Comparison testing of methods for gel permeation ...

Cellulose DOI 10.1007/s10570-015-0586-2

ORIGINAL PAPER

Comparison testing of methods for gel permeation chromatography of cellulose: coming closer to a standard protocol Antje Potthast • Sylvia Radosta • Bodo Saake • Sascha Lebioda • Thomas Heinze • Ute Henniges • Akira Isogai • Andreas Koschella • Paul Kosma • Thomas Rosenau Sonja Schiehser • Herbert Sixta • Matija Strlicˇ • Grazyna Strobin • Waltraud Vorwerg • Hendrik Wetzel

•

Received: 2 January 2015 / Accepted: 18 February 2015  Springer Science+Business Media Dordrecht 2015

Abstract A round robin on GPC of a wide range of different pulp samples was conducted among leading groups in cellulose analysis. The aim was to survey the status quo of the methods available to date. The pulp samples covered not only fully-bleached dissolving pulps but also bleached paper pulps and one unbleached sample. The methods applied were current state-of-the-art GPC with RI, MALLS, and viscosimetry detectors. Different dissolution protocols were compared as well. Following from the obtained results, more standardized protocols were proposed for

approaches with different equipment (RI or MALLS/ RI) and solvent systems (direct dissolution or derivatization). Major influencing factors, such as derivatization compared to direct solution, calibration versus light scattering and in-between lab variation, were discussed.

A. Potthast (&)  U. Henniges  T. Rosenau  S. Schiehser Department of Chemistry, Division of Chemistry of Renewable Resources, University of Natural Resources and Life Sciences, Konrad-Lorenz-Str. 24, 3430 Tulln, Austria e-mail: [email protected]

A. Isogai Department of Biomaterial Science, Graduate School of Agricultural and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

S. Radosta  W. Vorwerg  H. Wetzel Fraunhofer Institute for Applied Polymer Research IAP, Geiselbergstraße 69, 14476 Potsdam-Golm, Germany B. Saake  S. Lebioda Department of Wood Science, University of Hamburg, Leuschnerstr. 91 B, 21031 Hamburg, Germany T. Heinze  A. Koschella Kompetenzzentrum Polysaccharidforschung, FriedrichSchiller-Universita¨t Jena, Humboldtstrasse 10, 07743 Jena, Germany

Keywords Round robin  Calibration  Carbanilation  Dimethylacetamide–lithium chloride (DMAc/LiCl)  Multi-angle laser light scattering (MALLS)  Refractive index

P. Kosma Department of Chemistry, Division of Organic Chemistry, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria H. Sixta Department of Forest Products Technology, Vuorimiehentie, Aalto University, 02150 Espoo, Finland M. Strlicˇ The Bartlett School of Graduate Studies, University College London, Gower Street, London WC1E 6BT, UK

123

Cellulose

Introduction Cellulose, as the most abundant and most important natural resource, is the prime example of a natural polymer. Consisting of 1,4-glycosidically linked b-Dglucopyranoside units—usually denoted anhydroglucose units (AGUs)—in strictly linear fashion, its chemical structure appears quite simple. Despite the proximal 4-OH-group and the hemiacetal at the terminal AGU, the so-called reducing end, the only functionalities of cellulose are three hydroxyl groups per AGU. Thus, from the viewpoint of its chemistry, cellulose appears perfectly straightforward. However, there are two factors which render cellulose highly complex and still challenging for polymer and analytical chemists. First, there is a complicated network of hydrogen bonds that undergoes dynamic, reversible and irreversible changes upon cellulose processing. This network governs all aspects of the properties and chemical behavior of celluloses, such as its occurrence in different polymorphs, the presence of different ratios of crystalline and amorphous regions, its swellability, its insolubility in standard solvents, and its strength properties. Second, there is a variability in chain length (observed in almost all natural and synthetic polymers), which has similar effects on properties and chemical behavior. This variability is portrayed by polymer-related molar mass data, such as Mw and Mn, but is best described by the molar mass distribution function (MMD) of the material. For polysaccharides in general, all being polydisperse polymers, the MMD is an important parameter. The actual situation in cellulose is as complicated as for other polysaccharides because cellulose samples might have variable natural origins, different processing histories, and many provenances, all of them influencing, changing, and governing the MMD in different ways. As a consequence, a reliable determination of this important cellulose parameter remains elusive today, and approaches, qualities, results and protocols for MMD determination of celluloses are almost as diverse as the celluloses themselves. The standard method of determining the MMD of celluloses is size exclusion chromatography (SEC). For established analytical techniques, such as nuclear G. Strobin Institute of Biopolymers and Chemical Fibres, ul. Sklodowskiej-Curie 19/27, 90-570 Lodz, Poland

123

magnetic resonance (NMR), infrared spectroscopy (IR) or mass spectrometry (MS), measurements of a defined sample are expected to provide results largely independent of external parameters, such as instrument type, operator, and lab environment, and they can be unambiguously repeated and verified. By contrast, gel permeation chromatography (GPC) of celluloses remains in a somewhat developmental stage. Neither commonly-accepted protocols nor standard operation procedures (SOPs) for either polymer dissolution and measurement protocol or the appropriate equipment and data evaluation exist. An earlier small-scale round robin test on cellulose GPC carried out by the German Zellcheming (Schelosky et al. 1999) showed large discrepancies between different labs and different methods (e.g. derivatization vs. direct dissolution). Similar tests have been carried out on water soluble barley glucans and SEC (Christensen et al. 2001). A large potential for errors lay not only in sample preparation and instrumentation, but also in the measurement protocol and the data treatment. The comparability of GPC results of (less diverse and complex) synthetic polymers between different setups and laboratories has already proven to be noticeably difficult (Mori 1998; Barth et al. 1998), which is also true for cellulose. GPC instrumentation has made further improvements since those studies, and more research institutions now apply GPC of cellulose, which has consequently resulted in broader awareness of the method and more practical expertise in its execution. Still, very different approaches, instrumentations, and experimental setups exist, and a comparison is impossible to elaborate solely from the literature data. Typically, the GPC system, solvent, detection mode and experimental protocol for cellulose analysis are selected according to subjective factors like previous experience of the operator with the system, preference for uncomplicated solvents and rapid methods, but no detailed evaluation or comparison of different systems, detectors, solvent systems, and analytical approaches yet exists. In summary, it is evident that a more detailed understanding of main sources of error and pitfalls in cellulose GPC is long overdue. In response to that need, a round robin test was performed within the framework of the European Polysaccharide Network of Excellence (EPNOE), comprised of eight international research groups concerned with GPC of celluloses. Each of the partners received identical samples and analyzed a set of six pulps according to their respective

Cellulose

approaches and methods. The results were comparatively evaluated and discussed among the partners. The combined study had the following primary goals: • • •

• •

appraisal of existing methodologies and protocols; comparison of results and identification of major sources of difference; comparative evaluation of significant influencing factors: (a) methods of data processing (calibration vs. light scattering, calibration line functions within light scattering data processing), (b) solvent systems, (c) detection systems, and (d) direct dissolution versus derivatization; identification of possible weaknesses and general agreement on those weak points; elaboration of a common protocol for best practices of GPC on celluloses based on the different arrays of available instrumentation.

The direct comparison of different methods will support researchers in determining which approach is best for their specific problem and to identify clearly the pro and cons of each particular method. Even though different operators will have to learn from their own experience, we have proposed a number of basic rules to follow in order to minimize variations among analysts, experimental setups, and data evaluation techniques in cellulose GPC. Addressing the points above resulted in the recommendation of a better comparable protocol for analyzing the molar mass distribution of cellulose, implementing state-of-the-art analytical technology and chemistry. This protocol should serve as a basis for further improvements within this field.

average molar mass1 (Mw Eq. 2), and the z- and (z ? 1)-average molar masses (Mz, Eq. 3, Mz?1, Eq. 4). The magnitude of Mn largely reflects the presence of low molar mass material, whereas Mw is more sensitive to the high molar mass parts. The Mz and Mz?1 values emphasis changes in the highest molar mass regions. The number-average molar mass (Mn) is the most commonly used parameter, because it is directly related to the degree of polymerization. It is highly influenced by the separation efficiency of the column in the low molar mass region and is also dependent on the way baselines are aligned and peak limits are set in the raw chromatograms. The weightaverage molar mass (Mw) is more robust and less influenced by such factors. As Mn would have been overly complex in inter-lab comparisons due to the number of influencing factors, the weight-average molar mass Mw was used as the main characteristic of the MMD in this round robin test: P ðni Mi Þ ð1Þ Mn ¼ P ni P ðni Mi2 Þ Mw ¼ P ðni Mi Þ

ð2Þ

P ðni Mi3 Þ Mz ¼ P ðni Mi2 Þ

ð3Þ

Mzþ1

P ðni Mi4 Þ ¼P ðni Mi3 Þ

ð4Þ

The ratio of Mw and Mn is a measure of the nonuniformity of the sample, expressed as the dispersity index (ÐM), as given by Eq. 5. If the value for ÐM approaches 1, the sample is uniform; a number close to 1 indicates a narrow MMD, while higher numbers reflect increasing non-uniformity, i.e. broad or stretched molar mass distributions.

Fundamentals of cellulose MMD determination ÐM = Mw /M n

ð5Þ

Parameters of molar mass distribution In order to describe the molar mass distribution of a polymer, different statistical averages of the distribution are used, measured either independently by chemical or physical methods or calculated statistically in size-exclusion chromatography by different Eqs. (1–4). Typically, four values are calculated: the number average molar mass (Mn, Eq. 1), the weight-

1

Mw, Eq. 2, in order to better reflect the meaning of this statistical parameter it could be called weighted-average, as the average is a weighted average.

123

Cellulose

Detection modes in cellulose GPC Three detection systems are commonly applied to analyze the fractions eluting from GPC columns: concentration-responsive detectors, structure-responsive detectors, and molar mass-responsive detection systems. Refractive index (RI) detectors are generally used for determining the concentration of the analyte employed. An additional UV signal can be used, for example, to detect residual lignin or chromophores in unbleached cellulose material (Westermark and Gustafsson 1994), to detect UV or fluorescence labels, or to monitor UV- or fluorescence-active derivatives. Structure-sensitive detection systems rely mainly on UV, IR, or fluorescence. The use of mass spectrometry as a detection technique in cellulose GPC is relatively uncommon. Several choices for molar mass-sensitive detection are available, the most widely used ones being viscosity, calibration with narrowly distributed standards such as pullulan or polystyrene, universal calibration (log [g] 9 M) vs. retention volume), and laser light scattering. Among those methods, only light scattering detection is an absolute method almost free of additional (and possibly defective) assumptions, which is a major justification for the high costs of the detectors. Lightscattering detectors are distinguished by the number of angles of the scattered light detected: multi-angle laser light scattering: MALLS (Fig. 1); right-angle light scattering (RALS); low-angle light scattering (LALS); and triple-angle light scattering are currently the prevalent choices. Molar-mass-sensitive detection in cellulose GPC—determination of the molar mass distribution (MMD) The different approaches towards molar mass-sensitive detection are detailed in the appendix. There are different protocols available to obtain molar mass data after GPC separation of the polymer. Different calibration modes can be applied. The standard or conventional calibration relies on the availability of a set of narrow standards of the same molecule to be analyzed covering the whole molecular weight distribution. Due to the lack of narrow-distributed cellulose standards, a straightforward standard-based GPC calibration cannot readily be performed. The use of

123

polystyrene or pullulan standards as a substitute is hampered by those two polymers’ different hydrodynamic radii when compared to cellulose, so that independently determined correction factors must be introduced. Thus, standard calibration, light scattering detection or viscosity detection are the methods of choice. Light scattering relies on constants that, although they might be error-prone, can be determined within a reasonable limit of certainty and are far more reliable than GPC based on standard calibration. Light scattering requires a correct refractive index increment at constant chemical potential, i.e. obtained at thermodynamic equilibrium with the solvent, (dn/dc)l of the polymer in the respective solvent. Using multiangle detection enables detailed statements on physicochemical properties of the dissolved polymer, such as shape in solution, cross-linking, branching, and substitution effects. Among other factors, universal calibration relies on correct Kuhn–Mark–Houwink– Sakurada (KMHS) constants. Solvents, columns, and sample dissolution in cellulose GPC The separation of cellulose molecules on a suitable column material requires the polymer to be dissolved beforehand in an appropriate solvent, which should preferably be a thermodynamically good solvent to achieve a molecularly-disperse solution of cellulose molecules (Arndt and Mu¨ller 1996; Mori and Barth 1999). At present, two methods have evolved from a limited number of options for cellulose-GPC measurements: (a) derivatization of cellulose hydroxyl groups with phenylisocyanate to the corresponding cellulose tricarbanilates (CTCs), which are dissolved in common organic solvents (usually THF) and analyzed, and (b) direct dissolution of the cellulose, most commonly in N,N-dimethylacetamide (DMAc) containing LiCl (8–9 %) and subsequent GPC analysis in DMAc/LiCl (0.5–0.9 %). Both principal approaches and their variations are represented in the present round robin study. Analyzing the lignocellulosics directly as solution in ionic liquids (IL) appears to circumvent problems with solubility from the outset, and first reports (Fukaya et al. 2011; Kuroda et al. 2013; Engel et al. 2012; Qiu et al. 2012) on the use of IL as an eluant in chromatography are available, but application of pure IL eluants on

Cellulose

(2012). In general, to achieve dissolution of cellulose, its highly complex network of hydrogen bonds has to be loosened and broken, and has to be replaced by hydrogen bonds between solute and solvent or solvent components. Several steps are necessary to obtain a solution suitable for GPC measurements (Morgenstern and Kammer 1998). Derivatization of cellulose hydroxyl groups with phenylisocyanate Fig. 1 GPC set-up with multi-angle light scattering viscosity and RI/UV detection

a routine bases has thus far not found broad application due to high viscosities of the solutions and economic constraints. In GPC measurements, the polymer molecules are separated on a porous gel according to their hydrodynamic volume. Other interactions, such as adsorption, ion exchange, or electrolyte effects, so-called secondary effects, are less decisive, but cannot be neglected in the case of the highly-polar analyte cellulose. Possible non-entropic interaction in the DMAc/LiCl system, which do play a role in the variations visibly in the present round robin have been discussed by Strlic et al. (2002) and Strlic and Kolar (2003). The column material itself is a semirigid, highly cross-linked polystyrene gel (styrene–divinylbenzene copolymer) that can be used with common organic solvents such as THF, toluene, and acetone, but also with cellulose solvents, such as DMAc/LiCl. While GPC columns are available with different gel porosities, it is still difficult to control the pore size during production. For GPC measurements a complete dissolution of cellulose is required. The solvent should have no significant UV absorption in the visible region so as not to interfere with light scattering or UV detection systems. Its viscosity at room temperature should be sufficiently low to be pumped through columns and multi-detector GPC setups. Furthermore, to the extent possible, the solvent should be nontoxic, noncorrosive, and available in larger amounts at reasonable prices. A detailed description of solvents for cellulose, many of them developed only within the past three decades, appears in the work of Klemm et al. (1998) and in the references cited therein, while IL and cellulose are covered by El Seoud et al. (2007) and Wang et al.

The first general method for cellulose dissolution prior to GPC analysis is chemical derivatization with phenylisocyanate that proceeds to the corresponding cellulose tricarbanilate (CTC) (Fig. 2). Carbanilation was long the method of choice, after cellulose trinitrates, to dissolve cellulose in a solvent compatible with standard GPC systems, as no severe degradation was detected (Hall and Horne 1973; Terbojevich et al. 1995). The derivatization with phenylisocyante also has the advantage that the final cellulose derivative becomes UV-active and can be detected by UV detectors, which are more sensitive. Even micro-methods for the GPC separation of cellulose tricarbanilates in filled capillaries for capillary electrophoreses have been described (Stol et al. 2002). The prerequisite to a derivatization step prior to GPC is that the chemical reaction proceeds with quantitative yields, which is the case for the CTC derivative and pure cellulose. The derivatization step is complicated by the presence of large quantities of lignin; small amounts do not interfere. At this stage, whether the CTC will be completely soluble or not depends largely on the nature of the pulp or paper. The derivatization protocol normally includes a precipitation step in order to remove excess derivatization chemicals which, if injected with the polymer, can lower column lifetime and performance in GPC in the long run. This precipitation step carries the danger of keeping low molar mass fragments in solution (see ‘‘Results and discussion’’ section below). Another issue connected to the CTC derivative was the possibility of changing the state of oxidation during derivatization when DMSO was used as the solvent or as part of the solvent system. Cellulose oxidation and degradation by DMSO-derived intermediates during carbanilation can be minimized but cannot be avoided completely, which can be a critical issue if CTC is prepared in DMSO prior to GPC (Henniges et al. 2007).

123

Cellulose Fig. 2 Carbanilation of cellulose by phenylisocyanate

H

O OH

HO

N

O-Cell

Cell-O

N

O

=R

O

O OH

C

2d, Py

RO

O-Cell

Cell-O

O OR

Direct dissolution of the cellulose The solvent system DMAc/LiCl has evolved to the predominant system for analytical investigations in homogeneous solution over the last 20 years. DMAc/ LiCl was first applied to polysaccharides to dissolve chitin in 1977 by Austin. McCormick and Lichatowich (1979) and Turbak et al. (1980, 1981) reported the homogeneous dissolution of cellulose in DMAc/LiCl with little or no degradation. Other solvent systems containing a dipolar aprotic liquid and LiCl or LiBr have been summarized by Morgenstern and Kammer (1996). DMI/LiBr is a comparable solvent system and was first applied by Yanagisawa et al. (2004) to cellulose in a GPC system. Dissolution of cellulose in DMAc/LiCl requires a so-called activation step. This activation procedure is usually necessary to facilitate dissolution. Activation of cellulose can be achieved by solvent exchange from the swollen state—either in water, liquid ammonia, or sodium hydroxide—to methanol, ethanol, or acetone to finally (dry) DMAc (Dawsey and McCormick 1990). Freeze-drying (Ro¨hrling et al. 2002) or thermal treatment in solvents like DMAc or DMAc/LiCl (McCormick and Shen 1982) have also been proposed as means of activation, on the premises that such activation procedures lead to a more efficient transport of the actual solvent and to an effective change in the cellulose morphology and supermolecular structure, which is accompanied by an opening of pores and thus better accessibility for the solvent. For the influence of different activation steps on solubility, see Ro¨der and Morgenstern (1999) and Morgenstern and Berger (1993). The use of high temperature during dissolution (Klemm et al. 1998), or the hot slurry method, is certainly problematic; it leads to dissolution of cellulose, but at the expense of severe molar mass degradation. This phenomenon might be acceptable for subsequent modification in synthesis, but not for analysis of unaltered cellulose in GPC. The

123

mechanism for the sometimes extensive degradation of cellulose has been clarified (Rosenau et al. 2005; Potthast et al. 2002). In DMAc as a dipolar aprotic solvent the lithium cation is solvated whereas the chloride anion is poorly shielded. A common feature in most models for the DMAc/LiCl solvent structure is the coordination of the Li? ion to the carbonyl oxygen of DMAc, and the formation of macrocations [(DMAc)xLi]?. The complex stability of these macrocations, which must be neither too low nor too high, seems to be a crucial feature for the ability to dissolve cellulose (McCormick et al. 1985; Morgenstern 2001). Numerous models exist for the solvation structure of cellulose in DMAc/LiCl, which were reviewed by Morgenstern (Morgenstern and Kammer 1996; Morgenstern 2001). A more recent view on how DMAc/LiCl interacts with cellulose has been proposed by Zhang et al. (2014) based on isotopic labelling of the solvent and NMR measurements.

Results and discussion The samples distributed between the round robin participants consisted of six cellulose pulps of different provenance, comprising hardwood and softwood pulps, kraft and sulfite pulping, and both high-molar mass paper-grade pulps and lower-molar mass dissolving-grade pulps (see Table 1). Only softwood kraft pulps have been excluded, since their severe solubility problems are already understood. Thus, relevant and representative pulps were chosen, at the same time covering as wide a range of influencing parameters as possible. Pulp sample 4 had residual lignin present (kappa number 4.7) in order to evaluate the effect of lignin during derivatization and dissolution. The pulps deliberately include not only samples that form solutions nearly completely, in a short timeframe and leaving no larger insoluble parts, such

Cellulose

as dissolving pulps, but also harder-to-dissolve pulps, such as paper-grade pulp containing hemicelluloses, which require longer dissolution times and special pretreatments. The main parameters to be discussed are the influence of the calibration technique, interand intra-laboratory variations, and molar mass distribution. The detrimental impact of hemicelluloses, especially for softwood kraft pulps, on the solubility of cellulose is well-known (Sjo¨holm et al. 2000), so Table 2 summarizes the carbohydrate content of the six chosen pulps. Two approaches for dissolution were included: derivatization as cellulose tricarbanilate subsequently analyzed by GPC-MALLS, and direct dissolution in appropriate solvents (DMAc/LiCl except for one lab where DMI/LiCl was used). These two methods reflect the current state of the art since no other competitive and widely-used dissolution methods in cellulose GPC exist; the nitration of cellulose and GPC determination of the resulting cellulose nitrates is infrequently used and posed too many drawbacks and pitfalls to become a general protocol. Also, dissolution in ionic liquids (IL) an increasingly popular cellulose solvent, still relies on precipitation and subsequent dissolution in DMAc/LiCl for analysis, as ILs are still too costly and

Table 1 Six pulp samples, all fully bleached except pulp 4, used in the round robin test Provenance

Description

Pulp 1

Eucalyptus, kraft

Paper grade pulp

Pulp 2 Pulp 3

Spruce, sodium sulfite Beech, sulfite

Dissolving pulp Dissolving pulp

Pulp 4

Beech, sulfite, unbleached

Dissolving pulp, j = 4.7

Pulp 5

Birch, kraft

Paper grade pulp

Pulp 6

Cotton linters

Hemicellulose-free

Table 2 Relative amounts of carbohydrates* in the six pulp samples determined according to (Sinner and Puls 1978; Willfo¨r et al. 2009)

* Neither arabinose nor rhamnose was detected

too high in viscosity to be used as GPC eluent on a routine basis. All pulps were analyzed by all round robin partners according to the methods described in Table 3. The operating conditions vary almost as widely as the hardware components used. The first major influencing factor was the sample preparation of the cellulose, either derivatization or direct dissolution. The second issue came from the GPC system depending on the eluants chosen. Four different solvents or solvent systems were used: THF; DMAc/LiCl (0.9 %); DMAc/LiCl (0.5 %); and DMI/LiCl (1 %). The number of separation columns in series was either three or four with one exception where only one column was used. The column temperature varied between 25 and 80 C, with most partners using temperatures between 25 and 40 C. With laser light scattering as the detection mode, the laser wavelengths were 488 nm (three partners), 658 nm (one partner) and 690 nm (one partner). Table 3 summarize the operating parameters used by the different test partners, including values for the refractive index increment (dn/dc)l used, eluant flow, injected volume and sample concentration, and information on applied calibration (standards, KMHS constants). As some partners provided results for more than one analytical method, each pulp was measured a total of twelve times, providing a sufficiently reliable basis for evaluating methods and statistics. As unanimously reported by the partners, pulps of higher molar mass (pulps 1, 4, and 5) proved to be more difficult to dissolve and also caused problems during analysis (some labs reported an increase in column pressure), whereas the other three samples posed no significant obstacles to a rapid and straightforward GPC analysis. This was equally true for both direct dissolution and derivatization with phenyl isocyanate. Table 4 summarizes the different partners’ measurement results, with comparison and discussion limited to Mw values. Mn

Pulp

Xylose (%)

Glucose (%)

Mannose (%)

Galactose (%)

4-O-MeGlcA (%)

1

17.7

81.1

0.3

0.4

0.5

2

1.1

97.0

1.9

–

–

3

2.9

96.1

1.0

–

–

4

6.4

92.1

1.5

–

–

5

24.2

75.3

0.5

–

–

6

0.5

99.5

–

–

–

123

Cellulose Table 3 Operating parameters of the GPC systems used Partner

A

Type

Solvent

Column

CTC

THF

a

3 PSS

B

CTC

THF

4 PLA

Cb

CTC

DMAc/LiCl 0.5 %

3 PLA

Cc

Direct dissol.

DMAc/LiCl 0.9 %

3 PLA

D

Direct dissol.

DMAc/LiCl 0.9 %

4 PLA LS

Ee

Direct dissol.

DMAc/LiCl 0.5 %

F

Direct dissol.

Gd He

Tcolumn C

kMALLS nm

Convent. Calib.

(dn/ dc)l

30

–

UV254, RI, PS

20 lm

25

488

UV254, RI, PS

20 lm

40

658

0.147

20 lm

40

658

0.136f

25

488

–

0.136

3 PLA

80

–

–

–

DMAc/LiCl 0.9 %

4 PLA LS

25

488

–

0.136

Direct dissol.

DMI/LiCl 1 %

1 KD 806 M

60

690

–

0.062

Direct dissol.

DMAc/LiCl 0.5 %

PLB

40

–

RI Pullulan

–

0.182

Remarks

cinj mg/ml

a3 = 0.714

0.175

Conv. PS

0.300

0.250

20 lm

Univ. RI

0.400 0.200

20 lm

10 lm

–

0.100

Flow: 1.000 ml/min, injection volumne in all cases 100 ll except for A (50 ll) and H (200 ll) a

For polystyrene: a = 0.642 and K = 17.35 9 10-3 ml/g, according to Timpa (1991)

b

For cellulose a = 0.957 and K = 2.78 9 10-3 ml/g, according to Bikova and Treimanis (2002)

c

For polystyrene: a = 0.714 and K = 0.0136 (Poly 7)

d

Conditions taken from Yanigasawa et al. (2005)

e

Universal calibration (RI): a1PS = 0.642 a2Cellulose = 0.957

f

This is the (dn/dc)l used, it does not necessarily mean that this value is correct as it was not determined for a 658 nm laser

was not further used in the discussion as it was highly prone to errors in both measurement (significant influence of separation quality in the low-molar mass region) and data processing (e.g. setting of base lines in the chromatograms). General observations for the pulps analyzed High relative standard deviations (RSDs) were obtained by calculating the average Mw of each pulp, ranging between 25 and 47 % (Table 4; Fig. 3). Even in the case of cotton linters (pulp 6), the purest in terms of hemicellulose and lignin residues, values were measured between 1.47 9 105 and 3.25 9 105 g mol-1. As expected, the deviations were largest for pulps with higher Mw (pulps 1, 4, and 5), which were also those that showed some difficulties in dissolution and were the most challenging samples in this study. Samples 1 and 5 were both paper grade kraft pulps from hardwoods (eucalyptus and birch) with 18 and 24 % xylose content, respectively. Pulp 1 had the highest molar mass of all samples. In CTC derivatization, pulps 1 and 5 showed

123

dissolution problems, as dissolution was severely hampered by the larger molar mass of these pulps. Whether the Mw parameters obtained characterize the pulp only incompletely or still yields the correct MMD depends on the actual pulp sample (Henniges et al. 2011). The high RSD in the overall comparison of the data resulted from obvious problems with conventional calibration modes. This trend was found independently in two labs (A and B). If only the LS data were considered and conventional calibration was disregarded, the RSD of Mw of all pulps improved from 32.7 % on average to 15.7 % on average. The impact of the evaluation method on molar mass data Overall, conventional calibration techniques yielded significantly overestimated Mw data. The molar mass determined was between 10 and 29 % higher than the mass obtained from light scattering (Table 5). The moderate lignin content might be assumed to be a factor complicating the analysis, but pulp 5, which

Cellulose Table 4 Overview of Mw data in (103 g mol-1) of the six test pulps Lab

No

Type of sample preparation and detection

Pulp 1

Pulp 2

Pulp 3

Pulp 4

Pulp 5

Pulp 6

D

1

Direct MALLS

570

247

314

498

396

154

B1

2

CTC MALLS

500

210

152

460

420

147

B2 B3

3 4

CTC UV convent. CTC RI convent.

700 730

400 390

260 262

1410 1430

760 760

237 227

C1

5

Direct MALLS

660

289

208

618

441

171

C2

6

CTC MALLS

649

264

187

673

410

175

A

7

CTC convent.

1317

602

290

956

748

270

F

8

Direct MALLS

454

275

292

531

347

202

G

9

Direct MALLS

519

243

291

577

284

166

H1a

10

Direct convent.

864

413

468

706

537

325

H2a

11

Direct convent.

474

264

288

397

321

228

E

12

Direct univ.

721

323

339

667

513

239

All

680

327

297

713

487

222

222

101

87

320

160

55

Average SD RSD

32.5

Averageb

1,2,5,6,8,9

559

30.8 255

29.4 241

44.9 560

33.9 383

24.9 169

SDb

83.1

27.8

66.9

78.9

57.8

19.2

RSDb

14.9

10.9

27.8

14.1

15.1

11.4

Convent: refers to conventional/standard calibration Contrary to the other numbers in the table, numbers in italics are relative standard deviations (RSD) in percent a

H1: calculated according to pullulan Mp and H2: calculated according to pullulan Mp corrected according to Berggren et al. 2003

b

Only with MALLS detection 50 45

ALL methods MALLS detection

40

RSD in %

35 30 25 20 15 10 5 0 PULP 1

PULP 2

PULP 3

PULP 4

PULP 5

PULP 6

Fig. 3 Evaluation of the standard deviation of the six pulps after molar mass determination with all methods used and MALLS detection independent from dissolution strategy. The mean RSD values for conventional calibration are 32.7 % for all measurements (n = 12) and 15.7 % for detection with light scattering (n = 6)

had no lignin content, showed the same trend of exaggerated Mw values in the same labs using the same measurements. Lignin content might thus be a

contributing factor responsible for the overestimated Mw values in CTC derivatives with conventional calibration, but it cannot be the sole or decisive one. In actuality, this overestimation of molar mass can be attributed mainly to the differences in hydrodynamic radii in solution of the cellulose derivatives (CTC) as compared to the calibration samples, such as pullulan or polystyrene. If the data of molar mass were corrected for the difference in hydrodynamic volume according to Berggren et al. (2003), values more comparable to LS were obtained (Table 6). For low-Mw samples the data were well comparable, whereas the data for high-Mw samples returned calculations that were up to 47 % lower. The reason for this difference could be the missing high-Mw pullulan standards, which only recently have become commercially available, but were not yet accessible at the time of the round robin test. Next to conventional calibration, universal calibration may be used to determine the molar mass provided that a viscosity detector is available. In

123

Cellulose Table 5 Comparison between average molar mass data Average of MW (103 g mol-1)

Pulp 1

Pulp 2

Pulp 3

Pulp 4

Pulp 5

Pulp 6

All (n = 12)

680

327

279

744

495

212

Without convent. calib. (n = 7)

582

264

255

575

402

179

17

24

10

29

23

18

D MW (%)

Table 6 Comparison between corrected conventional calibration and MALLS detection Type

Pulp 1

Pulp 2

Pulp 3

Pulp 4

Pulp 5

Pulp 6

Dir. convent. corr. H2 (n = 1) in kg mol-1a

474

264

288

397

321

228

Mean all except 11 (n = 11) in kg mol-1

696

333

297

735

499

221

32

20

3

54

35

3

551

264

276

556

367

173

14

0

4

29

13

32

Difference in % Direct MALLS (n = 4) in kg mol-1 Difference in % a

The pulp used by Berggren et al. (2003) for the correction was a paper grade birch kraft pulp

Table 7 Comparison between universal calibration and MALLS detection Type

Pulp 1

Pulp 2

Pulp 3

Pulp 4

Pulp 5

Pulp 6

Direct MALLS (n = 4) in kg mol-1

551

264

276

556

367

173

Mw direct univ (n = 1) in kg mol-1

721

323

339

667

513

239

31

22

23

20

40

38

Difference in %

general, universal calibration produced values on average 20 % higher compared to the average data of all methods. If compared only to directly dissolved samples evaluated by light scattering, the difference became even more pronounced (Table 7). To answer whether a correction factor would also be applicable for universal calibration to obtain more reliable numbers requires more data for universal calibration with cellulose samples. The impact of derivatization on molar mass determination The CTC samples analyzed by MALLS detection in tetrahydrofuran (THF) as the eluent by different labs gave a good comparability for most pulps compared to the same samples dissolved directly in DMAc/LiCl with

123

MALLS detection (Table 8). Only pulp 3 gave a large deviation (-62 %). The reason is not entirely clear, but the derivatization approach has two error-prone factors, one is the derivatization as such, which can cause discrimination effects (see section ‘‘Molar mass distributions of derivatized cellulose’’) and the other is the often insufficient state of dissolution in THF (cf. Fig. 8). The carbanilation procedure increases the molar mass of the sample material by a factor of 3.2, which is eventually corrected for pure cellulose. After this correction the molar mass is in most cases comparable to that of directly dissolved cellulose. If the sample contains large amounts of xylans which contains one hydroxyl group per unitless for derivatization the error is naturally larger. The same applies to lignin. Samples that already have high molar mass can experience problems with carbanilation because of the exclusion volume of the columns. The samples

Cellulose Table 8 Comparison between mean values from MALLS detection and CTC dissolution in THF followed by MALLS detection Type

Pulp 1

Pulp 2

Pulp 3

Pulp 4

Pulp 5

Pulp 6

Via CTC MALLS (n = 2) in kg mol-1

575

237

170

567

415

161

Direct MALLS (n = 4) in kg mol-1

551

264

276

556

367

173

4

-11

-62

2

12

-7

Difference in %

distributed in this round robin do not exceed the valid area for column separation after carbanilation; however, high molar mass samples such as bacterial cellulose sometimes show a very steep slope in the high molar mass area, which indicates a separation limit in the high Mw area. The more compact structure of CTC in solution compared to underivatized cellulose in DMAc/LiCl partially compensates for this effect (Rosenau et al. 2014). Principal component analysis The similar structure of the different analytical protocols is further elucidated in the ordination biplot as a result of a principal component analysis (Fig. 4). Here, each arrow denotes a single pulp and each symbol represents another analytical procedure characterized by the measured molar mass of each pulp. The first ordination axis explains 75.6 % and the second axis 18.9 % of the variance in this sample set. In this graph, similarity between procedures is

expressed by proximity of the symbols; the closer the symbols are, the higher the similarity of the measured molar mass. The length of a single arrow represents the contribution of the respective pulp to the dissimilarity of the full dataset. A small angle between a particular arrow and an axis represents a high linear correlation between the two. Different analytical protocols, especially protocols A and B (CTC-THF and conventional calibration), lead to divergent results that are reflected by both the first and second principal components. Compared to those two, all other workup procedures appear similar to each other, separated mainly by the first principal component (x-axis) with little influence from the second principle component (y-axis). It should be noted, however, that one approach with direct dissolution and DMAc as solvent (H1) also leads to diverging results as soon as conventional calibration is used. Therefore, conventional calibration results in substantially different results than light scattering or universal calibration for molar mass determination.

Fig. 4 Ordination-biplot of a principal component analysis (CANOCO)

123

Cellulose

Variation within one laboratory The variation between labs remains rather large (see Fig. 3) for the pulps analyzed, even though the RSDs are lower compared to a previous round robin performed about 15 years ago, when RSD ranged between 24 and 45 % for dissolving pulps (Schelosky et al. 1999). An important question in this context was how much of this deviation resulted from the different approaches chosen by the partners and how much arose from deviation within the same laboratory. The identification of major influencing factors of deviations is crucial for future improvements. In order to see the variation within one laboratory, B and D performed multiple analyses of all samples. The average Mw RSD ranged between 2 and 6 %, while for Mn it was a marginally higher and ranged between 3 and 16 % (Fig. 5, left). When analysing the samples with different analytical setups (Fig. 5, right), the lowest RSD (5 % on average) was generally observed when using MALLS for calibration, compared to an average of 6 % RSD for the other two detectors. All RSDs ranged between 1 and 12 %.

16 14

In order to observe differences within the derivatization procedure for CTC formation, the samples synthesized by different labs were directly dissolved in DMAc/LiCl (0.9 %) in one lab and measured on a single GPC system with MALLS detection. That result is compared to the data obtained at individual labs in Fig. 6. When CTC samples are analyzed in the same lab, the RSD is 8 %, but the inter-lab RSD is higher, with a 15 % average. As expected, the derivatization of pure cellulose (pulp 6) produced the most homogeneous derivative (SD 1 %), the unbleached dissolving pulp caused fewer problems compared to the bleached version (SD 4.5 vs. 12 %) while the papergrade pulp did not have a higher SD than dissolving pulps. CTC in different labs

48

with different GPC conditions

44

CTC in the same lab

40

RSD % of Mw

No other factor included in this study has a comparable influence on the result. Further insight into the data can be obtained by interpreting the impact of the different pulps on the resulting molar masses. From the length of the arrows, it becomes clear that especially pulp 1 and 4 have a strong impact on the divergence between the results.

with similar GPC conditions

36 32 28 24 20 16 12 8 4 0

Pulp1

Pulp2

Pulp3

Pulp4

Pulp5

Pulp6

Fig. 6 Inter- and intra-lab comparison of the relative standard deviation after cellulose tricarbanilation. The mean values are 8 % RSD for measurement in the same lab and 34 % for analysis in different labs

Mn

16

Mw

14

12

UV RI MALLS

12 10

SD [%]

SD [%]

10 8 6

8 6

4

4

2

2

0

0

Pulp1

Pulp2

Pulp3

Pulp4

Pulp5

Pulp6

Pulp1

Pulp2

Pulp3

Pulp4

Pulp5

Pulp6

Fig. 5 Left: SD of Mw and Mn data of directly dissolved pulp samples—data from lab D. Right: SD from Mw data according to conventional calibration (UV and RI as concentration detectors) and MALLS from lab B

123

Cellulose

Molar mass distributions of derivatized cellulose While the Mw values are reasonably stable, the variation increases considerably for the Mn values, for example pulp 4 (see Fig. 5, left). This is also clear from the MMD, where the main deviation is found in the low Mw region. Even within the same lab, these deviations cannot be suppressed completely, though they remain marginal compared to the differences found in inter-lab comparisons. The molar mass distributions shown in Fig. 7 deepen the understanding of this observation. Three labs have performed a tricarbanilation of pulp 4. They began by analysing the CTC samples themselves, then sent the CTC pulp to one lab where they were analyzed again, but on only one instrumental setup. Predictably, the single-lab analysis results are more similar to each other, but even for the same pulp that has been derivatized according to the same method and analyzed with the same equipment some differences remain. These differences occur mostly in the low molar mass area, underlining the risk of losing low molar mass fractions during the derivatization workup and hence a slight discrimination of the low Mw part (Fig. 7, right). Another factor that influences the results of the molar mass determination with CTC is the solvent used for the analysis of the derivatized cellulose. The notion that common solvents such as THF can be used when derivatizing cellulose is largely true only for easily-handled samples like cotton linters. However, Fig. 8 plots the volume versus concentration

Molar mass distribution after direct dissolution of cellulose The chosen pulps have characteristic molar mass distributions that help in comparing the column performance and evaluation of the project partners (Fig. 9). Pulp samples 2 and 3 are both sulfite dissolving pulps with some hemicellulose present, though only in small amounts. They were largely characterized by the typical multimodal distribution pattern of sulfite pulps produced under acidic conditions, being easily soluble in DMAc/LiCl and causing no difficulty during derivatization steps. Dissolution

Pulp 4 CTC derivatization and analysis in different labs

0.6

lab B lab C lab A

0.5 0.4 0.3 0.2 0.1

Pulp 4 CTC analyzed at GPC system of lab D

0.6

Differential weight fraction

Differential weight fraction

(RI) in addition to the corresponding molar mass fit to compare the solvents THF and DMAc/LiCl for CTC. The quality of molar mass determination depends on the fit that enables the transformation of the raw data into the molar mass distribution or molar mass data. This fit needs to be linear for the same kind of polymers if linear (mixed) columns are used. When the derivatized cellulose is dissolved in THF, a linear fit cannot be adapted to the light scattering data. By contrast, CTC-cellulose is DMAc/LiCl can be fitted linearly over a larger area. This refers mainly to the cellulose part, whereas differences are observed for the hemicelluloses area, possibly also due to differences in the (dn/dc)l values within that region. Therefore, the resulting data are in general more reliable and data evaluation is more stable.

derivatization at lab B derivatization at lab C derivatization at lab A

0.5 0.4 0.3 0.2 0.1 0.0

0.0 3

4

5

6

log Mw

7

8

3

4

5

6

7

8

log Mw

Fig. 7 Molar mass distribution of pulp 4 after tricarbanilation performed by different labs. Left side: comparison between measurements in different labs and with different GPC systems. Right side: measurement within one lab on one GPC system

123

Cellulose 8

Pulp 5 CTC in THF

0

1x10

7

-1

7 6

-1

6x10

6

-1

4x10

log Mw

RI [a.u.]

8x10

5 -1

2x10

5

0 20

22

24

26

28

30

32

34

4

36

retention volume [ml]

Pulp 5 CTC in DMAc/LiCl

8x10-5

8 7

RI [a.u.]

6 4x10-5

6

log Mw

7

6x10-5

5

2x10-5

5 0 20

22

24

26

28

30

32

34

4 36

retention volume [ml]

Fig. 8 Plot of retention volume versus RI to demonstrate the influence of the solvent to the state of dissolution. Left: Pulp 5 as CTC in THF. Right: Pulp 5 as CTC in DMAc/LiCl. Line: RI signal; Dotted curve: Relationship of retention volume and log molar mass from light scattering; dotted line: Linear calibration curve

was thus not a critical factor. The evaluation of the multimodal distribution influenced the mass average data to some extent (see Fig. 9, pulps 3 and 4). The two paper-grade pulps (1 and 5) and both beech sulphite pulps (3 and 4) still contained considerable amounts of hemicelluloses and/or smaller cellulose fractions and thus exhibited strong shoulders in the MMD. Pulps 3 and 4 even contained a second shoulder that proved more difficult to determine. Most round robin partners were able to monitor these distributions, but the partner using universal calibration obtained a different proportion between the single fractions, considerably overestimating the smaller fractions. Possible improvements by using DMI/LiCl Yanagisawa et al. suggested replacing DMAc with DMI for an increased solubility with different pulps

123

(2004, 2005), and DMI is actually less toxic and therefore preferred over DMAc. A comparison between DMAc and DMI for cellulose solutions was provided by Yanagisawa and Isogai (2008). That DMI setup was based on only one separation column, and the loss of separation power can be seen easily for pulp 3 in Fig. 10, right. The low molar mass shoulder was separated, but the medium molar mass shoulder that was found with DMAc/LiCl on three or four columns was lost. When comparing the MMD of pulp 6 without any hemicelluloses, the two distributions look identical. Therefore, the loss in separation power is mainly ascribed to the fact that in the DMI/LiCl approach only one column was used. As far as solubility is concerned, the samples were all soluble in DMI/LiCl within several days. The dissolution protocol is described in Yanagisawa et al. (2004, 2005).

Repeatability of molar mass determination in cellulose GPC and influence of the age of the columns on molar mass Figure 11 plots the data of one pulp (similar to pulp 6) in a process control chart over the course of several years. Process control charts are developed to monitor the stability and repeatability of industrial processes. Prior to establishing the process control chart, the limits of the standard deviation and the average value of this pulp were determined by more than 50 independent measurements. Based on these data, the repeatability of the determination of the molar mass was monitored over a period of 6 years in one lab. With the exception of a few outliers, the majority of the samples scattered around the mean value with small fluctuations in both directions. Beyond different solution protocols and calibration systems discussed above, the columns used for separation were also checked for their influence on MMD. Separation occurs depending on the hydrodynamic volume of the cellulose molecules in solution, so column performance is linked to the accessibility of the pores. The data given in Fig. 12 were measurements from lab D averaged from eight single measurements (n = 8) obtained through four different batches with duplicate injections on the same set of new (\100 injections) columns. Plotting Mn and Mw obtained from new columns versus data

Cellulose Pulp 1 lab D lab C lab F lab E

0.8 0.6 0.4 0.2 0.0 3

Pulp 2

0.8

Differential weight fraction

Differential weight fraction

1.0

4

5

6

7

lab D lab C lab F lab E

0.6 0.4 0.2 0.0 3

4

5

log Mw 0.8

Pulp 3

Differential weight fraction

Differential weight fraction

0.8

lab D lab C lab F lab E

0.6

0.4

0.2

0.0

7

Pulp 4 lab D lab C lab F lab E

0.6

0.4

0.2

0.0 3

4

5

6

7

3

4

log Mw 0.8

Pulp 5

0.4

0.2

0.0 3

6

7

Pulp 6

lab D lab C lab F lab E

0.6

5

log Mw

Differential weight fraction

Differential weight fraction

6

log Mw

4

5

6

7

log Mw

1.6

lab D lab C lab F lab E

1.2 0.8 0.4 0.0 3

4

5

6

7

log Mw

Fig. 9 Molar mass distributions of pulps 1–6 after direct dissolution in DMAc/LiCl comparing the results of four laboratories

from old columns, the impact on Mn and lower molar mass in general was not noticeably pronounced. The data for Mw of high molar mass pulps, in this case higher than 250,000 g mol-1, proved to be higher on the old columns than on the new columns.

A possible explanation is that upon prolonged use, column pores are expected to collapse due to an increase in local pressure or be blocked due to low molar mass impurities, it is likely that old columns lose their ability to separate smaller molecules,

123

Cellulose

Pulp 6

Pulp 3

0.8

1.4

Differential weight fraction

Differential weight fraction

1.6

DMAc/ LiCl DMI/ LiCl

1.2 1.0 0.8 0.6 0.4 0.2 0.0

DMAc/ LiCl DMI/ LiCl 0.6

0.4

0.2

0.0

3

4

5

6

7

3

4

5

log Mw

6

7

log Mw

Fig. 10 Comparison of two pulps and two dissolution approaches 5

8x10

Mw

165

135 120

11

5

4x10

5

2x10

18

.0

4.

20 28

.0

6.

1.

6x10

0

Fig. 11 Process control chart monitoring the long-term repeatability of the molar mass determination of a pulp similar to pulp 6

A round robin test of cellulose GPC was performed on six pulps with different provenances and pulping technologies at eight different labs with substantial experience in cellulose GPC. The weight-average molar mass obtained showed a variation of 36 % across all methods. The two major influencing parameters were sample preparation, i.e. derivatization and dissolution methods, and the type of molar mass evaluation in GPC, i.e. through calibration or

5

2x10

5

5

4x10

5

6x10

8x10

Mn

5

2x10

Mn [old columns]

Conclusion

0

Mw [new columns]

leading to an overestimation of higher molar mass fractions.

123

slope 1.28

5

20

10

10

09

20

20 .0

.0 11

02

6. .0 05

2.

20

20

5.

9. .0

.0 09

19

75

08

06

90

07

105 20

Mw [kg/mol]

MW - 3 SD MW - 2 SD single data MW + 2 SD MW + 3 SD mean value

Mw [old columns]

slope = 1 150

slope = 1

5

2x10

slope: 0.92 5

1x10

4

5x10

0

0

4

5x10

5

1x10

5

2x10

5

2x10

Mn [new columns] Fig. 12 Comparison of Mw and Mn data measured on new (\100 injections) and old ([2000 injections) columns

Cellulose

absolute measurements applying light scattering techniques. Both dissolution approaches led to acceptable results when correct procedures were applied. The derivatization of cellulose to CTC had both advantages and disadvantages. From the viewpoint of practicality and handling, the preparation of CTC was more time-consuming than direct dissolution, and required the use of toxic chemicals. During workup, low-molar mass fractions could be slightly discriminated. Furthermore, correct conditions during derivatization are mandatory, because CTC samples show background fluorescence. The approach is thus largely inappropriate for the analysis of lignin-containing samples. On the other hand, the CTC approach was distinguished by good resolution and a sensitive UV signal, as opposed to less sensitive RI-detection, and it could be employed advantageously when only a small amount of sample is available. The tricarbanilation was also reported to improve the solubility of difficult cellulose samples. In conclusion, CTC derivatization is recommended in the following instances: • • •

DMAc/LiCl is not available due to GPC hardware; Samples are difficult to dissolve; The available sample amount is very low (below 2 mg of cellulose).

The carbanilation protocol per se is reproducible, but can slightly discriminate the low Mw part (see above). In addition it is suggested to avoid DMSO and use only pyridine during derivatization to suppress cellulose degradation (see ‘‘Appendix’’ for a standard protocol). Direct dissolution in DMAc/LiCl has evolved into the most commonly used approach for cellulose GPC. However, DMAc is also a toxic chemical and DMAc/ LiCl is an aggressive solvent on common lab surfaces and coatings. Direct dissolution requires a timeconsuming activation of cellulose, and heating of cellulose solutions in DMAc/LiCl has to be avoided. Drying of the two chemicals (DMAc, LiCl) was in most cases not necessary. Cellulose, even in oxidized forms was stable in DMAc/LiCl. DMAc could be substituted for DMI if necessary (see ‘‘Appendix’’ for a standard dissolution protocol). A major influencing parameter was the way the molar mass was determined. The main differences in Mw found in this round robin resulted from the use of conventional calibration to determine the molar mass of the analyzed

pulp and paper samples. Data thus obtained need to be corrected due to large differences in hydrodynamic radii, because otherwise the molar mass would be considerably overestimated. Universal calibration also might cause considerable deviations. These differences followed the same tendency across the pulps, so that a correction factor similar to that used in conventional calibration could be introduced. The lowest analytical error was observed for absolute molar mass determination, i.e. by light scattering detection, which renders this technique the detection mode of choice whenever it is available. Another important factor for the determination of the molar mass is the solvent used as the eluant for GPC separation. In this study, CTC showed a better state of dissolution for paper-grade pulps when analyzed with DMAc/LiCl as eluant as compared to THF. Sufficient separation could be achieved only by a set of three columns, but four columns in series were recommended for optimum results. In order to improve the inter-lab comparability for cellulose GPC, it was proposed to introduce standard protocols in cellulose analytics for cellulose carbanilation, cellulose dissolution in DMAc/LiCl, and GPC analysis. A standard would also help in making literature data more comparable and make cellulosic pulp data more consistent than it is today. Acknowledgments The authors are grateful to the EPNOE association which provided frame and travel support for the present study. In addition we like to thank the association Zellcheming (subcommittee pulp analysis). The authors appreciate the help of Alexander Tischer with statistics.

Appendix: Round robin test on gel permeation chromatography of cellulose Dissolution of cellulosic pulps for measurement The following describes two representative procedures for the process of dissolving celluloses, the first by derivatization as tricarbanilate and the second by direct dissolution in DMAc/LiCl. Optimized carbanilation and measurement in THF Pulp sheets were disintegrated in deionized water by high-speed homogenisation (20,000 rpm). The sample was recovered on a glass frit (G3), applying only slight vacuum. The pulp was soaked in acetone two times

123

Cellulose

followed by filtration, and the sample was dried in vacuo at 30 C. The sample was fluffed in a non-cutting laboratory mill and further dried in a desiccator over phosphorus pentoxide. 50 mg of pulp were transferred into a 100 ml Duran bottle, anhydrous pyridine (50 ml) was added, and the bottle was placed in a vacuum oven for 10 min, in order to desorb air from the fibres. After addition of phenyl isocyanate (3.5 ml) the bottle was closed with a thermo-stable PTFE cap and the mixture was allowed to stand in order to react in a heating oven at 80 C for 48 h. During that time the bottles had to be shaken manually approximately eight times. The reaction was stopped by addition of methanol (10 ml). The mixture was cooled to room temperature and added dropwise into a mixture of methanol (250 ml), water (150 ml), and glacial acetic acid (5 ml). To improve the precipitation, the mixtures were kept in a refrigerator overnight. The precipitate was recovered by centrifugation in 500 ml beakers at 20,000 g (centrifuge with 11,000 U min-1). The supernatant was decanted and the precipitate was dissolved in acetone (50 ml) in a laboratory shaker overnight. The resulting solution was added dropwise to a solution of methanol (250 ml) and water (150 ml). The mixture was again stored overnight in the refrigerator, the precipitate was recovered by centrifugation as described before, and the products were dried in vacuo at 30 C. Cautionary notes: •

•

Before injection, the sample solution should be filtered to remove particles that might block the columns; Isocyanate is toxic and therefore care should be taken when handling.

Optimized direct dissolution and measurement in DMAc/LiCl To achieve a molecularly disperse dissolution in DMAc/LiCl (9 %, m/V) overnight at room temperature, pulp samples must be activated using the following steps. The pulp samples (20 mg air dried) are disintegrated in a kitchen mixer with 250 mL of deionized water by mixing three times for 10 s. Extended mixing, milling, or the use of ultra-turrax or ultrasound all must be avoided as they cause cellulose degradation. The water is filtered off and exchanged for ethanol or acetone and finally DMAc. The DMAcwet sample is placed into excess DMAc (4-5 ml) and

123

agitated overnight in a shaker at room temperature. The excess DMAc is filtered off using black ribbon cellulose filter paper and the sample dissolved in DMAc/LiCl 9 % applying gentle shaking and few times vortexing (the DMAc and the LiCl are used as received; no additional drying is necessary). DMAc is highly hygroscopic, and even a few seconds’ exposure causes rewetting of the dried solvent and renders previous drying useless (Chrapava et al. 2003). Traces of water will not interfere with the dissolution. The DMAc-wet sample is dissolved in 2 ml of DMAc/LiCl (9 %). Dissolution should proceed at room temperature; for special samples, such as highly oriented fibers like viscose, moderate heating (up to 40 C) may be applied. The samples cannot be heated above that levels, since the cellulose integrity would be severely affected (Potthast et al. 2002). Dissolution times vary depending on the material; for a study on different pulp samples and their dissolution times, see Henniges et al. (2011, 2013). In general, sulfite pulps dissolve very quickly, whereas annual plant fibers need longer dissolution times. Normally, dissolution times beyond 24 h offer no additional improvement to the MMD profile. For measurement, the dissolved cellulose is diluted with pure DMAc (3:1), filtered through a 0.45 lm disk filter and injected. For dissolution of viscose or lyocell fibers different protocols have to be applied (Siller et al. 2014). This is also true for nano-fibrillated cellulose samples (Hasani et al. 2013), but this is beyond the scope of this protocol. Cautionary notes: •

• •

•

•

DMAc is toxic and should be handled with due caution, refer to the MSDS (Material Safety Data Sheet) for safety precautions; Insufficient activation of the cellulose will lead to difficulties in dissolution; DMAc/LiCl is a powerful solvent. Careless handling or leaks will lead to damage of the analytical hardware; Not all analytical devices (e.g. stainless steel pistons in pumps) can withstand DMAc/LiCl and might be rapidly damaged due to corrosion. Check with the beforehand manufacturer if the system is compatible. Stainless steel capillaries should not be used to connect chromatography equipment (pump, autosampler, or detectors), use PEEK instead.

Cellulose

•

Operating a chromatography system in DMAc/ LiCl at higher temperature (T [ 30 C) will increase the risk of corrosion. Operation at room temperature is recommended and does not cause back pressure problems.

Calibration with standards The classical calibration approach in GPC of (synthetic) polymers—the use of narrow distributed polymer standards and the direct correlation of their retention time to the molar mass of the analytes—is not readily applicable in the case of cellulose, simply because of a lack of appropriate standards. For cellulose tricarbanilates, the chemical similarity likely justifies the use of polystyrene standards. However, retention times naturally differ for different polymers and thus the validity of using polystyrene standards is also limited. For cellulose being directly dissolved without preceding derivatization, pullulan standards are available up to a molar mass of 2.5 9 106 g mol-1, which can be used with the solvent system DMAc/ LiCl. Pullulan is a polysaccharide composed of a-1,6glycosidic linked a-1,4-maltotriose units with a small number of randomly distributed a-1,4-maltotetraose subunits (Carolan et al. 1983; Leathers 2004; Taguchi et al. 1973; Catley et al. 1986) and cellulose is a b-1,4linked glucan. Therefore these polysaccharides have different structures in solution and likely different hydrodynamic radii at specific molar masses. Hence, the direct correlation of molar mass and retention volume based on pullulan standards gives only relative molar masses (pullulan-related). A possible approach to improve the calibration with standards was introducing correction factors for the pullulan calibration to better reflect the molar mass of celluloses. Such factors have to be determined beforehand by an absolute method, such as light scattering (Sjo¨holm et al. 2000). A set of pullulanto-cellulose conversion factors is available from Berggren et al. (2003).

of the polymer through the Kuhn–Mark–Houwink– Sakurada equation (KMHS, Eq. 6): ½g ¼ KMa

ð6Þ

with K and a being constants which have to be determined from a double logarithmic plot of [g] versus Mw. These constants vary with solvent type, temperature, and type of polymer. They reflect the hydrodynamic interaction between polymer and solvent. In plots of log[g] 9 Mw versus retention time, data points from different polymers in the same solvent merge into a single curve (Gallot-Grubisic et al. 1967). This plot is called the universal calibration curve and can be used for both cellulose solutions and solutions of cellulose derivatives (such as cellulose tricarbanilates in THF), provided that the corresponding KMHS constants are available. Such sets of constants for polymers in solution can be obtained from literature (Ganster and Fink 1999); for solutions of cellulose in DMAc/LiCl the KMHS constants were reviewed by Bikova and Treimanis (2002). Further a and K values are listed in Mori and Barth (1999). However, caution has to be exercised when values for a and K from the literature are used. These values may be faulty, and it is advisable to double-check that the solvents and conditions match properly. The use of a viscosity detector also allows obtaining additional information on the shape of the molecule in solution, because the viscosimetric radius Rg can be derived from the intrinsic viscosity. The viscosimetric radius is reported to resemble the hydrodynamic value (RH) as determined by dynamic light scattering (Burchard 1999; Mourey 2004). Possible pitfalls are that KMHS parameters have to be known for identical conditions. Literature data to apply the KMHS-equation might not be suitable for the equipment available in the respective laboratory. Thoughtless application of available parameters from literature and/or inappropriate molecules for calibration might lead to under/overestimation of the samples’ molar mass.

Viscosity detection and universal calibration

Determination of the refractive index increment (dn/dc)l in DMAc/LiCl

Another way to obtain information on the molar mass of celluloses by GPC is the use of a viscosity detector. The value of the intrinsic viscosity [g] for a polymer in a specific solvent is directly related to the molar mass

It is evident that knowledge of the exact specific refractive index increment (dn/dc)l is an essential prerequisite to precise measurement of the molar mass by light scattering. As the (dn/dc)l parameter is also

123

Cellulose

wavelength-dependent, its determination should be performed at the same wavelength as the actual GPC light scattering experiment, i.e., the light source of the RI detector should be a laser of the same wavelength as that used for the light scattering. In practice, tungsten lamps do the job just as well. There is a dependence on the molar mass, which comes into play especially at low molar masses below 2 9 103 g mol-1, and a dependence on the solute composition, which is important for copolymer analysis. In the analysis of cellulosic substrates, the latter might be significant for the analysis of non-uniformly substituted cellulose derivatives. n  no ¼ Dn ¼ kDU

ð7Þ

Dn Dc ¼ dn

ð8Þ

dc

n = refractive index; no = refractive index of pure solvent; k = detector constant; U = output voltage of RI detector; c = concentration of the polymer. The (dn/dc)l value is the only constant required by a GPC-MALLS system in order to measure the molar mass in an absolute way, without calibration by standards. The (dn/dc)l value is independent of concentration and molar mass at very dilute solutions and sufficiently high Mw (Mori and Barth 1999). At low Mw values (monomer to oligomers up to *2000 g mol-1), (dn/dc)l depends highly on Mw. In addition, (dn/dc)l is required to measure the concentration of the sample with an RI detector (Eqs. 7 and 8). The (dn/dc)l value can be a measure of the polarisability of the respective molecule which influences the interaction with the electromagnetic waves of the laser and hence the light scattering intensity. The (dn/dc)l value has a large effect on the final data obtained for the absolute values of the molar mass, as (dn/dc)l is part of the Zimm equation where (dn/dc)l2 is part of the denominator, e.g. with a (dn/dc)l being 0.077 ml g-1 in 0.5 % LiCl/DMAc (Dupont and Harrison 2004) compared to 0.140 ml g-1 (Ro¨hrling et al. 2003) in 0.9 % LiCl/DMAc, the final value for Mw will deviate to a large extent (compare Eqs. 9 and 10). Kc 1 ¼ þ 2A2 c þ 3A3 c2 þ    RðhÞ Mw PðhÞ K¼

4p2 n2o

dn2 dc

NA k4o

123

ð9Þ

ð10Þ

The refractive index increment depends on the solvent used and the laser wavelengths applied. However, there is a linear relationship between the laser wavelength and (dn/dc)l in the same solvent. If the (dn/dc)l values are known for two wavelengths in the same solvent, the third wavelength’s (dn/dc)l can be estimated with sufficient accuracy. The (dn/dc)l value also relies on a number of other parameters, such as temperature, solvent, pressure, and salt concentration in the case of salt-containing eluants. Some specific problems arise from the salt dependence in determining the (dn/dc)l value for cellulose dissolved in DMAc/LiCl. In this specific system, the rather high salt concentration compared to the concentration of cellulose and the polyelectrolyte character of the net solvent system combine to render a straightforward offline measurement of (dn/dc)l inapplicable for cellulose in DMAc/LiCl. The change in the (dn/dc)l value is much more pronounced for polymers dissolved in organic polymers as compared to polymers in water. For polysaccharides a (dn/dc)l of 0.146 ml g-1 is more or less valid for all polysaccharides dissolved in aqueous solution including those containing different salt concentrations. For many synthetic polymers, the (dn/dc)l constant can be found in the literature for a given solvent and given wavelength. When it comes to biopolymers, the situation is unfortunately quite different. Some data are available for cellulose derivatives in THF. However, incorrect (dn/dc)l values have often been published, so values solely from the literature should never be relied on without corroboration. This is especially critical when it comes to the solvent system DMAc/LiCl. Here two different concentrations influence the (dn/dc)l value, the concentration of the salt (LiCl) and the concentration of the cellulose which is varied in the determination of the (dn/dc)l value. The concentration of the salt is usually in the range of 0.5–0.9 % and hence an order of magnitude larger compared to the concentration of the cellulose itself. The ordinary procedure to determine a (dn/dc)l value would consist in the direct injection of different concentrations of the polymer (e.g. cellulose) in the respective solvent into the cell of the refractive index detector in order to obtain a plot of dV over dc in which the slope corresponds to (dn/dc)l for a known RI-detector constant (this constant is measured with the same procedure for a compound

Cellulose

with a known (dn/dc)l, often saccharose, with 0.15009 ml g-1, see Fig. 13). For a thorough discussion on that topic refer to Kes and Christensen (2013) and references sited therein. This offline procedure is only applicable, however, if the solvent does not contain any salt or if these differences are levelled out. If the solvent contains salt, a solvent gradient between the polymer and the surrounding solution is generated, even if the polymer is not a polyelectrolyte and hence not charged per se (Fig. 14). An indication of such a process occurring may be the fact that the slope of the dV over dc curve used to determine the (dn/dc)l with the offline RI detection does not cross the dV axis close to zero (see right Graph in Fig. 13). To level the concentration gradient induced by the salt and the polymer, dialysis is normally applied. However, this is not possible or very tedious with DMAc/LiCl, as the cellulose solvent will dissolve the dialysis membranes as well. Beyond the high concentration of LiCl that has a large influence on the (dn/dc)l value itself, the determination of (dn/dc)l requires very precise concentrations of cellulose. Before cellulose can be dissolved in DMAc/LiCl, it has to go through an activation step comprising a solvent exchange, which significantly hampers accurate determination of the cellulose concentration. To overcome this problem, an easily-soluble cellulose, e.g. a sulfite dissolving pulp may be used after activation followed by freeze-drying. These pulps dissolve instantly and can be used directly as test substrates.

In order to overcome the dialysis problem, the column approach can be employed (Podzimek 2011); the levelling effect of the column material can be used in the same way that dialysis operates. The only prerequisite is the knowledge of the exact cellulose concentration in the solution injected. The approach noted above with freeze-dried cellulose may be applied to ensure a correct concentration in the solution. The major hurdle consists in knowing whether the injection system (e.g. autosampler or manual valve) actually meets the nominal setting. This should be tested in any case (e.g. by calibration with a UV- or fluorescence-active substance without columns). With a known RI detector constant and a reliable concentration of injected cellulose and assuming 100 % recovery after the columns, the (dn/ dc)l value can be determined with light scattering

Saccharose in water Shodex RI-71, 35°C dn/dc = 0.15009 ml/g

250 200

160 140

U=253.99373c-4.63846 R=0.99957

voltage [mV]

voltage [mV]

Fig. 14 Gradient of charged solvent-complex molecules, e.g. in DMAc/LiCl solutions, around a dissolved cellulose chain

150 100

Pulp 3 in DMAc/LiCl 0.9 (m/V) AUX1=5.9092e-4 g/(ml V) offline Shodex RI-71, 35°C

120

U=161.7638c-12.35147 R=0.99824

100 80 60

dn/dc=AUX1*k=0.0956 ml/g

40

50

AUX1=dn/dU=dn/dc/k=5.9092e-4 1/V

20 0

0 0.0

0.2

0.4

0.6

0.8

saccharose concentration [g/l]

1.0

0.0

0.2

0.4

0.6

0.8

1.0

1.2

cellulose concentration [g/l]

Fig. 13 Left: Determination of the RI-detector constant for a substance with a well-known dn/dc value (saccharose). Right: Offline measurement of cellulose dn/dc in DMAc/LiCl leading to incorrect (dn/dc)l values (dn/dc)l obtained here 0.095 ml g-1)

123

Cellulose

software. Experience with (dn/dc)l determination by the column approach shows that the value obtained also depends on the age of the columns, obviously due to decreased recovery ratios of older columns.

References Arndt KF, Mu¨ller G (1996) Polymercharakterisierung. Carl Hanser, Mu¨nchen, Wien Austin, PL (1977). US patent, 4,059,457 Barth HG, Boyes BE, Jackson C (1998) Size exclusion chromatography and related separation techniques. Anal Chem 70(12):251–278 Berggren R, Berthold F, Sjo¨holm E, Lindstro¨m M (2003) Improved methods for evaluating the molar mass distributions of cellulose in kraft pulp. J Appl Polym Sci 88(5):1170–1179 Bikova T, Treimanis A (2002) Problems of the MMD analysis of cellulose by SEC using DMA/LiCl: a review. Carbohydr Polym 48(1):23–28 Burchard W (1999). Solution properties of branched macromolecules. Advan Polym Sci (Branched Polymers II) 143:113–194 Carolan G, Catley BJ, McDougal FJ (1983) The location of tetrasaccharide units in pullulan. Carbohydr Res 114(2):237–243 Catley BJ, Ramsay A, Servis C (1986) Observations on the structure of the fungal extracellular polysaccharide, pullulan. Carbohydr Res 153(1):79–86 Chrapava S, Touraud D, Rosenau T, Potthast A, Kunz W (2003) The investigation of the influence of water and temperature on the LiCl/DMAc/cellulose system. Phys Chem Chemi phy 5(9):1842–1847 Christensen BE, Ulset A-S, Beer MU, Knuckles BE, Williams DL, Fishman ML, Chau HK, Wood PJ (2001) Macromolecular characterization of three barley b-glucan standards by size-exclusion chromatography combined with light scattering and viscometry: an inter-laboratory study. Carbohydr Polym 45(1):11–22 Dawsey TR, McCormick CL (1990) The lithium chloride/ dimethylacetamide solvent for cellulose: a literature review. J Macromol Sci Part C Polym Rev 30(3&4):405–440 Dupont AL, Harrison G (2004) Conformation and dn/dc determination of cellulose in N,N-dimethylacetamide containing lithium chloride. Carbohydr Polym 58(3):233–243 El Seoud OA, Koschella A, Fidale LC, Dorn S, Heinze T (2007) Applications of ionic liquids in carbohydrate chemistry: a window of opportunities. Biomacromolecules 8(9):2629–2647 Engel P, Hein L, Spiess AC (2012) Derivatization-free gel permeation chromatography elucidates enzymatic cellulose hydrolysis. Biotechnol Biofuels 5(1):8 Fukaya Y, Tsukamoto A, Kuroda K, Ohno H (2011) High performance ‘‘ionic liquid’’ chromatography. Chem Commun 47(7):1994–1996 Gallot-Grubisic Z, Rempp P, Benoit H (1967) Universal calibration for gel permeation chromatography. J Polym Sci Polym Let Ed 5(9):753–759

123

Ganster J, Fink HP (1999). Physical constants of Cellulose. In: Brandrup J, Immergut EH, Grulke EH (eds) Polymer Handbook. 4th edn. John Wiley and Sons: New York, Chichester, Weinheim, Brisbane, Singapore, Toronto pp 135–157 Hall DM, Horne JR (1973) Preparation of cellulose triacetate and cellulose tricarbanilate by nondegradative methods. J Appl Polym Sci 17(12):3729–3734 Hasani M, Henniges U, Idstro¨m A, Nordstierna L, Westman G, Rosenau T, Potthast A (2013) Nano-cellulosic materials: the impact of water on their dissolution in DMAc/LiCl. Carbohydr Polym 98(2):1565–1572 Henniges U, Kloser E, Patel A, Potthast A, Kosma P, Fischer M, Fischer K, Rosenau T (2007) Studies on DMSO-containing carbanilation mixtures: chemistry, oxidations and cellulose integrity. Cellulose 14(5):497–511 Henniges U, Kostic M, Borgards A, Rosenau T, Potthast A (2011) Dissolution behavior of different celluloses. Biomacromolecules 12(4):871–879 Henniges U, Vejdovszky P, Siller M, Jeong MJ, Rosenau T, Potthast A (2011) Finally dissolved! activation procedures to dissolve cellulose in DMAc/LiCl prior to size exclusion chromatography analysis: a review. Curr Chromatogr 1(1):52–68 Kes M, Christensen BE (2013) A re-investigation of the Mark– Houwink–Sakurada parameters for cellulose in Cuen: a study based on size-exclusion chromatography combined with multi-angle light scattering and viscometry. J Chromatogr A 1281:32–37 Klemm D, Philipp B, Heinze T, Heinze U, Wagenknecht W (1998) Comprehensive cellulose chemistry, vol 2. Wiley, Weinheim Kuroda K, Fukaya Y, Ohno H (2013) Direct HPILC analysis of cellulose depolymerisation in ionic liquids. Anal Methods 5(13):3172–3176 Leathers TD (2004) Chapter 1: Pullulan. In: biopolymers, vol. 6, Polysaccharides II: Polysaccharides from Eukaryotes. De Baets S, Vandamme E, Steinbu¨chel A. (eds) Wiley VCH McCormick CL, Lichatowich DK (1979) Homogeneous solution reactions of cellulose, chitin, and other polysaccharides to produce controlled-activity pesticide systems. J Polym Sci Polym Lett Ed 17(8):479–484 McCormick CL, Shen TS (1982) In: Seymour RB, Stahl GS (eds) Macromolecular solutions. Pergamon: New York, p. 101 McCormick CL, Callais PA, Hutchinson BH (1985) Solution studies of cellulose in lithium chloride and N,N-dimethylacetamide. Macromolecules 18(12):2394–2401 Morgenstern B (2001) Habilitation thesis, Dresden University of Technology, Germany Morgenstern B, Berger W (1993) Investigations about dissolution of cellulose in the LiCl/N,N-dimethylformamide system. Acta Polym 44(2):100–102 Morgenstern B, Kammer HW (1996) Solvation in celluloseLiCl-DMAc solutions. TRIP 4:87–92 Morgenstern B, Kammer HW (1998) On the particulate structure of cellulose solutions. Polymer 40(5):1299–1304 Mori S (1998) Problems associated with SEC measurements of polymers among laboratories: summary on cooperative determination of molecular weight averages of

Cellulose polystyrenes by SEC in Japan. Int J Polym Anal Charact 4(6):531–546 Mori S, Barth HG (1999) Size exclusion chromatography. Springer Laboratory Series, Berlin Mourey T (2004) SEC molecular-weight-sensitive detection. Intl J Polymer Anal Char 9(1–3):97–135 Podzimek S (2011) Light scattering, size exclusion chromatography and assymetric field flow fractionation. Wiley, New York Potthast A, Rosenau T, Sartori J, Sixta H, Kosma P (2002) Hydrolytic processes and condensation reactions in the cellulose solvent system N, N-dimethylacetamide/lithium chloride. Part 2: degradation of cellulose. Polymer 44(1):7–17 Qiu H, Mallik AK, Takafuji M, Jiang S, Ihara H (2012) Ionic liquids in liquid chromatography. In: Handbook of ionic liquids: properties, applications and hazards, pp 325–350 Ro¨der T, Morgenstern B (1999) The influence of activation on the solution state of cellulose dissolved in N-methylmorpholineN-oxide-monohydrate. Polymer 40(14):4143–4147 Ro¨hrling J, Potthast A, Rosenau T, Lange T, Ebner G, Sixta H, Kosma P (2002) A novel method for the determination of carbonyl groups in cellulosics by fluorescence labeling. 1. Method development. Biomacromolecules 3(5):959–968 Rosenau T, Potthast A, Hofinger A, Sixta H, Kosma P (2005) Hydrolytic processes and condensation reactions in the cellulose solvent system N, N-dimethylacetamide/lithium chloride. Part 1. Holzforschung 55(6):661–666 Rosenau T, Potthast A, Krainz K, Hettegger H, Henniges U, Yoneda Y, Rohrer C, French AD (2014) Chromophores in cellulosics, XI: isolation and identification of residual chromophores from bacterial cellulose. Cellulose 21(4): 2271–2283 Schelosky N, Ro¨der T, Baldinger T (1999) Molecular mass distribution of cellulosic products by size exclusion chromatography in DMAc/LiCl. Das Papier 53(12):728–738 Siller M, Ahn K, Pircher N, Rosenau T, Potthast A (2014) Dissolution of rayon fibers for size exclusion chromatography: a challenge. Cellulose 21(5):3291–3301 Sinner M, Puls J (1978) Non-corrosive dye reagent for detection of reducing sugars in borate complex ion-exchange chromatography. J Chromatogr 156:197–204 Sjo¨holm E, Gustafsson K, Eriksson B, Brown W, Colmsjo¨ A (2000) Aggregation of cellulose in lithium chloride/N,Ndimethylacetamide. Carbohydr Polym 41(2):153–161 Stol R, Pedersoli JL Jr, Poppe H, Kok WT (2002) Application of size exclusion electrochromatography to the microanalytical determination of the molecular mass distribution

of celluloses from objects of cultural and historical value. Anal Chem 74(10):2314–2320 Strlic M, Kolar J (2003) Size exclusion chromatography of cellulose in LiCl/N,N-dimethylacetamide. J Biochem Biophys Methods 56:265–279 Strlic M, Kolenc J, Kolar J, Pihlar B (2002) Enthalpic interactions in size exclusion chromatography of pullulan and cellulose in LiCl–N,N-dimethylacetamide. J Chromatogr A 964:47–54 Taguchi R, Kikuchi Y, Sakano Y, Kobayashi T (1973) Polysaccharide production by pullularia pullulans. I. Structural uniformity of pullulan produced by several strains of pullularia pullulans. Agric Biol Chem 37(7):1583–1588 Terbojevich M, Cosani A, Camilot M, Focher B (1995) Solution studies of cellulose tricarbanilates obtained in homogeneous phase. J Appl Polym Sci 55(12):1663–1671 Timpa JD (1991) Application of universal calibration in gel permeation chromatography for molecular weight determinations of plant cell wall polymers: cotton fiber. J Agric Food Chem 39(2):270–275 Turbak AF, Hammer RB, Davies RE, Hergert HL (1980) Cellulose solvents. Chemtech 10(1):51–57 Turbak AF, El-Kafrawy A, Snyder FW, Auerbach AB (1981) US patent 4,302,252 Wang H, Gurau G, Rogers RD (2012) Ionic liquid processing of cellulose. Chem Soc Rev 41(4):1519–1537 Westermark U, Gustafsson K (1994) Molecular size distribution of wood polymers in birch kraft pulps. Holzforschung 48(Suppl.):146–150 Willfo¨r S, Pranovich A, Tamminen T, Puls J, Laine C, Suurna¨kki A, Saake B, Uotila K, Simolin H, Hemming J, Holmbom B (2009) Carbohydrate analysis of plant materials with uronic acid-containing polysaccharides: a comparison between different hydrolysis and subsequent chromatographic analytical techniques. Ind Crops Prod 29:571–580 Yanagisawa M, Isogai A (2008) SEC-MALS-QELS study on the molecular conformation of cellulose in LiCl/Amide solutions. Biomacromolecules 6(3):1258–1265 Yanagisawa M, Shibata I, Isogai A (2004) SEC–MALLS analysis of cellulose using LiCl/1,3-dimethyl-2-imidazolidinone as an eluent. Cellulose 11(2):169–176 Yanagisawa M, Shibata I, Isogai A (2005) SEC-MALLS analysis of softwood kraft pulp using LiCl/1,3-dimethyl-2imidazolidinone as an eluent. Cellulose 12(2):151–158 Zhang C, Liu R, Xiang J, Kang H, Liu Z, Huang Y (2014) Dissolution mechanism of cellulose in N, N-dimethylacetamide/lithium chloride: revisiting through molecular interactions. J Phys Chem B 118(31):9507–9514

123