characterisation of the substituent distribution in starch and cellulose derivatives

Analytica Chimica Acta 497 (2003) 27–65

Review

Characterisation of the substituent distribution instarch and cellulose derivatives

Sara Richardson1, Lo Gorton∗Department of Analytical Chemistry, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden

Received 13 December 2002; received in revised form 8 July 2003; accepted 5 August 2003

Abstract

Derivatised polysaccharides are polymers from renewable sources of great importance in a whole range of different indus-tries. However, only until recently have their characterisation been hampered by the lack of suitable instrumental methods orrather combination of suitable instrumentation. This review gives details on the state-of-the-art on polysaccharide analysis,especially focussing on starch and cellulose derivatives and the use of specific hydrolysing enzymes facilitating their analysis,reflecting recent work in the author’s respective laboratories.© 2003 Elsevier B.V. All rights reserved.

Keywords:Hydrolysing enzyme; Instrumentation; Polysaccharide analysis

1. Introduction

1.1. General introduction

Starch and cellulose are naturally occurring poly-saccharides and the most abundant renewable re-sources available to man. They are both glucosepolymers photosynthesised by solar energy in variousplants, where starch serves as the main energy re-serve, and cellulose as the structural basis of the plantcell wall. These polymers also constitute a major en-ergy source in human and animal diets. In addition,starch and cellulose are widely used as raw materialsin numerous industrial applications, e.g. in the paper,paint, textile, food and pharmaceutical industries. Thepolymers have great potential for providing a broad

∗ Corresponding author. Tel.:+46-46-2227582;fax: +46-46-2224544.E-mail address:[email protected] (L. Gorton).

1 Reflecting recent work in the authors respective laboratories.

range of important functional properties and possessseveral advantages that make them excellent materi-als for industrial use; they are non-toxic, renewable,biodegradable and modifiable[1–3]. As environmen-tal requirements have become of great importancein today’s society, there is an increasing interest inthe industrial use of renewable resources, and con-siderable efforts are now being made in the researchand development of starch and cellulose as the basicmaterial in new applications.

In order to increase their industrial use and to ful-fil the various demands for functionality of differentstarch and cellulose products, they are often modifiedby physical, chemical, enzymic or genetic means.Modification leads to changes in the properties andbehaviour of the polymer and consequently, improve-ment of the positive attributes and/or reduction of thenegative characteristics can be achieved[4,5]. Chem-ical modification implies the substitution of free hy-droxyl groups in the polymer with functional groups,yielding different starch and cellulose derivatives.

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2003.08.005

28 S. Richardson, L. Gorton / Analytica Chimica Acta 497 (2003) 27–65

The properties of a modified polysaccharide dependon several factors, such as the modification reaction,the nature of the substitution group, the degree ofsubstitution (DS) and the distribution of the substitu-tion groups. To direct a modification reaction towardsa certain product with the desired properties, it isof importance to have knowledge of the correlationsthat exist between the modification process, chemicalstructure and functional properties of the final prod-uct. However, the relationships, if any, between theseparameters are still far from fully understood, largelydue to difficulties in the elucidation of the modifiedpolymer structure, including the distribution of sub-stituents. Therefore, there is a great need for andinterest in the development of sensitive and selectivemethods for the analysis of the chemical structure ofstarch and cellulose derivatives.

2. Starch and cellulose

2.1. Native starch

After cellulose and hemicellulose the two mainpolysaccharide components in lignocellulose, starchis the principal carbohydrate found in nature. Nativestarch occurs in the amyloplasts of seeds, roots andtubers, and in the plastids of green plant leaves asdiscrete, partially crystalline granules. The major con-stituent (∼99%) of the starch granule is�-d-linkedglucose, which occurs in two different polymericforms; amylopectin and amylose, but there are alsosmall amounts of phosphorus, lipids and proteinspresent in the granule[6]. Furthermore, starch gran-ules differ with regard to size, shape, morphology andconstituent composition, mainly depending on theirbotanical origin, but also on the degree of maturity,weather, soil, etc.[7].

The total annual starch production worldwide is es-timated to be about 45 million tonnes and the dom-inant raw materials used for the extraction of starchare maize, potato, tapioca and wheat[8]. Most of thestarch produced is intended for food purposes; otheruses are in the paper and textile industries.

2.1.1. AmyloseAmylose is considered to be an essentially lin-

ear macromolecule consisting of several hundreds of

(1 → 4)-�-linked d-glucose residues. However, it isnowadays well established that there exists a minordegree of branching in amylose, as the chains maycontain a small number of (1→ 6)-�-d-linkages[7]. The (1 → 4)-�-d-glucosidic linkages in amy-lose allow the molecule to rotate in such a way thatthe chains form left-handed helices with six glucoseunits in each full turn. The weight-average molecularweight (MW) of amylose has been estimated to be 105

to 106 Da [7,9]. The amylose content of most starchesis generally about 20–30%[4,10], although there arestarches that are comprised mainly of amylose[7].

2.1.2. AmylopectinAmylopectin is the major component of starch with

a weight-average MW of 107 to 109 Da [7,9], makingit one of the largest molecules in nature. Commonstarches normally consist of 75–80% amylopectin,although there are natural mutants of, for example,maize, rice, sorghum and barley, which contain al-most exclusively amylopectin (waxy starches)[7].Furthermore, amylose-deficient potato amylopectinstarch (PAP) has been produced by genetic mod-ification [11,12]. Amylopectin, unlike amylose, isa highly branched molecule composed of (1→4)-�-glucosidically linked chains with an averagechain length (CL) of 15–25 glucose residues, butwith 4–6% (1 → 6)-�-d-glucosidic bonds at thebranching points[4,10,12]. The branching structureof amylopectin has been studied extensively and avariety of models of the molecular architecture havebeen proposed. The first so-called cluster model waspresented independently by Nikuni[13] and French[14], and is based on the suggestion that the branch-ing points occur in groups (clusters) (seeFig. 1).Today, this cluster model of the amylopectin struc-ture is generally accepted[10], and describes thebranched structure as three different chains: A, Band C, which are grouped according to their locationin the amylopectin molecule[15] (Fig. 1). A chainsare linked to B chains only through the reducingend and do not carry any other chains. The B chainscarry A chains and/or other B chains through oneor more (1→ 6)-�-d-glucosidic linkages. They arelinked via their reducing ends to other B chains or thesole C chain, which carries the only reducing end ofthe whole molecule[15]. The A chains and short Bchains form left-handed, parallel double helices that

S. Richardson, L. Gorton / Analytica Chimica Acta 497 (2003) 27–65 29

Fig. 1. The revised cluster model of the amylopectin organisation; (∅) denotes the single reducing end of the molecule.

constitute the clusters, whereas the longer B chainsinterconnect the clusters into larger structures[10].

2.1.3. Starch granulesThe early cluster models described above have de-

veloped into research that is more focused on theamylopectin organisation on different and/or higherstructural levels, which is of importance as the amy-lopectin structure, to a large extent, determines thephysicochemical properties of starch. The clusters arepacked together to form alternating crystalline andamorphous lamellae (∼9 nm in thickness) perpendic-ular to the helix axis. The crystalline regions con-sist of the short chains in amylopectin that form thedouble helices, whereas the amorphous lamellae arecomposed of the branching parts of the clusters[16].The lamellae are further organised on a higher level,where stacks of these lamellae form semi-crystallineblocklets (20–500 nm in diameter)[17]. On an evenhigher structural level, the blocklets are the unit con-stituents of the growth rings that are observed withmicroscopic techniques as alternating semi-crystallineand amorphous layers (120–400 nm in thickness). Itis suggested that the amylose component is to befound mainly in the amorphous zones of the gran-ule, whereas amylopectin is thought to be present inboth the semi-crystalline and amorphous regions ofthe growth rings[6,7,17].

The crystalline nature of amylopectin allows starchto be classified according to the packing arrange-ment of the double helices in amylopectin, which isdetermined by their X-ray diffraction pattern[6,13].The packing arrangements give rise to three differ-ent X-ray patterns, namely A-, B- and C-type pat-terns. The A-type crystals are found mainly in cerealstarches, e.g. maize, barley and wheat, which have

relatively short amylopectin chains. Longer chainsare associated with B-type crystallinity that is ob-served in starch from tubers, such as potato, andhigh-amylose varieties of cereals. C-type crystallinityis an intermediate between A- and B-type patternsand is found primarily in legumes, e.g. beans and peas[6,13,18].

2.1.4. Starch characteristicsNative starch granules are insoluble in cold water,

due to their semi-crystalline structure. Heating of a di-lute aqueous suspension of starch results in a swellingof the granules, disruption of the crystalline parts andloss of birefringence. As the temperature increases, ir-reversible swelling occurs, and the granular order isdestroyed[6,19]. In addition, the viscosity increaseswith increasing swelling of the granules. This processis known as gelatinisation and is an important prop-erty of starch, resulting in the use of starch as thicken-ers in foods. Another important physical property ofstarch is retrogradation, which occurs when the poly-mer chains, after gelatinisation, re-associate and returnto a more ordered state[6,19]. Retrogradation resultsin gels or precipitates and is related to storage sta-bility properties. For example, the aggregated struc-tures that are formed when amylose self-associates inan aqueous dispersion are utilised in the formation offilms. Another example is the retrogradation of amy-lopectin, which is partially responsible for staling ofbread[6].

2.2. Native cellulose

Cellulose is a uniform, linear glucose polymerand the most abundant of all naturally occurringsubstances. The polymer constitutes approximately


one-third of all vegetable matter as it is the principalstructural cell wall component of all major plants,such as trees, annual plants, mosses, seaweeds andcotton, in which cellulose occurs in varying amounts.The structural strength of plant cell walls is due to theease with which cellulose molecules form stabilisingnetworks of intra- and interchain secondary forces,resulting in straight, stable supramolecular fibres ofgreat tensile strength[20].

Commercial cellulose supplies an annual world con-sumption of about 150 million tonnes of fibrous rawmaterials. The greatest portion of this amount, derivedmostly from pulped wood, is used for the productionof paper. Another significant amount, mostly from cot-ton, goes into the manufacturing of textiles[20,21].

2.2.1. Molecular structureCellulose is a polysaccharide consisting of anhy-

droglucose units (AGU) linked together by (1→4)-�-d-glucosidic bonds. Unlike the�-d-glucosidiclinkages in starch, the�-d-linkages in cellulose can-not be broken by the human digestive system. Due tothese�-d-glucosidic linkages, the most stable confor-mation for cellulose is that in which each glucose unitis rotated 180◦ relative to the preceding unit. Conse-quently, the smallest repetitive unit is cellobiose (twoglucose units). It has been suggested that there existsa slight helical twist along the cellulose chain. Thistwist is caused by intramolecular hydrogen bondsprimarily between the hydroxyl group on C-3 andthe pyranose ring oxygen in the adjoining glucosemonomer, but also between the hydroxyl groups onC-6 and C-2 of the adjacent glucose unit. The in-tramolecular bonds are responsible for the rigidity ofthe cellulose molecule[20,22].

The average molecular length in native cellulosevaries with its origin, but most celluloses have adegree of polymerisation (DP, average number ofglucose residues per molecule) from 1000 to 15,000glucose units. Native cellulose is always polydis-perse, i.e. it consists of a mixture of molecules withthe same chemical constitution but large variations inaverage CL[20,22].

2.2.2. Crystalline structureSolid cellulose shows a highly ordered microcrys-

talline structure alternating with regions of distinctlylower order (amorphous regions). The crystalline na-

ture of cellulose originates from intermolecular forcesbetween neighbouring cellulose chains over longlengths. All native celluloses show the same crystallattice structure, called cellulose I. However, variousmodifications of native cellulose can alter the latticestructure to yield other types of crystals[20,23]. Theintermolecular forces in the crystalline domains aremainly hydrogen bonds between adjacent cellulosechains in the same lattice plane, which results in asheet-like structure of packed cellulose chains. Inaddition, the sheets are probably connected to oneanother by hydrogen bonds and/or van der Waal’sforces. The organisation of cellulose molecules intoparallel arrangements is responsible for the formationof crystallites. The length of an elementary crystalliteranges from 12 to 20 nm (∼24–40 glucose units) andthe width from 2.5 to 4 nm[20,22].

2.2.3. Supermolecular structureThe tendency of hydroxyl groups to form inter-

molecular hydrogen bonds between neighbouring cel-lulose chains, in combination with the stiff, straightnature of the cellulose molecules, results in the for-mation of crystallite strands. Such a strand of severalcrystals linked together by segments of long cellu-lose molecules constitutes the so-called elementaryfibril, which is the basic structural component of thecellulose fibre. The interlinking regions between thecrystalline areas in the strands have a distinctly lesspronounced organisation and thus constitute the amor-phous cellulose. The elementary fibrils are packedtogether to form larger aggregates called micro- andmacrofibrils, these aggregations are then further or-ganised in a typical manner (depending on source) inthe cell wall[20,21].

2.2.4. Cellulose characteristicsCrystalline cellulose is a relatively inert substance,

due to the strong binding between adjacent cellulosemolecules. Native cellulose swells in water, but is in-soluble in both water and dilute acids. In order to ob-tain a solution of cellulose, concentrated acids thatcause extensive hydrolysis of the polymer can be used[20]. As an alternative, a Me2NAc–LiCl solvent sys-tem has been employed to dissolve cellulose[24].The crystalline parts of cellulose are rather resistant todegradation by enzymes (seeSection 3), and a systemof several synergistically acting enzymes is necessary


to obtain any significant hydrolysis[25]. However, thereactivity of cellulose can be greatly enhanced by var-ious forms of treatment, such as swelling, degradationor mechanical grinding, which break down the fibril-lar aggregations[20]. The cellulose fibre is straight,very stable and of high tensile strength, and has beenutilised by human civilisation for millennia, e.g. in themanufacturing of paper, cardboard, building materialsand rayon.

2.3. Chemical modification of starch and cellulose

Starch and cellulose belong to the family of bio-degradable, renewable polymers that provides a broadrange of important functional properties, and are thuswidely used in industry today. However, some of theinherent properties of these polysaccharides limit theirutility in certain applications. Therefore, native starchand cellulose are commonly modified by physical,chemical, enzymic or genetic means in order to obtainspecific functional properties[4,5,9,26]. In this reviewonly chemical modification will be described in moredetail.

Chemical modification is based on reactions ofthe free hydroxyl groups in the anhydroglucosemonomers, resulting in changes in the chemicalstructure of the glucose units and, ultimately, the pro-duction of starch and cellulose derivatives. Usually,these modifications involve esterification or etherifi-cation reactions of the hydroxyl groups. Each AGUis available for up to three sites of substitution; thehydroxyl group on C-2, C-3 or C-6 (in amylopectin,the hydroxyl groups on C-6 involved in the branchingpoints cannot be substituted) (seeFig. 2).

The degree of substitution describes the averagenumber of substituted hydroxyl groups per AGU andranges from 0 to 3. The term DS applies to derivatives

Fig. 2. Schematic illustration of an anhydroglucose unit.

in which the substitution group terminates the reactivehydroxyl sites. Substitution by chemical groups thatgenerate new free hydroxyl groups for further substi-tution is quantified by the molar substitution (MS).This value is defined as the average number of molesof substituent added per AGU[27]. The MS has notheoretical upper limit.

Starch and cellulose derivatives can be charac-terised by a number of factors, such as type and natureof substitution group, DS, MS, average CL and DP.These factors influence the functional properties ofthe derivative in various ways[2,26,27].

2.4. Starch derivatives

Some characteristics of native starch are unde-sirable in many applications. However, only slightmodification is required to change the behaviour andproperties of starch. Some of the principal reasonsfor starch modification are to modify cooking char-acteristics, increase freeze-thaw and process stability,decrease retrogradation and gelling properties, im-prove film formation properties or render the polymerelectrostatically charged[5].

Chemical modification of starch most commonlyinvolves cross-linking, hydrolysis, oxidation, esterifi-cation or etherification reactions. Cross-linking occurswhen a reagent introduces intermolecular bridges orcross-links between starch molecules[27,28]. Oxi-dised starch is obtained by the reaction of an oxidisingagent with the free hydroxyl groups in the glucosemonomer, resulting in the formation of carbonyland/or carboxyl groups. The oxidation process nor-mally causes depolymerisation of the starch moleculesby scission of some glucosidic linkages[27,29]. Es-terification and etherification involve substitution ofthe hydroxyl groups with, e.g. acetyl, hydroxyethylor succinate groups[1]. Some common commercialstarch derivatives and their fields of application arelisted in Table 1. The starch derivatives used in theexperimental studies in this work were starch ethers;therefore the contents of the following sections willbe focused on starch ethers.

2.4.1. Preparation and reaction mechanismsStarch ethers are produced mainly by substitution

with alkyl or hydroxyalkyl groups. Native starchcan be etherified by relatively simple processes as


Table 1Common industrial starch derivatives[1,27]

Substitution group Structure Fields of application

2-Hydroxyethyl –CH2CH2OH Paper industry, textiles, films, plasma extenders2-Hydroxypropyl –CH2CH(OH)CH3 Food products, filmsCationic –CH2CH(OH)CH2N+(CH3)3 Paper industry, textilesSuccinate –CO(CH2)2COO−Na+ Films, emulsionsAcetyl –C(O)CH3 Food products, textiles, paper industryCross-linked –PO(O−Na+)– Food products, emulsionsAnionic –PO(O−Na+)2 Paper, adhesives

it is dispersible in water and exhibits relatively highreactivity compared with native cellulose. Alkalinereagents, e.g. NaOH or KOH, catalyse the substitutionreaction by activation of the starch molecule, whichis achieved by deprotonation of the hydroxyl groups.The modification of starch can take place in differentreaction phases[27,30].

1. Heterogeneous phaseIn this reaction phase the starch is maintained

in its intact granular form throughout the entirereaction. Native starch is suspended in water ora solvent before the addition of the base catalystand modification reagent. To prevent swelling orpasting of the starch under the alkaline conditionssalts, such as NaCl or Na2SO4, may be added.When the reaction has reached completion, themodified starch product is recovered by filtration[30]. Another type of heterogeneous reaction isthe so-called “dry” reaction. In such a reaction,“dry” starch (around, 12–20% water) is thoroughlymixed with the alkaline catalyst. Subsequently, themodification reagent is introduced by dry blending,spraying reagent solution onto dry starch or purg-ing reagent gas at elevated temperatures, yieldingstarch derivatives in a dry, granular form[27,31].

2. Homogeneous phaseThis reaction phase results in the production of

starch derivatives with a disrupted granular struc-ture. The starch is dispersed in water at high tem-peratures or dissolved in aprotic polar solvents, e.g.DMSO, prior to the addition of the base catalyst andreagent. As a result of this treatment, the crystallineorder of the starch molecules is disorganised. Thisin turn leads to a higher molecular reactivity; sub-stitution takes place more uniformly and a higherDS can be obtained[30].

In general, commercial starch derivatives have aDS between 0.05 and 0.2 and are most commonlymanufactured in heterogeneous alkaline aqueous sus-pensions. The main advantage of producing starchderivatives in granular slurry reactions is that un-wanted by-products can be removed by simply fil-tering and washing the final starch product[27].However, recently, dry modification reactions havegained increasing interest due to their reduced envi-ronmental pollution, low energy costs and reducedwater consumption. The resulting derivatives oftencontain salts and reaction by-products, as the productsare usually not washed[27,31].

2.4.2. Properties and usesHydroxyalkylation of starch produces derivatives

with markedly improved dispersion stability (resis-tance to retrogradation), resulting in, for example,enhanced freeze-thaw and cold-storage stability. Asthe substituents prevent close alignment of the starchchains upon cooling of heated starch suspensions,this leads to suspensions with improved clarity. Fur-thermore, the clarity, solubility and flexibility of filmsare improved. In general, the effects increase withincreasing DS[1,27].

Chemically modified starches have a wide spec-trum of applications (seeTable 1), but the mostimportant areas are in the paper, food and textile in-dustries. Hydroxyethyl starch is extensively utilisedin paper manufacturing, especially for surface sizingand coating due to the superior clarity and flex-ibility of hydroxyethyl starch films. Furthermore,hydroxyethyl starches are used in the pharmaceu-tical industry as plasma volume extenders[1,27].Hydroxypropyl-modified starches are of great impor-tance in food and food-related products, primarilyas thickeners. It is the outstanding storage stability


and freeze-thaw properties of hydroxypropyl starchesthat make them so suitable for use in food products.Although no area has the same commercial impactas food applications, there are several non-food usesof hydroxypropyl starches, e.g. in sizing of paperand textiles, and as binders in building materials[1]. Cationic starches are very important commercialderivatives and in most applications it is the ionicnature of the product that is the property of prime in-terest. Cationic starches are used in large quantities inpaper manufacturing, where they act as very efficientwet-end additives, due to the electrostatic affinity forthe negatively charged cellulose fibres that results inirreversible adsorption of the cationic derivative. Theaddition of cationic starches improves the strengthand finish of the paper, the retention of fillers andfines, and the rate of drainage of the pulp[1,27].

2.5. Cellulose derivatives

The intrinsic lack of solubility of native cellulosein water and most organic solvent systems constitutesa major obstacle for utilising cellulose in many in-dustrial applications. Chemically modified celluloseswere developed primarily in order to overcome thisinsoluble nature of cellulose, thus extending the rangeof applications of the polymer. Commercial cellulosederivatives are usually ethers or esters that are solu-ble in water and/or organic solvents. They are pro-duced by reacting the free hydroxyl groups in theAGUs with various chemical substitution groups. Theintroduction of substituents disturbs the inter- and in-tramolecular hydrogen bonds in cellulose, which leadsto liberation of the hydrophilic character of the nu-merous hydroxyl groups and restriction of the chainsto closely associate[2,21,32]. However, substitutionwith alkyl groups reduces the number of free hydroxylgroups.

Table 2Common industrial cellulose ethers[2,32]

Substitution group Structure Abbreviations ofcommercial products

Fields of application

Methyl –CH3 MC, HPMC Building materials, paint removersEthyl –CH2CH3 EC, EHEC Paints, lacquers2-Hydroxyethyl –CH2CH2OH HEC, EHEC Paints, emulsions, drilling mud2-Hydroxypropyl –CH2CH(OH)CH3 HPC, HPMC Building materials, paints, tabletsSodium carboxymethyl –CH2COO−Na+ CMC Detergents, textiles, food products

Cellulose derivatives are used in a wide range ofindustrial fields and their availability, economic effi-ciency, easy handling and low toxicity are reasons fora continuously expanding worldwide market. Com-mon industrial derivatives and their fields of applica-tion are summarised inTable 2. Today, cellulose ethersare the most widely used derivatives, although thereare some commercial esters as well, e.g. cellulose ac-etates. The following sections will focus on celluloseethers.

2.5.1. Preparation and reaction mechanismsEtherification of cellulose proceeds under alkaline

conditions, generally in aqueous NaOH solutions.Treatment of native cellulose with NaOH causes thecellulose to swell, which makes it more readily ac-cessible to the modification reagent[2]. Thoroughmixing and stirring are of vital importance to en-sure uniform swelling and alkali distribution, whichare the most important conditions for homogeneousetherification. Uneven distribution of the substituentscauses severe loss in solubility due to the unetherifiedregions in the final product. Two types of reactionsdominate cellulose etherification[32,33].

1. William etherificationAn organic halide is used as the etherification

reagent and alkali in amounts that are stoichiomet-rically equivalent to the reagent are consumed. Un-reacted alkali must be washed out of the final prod-uct as a salt.

2. Alkaline-catalysed oxalkylationIn this reaction an epoxide is added to the

swollen alkali cellulose. Only catalytic amountsof alkali are required, thus, in principle no alkaliis consumed. The reaction may proceed furtheras new hydroxyl groups are generated during thisreaction.


Industrial modification processes normally takeplace in heterogeneous systems, i.e. both the cellulosestarting material and the corresponding final celluloseether are present as solids, either as dry matter or sus-pended in the reaction medium. Most commonly, theindustrial manufacture of cellulose derivatives is car-ried out by spraying NaOH solution onto dry cellulosepowder, after which the modification reagent is addedas a gas or condensed gas[2,32]. In a homogeneousderivatisation process the cellulose is dissolved in thereaction system. However, this process is not used inindustrial scale. The production of mixed ethers, i.e.derivatives with two or more different substituentsin the polymer chain, requires two reactions that arecarried out either simultaneously or successively[2].

2.5.2. Properties and usesThe functional properties of cellulose derivatives are

predominantly determined by the nature of the sub-stituent and the DS, but also by the substituent distri-bution along the polymer chain. Common to all typesof cellulose ethers is that they provide solubility inwater, aqueous alkali or organic solvents[2]. Deriva-tives with DS below 0.1 are generally insoluble, butif the DS is increased up to 0.2–0.5 (depending onthe type of ether group) the product becomes solublein aqueous alkali. Water solubility is obtained abovea DS of∼0.4 for ionic ethers; for non-ionic ethers aDS of ∼1.0 is required. Commercial cellulose etherswith neutral substituents are normally produced withDS high enough to make them soluble in cold water.Cellulose ethers give highly viscous solutions and therheological behaviour of the solutions is the most im-portant property relating to applications[2,32]. Froman industrial point of view, important functional prop-erties of cellulose derivatives are, e.g. thickening ofaqueous and organic solutions, stabilisation of suspen-sions or emulsions, water retention, binding action,film formation and adhesiveness.

Today, a variety of cellulose ethers with differentstructures, properties and functions are used in numer-ous industrial applications (Table 2). With regard toannual production, carboxymethyl cellulose (CMC)is the largest industrial cellulose ether. The majormarkets for CMC are in detergents, where it acts asa soil-suspending agent, in textiles as warp sizingagents and in paper manufacturing, where CMC pro-motes fibre hydration that increases the dry strength of

the paper[2,34]. Another cellulose ether much usedin industry is hydroxyethyl cellulose (HEC). Thisproduct acts as, e.g. a thickener in drilling fluids, amoisture-retaining agent and retarder in cements, andas thickener and suspension aid in paints[2,32–34].Ethyl(hydroxyethyl) cellulose (EHEC) is a mixedether that is widely used in the paint and buildingindustries; in the former as protective colloids, thick-eners and pigment suspension aids, and in the latteras dispersion agents in cement formulations[2,32].

3. Enzymes

Enzymes are frequently utilised in a variety ofindustrial applications, such as dairy products (coag-ulants), detergents, baking, brewing, distilling, fer-mentation, textiles and medical diagnostics. Anothersignificant application is the starch industry, whereenzymes are employed in starch conversion reactionsfor, e.g. liquefaction, dextrinisation, saccharificationand isomerisation, as well as in the biosynthesisof new starch products[35]. Furthermore, as en-zymes catalyse reactions that are both substrate andproduct-specific, they are extremely valuable as an-alytical reagents, in particular for the determinationof compounds present at low concentrations and inthe presence of other, chemically similar substances.Enzyme-based assay procedures are often rapid andoffer high selectivity and sensitivity. The optimalconditions for most enzyme reactions are at pH val-ues near neutral and around room temperature, whichensures that the analyte structures are not altered ordegraded by extreme pH values or high temperatures.The increasing number of newly discovered enzymes,in combination with efficient methods of enzymepurification, have contributed to the development ofenzymes as highly selective research tools. In thiswork, various starch and cellulose-depolymerisingenzymes (hydrolases) were used for investigation ofthe substitution pattern in chemically modified starchand cellulose, and also for studying different charac-teristics of the starch structure[36].

3.1. Starch-hydrolysing enzymes

Starch hydrolases are enzymes that catalyse thecleavage of glucosidic linkages by the consumption


α-1,6-linkage

α-1,4-linkage

reducing end

glucose unit

PULLULANASE, ISOAMYLASE

AMYLOGLUCOSIDASE

α -GLUCOSIDASE

β-AMYLASE α-AMYLASE

Fig. 3. Schematic illustration of the action of starch-hydrolysing enzymes.

of water, thereby yielding two or more hydrolysisproducts. These enzymes are numbered EC 3.2.1 andcan be subdivided into three groups[4]:

1. Enzymes specific for (1→ 4)-�-d-glucosidic link-ages.

2. Enzymes specific for (1→ 6)-�-d-glucosidic link-ages.

3. Enzymes specific for both (1→ 4)- and (1 →6)-�-d-glucosidic linkages.

In addition, depending on their action pat-tern, starch hydrolases are classified as endo- orexo-enzymes; endo-enzymes cleave linkages insidethe polymer chain (randomly or specifically), whereasexo-enzymes start acting from the non-reducingand/or reducing ends of the polymer[37]. The dif-ferent endo- and exo-enzymes employed in this workare listed below and also depicted inFig. 3.

3.1.1. α-Amylase�-Amylase (1,4-�-d-glucan glucanohydrolase, EC

3.2.1.1) is an endo-enzyme that catalyses the ran-dom hydrolysis of (1→ 4)-�-d-glucosidic linkagesin polysaccharides containing three or more (1→4)-�-linked d-glucose units[38] (see Fig. 3). Thisendo-hydrolysis results in the production of glucoseand oligosaccharides consisting of two to seven glu-cose residues. With amylose as substrate the end prod-ucts are essentially glucose and maltose, whereas withamylopectin there is an additional branched�-limitdextrin residue containing up to seven glucose units.�-Amylase acts both by multichain attack and multiple

attacks on the same chain. In addition to hydrolysinggelatinised starch, the enzyme can also attack the sur-face of the starch granule[4]. �-Amylase exhibitswidespread occurrence in plants, mammalian tissuesand microorganisms, and some common sources de-scribed in the literature are porcine pancreas, barleymalt, Aspergillus oryzaeandBacillus subtilis[4,39].

3.1.2. β-Amylase�-Amylase (1,4-�-d-glucan maltohydrolase, EC

3.2.1.2) is an exo-enzyme that starts acting from thenon-reducing end of the chain and successively hy-drolyses every second (1→ 4)-�-d-glucosidic bondin starch with the liberation of�-maltose (seeFig. 3).Maltose is the main product of the hydrolysis ofamylose; however, linear chains with an odd numberof glucose residues give, in addition, a maltotrioseresidue from the reducing end[37]. When actingon branched polysaccharides,�-amylase cannot hy-drolyse the (1→ 6)-�-d-glucosidic linkages at thebranching points, which leads to the production of, inaddition to maltose, a residual branched�-limit dex-trin. Consequently, the enzyme acts only on the exte-rior chains and the outer parts of the interior chainsin amylopectin. The A chains are hydrolysed untiltwo or three glucose units remain from the glucosemonomer involved in the branching point, dependingon whether the chain contains an even or odd numberof glucose residues, whereas B chains are hydrolyseduntil one (odd number of monomers in the chain) ortwo (even number of monomers) glucose units remainat the branching point[10]. �-Amylase is produced in


higher plants, e.g. sweet potato, barley and wheat, andin some microorganisms, such asBacillus polymyxaandBacillus megaterium[4].

3.1.3. AmyloglucosidaseAmyloglucosidase (1,4-�-d-glucan glucohydro-

lase, EC 3.2.1.3) acts in an exo-manner and catalysesthe successive hydrolysis of terminal (1→ 4) linked�-d-glucose residues from the non-reducing ends inmalto-oligosaccharides containing at least two glu-cose units (maltose). The enzyme action results inthe release of�-glucose[4] (see Fig. 3). In addi-tion, amyloglucosidase has the capacity to cleave(1 → 6)-�-d-glucosidic linkages, provided that thenext linkage is of the (1→ 4)-�-d-type, though atlower hydrolysis rates[37]. Consequently, branchedsubstrates should theoretically be completely de-graded to glucose by amyloglucosidase, however, inpractice, total degradation often requires the concomi-tant action of�-amylase[4]. Amyloglucosidase actsfaster on polysaccharides than on oligosaccharides;amylopectin is hydrolysed at three to eight times therate of maltose and twice the rate of maltotriose[37],depending on the source of the enzyme. Amyloglu-cosidase is predominantly present in moulds, e.g.As-pergillus niger, A. oryzaeandRhizopus delemar[4].

3.1.4. α-Glucosidase�-Glucosidase (�-d-glucoside glucohydrolase, EC

3.2.1.20), also called maltase, is an exo-enzymethat cleaves (1→ 4)-�-d-glucosidic linkages fromthe non-reducing end to produce�-d-glucose (seeFig. 3). The main substrate for�-glucosidase is mal-tose, but the enzyme also acts on glucose oligomersand polymers, although at lower hydrolysis rates[38]. Common sources of the enzyme areDrosophilamelanogasterandSaccharomyces carlsbergensis[40].

3.1.5. PullulanasePullulanase (pullulan-6-glucanohydrolase, EC

3.2.1.41) is a debranching enzyme, catalysing thehydrolysis of (1→ 6)-�-d-glucosidic linkages in thebranching points in pullulan, amylopectin, glycogen,and �- and �-limit dextrins. Pullulan is debranchedto form maltotriose units, while hydrolysis of amy-lopectin results in liberation of only linear chains[4](seeFig. 3). The enzyme can debranch maltosyl andmaltotriosyl stubs from�- and �-limit dextrins, but

not glucosyl stubs[10]. Pullulanase occurs in severaldifferent microorganisms and is purified from, e.g.Enterobacter aerogenesand Klebsiella pneumoniae[4].

3.1.6. IsoamylaseIsoamylase (glycogen 6-glucanohydrolase, EC

3.2.1.68), like pullulanase, is a debranching enzymethat catalyses the hydrolysis of (1→ 6)-�-d-glucosidiclinkages in branched polysaccharides (seeFig. 3).Isoamylase debranches amylopectin and glycogencompletely but do not show any action on pullulan.The enzyme readily debranches the maltotriosyl stubsin �- and�-limit dextrins, whereas maltosyl stubs areonly slowly hydrolysed and glucosyl stubs not at all[10]. Isoamylase is found in microorganisms, such asPseudomonasandCytophagastrains[4].

3.2. Analytical use of starch hydrolases

Starch hydrolases are employed in numerous en-zymic methods for the determination of different struc-tural characteristics of starch and starch derivatives.As enzymic methods for structural analyses of starchare based on the different catalytic selectivities of theenzymes in question, pure enzyme preparations are offundamental importance in order to obtain accurateresults[41].

3.2.1. Native starchDetermination of the total starch content has been

performed by means of simultaneous or successive�-amylase and amyloglucosidase hydrolysis, both insolution[42,43]and immobilised on a support material[44,45]. �-Amylase in combination with�-amylaseand phosphorylase hydrolysis has been used forinvestigation of the composition and chain arrange-ment in amylopectin clusters[46,47]. Furthermore,the amylose:amylopectin ratio can be determined bycomplete debranching of starch by pullulanase orisoamylase followed by separation of the hydrolysisproducts using liquid chromatography. From the re-sulting elution profiles the relative amount of amyloseand amylopectin can be calculated[48,49]. Enzymicdebranching of starch can also be employed to studythe CL distribution profile in amylopectin[18,50–52].Another characteristic of amylopectin is the�-limitvalue, which can be determined by the action of


�-amylase on the exterior chains of starch. The result-ing �-limit dextrin can be further hydrolysed by pul-lulanase or isoamylase for debranching of the exteriorchain stubs in order to calculate the A:B chain ratio[10,47,52,53]. In addition, from known�-limit andaverage CL values, the average length of the exteriorand interior chains can be determined[10,52,54,55].

3.2.2. Modified starchThe distribution of substitution groups along the

polymer chain in chemically modified starch canbe investigated by means of enzymic methods. Theability of starch-hydrolysing enzymes to attack theglucosidic linkages is altered by the presence of sub-stituted glucose monomers, and glucosidic linkagesadjacent to substituted monomers become resistantto or less accessible to enzymic hydrolysis. Exhaus-tive degradation hydrolysis of modified starch by�-amylase and amyloglucosidase with subsequentdetermination of the amount of glucose liberatedgives information on the hetero- and homogeneityof the substituent distribution[55–59]. Debranchingenzymes have been employed for studies of the lo-cation of substituents around the branching points[55,60,61]], and �-amylase for elucidation of thesubstitution of the exterior chains[55,59–61]. Thedistribution of substituents over linear and branchedregions has been studied through�-amylase hydrol-ysis [62], while investigation of the distribution in

β-1,4-linkage

reducing end

glucose unit

CELLOBIOHYDROLASE, CBH II CELLOBIOHYDROLASE, CBH I

ENDO-1,4-β-GLUCANASE

β-GLUCOSIDASE

Fig. 4. Schematic illustration of the action of cellulose-degrading enzymes.

crystalline and amorphous parts has been performedby various combinations of pullulanase or isoamylase,�-amylase,�-amylase and amyloglucosidase hydrol-ysis [61,63,64](for further details, seeSection 5.2).

3.3. Cellulose-hydrolysing enzymes

Cellulose-hydrolysing enzymes are denoted cellu-lases and catalyse the hydrolysis of�-d-glucosidiclinkages in cellulose and other�-d-glucans[39]. Theybelong to the EC 3.2.1 class and are usually dividedinto exo-glucanases (also called cellobiohydrolases(CBH)), endo-glucanases (EG) and�-glucosidases(seeFig. 4). For complete depolymerisation of naturalcellulose, the synergistic action of this whole set ofcellulolytic enzymes is required[25]. Cellulases areproduced by microorganisms including bacteria andfungi, for example, the fungiTrichoderma reeseiandHumicola insolens[39].

3.3.1. Endo-1,4-β-glucanaseEndo-1,4-�-glucanase (1,4-(1,3; 1,4)-�-d-glucan

4-glucanohydrolase, EC 3.2.1.4), often called cellu-lase, is an endo-enzyme that catalyses the hydrolysisof (1 → 4)-�-d-glucosidic linkages in cellulose,lichenin and cereal�-glucans [39] (see Fig. 4).Endo-1,4-�-glucanase shows low activity on crys-talline cellulose and it is generally believed that theenzyme acts through random hydrolysis of the internal


bonds in the amorphous regions of the polymer. Thus,new chain ends are produced that become available forfurther enzymic hydrolysis (see below)[25]. The ma-jor hydrolysis products are cellobiose and cellotriose,but also longer cello-oligosaccharides with differentCLs depending on the source of the enzyme[37,39].

3.3.2. β-Glucosidase�-Glucosidase (�-d-glucoside glucohydrolase,

EC 3.2.1.21) is an exo-enzyme that hydrolysesterminal non-reducing �-d-glucose residues in�-d-oligosaccharides with the release of�-glucose.The enzyme is also called cellobiase, as it readily hy-drolyses cellobiose to glucose[39] (seeFig. 4). Thisactivity on cellobiose is crucial in order to achievetotal hydrolysis of cellulose, as cellobiose is the maindegradation product from the concomitant action ofEG and CBH on cellulose. Furthermore, no othercellulose-hydrolysing enzyme has the capacity toefficiently split cellobiose to glucose[65].

3.3.3. CellobiohydrolaseCellobiohydrolase (1,4-d-glucan cellobiohydrolase,

EC 3.2.1.91) is an exo-glucanase that catalyses the hy-drolysis of (1→ 4)-�-d-glucosidic linkages in cellu-lose with the release of cellobiose. Various forms ofthis enzyme are thought to hydrolyse cellulose chainseither from the reducing (CBH I) or the non-reducingend (CBH II) [25,66] (seeFig. 4). CBH acts syner-gistically with EG in the depolymerisation of highlyordered cellulose. As the major hydrolysis product ofCBH action on cellulose, cellobiose, inhibits the activ-ity of both EG and CBH, the depolymerisation reactionis enhanced by the presence of�-glucosidase[37,65].

3.4. Analytical use of cellulases

Although cellulases have been available and stud-ied for many years, enzymic methods for the structuralanalysis of cellulose are far from as widespread asthose for starch analysis. This is partly due to the factthat cellulose is a very long polymer with an alignedstructure firmly held by hydrogen bonds, which re-duces the accessibility to enzymic attack. On the otherhand, from a structural point of view, the cellulosemolecule is less complex than that of starch, as itconsists of only one, linear component. Consequently,there are less parameters that describe different struc-

tural characteristics of cellulose to be analysed. Still,enzymes provide an efficient tool to aid our under-standing of the biodegradation and structure of cellu-lose and its derivatives.

3.4.1. Native celluloseThe most common application of cellulases for an-

alytical studies of native cellulose is in the estimationof cellulose in biological material. The synergistic ac-tion of EG, CBH and�-glucosidase results in solubil-isation and subsequent degradation of native celluloseto monomers and oligomers. The hydrolysis productsare then determined by, for example, colorimetric orchromatographic methods. Apart from the selectivityand mildness in avoiding destruction of the celluloseand hydrolysis products, one of the main advantagesof enzymic methods is the solubilisation of native cel-lulose to soluble sugars, which are then removed frominsoluble non-cellulosic material (cellulose extraction)[67]. Cellulases have also been used to investigate theamorphous structures in cellulose by kinetic studies ofthe enzymic hydrolysis of cellulose[68].

3.4.2. Modified celluloseCellulose derivatives have been hydrolysed by

different cellulases in order to study the substituentdistribution along the cellulose chain. Indirect infor-mation on the length of cleaved chains and the relativeamount of modified cellulose fragments is providedby analysis of the enzymic hydrolysates, which isusually performed by measurement of reducing sugarsand/or changes in viscosity[69–71]. The heterogene-ity of the substituent distribution can be investigatedby determination of the amount of glucose liberatedfrom cellulase hydrolysis of the derivative[72]. Inrecent times, purified EG preparations instead of cel-lulase mixtures have been utilised for characterisationof the substitution in cellulose derivatives[73–76].As an example, the sole action of CBH I or II hasbeen employed to investigate the substitution at boththe reducing and non-reducing end[77] (for furtherdetails, seeSection 5.2).

3.5. Enzyme action on starch and cellulosederivatives

In general, the accessibility to the substrate ofstarch and cellulose-hydrolysing enzymes is altered


upon chemical modification of the polymer. The in-troduction of substitution groups makes the glucosidicbonds adjacent to substituted glucose monomers re-sistant or less accessible to enzymic attack, and as aconsequence the hydrolysis rate is retarded. The re-quirement for linkage hydrolysis to occur is that theactive site of the enzyme can bind a minimum of ad-jacent, amenable (unmodified) glucose units in orderto form the essential enzyme–substrate complex. Thecleavage of a glucosidic bond then occurs somewherein the middle of this sequence of glucose units. Sub-stituents bound to the glucose units constitute sterichindrance to the formation of the enzyme–substratecomplex, thus the capacity of the enzyme to hydrol-yse the glucosidic linkages in a modified polymer isreduced[78].

Enzymic methods for the determination of the sub-stituent distribution in starch and cellulose derivativesare based on the selective degradation of the modifiedpolymer; non-substituted regions are easily hydrol-ysed compared with low-substituted regions, whereashighly substituted areas are not hydrolysed at all andremain intact. However, one limitation of this enzymicapproach is that the hydrolysis of a certain glucosidiclinkage does not simply result in cleavage or not,but the hydrolysis rate may differ by some orders ofmagnitude. The rate of enzymic hydrolysis seems todepend on several factors, such as the DS, the natureof the substituent and position of the substituents inthe neighbouring glucose unit, but also on the sub-stituent distribution further along the chain, as anoligomeric sequence is usually involved in the forma-tion of the enzyme–substrate complex[25,70,79,80].In addition, the accessibility to enzymic hydrolysisis dependent on the physical features of the polymer,such as crystallinity, degree of swelling and solubil-ity. In many cases, chemical modification makes thepolymer less crystalline and enhances its water solu-bility, thus increasing the susceptibility of starch andcellulose derivatives to enzymic attack[25].

3.5.1. Starch derivativesThe enzymic digestibility of various starch deriva-

tives has been studied in a number of investigations,from which it was concluded that substituted glu-cose units hamper the cleavage of adjacent gluco-sidic bonds [55,56,59,62,63,79,81]. In a study onmethylated starch it was suggested that at least two

adjoining unsubstituted glucose monomers are re-quired for �-amylase (Bacillus amyloliquefaciens)and amyloglucosidase (A. niger) hydrolysis of gluco-sidic linkages to occur[56]. However, results fromanother investigation by Mischnick on methylatedamylose indicated that�-amylase (Bacillus licheni-formis) in combination with amyloglucosidase (A.niger) can hydrolyse bonds next to 2-O-, 6-O- andeven 2,6-di-O-methylated glucose residues from thenon-reducing end, whereas 3-O-methylation preventsbond scission[82]. Investigation of pig pancreas�-amylase hydrolysis of hydroxyethyl starch hasshown that 2-O-hydroxyethylation plays a more sig-nificant role on the resistance to�-amylase attackthan does 6-O-hydroxyethylation[79]. Chan et al.studied the binding of�-amylase (pig pancreas) to hy-droxyethylated amylose and found that the active siteof the enzyme tolerated 3-O-substitution before 2- or6-O-substitution[83]. In an investigation on hydrox-ypropylated starch it was indicated that�-amylasefrom A. oryzae and/or amyloglucosidase fromA.niger seem to have the capacity to cleave bonds nextto substituted glucose units, although to a limited ex-tent [81]. Furthermore, Kavitha and BeMiller foundevidence that�-amylase is not completely blockedby the substituents in hydroxypropylated starch, butcan, to some minor extent, hydrolyse linkages nextto hydroxypropylated monomers[64]. �-Amylase(B. subtilis, B. licheniformis, A. oryzae) hydrolysisof acetylated starch has proven to be more efficienton 2-O/3-O-acetylated than on 6-O-acetylated starch[84]. To summarise, results with respect to enzymicselectivity cannot simply be transferred from onetype of amylase to another, as the shape of the ac-tive site of enzymes from different sources varies.Regarding the DS, the extent of enzymic degradationof starch derivatives decreases and the average DP ofthe hydrolysis products increases with increasing DS[56,62,79,82]. This fact is explained by the enhancedsteric hindrance caused by the substituents, prevent-ing the enzyme from binding the minimum numberof glucose units to the active site, which is necessaryfor cleavage of the glucosidic linkages to occur.

3.5.2. Cellulose derivativesResearch on enzymic degradation of cellulose

derivatives has been in progress for several decades,and already in the 1950s it was concluded that, in


general, glucosidic linkages next to substituted glu-cose units are resistant to enzymic attack[85]. Wiricket al. reported that enzymic cleavage of the gluco-sidic bonds in CMC requires two or more contiguousunsubstituted glucose units[70], and according toresults obtained by Kasulke et al., three adjacent un-substituted monomers are needed for bond scissionto occur [86]. However, Bhattacharjee and Perlinsuggested that although cleavage occurs preferablybetween unsubstituted units, scission of a glucosidiclinkage in between an unsubstituted glucose unit anda unit substituted at C-2 or C-6 also seems possi-ble [87]. Likewise, Puls et al. found that cellulasehydrolysis of CMC can take place at linkages nextto 6-O-substituted units[77]. MC seems to be moreeasily degraded by cellulases. Nojiri and Kondodemonstrated that cellulases could cleave glucosidicbonds between unsubstituted and 2,3-di-O-methylatedmonomers. Scission also occurred between two6-O-substituted glucose units, but not between twocontiguous 2,3-di-O-methylated units[80]. Inves-tigations on the cellulase hydrolysis of HEC haveshown that cleavage between unsubstituted and 6- or2-O-substituted units is possible[69,88]. The DS in-fluences the enzymic hydrolysis in such a way that anincrease in DS leads to a decrease in depolymerisa-tion of cellulose derivatives, which has been proven inseveral investigations on various cellulose derivatives[25,69,73,74]. Similar to starch-hydrolysing enzymes,cellulases from different sources may show differentselectivities towards the same substrate.

4. Analytical techniques

Structural characterisation of starch and celluloseand their derivatives is commonly performed afterdegradation of the polymer, as the intact polymer,in general, is considered far too large and too com-plex to be analysed by most analytical techniques.In order to determine the substituent distribution orother structural parameters of these polysaccharides,accurate analysis of the degradation products is a pre-requisite. Therefore, various techniques for samplehandling, separation and detection of these productsare needed. The techniques should preferably giveboth qualitative and quantitative information and, inaddition, be reproducible, fast and easy to handle.

This chapter describes the analytical techniques thathave been used for investigations of native and mod-ified polysaccharides throughout the present work.

4.1. Microdialysis sampling

Microdialysis is a sampling technique in which asemi-permeable dialysis membrane is used to sampleanalytes from a surrounding matrix. It is, as the nameimplies, a miniaturised dialysis unit in which a mem-brane separates a flowing perfusion liquid from thematrix solution. As there is a concentration gradientover the membrane, analytes are driven by diffusionfrom the matrix, across the membrane and to theperfusion liquid. Small molecules, as defined by themembrane, diffuse through the membrane and aresubsequently carried by the perfusate for analysis.Large molecules are excluded from diffusion acrossthe membrane, thus providing a sample clean-upstep. The technique was introduced in the late 1960sfor in vivo sampling of extracellular fluids[89] andsoon developed into a widespread technique usedin neuroscience and pharmacokinetic studies for invivo sampling of various analytes in, e.g. the brain,blood and liver[90–94]. In recent years, microdial-ysis has found increasing application in the area ofbiotechnology, especially in fermentation[95–97]and enzymic bioprocesses[41,52,98–101], in bothin vivo (sampling from a bioprocess) and in vivo(sampling from a bioreactor without active enzyme)situations.

The microdialysis sampling device depicted inFig. 5, which has been used for sampling bioreactorsolutions[41,52,101], consists of an in-house-madetuneable concentric probe, with an outer cannulaand a lengthwise adjustable inner cannula[102]. Thedialysis membrane is mounted on the outer cannulaand the adjustable inner cannula makes it possibleto change the effective dialysis length (EDL). Theability to adjust the EDL offers a tuneable extractionfraction (EF), which enables optimisation of the an-alyte concentration to be in the linear range of anon-line sensor/detector while running the experiment.Furthermore, the EDL can be adjusted to a specificbioreactor volume. The membrane can be easily andrapidly removed from the probe and replaced by anew membrane, which makes microdialysis a flexibleand low-cost sampling technique.


Fig. 5. Schematic presentation of the experimental set-up of a microdialysis–chromatographic system (A); the microdialysis probe (B); andthe membrane configuration and analyte flow pathway in the probe (C) (from[101] reproduced with permission from Elsevier).

The dialysis process is based entirely upon diffu-sion of an analyte through the pores of a selectivemembrane that acts as a barrier between two solu-tions. Diffusion is the motion of a molecule driven bya chemical potential, i.e. the concentration gradient,and can be described by Fick’s first law of diffusion(Eq. (1)) [103]:

J = −Ddc

dx(1)

where J is the flux, expressed as the number ofmolecules per unit area per unit time,D the diffusioncoefficient and dc/dx the concentration gradient. Thismodel provides a simple description of diffusion understeady-state conditions. However, in the microdialy-sis process, a diffusion model must take into accounta number of additional variables, such as geometryand tortuosity of the membrane, perfusion flow rate(Qd) and the additive mass transfer resistances ofthe dialysate (Rd), membrane (Rm) and bioreactor

solution (Rext). The quantitative aspects of the masstransfer in microdialysis can be described as inEq. (2)[104]:

EF=Coutd − Cin

Cb − Cin=1 − exp

[ −1

Qd(Rd + Rm + Rext)

]

(2)

where Coutd is the concentration of analyte in the

dialysate, Cin the concentration of analyte in theperfusion liquid andCb the concentration of analytein the bioreactor. If pure water is used as perfu-sion liquid, Cin equals zero, and for highly soluble,low-molecular mass analytes in well-stirred solutions,Rext will also equal zero,Eq. (2) then becomes:

EF = Coutd

Cb= 1 − exp

[ −1

Qd(Rd + Rm)

](3)

Simplified, the EF can be calculated as the ratio ofthe concentration of analyte in the dialysate (Cout

d )


to that in the bioreactor (Cb) [52,101]. According toEq. (3), the EF decreases with increasing flow rate, asthe collected dialysate will be more diluted, which isdemonstrated in work by Torto et al.[52,101]. In theseinvestigations it was also shown that the diffusion ofanalytes is controlled by the concentration gradientand is independent of the substance concentration inthe bioreactor, as the EF was unaffected by a 250-foldchange in analyte concentration, as well as changes inthe complexity of the biomatrix[101]. Furthermore,the EF for membranes of the same geometry, at aconstant perfusion flow rate should depend entirely onthe resistance of the membrane to the diffusion of an-alytes, which is inversely proportional to the effectivediffusion coefficient of the analyte (Deff ) [105]. TheEF of different CLs of debranched amylopectin (andthus with different values ofDeff ) has been determinedby Nilsson et al.[52]. The results showed that the EFdecreased with increasing CL, which is in agreementwith lower diffusion coefficients for larger analytes.However, in most applications of microdialysis sam-pling in bioprocesses, enzymes or proteinaceous ma-terial in the matrix interact with or are adsorbed ontothe membrane surface or the inner tortuous structureof the membrane, which might reduce the diffu-sion coefficient of the analyte. This is called mem-brane fouling and results in an initial decrease in EFwith time.

Microdialysis membranes are made of polymericmaterials, preferably with a porous, asymmetricstructure. There is a variety of membranes avail-able on the market, differing in polymeric material,morphology, surface structure, molecular weightcut-off (MWCO), porosity and pore size distribu-tion. Polysulphone, polyethersulphone, polyamide,polycarbonate and regenerated cellulose are someof the membrane materials that are used for micro-dialysis sampling in bioprocesses. The membranedesign affects its performance and function in differ-ent applications, especially the surface chemistry andmorphology. In bioprocesses, sterilisable and biocom-patible membranes are desired, i.e. they should notaffect any on-going processes in the bioreactor, andanalyte–membrane interactions should be negligible[106]. Furthermore, protein–membrane interactionsthat cause membrane fouling are usually undesirable,as fouling alters the surface in an uncontrolled man-ner and mechanically blocks the pores. However, in

certain applications membranes could deliberately beimmobilised with enzymes so as to be used as enzymereactors.

Torto et al. reported on the performance and charac-teristics of membranes of different polymeric materialand MWCO were investigated with respect to theirEF, permeability, temperature stability and protein in-teraction[101]. It was shown that membranes with thesame MWCO, but from different manufacturers, didnot necessarily perform in similar manners, partly be-cause the MWCO is a nominal rather than an abso-lute parameter. Furthermore, the results demonstrateda significant protein–membrane interaction, althoughthe degree of this interaction varied depending on thepolymeric material of the membrane.

There are several advantages of microdialysis sam-pling in bioprocesses; the ability to sample in situwithout perturbing the bioreaction under investigation,sample clean-up, continuous sampling and on-linecoupling to chromatographic/mass spectrometric sys-tems. In comparison with the other main membrane-based sampling technique, filtration, where pressureis the driving force for analytes and solvent moleculesto cross the membrane, dialysis does not suffer fromany serious membrane fouling. Furthermore, in theideal case, no fluid transfer occurs, thus only slightchanges in the bioreactor are expected, althoughsampling of analytes from very small volumes mayvery well affect the system. The use of microdialy-sis for sampling of enzymic hydrolysates of starchprior to analysis by liquid chromatography enableson-line sampling, automated injection and computercontrol for unattended reactions, elimination of largepolysaccharides resulting in reduced separation timeand the removal of enzymes that could impair thecolumn and detector (sample pre-treatment/clean-up)[41,52,101]. As microdialysis sampling allows con-tinuous and on-line monitoring of products releasedfrom enzyme reactions, various characteristics of en-zymes may well be investigated with this samplingtechnique. In the investigation described by Richard-son et al.[41], the purity of different preparationsof starch-hydrolysing enzymes was studied using amicrodialysis-based system coupled on-line to ananion-exchange chromatographic system. The resultsobtained showed that several commercial enzymepreparations contained hydrolytic activity originatingfrom contaminations of other hydrolases.


4.2. Gas chromatography

Capillary gas chromatography (GC) is a classicalmethod for analysis of the monomer composition inpolysaccharides and their derivatives[55,56,107–110].GC analysis requires volatile and thermally stable an-alytes, thus polymers are always hydrolysed (usuallyto monomers) and derivatised by, e.g. methylationanalysis or reductive cleavage, before analysis (seeSection 5.1). The high separation efficiency of GCcolumns allows the separation of monomers with onlyminor structural differences, e.g. glucose monomerswith the same DS but substituted at different positions(i.e. C-2, C-3 or C-6) can be separated. Subsequent de-tection is most commonly accomplished by both flameionisation detection (FID), which provides quantita-tive results, and mass spectrometry (MS), which al-lows identification of the peaks[78,107,108]. The FIDresponse of a molecule depends on the number of car-bon atoms and the type of carbons in the chemical en-vironment (e.g. hydrocarbon, carbonyl or ether) in themolecule. By summing all the contributions from thevarious carbons to the flame response, the “effectivecarbon response” (ECR) for certain structural featurescan be calculated[111]. Thus, in order to make quan-titative evaluations, the peak areas must be correctedusing the ECR concept. The coupling of GC to MSallows identification of analytes without the need forstandards, which is of vital importance as very com-plex monomer mixtures can be obtained from certainpolysaccharides.

4.3. High-performance anion-exchangechromatography

The development of high-performance anion-exchange chromatography (HPAEC) with pulsed am-perometric detection (PAD) for carbohydrate analysisin the mid 1980s greatly revolutionized carbohy-drate chromatography[112]. Prior to the inventionof HPAEC–PAD, the separation of saccharides byhigh-performance liquid chromatography (HPLC)was carried out on reversed phase, metal-loadedcation-exchange or amine-bonded silica columns,usually coupled to differential refractive index (RI)detection. Carbohydrates were also separated as bo-rate complexes on anion-exchange columns. However,these techniques were found to suffer from inadequate

separation, pre-column derivatisation, non-specific re-sponse or incompatibility with gradient elution in theanalysis of mixtures of oligo- and polysaccharides.The combination of HPAEC with PAD resulted in atechnique that has proven to be fast, highly sensitive,specific and compatible with gradient elution and fur-thermore, with the capacity to separate carbohydrateswith high resolution[113–115].

4.3.1. SeparationCarbohydrates are weak acids that behave as anions

in basic solutions (pKa values of 12–14)[116]. Untilrecently, this property could not be utilised for separa-tion, due to the fact that the anion-exchange columnsavailable on the market were packed with silica parti-cles, which are unstable at pH> 8.5. However, the in-troduction of pellicular, polymer-based resins, whichexhibit stability over a wide pH range, transformedanion-exchange chromatography into a versatile andefficient separation technique for carbohydrates. Theseparation mechanism is based on differences in thepKa of the ionisable hydroxyl groups in the sugarmonomer. These slight differences in acidity resultin selective interaction with the anion-exchange resinand thus, differences in retention. The retention timesare inversely proportional to the pKa values, althoughthe retention of oligosaccharides is not directly re-lated to the order of acidity of the monomer units. Fora homologous saccharide series it can be said in gen-eral that the retention time increases with the degreeof polymerisation[114,115]. HPAEC columns havebeen commercialised by Dionex, who specificallydesigns columns for the separation of mono-, di- andoligosaccharides. The columns exhibit rapid masstransfer, which normally leads to sharp peaks withno or only minor tailing, and the resolution is usuallyvery high, as small differences in pKa and molecu-lar conformation result in significant differences inaffinity for the anion-exchange resin[113].

The retention and selectivity are also significantlyinfluenced by the composition of the mobile phaseused in HPAEC. NaOH solutions are most common,but in order to reduce the elution times a competing an-ion, such as acetate or nitrate, can be added to the mo-bile phase. These anions interact more strongly withthe anion-exchange sites than do the hydroxide ions,therefore the analyte ions should be displaced fasterby such counter ions[117,118]. If higher selectivity is


required, mobile phases of lower alkalinity should beemployed[119]. Furthermore, the alkaline conditionsenable dissolution of larger polysaccharides that arepoorly soluble in water, and also facilitate anomericequilibration.

4.3.2. DetectionCarbohydrates are easily oxidised at gold or plat-

inum electrodes at high pH, without any derivatisation,and are thus very promising candidates for ampero-metric detection[120]. In order to overcome electrodesurface poisoning by oxidation products (fouling),pulsed amperometric detection is employed, in whichthe potential is applied in a triple-step waveform (seeFig. 6) [121].

Furthermore, as PAD is highly sensitive (the de-tection limit for glucose has been reported to be atthe picomole level[113]) and no pre- or post-columnderivatisation is required, PAD has proved to be themost promising and convenient technique for carbo-hydrate detection. The major drawback is the varia-tion in molar response for different compounds, whichconstitutes a problem in quantitative analyses wherestandard compounds are unavailable[114].

4.3.3. Applications in starch and cellulose analysisHPAEC–PAD is today widely used for the char-

acterisation of starch. The CL distribution of amy-lopectins from various starches has been determined

Fig. 6. Typical potential waveform for pulsed amperometric de-tection of carbohydrates in alkaline solutions at a gold workingelectrode.

by enzymic debranching of the amylopectin, fol-lowed by analysis of the unit CLs by HPAEC–PAD[50–52,113,122]. Furthermore, the differences in unitchain distribution of amylose-containing starches andamylose preparations have also been studied[52]. Ithas been shown that the technique can provide base-line separation of CLs up to a DP of 80[50]. However,the PAD response varies for individual chains, andfor quantitative analysis relative response factors haveto be determined for each individual chain in orderto compensate for the variation in detector response[51,122,123]. In an investigation by Ammeraal et al.,it was shown that the detector response per gram de-creased with increasing DP up to about 14, beyondthis no clear downward trend could be seen[123].Similarly, Koch et al. found that the detector responseon a weight basis decreased considerably for DPs of3–7 but then levelled out for DP> 15 [51].

HPAEC–PAD has been employed for determina-tion of the �-limit value of various starches[52].�-Amylase hydrolysates of the starch samples weresampled using a microdialysis unit and subsequentlyinjected into the HPAEC–PAD system, where themaltose liberated was detected and quantified. The re-sulting �-limit values were in agreement with resultsobtained with other methods. The same microdialysisHPAEC–PAD system was also used to study the A:Bchain ratio in several starches. This was performed bydetermination of the maltotriose concentration afterpullulanase debranching of the starch samples andtheir �-limit dextrins [52]. The method was foundto be faster and less laborious than the classicalwet-chemistry methods for A:B chain ratio determina-tion. Fig. 7 presents the HPAEC–PAD chromatogramof the debranching products obtained from isoamy-lase and pullulanase hydrolysis of�-limit dextrinsof potato amylopectin starch. The double peaks inFig. 7aclearly demonstrate the incomplete debranch-ing of the�-limit dextrin by isoamylase. In order todebranch the maltosyl stubs in�-limit dextrins, theaction of pullulanase is required (Fig. 7b).

In an investigation on the purity of some commer-cial starch-hydrolysing enzymes, HPAEC–PAD hasbeen used to characterise the enzymic hydrolysis prod-ucts[41]. Similarly, HPAEC–PAD has been used in thesame way to determine the purity and linkage speci-ficity of exo-glycosidase preparations[124]. Karlssonet al. have studied the hydrolytic properties of various


0 2 4 6 8 10 12

Time/min

CL 2

CL 3

a)

b)

Fig. 7. HPAEC–PAD chromatogram of hydrolysis products obtained from debranching of�-limit dextrins of potato amylopectin starch by(a) isoamylase and (b) isoamylase plus pullulanase (from[52] reproduced with permission from Elsevier).

cellulases on cellulose and cellulose derivatives byHPAEC–PAD analysis of the mono- and oligosaccha-rides produced upon enzymic digestion[125,126].

In a work by Richardson et al.[81], the substituentdistribution in hydroxypropylated starch was char-acterised by means of HPAEC–PAD in combinationwith MS. The hydrolysis products from�-amylaseand amyloglucosidase digestion of the starch sampleswere separated and detected using HPAEC–PAD,while the peaks were identified by MS. It was shownthat un-, mono- and disubstituted sugars with DPs of1–5 could be separated on the HPAEC column, withmonomers being eluted before oligomers, and mono-substituted products before disubstituted (seeFig. 8).

The same approach was applied for the investigationof enzymic hydrolysates of EHEC[76]. HPAEC–PADhas also been successfully applied to the determina-tion of the substitution pattern on a monomer levelin starch and cellulose derivatives[74,127–129](seeSection 5.1).

4.4. Size-exclusion chromatography

Size-exclusion chromatography (SEC), also re-ferred to as gel-permeation chromatography (GPC),is a very useful separation technique for determina-tion of molar mass distribution and average molarmass of polymers, both natural and synthetic, in therange of typically 102 to 108 Da. The analytes are

separated according to their hydrodynamic volumeand eluted in order of decreasing size. The stationaryphase comprises a porous, three-dimensional net-work into which the analytes penetrate to an extentdepending on the ratio of their dimension to the av-erage diameters of the pores. Thus, SEC is not onlya means by which information on the distribution ofmolecular size can be obtained, but also a method offractionation[130,131].

In the study of carbohydrates, SEC is widely usedfor molar mass distribution analysis of amylose, de-branched amylopectin and enzymic hydrolysates ofstarch and cellulose[47,49,52,132,133], but also fordirect determination of molar mass distribution andfractionation of intact starch and cellulose[134–137].Furthermore, the technique has been applied to thedetermination of molar mass distribution of hy-drolysates of starch and cellulose derivatives in inves-tigations of the substitution pattern in these polymers[55,56,64,74–76].

The stationary phases used in SEC analysis of oligo-and polysaccharides are in general silica-based matri-ces (Synchropak from Merck and TSK G SW fromTosoHaas), polymeric sorbents such as methacrylate(TSK G PW from TosoHaas) or cross-linked gelsbased on, e.g. agarose (Superose from Pharmacia) ordextran (Sephadex from Pharmacia)[130]. As starchand its components have limited solubility in wa-ter, rather harsh conditions are necessary in the SEC


40

60

80

100

120

140

160

0 10 20 30 40 50 60 70 8

Time/min

Res

po

nse

/nC

(a)

(b)

n1n2,r1n2,r2

n3,r1

n2,r1

0

n3,r1

n3,r1

n4,r1

n4,r1

n4,r2

Fig. 8. HPAEC–PAD chromatogram from analysis of�-amylase and amyloglucosidase hydrolysis of (a) native potato amylopectin starchand (b) hydroxypropylated potato amylopectin starch (n, number of glucose units;r, number of substituents) (from[81] reproduced withpermission from Elsevier).

analysis of such analytes. Elution is often carried outin aqueous alkaline solutions[133,138]and there arealso columns compatible with DMSO, a common sol-vent for starch[139]. In order to avoid electrostaticinteractions of starch with the stationary phase, theionic strength of the mobile phase must be sufficientlyhigh (e.g. 0.1 M). Intact cellulose is amenable to SECanalysis only if the stationary phase is stable in theorganic solvents that are usually required for cellu-lose solubilisation[134,137], whereas water-solublecellulose derivatives, such as HEC or CMC, are moreeasily analysed with SEC[140].

The most widely used SEC detector is the RIdetector, a universal and non-selective detector thatresponds to all changes in the eluate composition.Determination of molar mass distribution by SEC–RIrequires calibration of the system with standards,which constitutes a weakness, as standards withmolecular properties similar to the polymer beingcharacterised are not always available. Furthermore,the RI detector cannot be used with gradient elu-

tion. RI detection is nowadays frequently replaced bymore sensitive and/or selective detection systems in-cluding, for example, UV, viscosity and conductivitydetectors[130,131]. A versatile technique for molarmass distribution and average molar mass analysisof starch and cellulose samples is SEC–RI combinedwith multi-angle light scattering (MALS) detection[52,76,133,136,141,142]. Coupling of MALS to RIdetection allows direct determination of the molarmass by an absolute method without calibration stan-dards, as the MALS and RI detectors give signalsproportional to molar mass and concentration, respec-tively. Apart from polymer size, the MALS detectoralso provides the radius of gyration distribution,which yields information on polymer conformation[142]. This parameter is of significant importance inthe detection of large aggregates.

SEC–RI in combination with low-angle light scat-tering (LALS) has been employed to determine theaverage molar mass of different�-limit dextrins [52].In a study on hydroxypropylated potato amylopectin


starch, SEC–RI was employed for elucidation of thesubstituent distribution around the branching points.The resulting molar mass distributions of pullulanasehydrolysates of one native and two hydroxypropylstarch samples modified under different conditions re-vealed slight differences in the substitution close tothe branches[55].

4.5. Mass spectrometry

Mass spectrometry (MS) of carbohydrates has, inrecent years, become a field of enormous activity,with the numerous techniques already existing undercontinuous development and the area of applicationsconstantly growing. MS analysis includes three dis-tinct steps: (i) creation of gas-phase ions; (ii) sepa-ration of the ions according to their mass-to-chargeratio (m/z); and (iii) detection. The first step was fora long time the major limitation in MS analysis ofbiopolymers, due to the difficulties in transformingsuch molecules into gas-phase ions without extensivedecomposition. Until the mid 1970s, ionisation wasperformed by electron impact (EI), which resulted insevere fragmentation of the polymers. The develop-ment of “softer” ionisation techniques, which allowthe generation of gas-phase ions of labile, involatilebiomolecules with less pronounced fragmentation,partly solved this problem. The first soft ionisationtechniques to be introduced were plasma desorption[143], secondary ion mass spectrometry (SIMS)[144]and fast atom bombardment (FAB)[145]. EspeciallyFAB–MS has been successfully applied to carbohy-drate analysis; one example is the characterisationof the substituent distribution in starch and cellulosederivatives[72,146,147]. However, in many applica-tions these techniques suffer from low sensitivity and,still, extensive analyte fragmentation, which generatesspectra that are very difficult to interpret. The develop-ment in the late 1980s of electrospray ionisation (ESI)[148] and matrix-assisted laser desorption/ionisation(MALDI) [149], both considered to be even softerand more sensitive ionisation techniques, had consid-erable impact on MS analysis of carbohydrates, andare currently the most promising and dominant toolsfor structural analysis of carbohydrates and their com-ponents. However, as carbohydrates represent such awide range of structural types, no single MS methodis ideal for all compounds. Furthermore, the choice

of ionisation method and instrumentation depends onthe type of structural information that is desired.

Following the ionisation process, the analyte ionsare separated according to theirm/z ratio in a massanalyser. All types of mass analysers can be employedin carbohydrate analysis, the choice depending moreon ionisation technique, desired mass range, massaccuracy, resolution, sensitivity, cost, as well as de-sired application, than on the type of analyte[150].Common analysers used in modern mass spectrome-try are magnetic and electric sectors, quadrupoles andquadrupole ion traps, Fourier-transform ion-cyclotronresonance (FTICR) traps and time-of-flight (TOF)instruments. The MS techniques of main inter-est throughout this work were ESI-quadrupole iontrap-MS and MALDI–TOF–MS, therefore these willbe described in more detail below.

4.5.1. ESI-quadrupole ion trap-MSESI, first pioneered by Dole et al.[151] and later

developed by Fenn et al.[148] and Yamashita andFenn[152], has emerged as one of the most versatilesoft ionisation techniques, although the mechanism oftransforming species in solution to gas-phase ions isstill not fully understood. In a typical ESI experiment,the sample is dissolved in a polar solvent, usually wa-ter or mixtures of water/methanol or water/acetonitrile.The sample solution is then pumped through a conduc-tive capillary into the electrospray interface. When ahigh electric field is applied between the capillary anda counter electrode, the sample solution that emergesfrom the capillary tip generates a fine spray of dropletscontaining charged molecules assisted by a flow ofnebulizer gas (commonly nitrogen). If a negative po-tential is applied to the counter electrode, positive ionsin the droplets will drift towards and accumulate at thedroplet surface. Eventually, this results in a conicallyshaped fluid of positively charged droplets that mi-grates from the capillary tip towards the counter elec-trode due to the potential gradient. During migration,the charged droplets continuously decrease in size dueto evaporation of solvent molecules, assisted by thenebulizer gas. As a consequence of the continuouslydecreasing droplet size, the charge density at the sur-face increases. Finally, the electrostatic repulsion ex-ceeds the surface tension and the droplets disintegrateto produce positively charged gas-phase ions[153].For the analysis of negatively charged ions, a positive


potential is applied to the counter electrode. ESI ischaracterised by the extremely gentle and highly effi-cient generation of gas-phase ions and, in contrast toother ionisation techniques, the production of multiplycharged ions. Electrospray ionisation of oligosaccha-rides usually produces ions of the type [M + nH]n+or cationic adducts with alkali metal ions, e.g. [M +nNa]n+ or [M + nLi] n+.

ESI can in principal be coupled to anym/zanalyser,although traditionally, ESI has been used with triplequadrupoles[152]. The use TOF instruments[154]and especially quadrupole ion traps[155] have alsobeen successful. Briefly, in an ESI-quadrupole ion trapprocess, the ions formed in the ESI source (at atmo-spheric pressure) are guided into the ion trap (undervacuum), which consists of three electrodes. Duringion injection, a radio-frequency voltage is applied overthe electrodes to create a three-dimensional quadrupo-lar trapping potential. When the trap is filled with ions(injection time typically 1–500 ms depending on an-alyte concentration), the ion beam is deflected awayfrom the trap and the captured ions oscillate insidethe trap at frequencies characteristic of theirm/z ratio.It is these unique frequencies that enable the use ofquadrupole ion traps as mass spectrometers. Selectedions can be ejected from the trap by applying an ad-ditional resonance frequency that matches their oscil-lating frequency, in a vertical direction. In a tandemMS (MS/MS) experiment, a selected analyte ion massis isolated by ejecting all other ions from the trap, fol-lowed by fragmentation of the selected ions. Severalsteps of ion isolation and fragmentation (MSn) can beperformed successively in the ion trap. MSn experi-ments are often employed for detailed investigationsof the structure of specific analyte ions[155,156]. Thepossibility of performing MSn analysis, in additionto the high sensitivity and relatively low price of thequadrupole ion trap, makes it an attractive instrumentfor carbohydrate analysis.

The application of ESI-quadrupole ion trap-MS inthe structural analysis of carbohydrates can yield, forexample, sequence and branching information, link-age type and monosaccharide substitution patterns[157–160]. In the work described by Richardson et al.[81], ESI-quadrupole ion trap-MS was employedfor the study of enzymic hydrolysates of cationicpotato amylopectin starch. Unsubstituted as well assubstituted oligosaccharides released from enzymic

degradation could be identified, which made it possi-ble to draw conclusions about the enzyme action on,as well as the substituent distribution in, the cationicstarch samples. ESI–MS/MS was employed for fur-ther verification of the identity of each oligomericproduct. ESI-quadrupole ion trap-MS was found to bean efficient method for the identification of cationichydrolysis products, for which there are no standardsavailable. However, the absence of standards made itimpossible to obtain any quantitative data.

The on-line coupling of LC to MS appears to bea highly attractive method for carbohydrate analysis.Direct identification and structural characterisation ofmono- and oligosaccharides in solution has been per-formed without derivatisation and with considerablyreduced sample preparation[161–163]. Especiallythe on-line coupling of HPAEC to MS, introduced bySimpson et al.[164], has proved to be very usefulin several applications of oligosaccharide analysis[165–167]. The major problem encountered whencombining these two techniques is the mobile phaseconditions in HPAEC, which are not compatible withMS detection. This is solved by introducing a desalt-ing device prior to the MS interface, which consists ofan ion-exchange membrane suppressor, for exchangeof Na+ ions with H3O+ ions[162,164,167]. The suc-cessful use of an HPAEC–PAD/ESI-quadrupole iontrap-MS system with a cation-exchange membranefor desalting in the characterisation of mono- andoligosaccharides released from enzymic hydrolysisof starch and cellulose derivatives has been described[81]. With PAD detection alone, the substituted prod-ucts liberated could not be identified, as there are nocommercial standards for these substances. Further-more, their elution order is unpredictable. However,subsequent on-line MS detection revealed the iden-tity of both unsubstituted and substituted compounds.The presence of monosubstituted glucose could notbe seen in the HPAEC–PAD chromatogram, but couldonly be detected by ESI-quadrupole ion trap-MSafter isolation of them/z ratio of interest, which isillustrated inFig. 9.

4.5.2. MALDI–TOF–MSMALDI emerged in the late 1980s as a technique

for desorption/ionisation and subsequent analysis ofmolecules with high molecular weights[149], andwas first applied to carbohydrates in 1991 by Mock


Time/min20 40 60

Int.

4x104

2x104

0x104

O

OHOCH2CH(OH)CH3HO

HO

OH

Fig. 9. Mass chromatogram of 2-O-(2-hydroxypropyl)-glucose (m/z 261) from HPAEC–ESI–MS analysis of the�-amylase and amyloglu-cosidase hydrolysate of hydroxypropylated potato amylopectin starch (from[81] reproduced with permission from Elsevier).

et al. [168]. In order to obtain a signal, the analyte insolution is mixed with a large molar excess of a matrixcompound, usually a small organic compound withUV light-absorbing properties. The most popular ma-trix for carbohydrate analysis is 2,5-dihydroxybenzoicacid (2,5-DHB) [169,170], but other matrices, suchas 2-(p-hydroxyphenylazo)benzoic acid (HABA),�-cyano-4-hydroxycinnamic acid and 3-aminoquino-line, have also been employed with varying degreesof success[170,171]. The principal functions of thematrix are thought to be: (i) isolation of the analytemolecules from each other; (ii) absorption of the laserenergy and mediation of energy to the analyte dur-ing the desorption/ionisation process; and (iii) chargetransfer, thus allowing a “soft” transfer into gas-phaseions [172]. The analyte-matrix mixture is allowed tocrystallise on a target plate by evaporation of the sol-vent, followed by irradiation of the crystals with shortlaser pulses, usually a UV nitrogen laser (337 nm).Each pulse causes desorption of both matrix and an-alyte species from the surface into the gas phase. Inthis process, the laser energy is absorbed by the ma-trix molecules and transferred to translational energy.The result is ablation of matrix and embedded analytespecies from the surface. Furthermore, a portion ofthe ejected matrix and analyte species is ionised. Theanalyte ions are either preformed or created by chargetransfer between the matrix and the analyte in the ex-panding plume following the impact of the laser pulse.Through the ionisation process, which is complexand not yet fully understood[173,174], positively and

negatively charged analyte ions are created. In thepositive ion mode, ions are generally formed throughhydrogen, alkali metal or ammonium attachments.For neutral sugars, [M + Na]+ ions predominate inthe positive ion mode with no significant formationof protonated ions, whereas in the negative ion modeproton abstraction is the dominant process[170].

The pulsed nature of the laser ion source in MALDIand its ability to ionise very large molecules are idealfor combination with TOF mass analysers. The mainadvantages of TOF analysers are the theoretically un-limited m/z range and simultaneous detection over thewhole m/z range without scanning, resulting in highion transmission. Furthermore, MALDI–TOF–MS isvery sensitive as the detector can record almost allions generated by the ion source[175].

MALDI–TOF–MS has been employed in a vari-ety of applications on carbohydrate analysis[171].Wang et al.[176] and Broberg et al.[177] studied theCL profile of debranched amylopectin by means ofMALDI–TOF–MS. Off-line coupling of chromato-graphic systems with MALDI–TOF–MS for molarmass determinations of saccharides has also beendemonstrated[178–180]. Further, MALDI–TOF–MShas been employed in investigations of the substituentdistribution in starch and cellulose derivatives. Afterpartial acid hydrolysis of the polymer, the oligomersproduced were qualitatively and quantitatively anal-ysed by MALDI–TOF–MS to give results that werethen evaluated statistically[147,181,182]. Karls-son et al. characterised CMC and the hydrolytic


activity of various EGs on CMC by enzymic degra-dation and subsequent analysis of the hydrolysate byMALDI–TOF–MS [125].

The same approach was used in another inves-tigation [59], where the substituent distribution incationic potato amylopectin starch was studied. Dif-ferent enzymes successively degraded the starchsamples and the hydrolysis products, both unmodi-fied and cationically modified, were analysed usingMALDI–TOF–MS. The cationic products releasedfrom the enzymic hydrolysis gave rise to considerablystronger signals than did the unmodified ones, dueto the permanently bound positively charged groups.However, as there is a lack of reference compoundsfor these cationic oligomers and polymers, no reliablequantitative data could be obtained, which is an ob-vious drawback of the technique in this application.Fig. 10 presents the MALDI mass spectrum fromthe characterisation of the�-amylase and amyloglu-cosidase hydrolysate of cationic potato amylopectinstarch.

0

10000

20000

30000

40000

50000

400 600 800 1000 1200 1400 1600 1800 2000

m/z

Co

un

ts

n5, r

2

n3, r

1

n7, r

1

n6, r

1

n7, r

2

n6, r

2

n5, r

1

n4, r

1

n8, r

1

n7, r

3

n8, r

2

Fig. 10. MALDI mass spectrum of the products obtained from�-amylase and amyloglucosidase hydrolysis of cationic potato amylopectinstarch (n, number of glucose units;r, number of substituents).

4.6. Nuclear magnetic resonance spectroscopy

NMR is a very powerful and valuable techniquefor structural determination of carbohydrates as theyare rich in1H and 13C atoms, both with nuclei thathave spins.1H NMR spectroscopy is the more sen-sitive technique due to the high natural abundanceof protons in all organic matter, but does not pro-vide as much structural information as does13CNMR because of incomplete resolution of the pro-ton resonance signals. Although1H NMR and 13CNMR spectroscopy are closely related techniques,they give sufficiently different data to complementeach other in both quantitative and qualitative anal-ysis of mono-, oligo- and polysaccharides. Valuablestructural information, such as anomeric configura-tion of glucosidic linkages, monomer compositionand sequence of polysaccharides, solution conforma-tions and three-dimensional structures of polysaccha-rides, can be provided by NMR spectroscopy[183–186].


4.6.1. Native starchIn the structural analysis of native starch,1H and

13C NMR spectroscopy can be used to determine dif-ferent molecular characteristics, such as the degree ofbranching, average CL and molecular weights of de-graded starch[12,55,185,187]. The different protonsin the AGUs lead to resonance at typical frequencies,and high resolution1H NMR allows separation of theresonance signals derived from the anomeric protons(H-1) involved in (1 → 4)-�-linkages ([H-1(1 →4)]), branching points ([H-1(1→ 6)]) and terminalnon-reducing ends ([H-1(t)])[12,55,185]. As the areaof the proton signals is proportional to the numberof protons, quantitative as well as qualitative data areobtained. The average CL in amylopectin and�-limitdextrins have been calculated according toEq. (4)[12,55]:

average CL

= integral[H-1(1 → 4) + H-1(t) + H-1(1 → 6)]

[H-1(1 → 6)](4)

4.6.2. Chemically modified starch and celluloseNMR spectroscopic techniques have been widely

applied since the late 1960s as an analytical toolfor structural elucidation of chemically modifiedstarch and cellulose, although starch derivativeshave been less the object of NMR analysis due totheir amylose/amylopectin heterogeneity and usu-ally very low DS [78]. Polysaccharide derivativeshave been studied both in their intact polymericform and after acid or enzymic hydrolysis. As hy-drolysates of polymers have lower viscosity and thushigher mobility, solutions of degraded derivativesgive higher resolution than do polymeric solutions[78]. In addition, the practical use of polymericsolutions of starch and cellulose derivatives is re-stricted due to poor solubility in common NMRsolvents[188]. 1H and 13C NMR spectroscopy canprovide structural information on the MS and DS[55,59,188–190], as well as on the molar distribu-tion of substituents at the C-2, C-3 or C-6 positionsin the AGU [78,191,192] (for further details, seeSection 5.1).

In an investigation by Richardson et al.[59] the MSin two hydroxypropylated potato amylopectin starchsamples was calculated from1H NMR measurements,

using the intensities of the signals from the methylprotons in the hydroxypropyl groups [H(CH3)] and thesum of the intensities of the signals from the anomericprotons (H-1) according to:

MS = integral[H(CH3)] /3

[H-1(HP) + H-1(1 → 4)

+H-1(t) + H-1(1 → 6)]

(5)

where [1H-1(HP)] denotes the anomeric proton ina glucose residue substituted with a hydroxypropylgroup. The spectrum obtained from1H NMR analy-sis of hydroxypropylated potato amylopectin starchis shown inFig. 11. The MS values obtained from1H NMR analysis were higher than those determinedwith colorimetric method or GC analysis.

1H NMR measurements of the average CL in�-limit dextrins of hydroxypropylated starch enabledevaluation of the average exterior and interior chainlength (ECL and ICL, respectively)[59]. Determi-nation of the ECL before and after�-amylase hy-drolysis revealed information on the localisation ofhydroxypropyl groups in the exterior chains (see alsoSection 5.2).

4.7. Classical wet-chemistry methods

There are numerous methods based on wet-chemi-stry for the analysis and detection of oligo- andpolysaccharides. In this section, only methods forthe determination of reducing ends and degrees ofsubstitution in starch and cellulose derivatives will bedescribed.

4.7.1. Reducing powerQuantitative determination of the reducing power

can be performed by the copper sulphate method,which is based on the reduction of Cu(II) to Cu(I) bythe reducing aldehyde group in the sample molecule.Subsequently, Cu(I) partially reduces Mo(IV) that,in acidic solution, produces intensely blue colloidalproducts. The intensity of the colour, measuredspectrophotometrically, is proportional to the con-centration of reducing ends[193,194]. Similarly,reducing sugars can be determined in an assay con-taining 2,2′-bicinchoninic acid (BCA). Cu(I), pro-duced from the reduction of Cu(II) by the sugar,forms a deep-blue complex with BCA in alka-


Fig. 11. 1H NMR spectrum of hydroxypropylated potato amylopectin starch (from[52] reproduced with permission from Elsevier).

line solution. The concentration of the reducingsugar is proportional to the colour intensity[195].

The concentration of reducing ends can also be de-termined by the ferricyanide method, where the re-ducing sugar reduces Fe(III)CN to Fe(II)CN. Whenadding KI to the solution, free iodide is produced inan amount proportional to the reducing sugars present.The amount of iodide is determined spectrophotomet-rically or by titration with sodium thiosulphate[196].

4.7.2. Molar substitutionThe MS of hydroxypropylated starch is commonly

determined by the Zeisel method or modificationsthereof [197,198], in which hydroiodic acid hydrol-yses the ether bonds to produce propyl iodide andpropylene in stoichiometric quantities. The MS of thehydroxypropyl moiety can then be determined fromthe amount of propyl iodide and propylene produced,which is determined using a specially designed distil-lation apparatus.

Another method used for the determination of theMS in hydroxypropylated starch is a colorimetricmethod proposed by Johnson[199]. The ether link-ages are hydrolysed in concentrated sulphuric acid at100◦C, followed by reaction of the hydrolysis prod-

ucts with ninhydrin to produce a purple complex thatis measured spectrophotometrically.

The MS of cationic starch is usually analysed bydetermining the nitrogen content using the Kjeldahlmethod[200]. Commercial, automated equipment hasmade nitrogen analysis a very convenient and accuratetechnique.

5. Analysis of the substituent distribution

The main goal of the chemical modification ofstarch and cellulose is to alter the properties of thenative polysaccharide to produce new materials withdesired properties. Consequently, it is important toelucidate the factors that determine which propertiescan be achieved by chemical modification. It is knownthat the functional properties of starch and cellulosederivatives depend on the type and number of sub-stituents introduced[1,2,32,78]. However, we stillknow relatively little about the relationships, if any,between the modification reaction, substituent distri-bution and functional properties. The most difficultand challenging task has proven to be the determina-tion of the substituent distribution in the derivatives,


Fig. 12. Substituent distribution in starch and cellulose derivatives on different structural levels.

due to a lack of suitable methods. Therefore, the de-velopment of such methods is of great interest andis the motivation behind the research project sum-marised in this thesis.

The distribution of substituents in modified polysac-charides can be investigated on different structurallevels; in the monomer, along the polymer chain, inthe crystalline/amorphous regions and on the sur-face/interior of the granule (seeFig. 12).

The substitution pattern on the monomer levelis known to affect properties involved in molecularrecognition, for example, the binding of the activesite of an enzyme to the substrate[72,82,83]. Thesubstituent distribution along the polymer chain andin the whole granule has been reported to exert aninfluence on functional properties such as solubil-ity, flocculation, gel formation and biodegradability[109,201,202].

5.1. Distribution on a monomer level

For (1→ 4)-linked glucans, such as starch and cel-lulose, three hydroxyl groups are available for sub-stitution in the monomeric glucose unit, namely thehydroxyl group on C-2, C-3 and C-6 (seeFig. 2).If one type of substituent is introduced, substitutioncan result in up to 23 = 8 different monomer units,whereas in mixed derivatives with two different typesof substituents that have free hydroxyl groups, 33 =27 different monomer units are possible. The analyti-cal methods commonly used in the investigation of thesubstituent distribution of the hydroxyl groups on C-2,C-3 and C-6 in the AGU are NMR spectroscopy andGC/MS following complete degradation of the poly-mer to monomers.

5.1.1. NMR spectroscopyNMR spectroscopy, mainly13C NMR, has been

used to reveal the partial DS at the C-2, C-3 andC-6 positions. The13C NMR signals for the glucoseC atoms are shifted downfield if substituted[192],thus the partial DS can be calculated from the rel-ative intensities of the unsubstituted (original) andsubstituted (shifted) signals of the C-2, C-3 and C-6atoms. The substituent distribution in the AGU hasbeen successfully determined by NMR studies of nu-merous cellulose derivatives, e.g. CMC[75,191,203],MC [204,205], HEC [206,207], HPC [188,208] andcellulose acetate[209]. In most cases, the derivativeswere analysed following acid or enzymic hydrolysisin order to achieve low-viscous solutions that generatespectra with high resolution[75,204,206]. However,NMR analysis of cellulose derivatives in their intact,polymeric form has also been performed. Tezuka et al.have proposed an alternative method in which cellu-lose ethers retain their polymeric form by acetylationof the hydroxyl groups in the polymer before NMRanalysis. Thus the problem of poor solubility in theNMR solvent is avoided[188,207,210].

The substituent distribution in starch derivatives hasalso been determined by NMR spectroscopy. The par-tial DS at the C-2, C-3 and C-6 positions has beendetermined by1H and/or13C NMR in hydroxyethylstarch[211], hydroxypropyl starch[190] and acety-lated starch[212].

5.1.2. Sample preparation prior to gaschromatography

Analysis of the substituent distribution on themonomer level by means of GC/MS must be pre-ceded by suitable sample preparation, i.e. derivati-


sation and complete degradation of the modifiedpolymer to monomeric units. This sample preparationis, in general, achieved either by standard methyla-tion analysis or reductive cleavage, depending on thetype of derivative. Standard methylation analysis isa well-established method for structural studies ofpolysaccharides and their derivatives[107,110,181].It comprises four steps: permethylation, acid hy-drolysis, reduction and acetylation. The first step,permethylation, is performed to achieve etherificationof all free hydroxyl groups. One common method ofpermethylation of polysaccharides is that describedby Hakomori [213], where the base sodium dimsylin DMSO is used to ionise the hydroxyl function,followed by addition of methyl iodide to accom-plish methylation. An alternative method developedby Ciucanu and Kerek[214], employs solid NaOHdissolved in DMSO as base before methylation bymethyl iodide. Next, the glucosidic linkages of thefully methylated polymer are hydrolysed at high tem-perature by a strong acid, such as trifluoroacetic acid[110,215]or sulphuric acid[107]. The monomers inthe resulting hydrolysate are then reduced by NaBD4in order to achieve ring opening of the monomers.Finally, the monomeric units are acetylated by reac-tion with acetic anhydride and pyridine[107,215].The resulting partially methylated alditol acetates areseparated using GC and identified with MS, and fromthe results obtained the molar distribution of sub-stituents in the AGU can be calculated. The drawbackof methylation analysis is that complete permethyla-tion of the polymer can be difficult to achieve, andunder-methylation introduces small errors into thesubsequent determination[216].

An alternative type of methylation analysis is thereductive cleavage method[217], which has been suc-cessfully applied to the structural analysis of modifiedpolysaccharides, especially samples containing com-ponents sensitive to acidic conditions[181,218,219].This method is based on the methylation analysis de-scribed above, but departs from it significantly withregard to the cleavage reaction and the types of frag-ments obtained after cleavage. Here, the glucosidiclinkages of the fully permethylated sample are cleavedby ionic hydrogenation of the carbon–oxygen bonds,promoted by a Lewis acid, with triethylsilane as re-ducing agent[181,217,220]. The resulting partially an-hydroalditols are acetylated and finally analysed by

GC and GC–MS. The advantage over the methylationanalysis is that the reductive cleavage method enablesbond cleavage, reduction and acetylation to be per-formed in a “single-pot” reaction at room temperature.

5.1.3. Gas chromatography/mass spectrometryGC in combination with FID and MS detection has

been widely applied to the determination of the molardistribution of substituents on the monomer level innumerous starch and cellulose derivatives, e.g. HEC[108,109], EHEC [221], CMC [222], methyl starch[56,147], hydroxyethyl starch[223], hydroxypropylstarch[55] and cationic starch[58,224]. In order toachieve volatile analytes that are amenable to GCanalysis, neutral ether derivatives are pre-treated bysample preparation methods such as standard methy-lation or reductive cleavage (see above), whereasionic derivatives have to be transformed into neutralspecies[58]. As GC columns have the ability to sep-arate both mono-, di- and trisubstituted monomers, aswell as monomers substituted at C-2, C-3 or C-6, theeight different monomers that are possible to obtain(provided the derivative is substituted by one type offunctional group) can be individually analysed. Iden-tification of the monomeric analytes is accomplishedthrough MS with both chemical ionisation (CI) andelectron impact ionisation (EI). From the CI spec-trum, the molecular mass, and thus the number ofsubstituents of a glucose monomer, can be obtained.Identification of the position of a substituent in themonomer requires intense fragment ions, which canbe provided by EI[78,225]. The fragmentation pat-terns in the EI mass spectra of permethylated groundstructures have been thoroughly investigated[225].Based on the characteristic fragmentation behaviourit is possible to deduce the location of the substituentsbetween the C-2, C-3 and C-6 hydroxyl groups. Afteridentification, the relative molar distribution of sub-stituents in the glucose monomer can be calculatedfrom the FID response by correction of the peak areasusing the ECR concept (seeSection 4.2).

5.1.4. Liquid chromatographic methodsIn contrast to GC, samples for HPLC analysis

do not have to be volatile but just soluble in theeluent. Therefore, many polysaccharide derivativescan be analysed directly after complete hydrolysisto monomer units without any further derivatisation


step. A few starch and cellulose derivatives (MC[226]and hydroxypropyl starch[227]) have, after com-plete hydrolysis, been separated on reversed-phase oramine-modified silica columns, followed by RI or po-larimetric detection. Separation of un-, mono-, di- andtrisubstituted glucose monomers is achieved throughdifferences in mass and polarity. The monomers inacid hydrolysates of CMC have been separated bycation-exchange chromatography[228].

HPAEC–PAD is a well established and very ef-ficient technique in carbohydrate analysis[114,115](Section 4.3), and has proven to be useful alsofor analysis of the substituent distribution on themonomer level in certain polysaccharide derivatives.In contrast to the HPLC methods mentioned above, noproblem arises from�- and �-anomers, as anomeri-sation is very fast in the high-pH mobile phase usedin HPAEC–PAD. Kragten et al. have studied the sub-stituent distribution in CMC and sulphoethyl celluloseby separation of the monomeric components in theiracid hydrolysates by HPAEC–PAD[127,128]. Theseanionic derivatives are very convenient analytes forHPAEC separation as every CM and sulphoethyl groupenhance the retention markedly. All eight monomericcomponents in the CMC hydrolysate could be com-pletely separated, thus not only resolution of the un-,mono-, di- and trisubstituted monomers was obtained,but also of the positional isomers within each group[74,127]. Neutral monomers can also be separatedusing HPAEC due to the high acidity of the free hy-droxyl groups. Heinrich and Mischnick have separatedO-methyl glucose monomers in acid hydrolysates ofmethylated starch and cellulose by means of HPAEC[129]. In contrast to anionic derivatives, trisubstitutedglucose units are eluted first in methylated samples,as fewer deprotonated hydroxyl groups are availablewith increasing number of methyl groups. Hencethe retention decreases. The major drawback ofHPAEC–PAD analysis is the significant difference inelectrochemical response for mono- and disubstitutedregioisomers and for monomers with different DSvalues[57,78]. Thus, for quantitative evaluation, therelative response factor of the amperometric detectorhas to be determined for each monomeric analyte,which requires access to standard compounds. Stan-dard compounds or reference methods are also neededfor the correct assignment of the chromatographicpeaks.

5.1.5. Statistical modelsThe distribution of substituents in the monomer unit

of starch and cellulose ethers can be described by a sta-tistical, kinetic model originally proposed by Spurlin[229]. This model assumes that the substituent dis-tribution and monomer composition are governed bythe relative rate constants for reaction of the threehydroxyl groups,k2, k3 and k6 (these rate constantscan be obtained from the partial molar distribution ofthe C-2, C-3 and C-6 positions). Furthermore, to en-sure statistical random distribution in the monomer, itis assumed that the ratio of the reactivity constants,k2:k3:k6, remains constant during the whole modifica-tion reaction.

If the molar distributions obtained experimentallydeviate from the calculated, statistical ones, intra-monomeric effects during the course of reaction maybe suspected[57]. For instance, Reuben found that thereactivity of the hydroxyl group on C-3 was enhancedby methylation of the vicinal hydroxyl group on C-2,and the model by Spurlin was therefore modified toinclude this effect[204].

Comparison of the statistical substituent distributionwith distributions obtained from experimental data cangive the initial indication of whether the substituentdistribution along the polymer chain is homogeneousor heterogeneous, as an observed intramonomeric ef-fect could propagate in the neighbouring areas dueto, for example, enhanced local solubility and thushigher accessibility for the modification reagents[57].

5.1.6. Substituent distribution in starch andcellulose derivatives

The DS of the individual hydroxyl groups in theglucose unit has been determined for numerous starchand cellulose derivatives. The substituent distributiondepends primarily on the conditions prevailing duringthe modification reaction, but also to some extent onthe type of substituent. In general, low alkali con-centration during the modification reaction favours2-O-substitution above 3-O- and 6-O-substitution,as the hydroxyl group on C-2 exhibits the highestacidity and is therefore the most reactive. This highrelative acidity is due to the proximity to the electronwithdrawing anomeric centre. At higher alkali con-centrations, often used for cellulose activation, thesterically less hindered hydroxyl group on C-6 is the


most reactive[230,231]. Starch ethers seem to have ahigher preference for 2-O-substitution than celluloseethers modified under similar alkali conditions. It hasbeen suggested that this depends on the difference inacidity of the hydroxyl group on C-2 in starch andin cellulose, due to different configurations at theanomeric centre (� and�, respectively).

The distribution of substituent groups in numerouscellulose derivatives, e.g. MC[204,205], EC [232],HEC [108,109,233], HPC [188,208], EHEC [221],HPMC[234] and CMC[127,191,203], has been deter-mined. In general, OH-3 shows a significantly lowerreactivity than OH-2 and OH-6, which is explained bythe engagement of OH-3 in an intramolecular hydro-gen bond in the crystalline cellulose. Thus, substitu-tion at this position is greatly hindered relative to thehydroxyl groups on C-2 and C-6. For some cellulosederivatives (e.g. MC, EC and HEC), it has been foundthat the reactivity of OH-3 is enhanced by substitu-tion of OH-2. This is thought to originate from inter-ruption of the intramolecular hydrogen bond that theOH-3 is involved in upon etherification of the OH-2in the same AGU[204,232,233].

Starch ethers that have been studied regarding thesubstituent distribution in the AGUs are methyl starch[56,62,147], hydroxyethyl starch[211,223], hydrox-ypropyl starch[55,190] and cationic starch[58,224].A common feature of the results obtained in these in-vestigations is the relatively high reactivity of the hy-droxyl group on C-2 compared with those on C-3 andC-6. Starch derivatives are usually modified at low al-kali concentrations, thus the relatively high substitu-tion of OH-2 observed is expected. For example, themolar distribution on the monomer level in cationicstarch, investigated by Wilke and Mischnick, showeda strong preference for 2-O-substitution, followed byalmost equal amounts of 3-O- and 6-O-substitution[58,224]. Furthermore, the results were compared withstatistical models[204,229], which indicated a de-crease in the reactivity of the hydroxyl group on C-3when C-2 was substituted. This intramonomeric ef-fect was explained by electrostatic repulsion of thecationic reagent and/or intramolecular masking of thedeprotonated hydroxyl group on C-3[58]. In a studyon methyl amylose[147] it was observed that the or-der of reactivity in samples methylated in homoge-neous solution was OH-2 OH-6 ≈ OH-3, whereasthe sample modified in a concentrated slurry showed

the following order of reactivity: OH-6> OH-2 OH-3. No explanation of this result was given.

The substituent distribution of hydroxypropylgroups in the AGU of two different hydroxypropy-lated amylopectin starches, modified in granularslurry or in solution, were investigated using stan-dard methylation analysis followed by GC–FID andGC–MS analysis[55]. The peaks in the resultingGC–FID chromatogram were assigned by MS withCI and EI, and the molar ratio of the distribution ofhydroxypropyl groups was calculated from the peakareas, which were corrected according to the ECRconcept. As expected for reactions with low amountsof base, the hydroxyl group on C-2 showed the high-est affinity for the substituents, while the hydroxylgroups on C-3 and C-6 were substituted to a muchlower extent. These results are in agreement with anearlier study of hydroxypropylated starch[190]. Nosignificant difference in molar distribution betweenthe two different samples was observed.

5.2. Distribution along the polymer chain

5.2.1. General strategyThe substituent distribution along the polymer chain

in starch and cellulose derivatives has proven to bemore difficult to determine than the distribution inthe monomer unit, and relatively few investigations ofthe substitution pattern on a polymer level have hith-erto been published. The general strategy when study-ing the substituent distribution includes the followingsteps:

1. partial hydrolysis of the polymer;2. characterisation of the hydrolysis products;3. interpretation of experimental data.

First, well-defined partial hydrolysis of the polymermust be accomplished in order to determine how thedifferent monomer units are located in relation to eachother. In addition, smaller analytes are more amenableto detailed structural analysis than the intact poly-mer. Hydrolysis is carried out either randomly or se-lectively (enzymes). The subsequent characterisationof the hydrolysis products obtained requires appro-priate analytical methods and has so far been carriedout by techniques such as MS[59,62,81,146,147,182],HPAEC–PAD[76,81] or SEC[55,56,75,76]. Finally,the hydrolysis products can be compared with the


products obtained from the hydrolysis of the unmod-ified polymer to characterise the distribution of thesubstituents. Alternatively, the substituent distributionin the oligomeric products, which is assumed to reflectthe distribution in the entire polymer, can be comparedwith a calculated, statistical pattern that describes acompletely random distribution of substituents[78].

A random distribution along the polymer back-bone will be the outcome if all glucose monomersare accessible for substitution with the same proba-bility. In oligomeric sequences, this random patterncan be calculated by binominal distribution statistics[78,146,147]. Random distributions are expected forpolysaccharide derivatives modified in homogeneousreaction phases, where the polymer is completelydissolved in the reaction medium. It is provided thatthe relative reactivities of all the glucose monomersremain the same during the whole reaction, in anal-ogy to the Spurlin model for a random distribution inthe AGU [229]. However, if all the glucose units inthe chain are not equally accessible for substitutionat all times, e.g. in a polymer with crystalline regionsremaining that have not been activated, then a moreheterogeneous pattern is expected than the calculatedrandom distribution, which is always considered tobe the reference model. Furthermore, deviation fromthe random distribution will occur if the reactivityof the glucose units changes as the modification re-action proceeds. For example, the local reactivity ofmonomers in the close vicinity of modified regionscould be enhanced due to changes in polarity or im-proved solubility of the modification reagent, whichincrease the local accessibility of substituents to themonomers. If, on the other hand, the substituentsconstitute a steric hindrance or cause electrostaticrepulsion, the reactivity of glucose monomers closeto substituted regions may be decreased. In this case,

Fig. 13. Schematic illustration of different substituent distributions in starch and cellulose derivatives.

a narrow or more regular distribution of substituentsis expected[78,147,234]. Overlapping of differentdistributions can be the outcome of competing re-actions in the modification process[147]. Differentsubstitution pattern are depicted inFig. 13.

5.2.2. Random/chemical depolymerisationAs mentioned earlier, the modified polymer must

be partially hydrolysed, either randomly or selec-tively, in order to obtain experimental data that can becompared with statistical models or data from hydrol-ysis of the unmodified polymer. Random hydrolysisof a modified polymer implies that the substituent dis-tribution in the oligomeric fractions obtained reflectsthe distribution along the entire polymer chain. Inorder to achieve completely random cleavage of theglucosidic linkages, the free hydroxyl groups in thepolymer should preferably be derivatised to obtain achemically uniform product. The subsequent partialhydrolysis can be performed by methanolysis, acidhydrolysis or mild reductive cleavage[72,146,147].

Arisz at al.[146] investigated the substituent distri-bution in MC modified under different heterogeneousconditions. The modified cellulose was partiallyhydrolysed by acid and the resulting oligomerswere analysed (quantitatively and qualitatively) byFAB–MS. The difference in detector response betweenmethylated and unmodified cello-oligomers can bevery large, thus, to obtain as similar response factorsof the different products as possible, the free hydroxylgroups in the oligomers were perdeuteromethylatedbefore analysis. The FAB–MS analysis revealed themolar distribution of methyl groups in the cellotrioses,i.e. the number of methyl groups in the trimers. Theexperimentally derived substituent distribution of thetrimers was then compared with a statistical, randomdistribution calculated with binominal distribution


statistics. According to the results obtained, a slightdeviation from the random pattern was observed, in-dicating heterogeneity in the substituent distributionalong the cellulose backbone. A similar approach wasapplied in a study on methyl amyloses modified inhomogeneous and heterogeneous reactions[147]. Inthat work, the methylated samples were permethy-lated prior to partial degradation by mild methanolysisor reductive cleavage, in order to obtain a chemicallyuniform product for random hydrolysis. Subsequently,the oligomeric fractions obtained were analysed byFAB–MS and MALDI–TOF–MS. The molar distri-butions of methyl groups in the dimers, trimers andtetramers were compared with the statistical distribu-tion in each of these oligomers. The homogeneouslymodified samples showed a distribution pattern verysimilar to the pattern calculated for a random distribu-tion of substituents. However, in the sample modifiedunder heterogeneous conditions, significant deviationfrom a random pattern was observed as the numberof oligomers with low and high DS was enhancedand the number of oligomers with an average DS wasdecreased. In addition, the distribution curve showedslight distortion, indicating a substituent gradient inthe material due to slower diffusion of the reagentsinto the starch granule than the reaction rate.

Cellulose sulphates have also been investigatedwith respect to the substitution pattern along thepolymer chain [72]. The cellulose sulphates wereconverted to methyl ethers and then randomly cleavedby methanolysis. The distribution patterns in the di-,tri- and tetramer fractions were derived from quan-titative FAB–MS analysis. Subsequent comparisonwith the calculated homogeneous distribution showedthat all samples, independent of reaction conditions(homogeneous or heterogeneous), had a random dis-tribution of sulphate groups. This can be explained bythermodynamic control of the modification reaction.In contrast to the kinetically controlled etherification,where no “correction” of the heterogeneity is possibleduring the course of reaction, the reversibility of thesulphation reaction allows an equilibration of the sub-stituent distribution, thus resulting in a homogeneoussubstitution pattern.

5.2.3. Selective/enzymic depolymerisationIn contrast to the random approach, partial hydroly-

sis of modified polysaccharides by enzymes provides

selective cleavage of the glucosidic linkages. A funda-mental characteristic of starch and cellulose-degradingenzymes is that they only hydrolyse certain gluco-sidic linkages in the polysaccharide. In addition, ifthe polysaccharide is substituted by chemical groups,these substituents constitute a hindrance to enzymichydrolysis in such a way that glucosidic linkages nextto substituted glucose units are resistant or less ac-cessible to enzymic attack (see Section 3.5). Conse-quently, the product spectrum obtained from enzymichydrolysis of native starch and cellulose differs fromthat obtained from starch and cellulose derivatives.These characteristics make the enzymes highly selec-tive tools for investigation of the detailed structure ofstarch and cellulose derivatives[25,56,61–64,74].

The rate of enzymic hydrolysis for various gluco-sidic linkages may vary depending on the substituentdistribution of adjacent glucose monomers, as the ac-tive substrate–enzyme complex usually involves anoligomeric sequence. Consequently, it cannot be as-sumed that all theoretically expected cleavages run tocompletion, thus the interpretation of the experimen-tal data becomes complicated. In contrast to enzymichydrolysis, a chemical hydrolysis process results ina statistically well-defined oligomeric mixture that isrepresentative for the intact polymer[57,78].

The use of various enzymes with different selectivi-ties provides different types of information on the sub-stituent location.�-Amylase followed by amyloglu-cosidase hydrolysis with subsequent determination ofthe amount of unsubstituted glucose liberated has beenapplied to study the heterogeneity of the substitutionpattern[55]. In a polymer with a homogeneous distri-bution of substituents there are less glucosidic bondsbetween two unmodified glucose units than in a poly-mer with a more heterogeneous distribution. Conse-quently, there are less glucosidic linkages available forenzymic hydrolysis and a lower amount of glucoseis liberated upon enzymic hydrolysis of the homoge-neously substituted polymer.

In a study of methylated starch, modified underheterogeneous and homogeneous conditions, the mod-ified samples were subjected to�-amylase followedby amyloglucosidase hydrolysis[56]. Subsequentanalysis of the hydrolysis products showed that moreglucose and larger fragments with enhanced DS wereobtained from the heterogeneously modified starch,indicating a more heterogeneous pattern than in the


sample modified in a homogeneous reaction. Similarobservations were made by Mischnick on methyl amy-loses modified under different conditions; the amountof glucose liberated from exhaustive�-amylase andamyloglucosidase hydrolysis was lower for methylamylose prepared under homogeneous conditionsthan for heterogeneously prepared amylose[235].This behaviour has also been observed for cationicstarch[58]. In the present work, hydroxypropyl andcationic potato amylopectin starch samples, modifiedunder different conditions, were hydrolysed by thesuccessive action of�-amylase and amyloglucosi-dase[55,59]. It was demonstrated that hydrolysis ofstarch modified in solution (homogeneous conditions)liberated less glucose than starch modified in a drystate and/or granular slurry (more heterogeneous con-ditions). It was concluded that the homogeneouslyprepared starch derivatives have a more random dis-tribution of substituents than the heterogeneouslyprepared ones, which is in agreement with resultsobtained elsewhere (see above).

The sole action of�-amylase has been employed ina study of the substituent distribution over branchedand linear regions in normal methyl starch andhighly branched amylopectin methyl starch[62]. Af-ter �-amylase hydrolysis of the methylated samples,branched and linear fragments were separated andthe distribution of methyl groups in the separatedoligomeric fractions was determined by FAB–MS.The experimental data were compared with a sta-tistically random distribution. The results providedevidence of a heterogeneous substitution pattern. Fur-thermore, it was found that substitution takes place

Fig. 14. Schematic illustration of the crystalline and amorphous regions of an amylopectin molecule.

preferentially in the branched regions in amylopectin,and that amylose had a higher DS than had the linearregions in amylopectin.

In order to investigate the substitution of the exteriorchains in starch derivatives,�-amylase hydrolysis hasbeen employed[55,59]. In the study of cationic potatoamylopectin starch, it was shown that starch sam-ples cationised under heterogeneous conditions hadslightly lower �-limit values than starch modified insolution, which could indicate that the former sam-ples were substituted slightly closer to the branchingpoints [59]. �-Amylase hydrolysis of hydroxypropy-lated potato amylopectin starch, modified in granularslurry and in solution, did not reveal any significantdifferences in the substitution of the exterior chains[55].

In the study presented by Richardson et al.[55],pullulanase hydrolysis of hydroxypropylated potatoamylopectin starch followed by SEC–RI analysis ofthe debranching products gave information on thesubstitution around the branching points. The averagemolar masses of the enzymic hydrolysates followedthe order: hydroxypropyl starch modified in granularslurry > hydroxypropyl starch modified in solution>native starch. Thus, native starch was completely de-branched, while starch modified in granular slurry wasthe least debranched sample. This was explained by ahigher degree of substitution in close proximity to thebranching points in the sample modified in granularslurry, due to a higher reactivity of the amorphousthan the crystalline regions (seeFig. 14). In the samplemodified in solution the crystallinity was destroyedduring the modification reaction, resulting in a more


uniform distribution of the hydroxypropyl groups overthe starch molecule.

Kavitha and BeMiller determined the location ofsubstituents in hydroxypropylated starch by enzymichydrolysis of the polymer with various combina-tions of isoamylase,�-amylase,�-amylase and amy-loglucosidase[64]. The different hydrolysates werefractionated by SEC and the elution profiles werecompared with those from enzymic hydrolysis of theunmodified starch. It was concluded that amylosewas substituted to a greater extent than amylopectin,and also that the substitution of both the amyloseand amylopectin fractions deviated from a uniformpattern. In amylopectin, the modification occurredpreferentially close to the branching points. A similarapproach has previously been reported in an inves-tigation of the substitution pattern in hydroxypropyldistarch phosphate[63]. The results obtained in thiswork indicated a higher reactivity and thus a higherDS in the amorphous regions of the starch moleculethan in the crystalline regions.

Manelius et al. investigated the substituent distribu-tion in the granules of cationised and oxidised starchby �-amylase,�-amylase and isoamylase hydrolysis.Dry-cationisation of starch resulted in local derivati-sation at the granular surface, while starch cationisedin solution had a more uniform distribution through-out the entire granule[61,236]. Oxidation of starchresulted in homogeneous modification of all starchcomponents[236].

Cellulose derivatives have also been investigatedwith respect to the substitution pattern on the polymerlevel by enzyme-catalysed degradation. Wirick[69,70]and Gelman[71] used a mixture of EGs and CBHs tohydrolyse water-soluble HEC and CMC. The amountof glucose released from enzymic digestion was mea-sured and related to the average sequence length ofunsubstituted glucose monomers.

Cellulose sulphates prepared under different homo-geneous and heterogeneous reaction conditions werehydrolysed by cellulase fromT. reeseiwith subsequentdetermination of the amount of unsubstituted glucosereleased. Samples that, according to results from anal-ysis using a statistical approach, showed significantheterogeneity in the substitution pattern released ahigher amount of glucose than did the samples with,according to the statistical analysis, a more uniformdistribution of substituents[72].

Recently, Horner et al. investigated the substitu-tion pattern in various CMC samples with differentDS values. The cellulose derivatives were hydrolysedby EG from H. insolens, followed by characterisa-tion of the hydrolysates by means of SEC–RI andHPAEC–PAD. It was found that all CMC samplescontained hydrolysis products of significantly higherDS than the average DS, but also high amounts ofproducts with low DS or no substitution at all. Thiswas interpreted as evidence of a structure with anon-uniform substitution pattern, where highly substi-tuted areas alternate with areas of low substitution[25,4,75].

The substituent distribution in EHEC has been stud-ied using an enzymic approach[76]. The EHEC washydrolysed by two different EGs (EG I or EG III) fromT. reeseiwith subsequent analysis of the hydrolysatesby means of SEC–MALS, HPAEC–PAD/MS and glu-cose determination. It was observed that the two EGshad different activities on the EHEC sample, althoughit was very low for both enzymes. However, it wasdifficult to draw any conclusions about the substitu-tion pattern along the cellulose chain, partly due tothe high DS and MS and partly due to the presence oftwo types of substituents.

6. Conclusions and future perspectives

The goal of this review was to present a summaryof the analytical methods and strategies that are usedfor structural analysis of starch and chemically mod-ified starch and cellulose, especially those for studiesof the substituent distribution in starch and cellulosederivatives.

The complexity of starch and cellulose derivativesrequires depolymerisation of the polymer into smallerfragments in order to study the structure. This hy-drolysis can be achieved either randomly (chemicalhydrolysis) or selectively (enzymic hydrolysis) andthe type of oligomeric products obtained and theirDS reflect the distribution of substituents in the in-tact polymer. In order to obtain information on thesubstitution pattern in the polymer, the hydrolysisproducts must be identified and quantified, which de-mands the use of various analytical techniques such aschromatography, NMR spectroscopy and mass spec-trometry. Well-defined depolymerisation, resulting


in a product spectrum that is known, can reveal thelocation of the substituent in the polymer. Further in-formation can be gained if the experimental data areevaluated using mathematical models.

It has been shown that selective depolymerisa-tion of the polymer by different enzymes has greatpotential to provide valuable information on the sub-stitution pattern along the polymer chain, althoughmore knowledge of the influence of various substitu-tion groups on the enzyme action is needed. Cloningand overexpression of pure enzymes to obtain newenzymes with known, and perhaps also desired, se-lectivities have been relatively unexplored but arevery interesting fields of bioengineering, which couldprovide extremely valuable tools in the research onmodified polysaccharides. Furthermore, ESI–MS andMALDI–TOF–MS were shown to be promising inthe analysis of both unsubstituted and substitutedhydrolysis products.

In the course of the work it became more and moreobvious that there is an urgent need for standard com-pounds with known structures, which are vital in orderto obtain quantitative results and, as a consequence, tobe able to interpret the results obtained from the vari-ous analytical methods. Development of mathematicalmodels and simulations that can assemble the charac-terised oligomeric fragments into the original, intactpolymer is also required. Initial work in this directionhas been carried out by Stokke et al. on enzymic hy-drolysates of chitosan[237,238].

Not covered here, but of great interest and thereforean important topic for future work, are methods forthe determination of the substituent gradient over theentire starch granule. It is possible that such a gradient,if present, could exert great influence on the propertiesof the final starch product.

Acknowledgements

The authors thank The Competence Centre forAmphiphilic Polymers (CAP) for financial support.

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characterisation of the substituent distribution in starch and cellulose derivatives

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