mapping the intrinsic viscosity of hyaluronic acid at high conce ...1114201/fulltext01.pdf ·...

17010

Examensarbete 15 hpMaj 2017

Mapping the intrinsic viscosity of hyaluronic acid at high concentrations of OH-

Mathias AxelssonAnton LindbladLovisa RingströmJohanna Munck af RosenschöldVictor Spelling

Teknisk- naturvetenskaplig fakultet UTH-enheten Besöksadress: Ångströmlaboratoriet Lägerhyddsvägen 1 Hus 4, Plan 0 Postadress: Box 536 751 21 Uppsala Telefon: 018 – 471 30 03 Telefax: 018 – 471 30 00 Hemsida: http://www.teknat.uu.se/student

Abstract

Mapping the intrinsic viscosity of hyaluronic acid athigh concentrations of OH-

Mathias Axelsson, Anton Lindblad, Lovisa Ringström, Johanna Munck afRosenschöld, Victor Spelling

Hyaluronic acid is commonly used in dermatological fillers in the form of gels. It is established how these gels' firmness is affected by the amount of cross linker and hyaluronic acid respectively. However, the effect of hydroxide ions in solution is rather unknown. This thesis examines how the alkalinity of the solvent affects the intrinsic viscosity of 3 MDa hyaluronic acid by using the method of Ubbelohde capillary viscometry. Sodium hydroxide solutions between 2 and 10 wt% were prepared to study the variation in intrinsic viscosity at concentrations relevant for cross linking (1<wt%). From these respective solutions, four solutions of different mass concentrations of hyaluronic acid were made. The flow time of respective samples were measured between two points in the capillary viscometer in a controlled temperature of 25 °C with an SI Viscoclock to ensure a high accuracy. From the resulting flow times, the intrinsic viscosity was calculated. The intrinsic viscosity varied between 0,55 and 0,70. The relation between intrinsic viscosity and hydroxide ion concentration had a correlation coefficient r < 0,001. No trend could be ensured as the confidence interval for the intrinsic viscosity at the different concentrations was too large.

Keywords: Alkaline solution, Degradation, Extrapolation, Hyaluronic acid, Intrinsic viscocity, Macromolecules, Polymer, Regression, Sodium hydroxide, Ubbelohde capillary viscometry.

ISSN: 1650-8297, TVE-K17 010Examinator: Enrico BaraldiÄmnesgranskare: Isak ÖhrlundHandledare: Åke Öhrlund

Acknowledgements

We would like to thank Uppsala University:Thank you, Jan Bohlin, for all the time, energy and commitment you haveinvested in us and this project. Thank you for making sure we had the bestequipment and information available. And thank you for always finding kindwords of encouragement and motivating us from start to finish.

Thank you, Isak Öhrlund, for giving us valuable second opinions, and forguiding us throughout the project.

Thank you, Marit Andersson, for taking the time to help us process our read-ings and making sure it was performed statistically correct.

Thank you, Christer Elvingsson, for taking the time to discuss the theory be-hind our readings with us and helping us strengthen our arguments.

Thank you, Tim Bowden, for also discussing our results with us and helping topoint us in the right direction.

Thank you, Jöns Hilborn, for taking the time to listen to our presentation anddiscussing our readings with Jan and us.

We would also like to thank Galderma: Thank you, Åke Öhrlund and Mor-gan Karlsson, for always being available to meet for a discussion, providingmaterial and for always showing interest in our work during the project.

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.1 Hyaluronic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.1.1 History of- and applications for Hyaluronic acid . . . . . . . . . . . . 51.1.2 Chemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51.1.3 Degradation of Hyaluronic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61.1.4 Hyaluronic acid in aqueous solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.2 Galderma AB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.3 Aim . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

2 Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.1 Viscosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.2 Viscosity in macromolecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102.3 Viscometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2.3.1 Rheometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.3.2 Capillary viscometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

2.4 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

3 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153.1 Choice of method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153.2 Procedure of method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

3.2.1 Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163.2.2 Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3.3 Method Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

4 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235.1 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235.2 HA in solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245.3 Degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255.4 Shear-thinning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255.5 Future research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Appendix 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

1. Introduction

1.1 Hyaluronic acid1.1.1 History of- and applications for Hyaluronic acidHyaluronic acid (HA) was discovered and named by Karl Meyer and JohnPalmer in 1934 when they extracted the molecule from bovine eyes. By thattime, they could not determine the exact structure of the molecule. The hy-pothesis of the structure was that it was a polymer contained an amino sugar,a uronic acid and a pentose. Therefore, they suggested to name the moleculehyaluronic acid (hyaloid = vitreous). Even though the structure was not deter-mined the newly found molecule was described as a "polysaccharide of highmolecular weight". Determining the structure took almost 20 years [1].

During the 1930’s and the 1940’s other extraction sources were found. Thesesources include everything from roosterâs comb, human tumour tissue, skinand umbilical cord, and even strains of bacteria. Today, HA is industriallyproduced from either gene modified bacteria or by extraction from rooster’scomb [1].

The many great properties of hyaluronic acid entail a lot of different areas ofapplication. The first one being eye surgery where it was used as a replacementfor vitreous loss in the eye. Other medical applications for HA is to preventadhesion during surgery and for healing wounds. Furthermore, the elastic andviscous properties of the hyaluronic acid makes it a great polymer for treatingarthritis and other joint disorders in humans and even horses [1,2].

Many of its properties makes it a great molecule for application in beauty andcosmetic market. As the polymer binds a lot of water it is very often usedas a hydrator in cosmetics and skin care products. The viscous and elasticproperties are instead utilized when making cosmetic fillers. By crosslinkingHA, gels with desired firmness can be created which can be used for fillingout wrinkles and lines. Fillers can also be used to enhance one’s appearanceby giving more volume to desired areas, such as lips or cheekbones. As thepolymer is a bio polymer, i.e. bio compatible, it is a great polymer to applicatein both medical areas and cosmetics [2].

1.1.2 Chemical propertiesHyaluronic acid (HA) is a bio polysaccharide found i.a. in vertebrate tissues inthe extracellular matrix. The name hyaluronic acid can be regarded as slightly

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misleading, since the hyaluronic acid rather is a glycosaminoglycan consist-ing two monosaccharide units; D-glucuronic acid and N-actyl-D-glucosamine,connected by b (1 ! 4) and b (1 ! 3) glucosidic bonds [3], see figure 1.1.

Figure 1.1. Hyaluronic acid.

At physiological conditions HA acts like a salt, i.e. sodium hyaluronan. Thestrongest acidic group, the carboxylic group, in HA has a pKa value of 2.87[4]. This means that the carboxylic group will be deprotonated under phys-iological conditions. The negative charge of the molecule will interact withpositively charged ions and molecules, principally Na+, within the body [5].Due to this polyanionic charge and because the HA molecule is not branched,the molecule has a rigid and extended conformation. However, in solutionsthe HA molecule tend to be in a random coil conformation and this is theconformation that has been found when extracting HA from tissues [1].

HA has an exceptional ability to bind water molecules, where the randomcoils contain almost 99% bound water. Even at highly diluted solutions theextended molecules get tangled together giving the solution great elasticityand viscosity [5]. There will naturally be a variation of elastic and viscousproperties of a HA solutions dependent on the average molecular weight, con-centration and the conformation of the polymer. The conformation can differat different conditions such as pH and temperature. This means that the prop-erties of the polymer are dependent on the conditions in solution [1].

1.1.3 Degradation of Hyaluronic acidThe hyaluronic acid degrades naturally in vivo and, in clinical usage as fillers,last for approximately six to twelve months depending on various circum-stances such as age, lifestyle, individual skin type etc. [6].

To be able to create products of cross linked hyaluronic acid for clinical usage,the knowledge of its physical properties is of interest. During the process ofcross linking hyaluronic acid elevated pH is a necessity, though, higher pHcause a degradation of the HA. This results in a decrease in molecular weightof the product in comparison to the raw starting material, which give rise todifferent physical properties. Prior studies of the degradation of hyaluronic

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acid present a linear behaviour of the degradation, depending on pH respec-tively of the temperature. In these prior studies the temperature dependentdegradation showed greater significance than that of high pH [7].

1.1.4 Hyaluronic acid in aqueous solutionsStudies done on the rheological behaviour of HA in aqueous solutions hasproven that there is a pH dependence of the viscosity. At very low and veryhigh pH values the formation of the HA molecule differ from the neutral con-formation of HA. At very low pH the molecule stiffens, making the polymermore voluminous. High pH interferes with hydrogen bonding, leading to asmaller volume of the polymer. As the conformation of the molecule changes,the viscosity will most likely change. This means that the viscosity of a solu-tion at a given concentration will vary with pH alterations.

Mutually for these studies there is a distinct increase in the viscosity of solu-tions at pH < 2.5 and a clear decrease as the pH exceed 11. At physiologicalpH, the polymer is less sensitive to pH change. At 6.5 < pH < 8.0 there isalmost a constant expansion of the HA chains which means the intrinsic vis-cosity is unchanged [3].

As mentioned earlier the carboxylic group will be deprotonated in neutral so-lution. Due to intramolecular hydrogen bonds the conformation of the poly-mer will be rigid. The rigidness increases as the pH reaches pH 2.5. Thisis due to a protonation of the carboxylic group leading to a formation of anintermolecular H-bond network. The result of this is a viscoelastic, gel-likemass is formed. The viscosity at this pH is therefore higher than at neutral pH.When decreasing the pH to about 1.6 the polymer is yet again soluble. Theexplanation for this is that the amido group in addition to the carboxylic groupprotonates causing an increase in the net charge of the polymer.

In alkali conditions (11 < pH) the viscosity of HA solutions seems to de-crease. This is a result of deprotonation of not only the carboxylic group butalso hydroxyl groups, which causes the hydrogen bonds to break. Conse-quently, the rotational freedom of the glucosidic bonds increase. This causes areduction in polymer volume and thereby the solution viscosity will decrease[3,8].

1.2 Galderma ABAs mentioned earlier hyaluronic acid is an eminent bio polymer to use formedical solutions and cosmetics. Because of these great dermatological quali-ties, Galderma use HA in many of their products. Galderma AB is a global der-matology company that provide medical solutions and treatment for patients

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with different dermatologic issues i.e. acne, rosacea, psoriasis, skin cancerand pigmentary disorders. Another area of expertise is beauty and solutionsfor aging skin by producing cosmetic fillers. The company was founded byL’Oréal and Nestlé in the year of 1981 and is today a worldwide companywith over 6000 employees, a wide product portfolio available in more than100 countries and 35 wholly-owned affiliates [9].

Galderma Nordic AB, with headquarters in the city of Uppsala, is a resultof the take-over of the company Q-Med AB the year of 2011. Q-Med wasfounded by the entrepreneur Bengt ågerup in 1987 [10]. With a PhD in phys-iology and valuable knowledge and experience of hyaluronic acid from hisearlier employers, Pharmacia and Biomatrix, ågerup founded Q-Med to de-velop and commercialise his research of hyaluronic acid [11]. With somesmall molecular modifications, cross linking of the molecule was made possi-ble and a firm gel was developed- a quality that came to be very useful. Thetechnology is called NASHA, Non-Animal Stabilized hyaluronic Acid, andthe patent was granted the year of 1995 [12].

1.3 AimThe gel in Galdermaâs fillers is constructed by cross linking HA with a crosslinker at high concentrations of OH�. To be able to produce gels of desiredqualities it is of interest to find out how the gel behaves under different con-ditions. One quality that is usefully adjustable is the firmness which variesaccording to the conditions during cross-linking.

It is known by the development department of Galderma Nordic, Uppsala,that the resulting firmness of the gel from cross linking will increase, as theconcentration of HA and the concentration of cross linker increases. However,it is less known how the concentration of OH� affects the molecular volume.

It has been suggested by Galderma that a greater amount of overlap betweenthe molecules during crosslinking may result in a firmer gel. This overlapis affected by the concentration of hyaluronic acid as well as the volume ofthe polymer. Furthermore, the volume of each polymer is dependent on thesurrounding environment, such as the pH. Therefore, it is of high interest tofind out how the volume of HA varies at different concentration of OH�.

One way of measuring the volume of HA is to study the intrinsic viscosity,where an increased intrinsic viscosity corresponds to an increased polymervolume, and vice versa. Thus, the aim of this project is to map how the intrin-sic viscosity of 3 MDa hyaluronic acid varies in aqueous solutions with highconcentrations of OH�.

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2. Theory

2.1 ViscosityThis section is concerned with the general background to viscosity. A moredetailed presentation of its application in macromolecules, is given in 2.2 Vis-cosity in solutions of macromolecules.The viscosity of a fluid is a measurement of the internal resistance that arisein a flux, which is defined as shear stress (t) over rate of shear (g). The rela-tionship is here given by Newton’s equation, eq. 2.1.

h =tg

(2.1)

Viscosity is a state dependent phenomenon that varies with temperature andpressure. In general viscosity increases with increasing temperature and de-creasing pressure, for solutions containing polymers [13,14].

A fluid can respond to shear stress in different ways. Based on the behaviourof a fluid it can be classified as Newtonian or non-Newtonian. For a Newto-nian fluid, there is a direct correlation between shear stress and deformation.This linear dependence means that viscosity remains constant regardless of theshear stress. The response is the opposite for a non-Newtonian fluid, as it cantypically behave shear thinning or thickening. In this context, viscosity caneither decrease or increase under stress.

For a solution containing macromolecules it is of relevance to compare theviscosity with that of the pure solvent. Relative viscosity hrel is defined as theratio between the solution’s viscosity and the pure solvent’s viscosity, see eq.2.2.

hrel =hx

h0(2.2)

Specific viscosity hsp is the relative viscosity subtracted by one, given by eq.2.3.

hsp =hx

h0�1 (2.3)

In reduced viscosity, hred is derived from specific viscosity with the only dif-ference that it takes concentration in to account, where the concentration isgiven in ml/g. This equation can be found in eq. 2.4.

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hred =hsp

c(2.4)

Intrinsic viscosity [h] is a parameter used to describe the hydrodynamic prop-erties of a macromolecule [14]. It is defined as 1/concentration; this can becompared with viscosity that has the unit Pa · s. To determine the [h], onecan measure the inherent or the specific viscosity. [h] on the other hand isestimated through extrapolation of these values to infinite dilution, with thedefinition of [h] being according to eq. 2.5.

[h ] = limc!0

hsp

c(2.5)

The relationship between h and inherent viscosity is given by Huggin’s equa-tion, eq. 2.6, where the specific viscosity is given by eq. 2.7.

hsp = k0[h ]c+ k1[h ]2c2 + k2[h ]2c2 + ... (2.6)

hsp

c= [h ]+ kH [h ]2c (2.7)

Where hsp, h , kH and C designate specific viscosity, Intrinsic viscosity, Hug-gin’s constant and concentration respectively. The constant kH is unique for aspecific molecule and solvent, in theta-solutions kH ranges between 0.5�0.7[15]. In this context theta-solution has favourable polymer-solvent interaction.The polymer chain becomes flexible and takes the configuration of a randomcoil [16].

2.2 Viscosity in macromoleculesHighly viscous liquids flow more slowly due to higher resistance compare tolow viscous liquids [13]. The size of a molecule has a large effect on the vis-cosity. Therefore, polymers highly affect the viscosity, even at very low con-centrations. Outstretched macromolecules cause higher viscosity comparedto coiled up molecules. This is due to outstretched molecules have a largercontact area, which increase the possibility to interact with other molecules.The interactions between macromolecules appears as overlaps, entanglements,inter-molecular bonds and electrostatic interaction, with one or many othermolecules [13].

Coiled up molecules give rise to lower viscous fluids, due to the possibility ofinteraction with other molecules decrease as the contact area decreases.

Different functional groups dissociate at a range of pH-values in the surround-ing environment. Depending on if respective functional group of the polymerare in a dissociated or protonated state, the molecule adopts different charges,

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which give rise to different intra- and/or inter-molecular bonding and elec-trostatic interactions. A polymer with charged side groups will repel intramolecularly, and the molecule will therefor become more stretched out withan increased volume consequently [16]. Though, in presence of counter ions,charged groups will be shielded and the repelling will therefore decrease. Theviscosity of a charged polymer is therefore dependent on both the pH valueand the salinity of the solution. Generally, for many polymers the viscosity ofa solution will increase as the charge of the polymer increases. The effect ofthe repellent obviously also affect the inter molecular interactions. Stretched-out polymers have a larger area of interaction which give rise to an increase inviscosity. Though, at the same time, charged molecules repel each other whichdecreases the viscosity due to increased, possible inter molecular movement[16].

However, other molecular features show a higher importance in the viscositybehaviour of hyaluronic acid, due to the commonly occurring hydrogen bonds.When going from neutral solution to high OH� concentration, hydroxy groupsdissociates. This results in breakage of intra- and intermolecular hydrogenbonds, giving the molecule free rotation at the glucosidic bonding. This causesthe molecule to a have less rigidity which increases the ability to coil up to asmaller volume. Even though there will be more charge within the moleculeleading to repelling, the effect of breaking the hydrogen bonds is of greaterimpact [1,3].

2.3 ViscometryTwo of the more general methods used to determine the viscosity of a fluid arecapillary viscometry and rotating rheometers. These two are used in differentsituations as they measure different things, especially rheometry, which hasa wider range of applications. In capillary viscometry, time is measured andviscosity can be calculated. In rheometry, one can apply a controlled force orstrain to measure the responding force or strain of the liquid; and this forceor strain can be varied stepwise and thus give more information from onesingle run. This makes rheometry suitable to measure non-Newtonian fluids[17]. Rheometry can also be used for measurements on solid and semi-solidmaterials.

2.3.1 RheometryIn rheometry the bob measures at a controlled torque or speed, and rotationalor oscillating movement. What setup is used depends on the substance and/orwhat one wants information about, e.g. the viscosity of ketchup is best mea-sured with controlled shear-rate test and the movement should be rotational

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instead of oscillating because ketchup is a non-Newtonian fluid. With thistest, the shear-rate could be changed stepwise and the changes in viscositycould be examined [18].

2.3.2 Capillary viscometryWhen using capillary viscometry there are different instruments to choosefrom. In this study the chosen capillary was a Ubbelohde viscometer, normalform (ASTM), Capillary No.I. The principle of Ubbelohde capillary is simple:Time is measured for an arbitrary volume of given solution to flow through acapillary of a specific length and diameter. The flow time can be registeredmanually or automatically. Automatic registration with a Schott Instrument(Hoffheim, Germany) ViscoClock system is a clear advantage over manualregistration as the manufacturer [19] states the measurement accuracy to be±0,01 s; with this automatic system, subject measurement error is eliminated.

To conduct measurement in an Ubbelohde capillary, specific requirementsneed to be met. First, temperature needs to be controlled; by praxis opera-tions are carried out in 25�C for capillary viscometry. Second, concentrationis another critical factor. At high concentration, there are multiple molecularinteractions between adjacent molecules. This is an associative phenomenonof HA [1], which means solutions in this project will assume non-ideal states.Both the reduced viscosity and inherent viscosity are concentration dependent[1]. To calculate these values the concentration needs to be defined with a highaccuracy. Intrinsic viscosity on the other hand is defined at zero concentration.This gives a theoretical value for the polymers volume that is independent ofthe concentration. Therefore, this value is suitable for comparison.

Capillary size is of importance when measuring as it must operate well forgiven solvent and solute. By changing to a capillary with a different diameter,it is possible to change the flow rate. It is essential that have the appropriatecapillary size to get a reliable measurement. In this project, the solvent usedwas water and therefore the capillary size needs to be large enough to avoidcapillary action propelling the sample and thereby affecting the flow time.The solute properties are also of importance when choosing capillary, as inthis project. HA has a non-Newtonian fluid behaviour. Under stress it behavesshear-thinning. Hereby a problem arises since the gravitational force, in thecapillary, may affect. In a publication [14] by Steve Harding, a theory is beingpresented that a non-Newtonian fluid can behave as a Newtonian fluid at lowflow rate.

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2.4 Data analysisTo calculate the intrinsic viscosity from the flow times, several calculationsmust be made. At first one calculates is the relative viscosity, eq. 2.2. Inthis experiment, the density of the solution and of HA were considered equal,giving the reduced form of eq. 2.8 as eq. 2.9, which is used in the calculations.

hrel =txt0· rx

r0(2.8)

hrel =txt0

(2.9)

The second step is to calculate the specific viscosity from the relative viscosity,using eq 2.10.

hsp =tx � t0

t0(2.10)

In order to estimate the intrinsic viscosity, [h], reduced viscosity hred 2.11 isplotted against mass procent HA (see figure 4.1.)

hred =tx � t0

ct0(2.11)

A regression line is fitted to the scatter plot and from the intercept on theordinate axis, the intrinsic viscosity is acquired[20]. In order to accuratelyestimate the true intrinsic viscosity, the uncertainty of the intercept needs tobe coincided. Correlation coefficient and regression analysis are two goodmethods that gives complementing information to the analysis.

Correlation coefficientAll the intrinsic viscosity values were plotted against their respective wt%NaOH, allowing the graph to display the mapping that was sought. A correla-tion analysis was made of the graph with eq. 2.17 [21].

Correlation coefficient r describes the linear ’co-relations’ between two quan-titative variables. Here, designated X and Y. It indicates on both direction andstrength in the relationship. For a high correlation coefficient, the variabilityis well explained. This is shown by a r-value close to -1 or 1. The sign simplyindicates whether the relationship is positive or negative. If Y increases withX, the relationship is said to be positive. The opposite is true for a negativerelationship.

Correlation coefficient can be derived from the covariance Cov(X,Y).

Cov(Y,X) =Â[(xi � x)(yi � y)]

n�1(2.12)

13

Covariance is closely related to correlation coefficient, with one mayor differ-ence. Covariance can not be used to estimate the strength in the measurement.Instead data needs to be standardised, by dividing them with the standard de-viation in x and y. Here follows a example of X.

zi =(x� xi)

sx(2.13)

where

sx =

r1

n�1 Â(xi � x)2 (2.14)

is the standard deviation in X. The same is preformed for Y. Finally, correlationcoefficient is given by covariance.

r =Cov(X ,Y )

sxsy(2.15)

Corelation coeffiecient can also be written as

r =1

n�1 Â (xi � x)2

sx

(yi � y)2

sy(2.16)

r = Â[(xi � x)(yi � y)]p[Â(xi � x)2][Â(yi � y)2]

(2.17)

.

Regression analysisLinear regression analysis can be used in descriptive purpose, as with correla-tion coefficient. In addition to this, regression analysis can be used to predict.For a given value of x, the correspondent y-value can be calculated with aassociated confidence interval. This makes it possible to extrapolate a trendline to the intercept on the y-axis. From this intercept the intrinsic viscosity isestimated with some uncertainty given by the confidence interval.

Different models are used in linear regression analysis eg. least square methodand maximum likelihood estimation etc. Fully developed mathematical de-scription could be found in a statistical textbook.

R-squareTo statistically secure that calculated values are close to the regression line,one can calculate an R2 value (goodness of fit). If R2 equal to 0 (0 %), themodel doesn’t explain the variability of the points along the regression. If R2

is equal to 1 (100 %), the model does explain all the variability of the points.In other words, the higher R2 value, more points variability will be explainedby the regression model. [22]

14

3. Method

3.1 Choice of methodTo obtain results precise enough to be able to calculate the relative viscosity,and thereafter the intrinsic viscosity, a method with high precision was sought.Capillary viscometry was chosen as method, seeing as an available SI Visco-clock had an accuracy of 0.01 % for time measurements, which was judged tobe the most superior method. The SI Viscoclock was connected to an Ubbe-lohde capillary, making it possible to measure the flow time automatically,which provides more accurate results compare to manual clocking. Beyondbeing easily accessible it is also an economically beneficial method for thistype of study.

A capillary size of 0.58 mm was chosen with the properties of water as asolvent and HA as a solute considered. The running terms for capillary vis-cometry of measuring at 25�C was facilitated in a temperature-regulated tankHeto-Holten A/S (Allerød, Denmark) type 21DT-2.

3.2 Procedure of methodThe procedure of the method is summarised in a flow chart below, presentedin figure 3.1.

15

Lye dilution

Reference run

Hyaluronic acid

dilution

40 minutes of stirring

10+2 minutes of tempering

Run thrice in capillary

Clean and dry the capillary

Repeat

Figure 3.1. Method illustrated as flowchart

3.2.1 SolutionsTo outline the variation in intrinsic viscosity at high [OH�], NaOH solutions of2, 4, 6, 7, 8, 9 and 10 wt% respectively were prepared. For each concentrationof NaOH a water solution of approximately 200 mL was prepared in a roundbottom flask, by first weighing milli-Q water followed by the required mass ofNaOH pellets to achieve the desired mass concentration. From this solutionfour Hyaluronic acid solutions, of different mass concentrations, were made.An example is given in Table 3.1, which presents the compositions of the fourdifferent HA-solutions at the NaOH-concentration of 2 wt%.

Table 3.1. Dilution series for the four HA solutions at 2 wt% of NaOHHA (g) NaOH-solution (g) [HA] (mg/g)

C1 1.40 32.60 0.800C2 1.05 33.95 0.600C3 0.700 34.30 0.400C4 0.350 34.65 0.200

Each solution was kept under magnetic stirring for 40 minutes, followed by10 minutes of tempering in 25�C thermostatic bath. Afterwards 17 mL oftempered solution was measured in a measuring cylinder from which it waspoured into a plastic syringe and therefrom dosed into the Ubbelohde vis-cometer through a PVDF membrane filter. Before starting measurements the

16

dosed viscometer, attached to the SI Viscoclock, was intentionally left for 2minutes of tempering in the 25�C thermostatic bath.

3.2.2 MeasurementsFor each measurement of the HA-solutions, the flow time in the capillary wasclocked with the SI Viscoclock. Each solution was repeatable measured thriceand a mean time was calculated. In addition to this a reference flow time wasalso clocked thrice, from the NaOH-solution without any HA, and later usedin the calculations. The time in between the repeated measurements of eachsample was kept as short as possible to minimise the time dependent degra-dation of the polymer while in the NaOH-solution. In between the differentsolutions the capillary was washed out with distilled water, followed by KOHsoap and acetone so that it could easily be dried with N2 gas.All the data processing and analysis processing was done with Microsoft Of-fice Excel 2016.

3.3 Method DevelopmentBefore the method was established, pilot-laboratorial work was made to de-terminate and predict difficulties throughout the process. The same laboratoryequipment was used as in the latter experiments. The measurement of each so-lution was also made repeatedly thrice. Though, the concentrations of NaOHvaried compare to the solutions of the latter experiments (3, 5, 7, 8.5 and 10wt% NaOH respectively).During the pilot project, observations made it clear that the time consistencyduring the sample preparations were of importance due to the degradation ofHA. The importance of tempering each sample for an equal amount of timewas also evaluated. The experience from the pilot project resulted in a higherknowledge of how important it was to keep the consistency throughout thelaboratorial work. From this knowledge, the decision to always stir and tempereach sample for an equal amount of time in latter experiments was made. Theresults from the pilot project was valued not to be credible enough to presentand will therefore be disregarded.To satisfy Galderma’s acceptance requirements of measurements of the vis-cosity of HA, the quota between the mean flowtime of the highest concentra-tion (C1) and the reference mean flow time was to fall within the range 1.6and 1.8. By varying the amount of HA in the solutions, the acquired rangecould be accomplished. During the pilot-laboratory work it was found thatthe searched range was implemented in the solution of 3 wt% NaOH at HA-concentrations of 0.8 [mg/g] for C1, respectively HA-concentrations of 1.0[mg/g] for solutions between 5-8.5 wt% NaOH.

17

4. Results

In this chapter our laboratory findings are presented. The results are given in achronological order, which ultimately leads to the intrinsic viscosity. Since theprocedure was the same for 2, 4, 6, 7, 8, 9 and 10 wt%, a detailed explanationis only given for 2wt%. The measured values for the others can be found inappendix 1.

The exact mass concentration of the 2 wt% NaOH solution is shown in table4.1. The table also shows the amount of water and NaOH(s) that were weighedto create the solution.

Table 4.1. The weighed masses of water and NaOH for the 2 wt% solution and itsexact concentration value.

Water (g) 185,16NaOH (g) 3,7793

Concetnration (wt%) 2,000

Four samples with different masses of Hyaluronic acid (HA) were prepared.The amount of HA and NaOH(aq) needed, and their resulting concentrationare presented in table 4.2.

Table 4.2. The weighed masses of HA and 2 wt% NaOH(aq) that were mixed and theresulting concentrations of the samples used in the experiments.

Cx HA (g) NaOH(aq) (g) Concentration (mg/g)C1 1,403 32,6829 0,8232C2 1,058 33,9505 0,6044C3 0,7037 34,3026 0,4020C4 0,352 34,6503 0,2011

The results of the 2 wt% NaOH solution measurements are found in tables 4.3and 4.4. The first one presenting the flow time of the different concentrationsand reference sample, along with the mean values, standard deviations and rel-ative standard deviations. The latter presents the relative, specific and reducedviscosities calculated from the flow times and concentrations (in table 4.2).

18

Table 4.3. Observed flow times in the Ubbelohde capillarry of the reference and thesamples, with a calculated mean, standard deviation and relative standard deviation.For each run, the flow time are clocked three times.

Flow time (s)

Ref C1 C2 C3 C4

110.32 189.59 161.44 140.73 124.48110.16 188.4 160.98 140.54 124.44110.46 187.47 160.65 140.70 124.39

Mean time (s) 110.31 188.49 161.02 140.55 124.44Std.dev. (s) 0.1501 1.063 0.3968 0.1801 0.04509Relativ std.dev. (%) 0.1361 0.5638 0.2464 0.1281 0.03624

Table 4.4. The relative, specific and reduced viscosities of samples of 2 wt% NaOHsolution. These values are calculated, based on the rawdata presented in table 4.3

hrel hsp hredC1 1,709 0,7086 0,8608C2 1,460 0,4597 0,7605C3 1,274 0,2741 0,6817C4 1,128 0,1280 0,6366

The calculated reduced viscosities were plotted against the different HA con-centrations, with the reduced viscosity on the y-axis and HA concentration onthe x-axis. This graph can be seen in figure 4.1.

19

Figure 4.1. Graph showing the resulting reduced viscosities [cm3/g] on the y-axisand the HA concentrations [mg/g] on the x-axis for measurements at 2 wt% NaOHsolution.

All the intrinsic viscosities were obtained at the intercept with the y-axis in thegraphs shown in figure 4.2.

20

Figure 4.2. All the graphs where the reduced viscosity is plotted against the NaOHsolutions (a-2, b,c-4, d-6, e-7, f-8, g-9, h-10 wt%) and where the intrinsic viscositiesare obtained at the intercept with the y-axis.

The intrinsic viscosities of HA at different NaOH concentrations are presentedin table 4.5, along with the corresponding NaOH concentrations and a 95,0%confidence interval’s upper and lower limits for the intrinsic viscosity values.The table’s values are calculated from the graphs in figure 4.2.

A graph showing the NaOH-concentration dependence of the intrinsic viscos-ity is found in figure 4.2. The error bars in the graph are the confidence intervalfor each point.

21

Table 4.5. All the estimated intrinsic viscosities, their upper and lower confidenceinterval limits, their corresponding NaOH solution concentrations, p-values and R-square values.

NaOHaq (wt%) IV (cm3/g) Upper 95.0% CI Lower 95.0% CI p-value t-value R-square2.000 0.5499 0.6403 0.4595 0.01051* 26.17 0.97914.006 0.6522 0.8105 0.4938 0.1583 17.72 0.70844.159 0.6537 0.7066 0.6009 0.02436* 53.19 0.95196.008 0.6091 0.6787 0.5396 0.02734* 37.68 0.94616.951 0.6136 0.7191 0.508 0.08171 25.01 0.84338.005 0.689 0.7897 0.5884 0.8588 29.45 0.019958.998 0.5826 0.6461 0.5191 0.01917* 39.48 0.962010.00 0.5743 0.6485 0.5191 0.04589* 33.29 0.9103

* Significant on a 5% level.

Figure 4.3. The intrinsic viscosity’s NaOH dependence are illustrated in the scatterplot with the confidence interval as error bars. Intrinsic viscosity [cm3/g] on the y-axisand NaOH solution [wt%] on the x-axis.

The relationship between IV and massprocent NaOH was evauated. Corre-lation coefficient was calculated of the estimated mean intrinsic viscosity, tor = 0.0006265.

22

5. Discussion

5.1 Data AnalysisAs can be seen in figure 4.3 the error bars have a wide range, meaning a wideconfidence interval. With such high uncertainty a final conclusion of a trendcan not be made. As of now a trend line could be drawn horizontally withinthe confidence interval of all the points, which would indicate that the intrinsicviscosity does not change at all with the NaOH concentration. To draw anyconclusions, this uncertainty needs to decrease. Even though two separatemeasurements were made around 4 wt% it might be a random coincident thatthey were roughly the same, seeing as one of them had a significantly largerconfidence interval.

If the degradation of the polymer could be avoided, the standard deviation(std.dev.) of the flow time would improve as the systematic loss in secondsbetween the measurements would not occur. This in turn should have resultedin a narrower spread around the true mean. If the std.dev. could be decreasedthe confidence interval would improve, e.g. at C2 for 7 wt% the mean dif-ference between t0 and tx is 77 [s] and tx has a std.dev. of 1,2 [s]. If thenthe std.dev. was added to the mean and then divided with the mean, the resultwould be 1.5 % increase. This 1.5 % difference in the mean of measured timewill follow through the rest of the calculations being made and giving them atleast the same uncertainty.

For the graphs a-h, in figure 4.2, 95.0 % confidence intervals have been calcu-lated and tabulated in table 4.5. These confidence intervals refers only to theintercept with the y-axis. No confidence lines around the regression have beenvisualised.

Overall, the calculated p-values are < 0.05, but the first 4 wt%, 7 wt% and the8 wt% shows a higher value and they all have a lower R-square value than theones with p<0.05. For these three measurement, Y does not depend on X in alinear way. The intrinsic viscosity calculated from these three measurement,are weak estimations.Why the first regression for 4 wt% NaOH show poor values and confidenceinterval might be a result of problems with the thermostatic bath during runs.C2 and C3 ran at 26�C, and not 25�C where C1 and C4 were run at. This couldhave resulted in a viscosity difference for those samples. Why 7 wt% valuesp > 0.05 is unknown. There might have been something wrong with C3 if onestudy the 7 wt% graph in figure 4.2. C3 is not as close to the regression line

23

as the other and causes the R-square to decrease. Why the 8 wt% values arepoor we do not know. The observed flow times are without any noteworthyoddities but the reduced viscosities do not follow a linear trend like the othersregressions do. This regression has an R-square value of 0.01995, meaningthat the model is bad at explaining the variability of the points. Said resultsmakes these graphs very questionable and thus also the extrapolation resultingin the intrinsic viscosity used in the final results. With all having a p>0.05 wecan not say that Y depends on X.

5.2 HA in solutionsData in documents provided by Galderma presents that the intrinsic viscosityfor this batch of HA in neutral solutions is 2.9 cm3/g. As mentioned earlierthe intrinsic viscosity should decrease once solution reaches pH < 11. In thisproject, the pH ranges from 13.7 < pH < 14.4 and the estimated intrinsicviscosity ranges between 0.55 and 0.69 cm3/g. Thus, there is a great decreaseof the intrinsic viscosity relative that at pH < 11, as predicted. This indicatesthat there has been a dissociation of the hydrogen within the hydroxy groups,leading to a disruption of the hydrogen bond network. This results in shrinkageof the polymers volume since there is free rotation of glucosidic linkages.Smaller volume of the tangled polymer leads to a decreased chance of overlapwith other HA molecules within the solution.

Even though the interval of the wt% is rather large, the corresponding intervalof pH is very small. This is because the scale of pH is logarithmic. It istherefore reasonable that the intrinsic viscosity would be approximately thesame for all samples due to lack of variation in the alkalinity. The results foundshow that there are two peaks at 4 wt% and 8 wt% respectively, albeit theseresults are not reliable since the confidence interval is very wide, as mentionedearlier. It is not likely that the true value of intrinsic viscosity would vary theway it seems with the diverging values of 4 and 8 wt%, since the interval ofpH is very narrow. These two diverging values are probably a result of randomexperimental errors.

Furthermore, if the resulting values of 4 and 8 wt% were to be ignored, aslightly decrease in the intrinsic viscosity could potentially be observed. Thiscould possibly be explained by the shielding effect of [Na+]. By increasingthe concentration of NaOH more cations will be available in the solution, in-tensifying this salt effect. The net charge of the polymer will thereby decreasewithout it regaining the ability to hydrogen bond. This will in turn lead to lessintra molecular repelling, resulting in a smaller volume. This theory is notconfirmed but could be tested by adding NaCl to the solution to see whetherthe viscosity would decrease even more. A decrease would strongly indicate

24

that the polymer is indeed affected by this salt effect. This is therefore some-thing that could be investigated in the future.

5.3 DegradationPrevious research show a degradation of HA when it is exposed to high tem-peratures or strongly alkaline solution. The degradation shows a linear be-haviour, where the temperature is contributing to a higher extent compared tothe alkalinity. Degradation of the hyaluronic acid leads to shorter length of themolecules, which in turn lowers the viscosity. The degradation is most likelythe reason why the three flow times for each solution decreases in descendingorder. This behaviour is observed in all samples, except from one (sample C2,4 wt%) which most likely is an error due to contamination of the equipment.It is difficult to say what exact effect the degradation has on the resultingintrinsic viscosity. It is probably the case that the entire graph is displacedvertically since all the observed reduced viscosity values are lower than thereal ones; this would result in a lower calculated [h] compared to a scenariowhere degradation does not occur. This displacement would not be propor-tional throughout the four points of the regression. Since C1 spends moretime in the capillary while being measured, the significance of the degradationis greater than it is for C4.Since the degradation is unavoidable, method developments were made to en-sure a small time variation, and thereby variation in degradation, as possiblebetween samples. By working under a consistent time frame during the prepa-ration of each sample, the time between the mixing of HA with the NaOHsolution, and the first run in the capillary is kept the same. Since every sampleis measured thrice, the procedure between measurements has been made asefficient as possible to avoid further time differences.However, since the flow time differs in between samples, as a result of changein concentration and thereby also in viscosity, the starting time of runs two andthree varied. Higher concentration of HA and NaOH in the samples increasedthe flow time. Even though the second and third run was started as soon asthe previous run was finished different extents of delays occurred, giving riseto a slight uncertainty throughout the method. One other possible method thatwould minimise the effect of the degradation is the idea of doing triplets ofeach sample that is being measured only once. However, this procedure wouldbe much more time consuming and acquire three times more work material.

5.4 Shear-thinningThe capillary viscometer has a time specific operating range that is appliedto Newtonian fluids. Though, uncertainties can occur when measuring non-

25

Newtonian fluids. Fluids that are shear-thinning such as the hyaluronic acidcan, if the capillary viscometer radius is too large, flow disproportionally fastwhich will entail to incorrect results of the intrinsic viscosity. Non-Newtonianfluids can however be approximated as Newtonian ones if the flow time is suf-ficiently low. It is therefore important to use a capillary with a suitable radiusfor the solute to avoid deceiving results. However, it has not been possible toinvestigate if shear-thinning of the hyaluronic acid take place in the capillaryused, which is presenting an experimental error to consider [14].

5.5 Future researchOur results found clearly indicate that a degradation of the polymer occursduring measurements as there is a consistent reduction in flow time. Accordingto a publication by Steve Harding [14], capillary viscometry should not beused for a polymer that degrades during measurements. However, capillaryviscometry is an established and well-tried method used to measure viscosity[13], and combined with a SI Viscoclock high accuracy is to be achieved.It is also an easily accessible and economically beneficial method. Though,a greater amount of measurements on each wt% must be made to ensure amore reliable result and to obtain a satisfactory confidential interval. Anyhow,if further investigations of an alternative method would be of interest, onesuggestion would be to use light scatter spectrophotometry, since it improvesthe time aspect and therefore the significance of degradation.

One very important thing to keep in mind is that the temperature has a bigimpact on the degradation [7]. It is therefore highly important to keep thetemperature stable. Furthermore, as the degradation of the polymer is timedependent, it is of interest to continue making sure that each solution spendsthe same amount of time in the process of stirring respectively tempering, aswell as in the capillary.

In further investigations it should, as a suggestion, be of interest to do the studyat a larger interval of pH, to be able to see at which exact pH the dissociationof hydroxyl groups occurs. Lastly it would be interesting to add NaCl to thesolution to establish whether there is a common ion effect in the solution ornot.

26

6. Conclusions

Given the results, no trend can be observed in the intrinsic viscosity between2 and 10 wt% NaOH. The degradation is great enough to be noticeable duringthe tests, which causes an uncertainty in the results. Using an Ubbelohdecapillary viscometer for the experiment has been difficult to use, and a bettersuited method may be found. If there is a variation in molecular volume of HAdepending on different concentration of OH�, it could not be detected in ourstudy due to the uncertainty of the results. However, one can with certaintysay that the intrinsic viscosity, and thereby also the molecular volume, is lowerat these higher concentrations of NaOH than in neutral solution.

27

References

[1] Garg H G, Hales C A, Hales C A. Chemistry and Biology of Hyaluronan[Internet]. Amsterdam: Elsevier Science; 2004. [cited 2017 May 15]. Avail-able from: ProQuest Ebook Central

[2] NE.se [Internet], Nationalencyklopedin. Hyaluronsyra. [cited 2017 May18]. Available from:http://www.ne.se/uppslagsverk/encyklopedi/lång/hyaluronsyra

[3] Cowman M K, Schmidt T A. , Raghavan P, Stecco A. Viscoelastic Prop-erties of Hyaluronan in Physiological Conditions [version 1; referees: 2 ap-proved]. F1000Res. 2015 Aug 25;(4)622:5-6

[4] Drugbank. Hyaluronic acid [Internet]. Place unknown. [cited 2017 May19] Available from: https://www.drugbank.ca/drugs/DB08818

[5] NE.se [Internet]. Nationalencyklopedin. Hyaluronsyra. [cited 2017 May17]. Available from:http://www.ne.se/uppslagsverk/encyklopedi/lå/hyaluronsyra

[6] Galderma. Frågor och svar Restylane [Internet]. Galderma; [cited 2017May 16]. Available from: http://www.restylane.se/behandlingar/Vanliga-fragor/

[7] Fagerström Troncoso J, Idjbara A, Kalrsson I, Lekander M, Lindgren T,Ström S. A kinetic study of the degradation of hyaluronic acid at high concen-trations of sodium hydroxide [Bachelor’s thesis on the Internet]. Uppsala: Up-psala Universitet; 2016 [cited 16 May 2017]. Available from: http://uu.diva-portal.org/smash/get/diva2:954372/FULLTEXT01.pdf

[8] Gatej I, Popa M, Rinauo M. Role of the pH on Hyaluronan Behavior inAqueous Solution. BIOMACROMOLECULES:2015(6);61-67.

[9] Galderma. About Galderma, a Dermatology Company [Internet]. Lau-sanne: Galderma S.A; [cited 2017 May 15]. Available from:http://www.galderma.com/About-Galderma

[10] Galderma. Om Galderma [Internet]. Uppsala: Galderma Nordic AB;[cited 2017 May 15]. Available from: http://www.galderma.se/Om-Galderma

[11] Entreprenörskapsforum. Entreprenör, forskare, uppfinnare, filantrop...[Internet]. Stockholm: Entreprenörskapsforum; [cited 2017 May 15]. Avail-able from:http://entreprenorskapsforum.se/natverk/globaliseringsforum/bengt-agerup/entreprenor-forskare-uppfinnare-filantrop%E2%80%A6/

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[12] Mellgren, E. Han tämjde molekylen [Internet]. Stockholm: Ny Teknik;2010 [Updated 2010 October 12; cited 2017 May 16] Available from:http://www.nyteknik.se/innovation/han-tamjde-molekylen-6424344

[13] Atkins P, de Paula J. Atkins’ Physical chemistry. 10 ed. Oxford: OxfordUniversity Press, 2014.

[14] Harding S.E. The intrinsic viscosity of biological macromolecules. Progressin meassurment, interpitation and application to structure in dilute solutions.Prog. Biophys. Molec. Biol. 1997; 68: pp. 207-262.

[15] Sakai T. Huggins constant k’ for flexible chain polymers. J. Polym. Sci.,Part B: Polym. Phys. 1968;8(6) pp. 1535-1549.

[16] Piculell L. "Polymerlösningar och geler". Handout. Lund university.Uppsala. 1997. Print.

[17] ATA Scientific Instrument [Internet]. [Place unknown]: ATA ScientificInstrument; [Publication unknown]; An introduction to viscosity and rheol-ogy; 2010 Feb 10 [cited 2017 May 15];[about 2 screens]. Available from:https://www.atascientific.com.au/an-introduction-to-viscosity-and-rheology/

[18] Anton Paar blog [Internet]. [Place unknown]: Jürgen Utz; 2016 June 9.Rheometer âWhat do They Measure and How are They Set Up?; 2016 June9 [cited 2017 May 17]; [about 3 screens]. Available from: http://blog.anton-paar.com/rheometers-what-do-they-measure-and-how-are-they-set-up/

[19] Schott-Geräte [Internet]. Allemagne: Schott-GerÃte GmbH; [Publica-tion unknown]; Operating Instructions Viscosity Measuring Unit ViscoClock;1997 May 20 [cited 2017 May 17].

[20] Harding S.E (2013) Intrinsic viscosity, in Roberts G.C.K. (ed) Encyclo-pedia of Biophysics, pp. 1123-1129, Berlin: Springer Verlag

[21] Miller N J, Miller C J. Statistic and Chemometrics for Analytical Chem-istry. 6 ed. Harlow: Prentice Hall; 2010. 278p.

[22] The Minitab Blog [Internet]. [Place unknown]: Jim Frost; [2013 May 30];Regression Analysis: How do I Interpret R-square and Assess the Goodness-of-Fit?; 2013 May 30 [cited 2017 May 31]; [about 4 screens]. Available from:http://blog.minitab.com/blog/adventures-in-statistics-2/regression-analysis-how-do-i-interpret-r-squared-and-assess-the-goodness-of-fit

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Appendix 1

Table 6.1. 4 wt% (1st): the weighed masses of water, NaOH(g), HA and NaOH(aq),the concentration of NaOH(aq) (wt%) and HA (mg/g) and the values of hrel , hsp andhred (cm3/g).

4 wt% NaOHaq (1st)H20(l) (g) NaOH(g) (g) Conc. (wt%)

182.94 7.939 4.1595HA (g) NaOH(aq) (g) Conc. HA (mg/g) hrel hsp hred (cm3/g)

C1 1.748 33.34 0.9965 1.751 0.7515 0.7542C2 1.431 31.39 0.6237 1.665 0.6649 0.7626C3 1.093 33.96 0.6237 1.434 0.4336 0.6952C4 0.6977 34.36 0.3981 1.281 0.281 0.706

Table 6.2. 4 wt% (2nd): the weighed masses of water, NaOH(g), HA and NaOH(aq),the concentration of NaOH(aq) (wt%) and HA (mg/g) and the values of hrel , hsp andhred (cm3/g).

4 wt% NaOHaq (2nd)H20(l) (g) NaOH(g) (g) Conc. (wt%)

192.06 8.016 4.007HA (g) NaOH(aq) (g) Conc. HA (mg/g) hrel hsp hred (cm3/g)

C1 1.757 33.3 1.002 1.755 0.7551 0.7533C2 1.405 33.62 0.8021 1.599 0.5987 0.7464C3 1.051 33.94 0.6008 1.431 0.4306 0.7167C4 0.7033 34.33 0.4016 1.278 0.2783 0.6931

Table 6.3. 6 wt%: the weighed masses of water, NaOH(g), HA and NaOH(aq), theconcentration of NaOH(aq) (wt%) and HA (mg/g) and the values of hrel , hsp and hred

(cm3/g).6 wt% NaOHaq

H20(l) (g) NaOH(g) (g) Conc. (wt%)196.83 12.58 6.008HA (g) NaOH(aq) (g) Conc. HA (mg/g) hrel hsp hred (cm3/g)

C1 1.75 33.25 0.9998 1.746 0.7458 0.7459C2 1.397 33.41 0.8028 1.566 0.5661 0.7051C3 1.053 33.94 0.6017 1.410 0.4104 0.682C4 0.7063 33.40 0.4141 1.277 0.2771 0.6691

30




C1 1.746 33.59 0.988 1.717 0.7176 0.7263C2 1.402 33.94 0.7935 1.559 0.5591 0.7047C3 1.049 33.99 0.5988 1.397 0.3966 0.6622C4 0.6957 34.31 0.3975 1.265 0.2655 0.6679




C1 1.751 33.26 1.001 1.706 0.7058 0.7054C2 1.398 33.61 0.7935 1.547 0.5467 0.6845C3 1.06 33.95 0.6054 1.413 0.4132 0.6826C4 0.6867 34.3 0.3925 1.275 0.2754 0.7016




C1 1.752 33.25 1.001 1.733 0.7325 0.7318C2 1.412 33.59 0.8069 1.556 0.5563 0.6894C3 1.048 33.97 0.5987 1.398 0.3976 0.6641C4 0.7092 34.31 0.4051 1.262 0.2615 0.6456

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C1 1.746 33.25 0.9978 1.678 0.6784 0.6799C2 1.407 33.6 0.8038 1.525 0.5248 0.6529C3 1.054 33.95 0.6022 1.391 0.3913 0.6498C4 0.7047 34.31 0.4025 1.246 0.2459 0.6109

Table 6.8. The measured times for all the experiments.

2 wt% 4 wt%(1st)

4 wt%(2nd) 6 wt% 7 wt% 8 wt % 9 wt% 10 wt%

t (s) t (s) t (s) t (s) t (s) t (s) t (s) t (s)110.32 118.7 116.56 129.78 136.38 144.51 150.13 163.86

Ref. 110.16 118.53 116.47 129.55 136.35 144.47 149.97 163.86110.46 118.6 116.36 129.5 136.28 144.48 149.92 163.88189.59 209.63 205.64 228.07 236.13 248.58 262.08 277.61

C1 188.4 206.8 204.37 226.16 234.1 246.48 259.7 275.06187.47 205.42 203.21 224.59 232.3 244.32 257.88 272.43161.44 198.45 186.55 204.21 213.82 224.76 235.04 251.6

C2 160.98 197.54 187.5 202.85 212.47 223.48 233.5 249.87160.65 196.44 184.51 201.87 211.43 222.18 231.84 248.13140.73 170.6 167.16 183.79 191.24 205.19 211.01 229.41

C3 140.54 170.12 166.6 182.68 190.35 204.19 209.51 227.92140.37 169.41 166.07 181.93 189.62 203.18 208.42 226.64124.48 153.13 149.22 166.05 173.08 185.13 190.08 204.71

C4 124.44 151.51 148.84 165.51 172.51 184.32 189.02 204.23124.39 151.15 148.57 165.02 172.00 183.37 188.61 203.55

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