Gel Permeation Chromatography - ACS Publications - American

20 Gel Permeation Chromatography Use in Estimating the Effect of Water Washing

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on the Long-Term Stability of Cellulosic Fibers Helen D. Burgess Conservation Processes Research, Canadian Conservation Institute, Ottawa, Ontario, Canada, K1A 0M8

A gel permeation chromatographic (GPC) procedure using agarose gel and tris(ethylenediamine) cadmium dihydroxide to give a high -resolution separation of cellulose is described. The GPC technique was used to compare the long-term stability of naturally aged cotton textile fibers washed in distilled-deionized water, tap water, calcium bicarbonate (20 ppm), or calcium sulfate (20 ppm). The results, which were verified by viscometry, showed that tap water or calcium sulfate washed fibers exhibited less degradation after accelerated thermal aging (70°C, 50% rh, 70 days) than the untreated control. The distilled-deionized water or calcium bicarbonate washed fibers showed greatly increased deterioration relative to the control.

^VATER WASHING IS OF CENTRAL IMPORTANCE

to both paper and textile conservation. The cleaning action of even a brief immersion in water improves the appearance of an artifact and removes any watersoluble materials that may be harmful to the chemical and physical stability of the fibers. However, the influence of the water quality, including such factors as p H and type and quantity of dissolved materials, upon the long-term stability of cellulosic fibers is not well understood, although recent research (1-3) has addressed a number of important questions arising from this problem. These studies provided the first step to understanding some of the factors that can influence the permanence and durability of fibers. However, many questions remain unanswered. One of the most imThis chapter not subject to U.S. copyright. Published 1986, American Chemical Society

In Historic Textile and Paper Materials; Needles, H., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1986.


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HISTORIC TEXTILE AND PAPER MATERIALS

portant considerations is whether these conclusions, drawn from experi ments performed on new materials, are valid for historic artifacts. The partially degraded state of naturally aged fibers, with its concomitant changes in morphological structure and functional group levels, can be expected to contribute considerably to the sensitivity of cellulose to oxidative and hydrolytic (both acid- and base-catalyzed) mechanisms of degradation. Evidence supporting this reasoning may be seen in the literature dealing with color reversion (4-7). In these papers, the importance of the degree of degradation in influencing the rate of change of some pertinent chemical or physical property is very well illustrated. Another point that must be raised is whether any experiments performed on paper fibers can be applied without reservation to linen or cotton textiles. If any differences exist they would relate to the influence of fiber length (textile fibers are generally longer) and structure (the random nature of the paper web versus the regularity imposed on textiles by the spinning and weaving process) upon chemical reactivity. The more extensive processing of paper fibers as well as the possible presence in the paper of undesirable components (e.g., lignin, hemicellulose, or extractives) would also impede valid comparison. The legitimacy of comparisons between paper and textiles, as well as the ability to apply the results of a limited series of experiments to general conservation recommendations, is going to be very much determined by the depth of our understanding of the chemical changes taking place. Therefore, the most valid method of determining the effect of water washing on fiber stability will be one that can be related to a degradative process that influences both the chemical and physical properties of cellulose. Probably the most suitable technique is one that measures polymer length. Any method that involves the determination of molecular weight or degree of polymerization (DP) will permit chain scission—the ultimate result of both oxidative and hydrolytic degrada tion. If one is mainly concerned with chemical changes, the viseometric average DP (DP) is an ideal technique because of the relative ease, accuracy, and sensitivity of the analytical procedure. However, physical parameters such as breaking strength, flex lives (fold endurance), and toughness are much more accurately described by knowledge of the molecular weight distribution than they are by DP (8). Therefore, the examination of a problem relating to fiber degradation (with its concern with both physical and chemical changes) can best be accomplished by the distribution analysis. Although this method of analysis is of obvious value on the basis of theoretical considerations, its useful application could only be achieved after solving a number of problems. The most important of these was to arrive at a suitable solvent system and to find a gel type that was



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compatible with the particular solvent. These difficulties have greatly inhibited progress in the field. Most of the available literature deals with systems in which the cellulose was first nitrated or acetylated so that a convenient organic solvent could be used. However, these modifications are very degradative and almost certainly have contributed to the problem of getting good chromatographic resolution. Therefore, the best answer to these difficulties was to use a chemically stable solvent that was able to dissolve cellulose without excessive deterioration of the fiber. The colorless, odorless cadmium ethylenediamine solvent, tris(ethylenediamine)cadmium dihydroxide (Cadoxen), seemed to be the most suitable solvent for the task. Cellulose solvents such as tris(ethylenediamine)cadmium dihydrox ide present difficulties with most conventional gel permeation chroma tographic (GPC) gels: carbohydrate-based gels such as the Sephadexes swell too strongly; the cross-linked polystyrene gels such as Styragel do not swell enough; and the porous silica or glass-based gels break down in the alkaline aqueous solvents used to dissolve cellulose (8). Considerably more success was shown to be possible with the very rigid cross-linked agarose gels, Sepharose CI (Pharmacia) (9-12). The analyses described in this chapter were based on this last choice. The carbohydrate being eluted from a GPC column can be detected by a number of physical or chemical means (e.g., variation in refractive index or viscosity and colorimetric or fluorometric spectroscopic anal ysis). For the purpose of these experiments, the cellulose was tagged with a fluorescent label, dichlorotriazinylaminofluorescein (DTAF), which permits easy detection of very small quantities. The chroma tographic system was set up to allow for convenient analysis of cellulose with a maximum resolution of the molecular weight distribution and a minimum of change to the sample. With the help of this powerful technique, an effort was made to answer a few of the questions that exist concerning the effect of water washing on the long-term stability of cellulosic fibers. These questions include the following: (1) Can the conclusions published in the existing literature be applied to textiles and to fibers that are partially degraded by natural aging? (2) Can the beneficial effect of washing with tap water or dilute calcium hydroxide be ascribed (a) to the formation of a complex between the calcium cation and some component of the cellulose fiber and thus make oxidative attack more difficult or (b) to the prevention of acidic hydrolysis by the alkaline nature of calcium carbonate or calcium hydroxide? These points were investigated by a series of experiments involving the water washing of a Peruvian cotton textile that was approximately 800 years old. Various treatments were tested by subjecting suitable size samples to washing with tap water, reagent grade distilled-deionized


366


water, calcium bicarbonate (20 ppm), or calcium sulfate (20 ppm). Suitable controls were included. The long-term stability of the fibers was assessed by comparing distribution data before and after ac celerated thermal aging. The data obtained by the GPC techniques were verified by the determination of the viscometric DP.


Experimental Treatment Sequence. The textile used for the experiments was unbleached and undyed cotton. The fragment was plain weave and had a thread count of 17 X 12/cm (warp X weft), and the yarns were spun with a "Z" twist. The textile was dated from about A.D. 1100 to A.D. 1350 and was attributed to the Peruvian central coast. It was only moderately dirty and was in excellent condition. All aqueous treatments were performed at 25 °C. Suitable size pieces were washed for 120 min in one of two sources of running water: The first source was a reagent grade water purified from tap water by a high-capacity Corning AG11 distillation apparatus that fed distilled water into a Barnstead Nanopure system. This deionizing purification setup consisted of high-capacity, ultrapure, and organic-removal cartridges set up in tandem. The second water source was Ottawa's tap water which had a water hardness of approximately 42 ppm made up of about 35 ppm of calcium and 7 ppm of magnesium. After this initial treatment, the piece washed in the distilled-deionized system was divided into three parts: one was not further processed, and the other two were washed for an additional 60 min in one of two solutions containing 20 ppm of calcium (0.5 mM). These solutions were prepared from neutral calcium sulfate or calcium hydroxide converted to calcium bicarbonate by bubbling carbon dioxide gas through the solution for 60 min. After the treatments were completed, the five pieces representing the five different washing sequences were cut into two halves: one was kept as a treatment control and was analyzed without further processing, and the other was subjected to accelerated thermal aging for 70 days at 70 °C and 50% rh. An untreated control was also included as part of both the unaged and aged sample groups. Each of the 12 pieces (unaged and aged) was at least 125 cm and weighed 2 g. 2

Determination of DP. DP was determined by using a Fenske viscometer equilibrated at 30 °C with tris(ethylenediamine)cadmium dihydroxide as the solvent for cellulose. This solvent was prepared in accordance with the method of Donetzhuber (13). All measurements were done in triplicate. The reported DP data were calculated from the mean intrinsic viscosities [η] by using the following relationship (14): [rç],. = 1.84 X HT (DP) 2

076

Preparation of Labeled Carbohydrate. Labeled cellulosic fibers were prepared by suspending approximately 100 mg of the cotton to be analyzed in 5 mL of 100 mM sodium carbonate that contained 3 mg of DTAF. The DTAF was prepared according to the method of Barskii et al. (15). The suspension was stirred for 18.0 h at room temperature. Unreacted label was removed by rinsing with distilled-deionized water. After drying at room temperature, the samples were stored in a desiccator in the dark.


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Labeled dextrans for calibration purposes were prepared by dissolving (in 15 mL of 100 mM Na2CU3) five dextran standards (100 mg each) of the follow ing average molecular weight distributions: 10,000; 40,000; 70,000; 500,000; and 2,000,000. After stirring overnight, unreacted label was removed by exhaustive dialysis of the solution with water. The solution was freeze-dried, and the modified yellow dextrans were reclaimed. Determination of Molecular Weight Distribution. A 1.6- X 95-cm column of Sepharose C1-4B in 50% tris(ethylenediamine)cadmium dihydroxide (1:1 with water) was prepared and equilibrated by elution with six column volumes of 50% tris(ethylenediamine)cadmium dihydroxide. Downward flow with a pressure head of 125 cm was used. Each sample to be analyzed was dissolved in tris (ethylenediamine) cadmium dihydroxide (1 mL) by stirring overnight, and then water (1 mL) was added. A 1-mL aliquot (concentration < 1.0%) was applied to the column, and elution proceeded with a pressure head of 100 cm and flow rate of 10 mL/h. A Turner 111 fluorometer (excitation filter 2A plus 47B and emission filter 8 plus 65A) fitted with a flow-through door allowed for automatic continuous monitoring of carbohydrates as they were eluted. Relative fluorescence was automatically recorded on a linear strip recorder. Fractions of 3 mL were collected on a FC-80K Gilson microfractionator. Typically, each sample was analyzed several times, usually at different concentrations, to ensure the reproducibility and accuracy of the data. A calibration run using the labeled dextrans was performed a minimum of one time per week.

Results and Discussion G P C Column Calibration. The chromatography of a typical cali bration run is shown in Figure 1. The successive elution of molecules of diminishing size is shown from left to right across the profile: peaks

Φ

ο c φ ϋ (β φ ο

Τ70 Τ2000

3

Φ >

_Ç3 Φ

10

20

30

Fraction Number Decreasing Polymer Length

Figure 1. GPC of Dextran Τ calibration standards.


40


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representing the T2000, T500, T70, T40, and T10 dextran standards (correlating to a molecular weight average of 2,000,000; 500,000; 70,000; 40,000; and 10,000, respectively) are clearly visible. This separation occurs because the rate of travel of the individual molecules down the GPC column is dependent upon their ability to pass through the pores within the gel beads. The result is that the carbo hydrate sample comes down the column at a rate proportional to the length of the polymer chain. The "apparent" size of an individual molecule (i.e., the size as observed by a G P C technique) is also going to be determined by the shape the polymer chain takes when it is dissolved in the solvent being used. Therefore, a column that is going to be used to fractionate cellulose should be calibrated with the best available polysaccharide standards. The Dextran Τ fractions from Pharmacia (mainly a-1,6 polymers of D-glucopyranose) have hydrodynamic pro perties in solution that are similar to those of cellulose and are the best commercially available materials for this task. The other synthetic polymer standards (e.g., polystyrene) will not give as accurate data, in absolute terms, unless the calibration is carried out according to Benoit's universal calibration concept (16-18). A plot of log molecular weight versus fraction number of the central point of each dextran peak (Figure 2) yielded a straight line with a statistical "measure of fit" greater than 0.99, which is very close to the theoretical value of 1.00. This calibration curve can be used to determine the molecular weight distribution of subsequent chromatographic runs of cellulose samples of unknown distribution. Only very slight shifts in

LOG. Mol

10 W t

10

20

30

40

Fraction Number

Figure 2. GPC calibration curve using Dextran Τ standards.



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this linear plot were observed when the successive calibration runs performed during the course of the experiments were compared. F o r ease of comparison, all the chromatograms reported in this chapter were normalized to the calibration run obtained approximately m i d w a y through the experimentation period. The data points for T500 and T70 fractions d i d not fall directly on the line as d i d the T2000, T40, and T10 dextrans. This pattern was consistendy noted i n every calibration curve obtained f r o m these experiments and also f r o m previous G P C data acquired b y us with this particular system. The uniformity of this observation suggests that there may be some p r o b l e m with the average molecular weight that the manufacturer attributes to the T500 and T70 fractions. It also illustrates the great difficulty that workers in the field w i l l have in obtaining wellcharacterized polymer fractions of narrow molecular size distribution. However, the extreme reproducibility of the chromatography of these standards offers considerable reassurance that comparisons among samples of cellulose of unknown molecular weight distributions w i l l be valid. Determination of Molecular Weight Distribution. A l l of the unaged material, including both untreated and treated textiles, gave similar chromatographic profiles. Therefore, the data for the unaged cotton control shown in Figure 3 can be considered to be representative of both washed and unwashed samples. The implication from these observations is that the washing processes had little immediate effect on the molecular weight distribution. The aged samples, however, had very significant differences which are very easily apparent. Also, all of the aged samples showed obvious signs of degradation relative to the unaged control. This result is expected and is a necessary prerequisite to drawing any conclusions concerning the long-term stability of the cellulosic fibers. The types of changes that were observed after the accelerated aging are illustrated b y the comparison of the molecular weight distribution of the untreated controls before and after aging (Figure 3). Comparison of all the chromatograms illustrated in this chapter is facilitated b y dividing the profiles into three areas of interest: (1) a high molecular peak section coming off the column at fraction numbers 11-15 having a molecular weight of 2,000,000 to about 850,000 ( D P = 12,350-5250), (2) a large central peak at fraction number 17 or 18 w h i c h corresponds to a molecular weight of about 500,000, and (3) a pair of peaks covering the lower molecular weight range at approximately fraction numbers 29 and 33 and correlating to a molecular weight of about 35,000 and 13,000, respectively ( D P = 216 and 80). Changes in the high molecular weight range were observed b y noting any alterations relative to the large central peak. Figure 3 clearly


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Molecular Weight

5

1*0

1*5

2Ό

2*5

3*0

35

40

Fraction Number

Figure 3. GPC of unwashed cotton samples before and after accelerated thermal aging.

shows how the aging process will result in the drastic loss of this high-DP material. Further evidence of changes due to thermal aging were noted by comparing the height of the two peaks in Area 3: in the unaged cellulose they were approximately the same, whereas in the aged materials the lower molecular weight peak, at fraction number 33, had increased in size relative to the other at fraction number 29. All of the changes evident in these observations may be interpreted as being the result of polymer-chain cleavage to produce shorter molecules of lower molecular weight (i.e., degradation of fibers). The four washed samples that were aged did not give molecular weight distribution data identical to the aged control. The conclusion is that the washing process was having an impact on the long-term stability of the fibers. This effect can be seen by careful interpretation of the chromatograms shown in Figures 4 and 5. The results for the tap water and calcium sulfate (20 ppm) are in Figure 4, whereas Figure 5 illustrates the data obtained for distilled-deionized water and calcium bicarbonate (20 ppm) washing methods. Figure 4 shows that very similar results were obtained from tap water and calcium sulfate washing. The distribution profiles show that the results fall somewhere between the unaged and aged controls depicted in Figure 3. The following observations are pertinent: (1) The


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Estimating the Effect of Water Washing Molecular Weight 10 I I I

10 l i l

e


I

έ

ι

lb

.

10 L.

3*0

3'5

6

ι

1'5

2*0

4

2'5

4Ό

Fraction Number

Figure 4. GPC of cotton samples washed in tap water or calcium sulfate (20 ppm) after accelerated thermal aging. Molecular Weight

I 10

ι 15

20

I 25

τ 30

1

35

1 40

Fraction Number

Figure 5. GPC of cotton samples washed in distilled-deionized water or calcium bicarbonate (20 ppm) after accelerated aging.



372


high molecular weight peaks at fraction numbers 11-15 were relatively large in comparison to the central peak at number 17. This result bore more similarity to the unaged control than to the aged fibers. (2) The height of the two peaks centered at fraction numbers 30 and 33 was slightly more than half of the height of the large peak at fraction number 17 (i.e., peak height ratio of Area 3 to Area 2 =0.5). The ratio for the aged, untreated control was also about 0.5, whereas the ratio for the unaged control was closer to one-third. These comments lead one to conclude that the aged fibers treated with tap water or calcium sulfate washing have deteriorated relative to the unaged material but have not deteriorated as much as the aged control sample. In terms of conservation choices, the implication is that these two types of washing will actually improve the permanence and long-term stability of cellulosic fibers. This statement must be qualified by the consideration that any artifact to be washed must meet the necessary criteria of having sufficient mechanical strength and an absence of water-sensitive components. The aged fibers that were previously washed with distilleddeionized water or 20 ppm of calcium bicarbonate showed similar results (Figure 5), but the degree of degradation was significantly higher than that seen with either tap water or calcium sulfate. The main difference between these two chromatograms and those in Figure 4 was in the elution of the high molecular weight material at fraction numbers 11-15: drastically reduced quantities were observed relative to the tap water or calcium sulfate treated cotton. The peak height ratio of Area 3 to Area 2 was slightly over 0.5, similar to that observed for all of the other aged samples. The conclusion is that the samples extensively washed with distilled-deionized water or the calcium bicarbonate system did not have as good long-term stability as cellulosic fibers washed with aqueous calcium sulfate or tap water. Determination of the Viscometric D P . The conclusions reached by the G P C experiments were verified by a separate determination of the viscometric D P (Table I). This analytical method does not give as full an understanding of the changes occurring during fiber deterioration as does a distribution analysis, but it will allow for a good quantitative comparison of samples. The main points that should be made from this data are the following: (1) The textile samples underwent a significant drop in average polymer length during accelerated thermal aging. (2) The aged samples that had been previously washed in either distilleddeionized water or 20 ppm of calcium bicarbonate showed DP values that were similar and drastically lower than the thermally aged untreated control. (3) The aged samples that had been previously washed in either Ottawa tap water or 20 ppm of calcium sulfate showed DP values that


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Table I. The Viscometric Average Degree of Polymerization (DP) of Cellulosic Fibers Washed in Water Sample

hh

DP

Unaged, untreated control Aged, untreated control Aged, Ottawa tap water Aged, calcium sulfate (20 ppm) Aged, distilled-deionized water Aged, calcium bicarbonate (20 ppm)

5.75 4.81 4.93 4.86 3.68 3.76

1918 1516 1566 1536 1064 1096

were similar and somewhat higher than the thermally aged untreated control. The most likely explanation for the deleterious effect of distilleddeionized water is that the very pure water used was able to solubilize and remove constituents of the fiber that protect against oxidative attack. The divalent cations such as magnesium and calcium were probably the vital components being removed. This conclusion cor relates well with published research in the pulp and paper field that shows that decreased degradation of cellulose occurs during oxygen bleaching if alkaline earth salts are added (19-23, 25, 26). Several mechanisms have been proposed to account for this effect: (1) The alkaline earth salts are able to complex free radicals (e.g., peroxides) and prevent them from taking part in the degradation phenomena (19, 20). (2) The alkaline earth salts are able to adsorb or complex transition metals (e.g., copper or iron) (21-23) which are known to catalyze the oxidative degradation of cellulose (24). (3) A specific interaction occurs between the salt and some easily oxidized portion of the glucose monomer. Oxidation is prevented by complex formation involving an unoxidized site (25) or via a direct stabilization of a primary oxidized derivative of cellulose (26). In either event, the swelling of the cellulose and the accessibility of the fibers may also be greatly affected and thus change the susceptibility of the cellulose to oxidation. The poor performance of the calcium bicarbonate treated fibers is not so readily understood. However, a very likely explanation is the greatly increased sensitivity of naturally aged (and hence partially degraded) cellulose to alkaline materials. One may speculate that the calcium carbonate that was formed in the fibers was acting as an initiator of depolymerization reactions that are base catalyzed. Alkaline degradation reactions are largely controlled by the carbonyl content of cellulose (27-29). Because the carbonyl levels go up as natural aging proceeds, one should expect that an 800-year-old textile would be much more likely to be adversely affected by alkali than would new, unoxidized cellulose. The vast majority of the conservation



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studies that show the beneficial effects of alkaline treatments such as deacidification have been done with new materials. The results described in this chapter indicate that serious difficulties could be involved in applying information obtained from contemporary cellulosic studies to conservation treatments of much older museum artifacts. A question must be raised as to w h y the tap water and the calcium bicarbonate washing treatments give such different results. The alkaline p H of the tap water suggests that calcium carbonate or bicarbonate is present and that the final product deposited in the fiber should be similar to that obtained with the pure bicarbonate solution. However, the chemical makeup of any city tap water is very complex and must contain a number of components that could affect the stability of cellulose. F o r example, the municipal treatment plant in Ottawa adds large amounts of alum (aluminum sulfate) to the water to settle particulate matter. Because alum makes the water very acidic, lime is then added to raise the p H . T h e result is that a large amount of calcium sulfate is present in the tap water and must affect the overall chemistry of the salts deposited in the fibers. One may further speculate that the anions present can influence the stability of cellulose as much as the cations. A n y comprehensive understanding of the factors involved must include aH parameters.

Conclurions The degree of sensitivity that the textile fibers showed (both to the accelerated aging and to the different washing treatments) demonstrated that cellulose f r o m cotton and linen textiles can be expected to be as susceptible to degradative processes as are the more extensively studied rag fiber papers. Therefore, much of the literature concerning treatments carried out on paper substrates should be applicable to textile conservation. H o w e v e r , it is still important to exercise caution in applying results f r o m w o o d pulp papers to textile artifacts. The results discussed in this chapter support the view that calcium or magnesium salts can be beneficial to the longevity of cellulosic materials. Removal of these components b y washing with distilled or deionized water w i l l be detrimental to a cellulosic artifact. However, conservators should not see these results as encouragement to use tap water in their washing treatments. E v e n areas having good tap water experience large seasonal variations of most constituents of the water as well as sporadic heavy metal contamination from pipes and water mains. These factors are not conducive to reliable high-quality water which conservation treatments require. The data showing a large difference in fiber stability between the calcium bicarbonate and calcium sulfate treated samples were contrary



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to what might have been expected on the basis of previously published studies. This disagreement can probably be ascribed to the age of the fibers used in the experiments. However, reliable general recommenda tions concerning the choice between neutral salts (of magnesium or calcium) and the traditional alkaline solutions cannot be based upon a single set of experiments performed on one type of cellulosic fiber. At the same time, revision is necessary of any previous conceptions regarding the complete safety of adding small amounts of magnesium or calcium bicarbonate (or hydroxide) to the wash water during the cleaning of historic artifacts. It is unlikely that the alkaline solutions could pose any problems with new materials, but evidence indicates that, in certain situations, the long-term stability of older fibers would be impaired.

Acknowledgments I would like to thank Charles E . Llewellyn for donating the Peruvian textile used in these experiments. I would also like to acknowledge the help of J. C . McCawley, Chief, Conservation Processes Research, Canadian Conservation Institute, both in the preparation of this manu script and in the performance of the experiments.

Literature Cited 1. Tang, L.; Jones, N. J. AIC 1979, 18, 61-81. 2. Tang, L. In "Preservation of Paper and Textiles of Historic and Artistic Value II"; Williams, J. C., Ed.; ADVANCES IN CHEMISTRY SERIES No. 193; American Chemical Society: Washington, D.C., 1981; pp. 63-86. 3. Nelson, J., et al J. AIC 1982, 21, 59-76. 4. Rapson, W. H.; Hakim, K. A. Pulp Pap. Mag. Can. 1957, 58, 151-57. 5. Rapson, W. H., et al Tappi 1958, 41(8), 442-47. 6. Hosokawa, J. et al; J. Jpn. Wood Res. Soc. 1978, 24(9), 638-42. 7. Corbi, J. C.; Rapson, W. H. Pulp Pap. Mag. Can. 1964, 65, 467-72. 8. Segal, L. In "Advances in Chromatography", Giddings, J. C. et al, Eds.; Dekker: New York, 1975; Vol. 12, pp. 32-59. 9. "Sepharose C1: For Gel Filtration and Affinity Chromatography"; Technical Publication; Pharmacia Fine Chemicals: Uppsala, Sweden, 1975; p. 8. 10. Burgess, H. D. "The Degradation of Cellulose in Conservation Bleaching Treatments"; M.A.C. Thesis, Art Conservation Programme, Queens Univ., Kingston, Canada, 1979. 11. Bose, A. "Aqueous Size Exclusion Chromatography of Non-Derivatized Cellulose: Application of Excluded Volume Concepts to Calibration"; Ph.D. Thesis, Purdue Univ., LaFayette, IN, 1980. 12. Burgess, H. D. Prepr. 9th Inter. Congr.IIC1982; pp. 85-88. 13. Donetzhuber, A. Sven. Papperstidn. 1960, 63, 447-48. 14. Segal, L.; Timpa, J. D. Sven. Papperstidn. 1969, 72, 656. 15. Barkii, V. E., et al Izv. Akad. Nauk SSSR Ser. Biol. 1968, 5, 744. 16. Benoit, H.; Grubisic, Ζ.; Rempp, P.; Decker, D.; Ziliox, J. G. J. Chim. Phys. 1966, 63, 1507.



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17. Grubisic, Z.; Reibel, L.; Spach, G. C.R. Hebd. Sceances Acad. Sci. 1967, C246, 1690. 18. Grubisic, Z.; Rempp, P.; Benoit, H., J. Polym. Sci. Part Β 1967, 5, 753. 19. Croon, I.; Andrews, D. H. Tappi 1971, 54, 1893. 20. Samuelson, O. DasPapier 1970, 24, 671. 21. Robert, Α.; Viallet, A. Rev. ATIP 1971, 25(3), 237-41. 22. Gilbert, A. F.; Paulovova, E.; Rapson, W. H. Tappi 1973, 56(6), 96-99. 23. Samuelson, O.; Stolpe, L. Sven. Papperstidn. 1974, 77, 16-26. 24. Ericsson, B.; Lindgren, B. O.; Theander, I.; Sven. Papperstidn. 1971, 74, 757. 25. Swan, B.; Akerblom, I. Sven. Papperstidn. 1977, 80(11), 346-49. 26. Defaye, J.; Gadelle, A. Pulp Pap. Mag. Can. 1974, 75(11), 394-97. 27. Golova, O. P.; Nosova, Ν. I. Russ. Chem. Rev. Engl. Transl. 1973, 42(4), 327-38. 28. Richards, G. N. In "Cellulose and Cellulose Derivatives"; Bikales, N., Segal, L., Eds.; Wiley-Interscience: New York, 1971; Vol. V, Part V; pp. 1007-14. 29. Haskins, J. F.; Hogsed, H. J. J. Org. Chem. 1950, 15, 1264. RECEIVED

for review November 26, 1984.

ACCEPTED

April 17, 1985.


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