gedi site, Israel, historically dated to the Early Roman Period ( 100 years– 100 years) by the Israel Antiquities Authority. We 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 Museum (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), Mr Ignat Tyshko, Keeper at the West Ukrainian Museum of Ethnography and Archaeology (Ternopol, Ukraine), Mr Kamil-Djan Youldashev, Keeper at the MiddleAsian Museum of Ethnography and Anthropology (Samarkand, Uzbekistan) for their remarkable courtesy in giving us a set of light, non-dyed, known-age samples of archaeological linen textiles. We thank Dr Alan Adler of the University of Western Connecticut at Danbury, CT, Professor Witold Brostow of the University of North Texas at Denton, TX, and Dr Alexander Volkov of Moscow State University for their critical remarks and participation in the discussion of our preliminary results. We especially thank Mr Timophey Palevich of the Moscow

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State University for his continued assistance in the preparation of samples for capillary electrophoresis.

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