Fundamental Investigations of Supercritical Fluid Chromatography

Martin Enmark

Martin E

nmark | Fundam

ental Investigations of Supercritical Fluid Chrom

atography | 2015:45

Fundamental Investigations of Supercritical Fluid Chromatography

This thesis aims at a deeper understanding of Supercritical Fluid Chromatography (SFC). Although preparative SFC has started to replace Liquid Chromatography (LC) in the pharmaceutical industry - because of its advantages in speed and its less environmental impact - fundamental understanding is still lacking. Therefore there is no rigid framework to characterize adsorption or to understand the impact of changes in operational conditions.

In Paper I-II it was demonstrated why most methods applied to determine adsorption isotherms in LC could not be applied directly for SFC. Methods based on extracting data from overloaded profiles should be preferred.

In Paper III a Design of Experiments approach was successfully used to quantitatively describe the behavior of several solutes in a separation system. This approach can be used to optimize SFC separations or to provide information about the separation system.

In Paper IV severe peak distortion effects often observed in SFC were carefully investigated and explained using experiments and simulations.

Finally, in Paper V, the prerequisites for performing reliable and predictable scale-up of SFC were investigated by small and large scale experiments.

DISSERTATION | Karlstad University Studies | 2015:45 DISSERTATION | Karlstad University Studies | 2015:45

ISSN 1403-8099

Faculty of Health, Science and TechnologyISBN 978-91-7063-663-9

Chemistry

DISSERTATION | Karlstad University Studies | 2015:45

Fundamental Investigations of Supercritical Fluid Chromatography

Martin Enmark

Print: Universitetstryckeriet, Karlstad 2015

Distribution:Karlstad University Faculty of Health, Science and TechnologyDepartment of Engineering and Chemical SciencesSE-651 88 Karlstad, Sweden+46 54 700 10 00

© The author

ISBN 978-91-7063-663-9

ISSN 1403-8099

urn:nbn:se:kau:diva-37913

Karlstad University Studies | 2015:45

DISSERTATION

Martin Enmark

Fundamental Investigations of Supercritical Fluid Chromatography

WWW.KAU.SE

1

Abstract

This thesis aims at a deeper understanding of Supercritical Fluid

Chromatography (SFC). Although preparative SFC has started to replace

Liquid Chromatography (LC) in the pharmaceutical industry - because of

its advantages in speed and its less environmental impact - fundamental

understanding is still lacking. Therefore there is no rigid framework to

characterize adsorption or to understand the impact of changes in opera-

tional conditions.

In Paper I we demonstrated, after careful system verification, that most

methods applied to determine adsorption isotherms in LC could not be

applied directly in SFC. This was mainly due to operational differences

and to the fact that the fluid is compressible which means that every-

thing considered constant in LC varies in SFC.

In Paper II we showed that the most accurate methods for adsorption

isotherm determination in LC, the so called plateau methods, do not

work properly for SFC. Instead, methods based on overloaded profiles

should be preferred.

In Paper III a Design of Experiments approach was successfully used to

quantitatively describe the retention behavior of several solutes and the

productivity of a two component separation system. This approach can

be used to optimize SFC separations or to provide information about the

separation system.

In Paper IV severe peak distortion effects, suspected to arise from in-

jection solvent and mobile phase fluid mismatches, were carefully inves-

tigated using experiments and simulations. By this approach it was pos-

sible to examine the underlying reasons for the distortions, which is vital

for method development.

Finally, in Paper V, the acquired knowledge from Paper I-IV was used

to perform reliable scale-up in an industrial setting for the first time.

This was done by carefully matching the conditions inside the analytical

and preparative column with each other. The results could therefore

provide the industry with key knowledge for further implementation of

SFC.

2

Contents

ABSTRACT .......................................................................................................... 1

CONTENTS .......................................................................................................... 2

LIST OF PAPERS ................................................................................................. 3

ABBREVIATIONS ................................................................................................ 7

1. INTRODUCTION ............................................................................................... 9

1.1 SUPERCRITICAL FLUID CHROMATOGRAPHY ......................................... 10

1.1.1 GENERAL OVERVIEW .............................................................................. 10

1.1.2 APPLICATION OVERVIEW ....................................................................... 12

1.2 AIM OF STUDY ............................................................................................ 14

2. THEORY AND METHODOLOGY ................................................................... 15

2.1 MEASUREMENT OF MASS FLOW, PRESSURE AND TEMPERATURE .... 15

2.2 CALCULATION OF DENSITY AND METHANOL VOLUME FRACTION ..... 18

2.3 ADSORPTION ISOTHERMS AND THEIR DETERMINATION ...................... 20

2.4 SIMULATION OF CHROMATOGRAPHIC PROCESSES ............................. 24

2.5 DESIGN OF EXPERIMENTS ........................................................................ 25

3. DISCUSSION OF PAPERS ............................................................................ 26

3.1 WORD ANALYSIS OF PAPER I-V ............................................................... 26

3.2 PAPER I ........................................................................................................ 27

3.3 PAPER II ....................................................................................................... 31

3.4 PAPER III ...................................................................................................... 34

3.5 PAPER IV ..................................................................................................... 37

3.6 PAPER V ...................................................................................................... 42

4. CONCLUDING REMARKS ............................................................................. 47

6. SWEDISH SUMMARY .................................................................................... 50

5. ACKNOWLEDGEMENT ................................................................................. 54

7. LIST OF REFERENCES ................................................................................. 56

3

List of Papers

The thesis is based on the following papers, hereby referred to by their

Roman numerals I-V

I M. Enmark, P. Forssén, J. Samuelsson, T. Fornstedt,

Determination of adsorption isotherms in super-

critical fluid chromatography, J. Chromatogr. A.

1312 (2013) 124–133.

II M. Enmark, J. Samuelsson, E. Forss, P. Forssén, T.

Fornstedt, Investigation of plateau methods for ad-

sorption isotherm determination in supercritical

fluid chromatography, J. Chromatogr. A. 1354 (2014)

129–138.

III D. Åsberg, M. Enmark, J. Samuelsson, T. Fornstedt,

Evaluation of co-solvent fraction, pressure and

temperature effects in analytical and preparative

supercritical fluid chromatography, J. Chromatogr.

A. 1374 (2014) 254 – 260.

IV M. Enmark, D. Åsberg, A. Shalliker, J. Samuelsson, T.

Fornstedt, A closer study of peak distortions in su-

percritical fluid chromatography as generated by

the injection, J. Chromatogr. A. 1400 (2015) 131-139.

V M. Enmark, D. Åsberg, H. Nelander, K. Öhlén, M.

Klarqvist J. Samuelsson, T. Fornstedt. Evaluation of

Scale-up From Analytical to Preparative Super-

critical Fluid Chromatography Submitted to J.

Chromatogr. A. In revision September 2015.

Reprints of Paper I-IV were made with permission from Elsevier.

4

My contribution to Paper I-V were as follows:

I: I did most of the planning, performed all experiments, made some

of the calculations and wrote most of the article. II: I did most of the

planning, performed most of the experiments and calculations and

wrote most of the paper together with my co-authors. III: I did most

of the planning, performed some experiments together with Dennis

Åsberg, made parts of the calculations except DoE analysis and wrote

most of the article together with Dennis Åsberg. IV: I did most of the

planning, performed some experiments except those related to

viscous fingering which were made by Jörgen Samuelsson and

Andrew Shalliker. I made preliminary calculations and wrote most of

the article together with my co-authors. V: I made most of the plan-

ning, experiments and calculations. I wrote most of the article togeth-

er with my co-authors.

5

Manuscripts VI-XVII not included in the thesis:

VI M. Enmark, R. Arnell, P. Forssén, J. Samuelsson, K. Kaczmar-

ski, T. Fornstedt, A systematic investigation of algorithm

impact in preparative chromatography with experi-

mental verifications, J. Chromatogr. A. 1218 (2011) 662–

672.

VII M. Enmark, J. Samuelsson, T. Undin, T. Fornstedt, Charac-

terization of an unusual adsorption behavior of race-

mic methyl-mandelate on a tris-(3,5-dimethylphenyl)

carbamoyl cellulose chiral stationary phase, J. Chroma-

togr. A. 1218 (2011) 6688–6696.

VIII M. Enmark, J. Samuelsson, P. Forssén, T. Fornstedt, Enanti-

oseparation of omeprazole—Effect of different packing

particle size on productivity, J. Chromatogr. A. 1240

(2012) 123–131.

IX J. Samuelsson, M. Enmark, P. Forssén, T. Fornstedt, High-

lighting Important Parameters Often Neglected in

Numerical Optimization of Preparative Chromatog-

raphy, Chemical Engineering & Technology. 35 (2012) 149–

156.

X D. Åsberg, M. Leśko, M. Enmark, J. Samuelsson, K. Kaczmar-

ski, T. Fornstedt, Fast estimation of adsorption isotherm

parameters in gradient elution preparative liquid

chromatography. I: The single component case, J.

Chromatogr. A. 1299 (2013) 64–70.

XI D. Åsberg, M. Leśko, M. Enmark, J. Samuelsson, K. Kaczmar-

ski, T. Fornstedt, Fast estimation of adsorption isotherm

parameters in gradient elution preparative liquid

chromatography II: The competitive case, J. Chroma-

togr. A. 1314 (2013) 70–76.

6

XII M. Enmark, D. Åsberg, J. Samuelsson, T. Fornstedt, The Ef-

fect of Temperature, Pressure and Co-Solvent on a

Chiral Supercritical Fluid Chromatography Separa-

tion, Chrom. Today. 7 (2014) 14–17.

XIII C.M. Vera, D. Shock, G.R. Dennis, J. Samuelsson, M. Enmark,

T. Fornstedt, et al., Contrasting selectivity between HPLC

and SFC using phenyl-type stationary phases: A study

on linear polynuclear aromatic hydrocarbons, Micro-

chem. J. 119 (2015) 40–43.

XIV C.M. Vera, D. Shock, G.R. Dennis, J. Samuelsson, M. Enmark,

T. Fornstedt, et al., A preliminary study on the selectivity

of linear polynuclear aromatic hydrocarbons in SFC

using phenyl-type stationary phases, Microchem. J. 121

(2015) 136 – 140.

XV M. Leśko, D. Åsberg, M. Enmark, J. Samuelsson, T. Fornstedt,

K. Kaczmarski, Choice of Model for Estimation of Ad-

sorption Isotherm Parameters in Gradient Elution

Preparative Liquid Chromatography, Chromatographia

2015, DOI: 10.1007/s10337-015-2949-0

XVI M.E. Fridén, F. Jumaah, C. Gustavsson, M. Enmark. T.

Fornstedt, C. Turner, P. J.R. Sjöberg, J. Samuelsson,

Evaluation and Analysis of Environmentally Sustaina-

ble Methodologies for Extraction of Betulin from

Birch Bark with Focus on Industrial Feasibility, Green

Chemistry 2015, DOI: 10.1039/C5GC00519A

XVII J. Samuelsson, M. Leśko, M. Enmark, K. Kaczmarski, E. Forss,

J. Högblom ,T. Fornstedt, Optimal column length and par-

ticle size in batch preperative chromatography: - Case

enantiomeric separation of omeprazole and etirace-

tam. Manuscript in preparation.

7

Abbreviations

BINOL (1,1'-binaphthalene)-2,2'-diol

BPR Back Pressure Regulator

CFM Coriolis Mass Flow Meter

DoE Design of Experiments

ECP Elution by Characteristic Point

FA Frontal Analysis

HPLC High Pressure Liquid Chromatography

IM Inverse Method

LC Liquid chromatography

MS Mass Spectrometry

PDA Photo Diode Array

PP Perturbation Peak

RTM Retention Time Method

SFC Supercritical Fluid Chromatography

SMB Simulated Moving Bed

TSO trans-1,2-diphenyloxirane

TTBB 1,3,5-tri-tert-butyl-benzene

UHPLC Ultra High Pressure Liquid Chromatography

Symbols

α Selectivity

C Solute concentration in mobile phase

ρ Density

F Phase ratio

k Retention factor

K Association equilibrium constant

L Column length

M Molecular weight

�� Mass flow rate

q Solute concentration in stationary phase

qs Monolayer saturation capacity (Langmuir)

t0 Void time

t Time

Vi Partial molar volume

x Mole fraction, column position

8

9

1. Introduction

Chromatography is the unified name for techniques to separate

groups of molecules or individual molecules, peptides or proteins

from more or less complex mixtures. The purpose can be to identify

and/or quantify and is then called analytical chromatography, or to

purify and is then called preparative chromatography. In 2003 it was

estimated that 5 % of all chemical research involved chromatography

[1]. The technique is generally classified as liquid chromatography

(LC), gas chromatography (GC) or supercritical fluid chromatography

(SFC), depending of the type of mobile phase. Chromatography is an

incredible versatile technique which finds its applications in most

fields, making it an indispensable tool in the realm of analytical chem-

istry. For example, the pharmaceutical industries rely on chromatog-

raphy for quality control and assurance using analytical chromatog-

raphy as well preparative chromatography for purification.

The outcome of the liquid or supercritical fluid chromatographic sep-

aration process is governed by the interactions between the solute and

the mobile and stationary phase. In liquid chromatography, solvent

mixtures of water, alcohols or organic solvents are used in combina-

tion with silica based stationary phases. Current trends in liquid

chromatography entails the use of smaller particles, higher flows and

pressures to reduce analysis times while maintaining efficiency [2].

SFC is another trend which offers decreased analysis time in analyti-

cal chromatography and increased productivity in preparative chro-

matography [3–6].

10

1.1 Supercritical Fluid Chromatography

1.1.1 General overview

Supercritical Fluid Chromatography is a chromatographic technique

which utilizes a supercritical or subcritical fluid as main solvent. His-

torically, nitrous oxide, ammonia and carbon dioxide has been used,

but also the noble gases argon and xenon as well as other hydrocar-

bons [6,7]. Chromatography using supercritical fluids was first de-

scribed in 1962 by Klesper et al. [8]. In that work, the authors used

mono- and dichlorodifluoromethane as mobile phase to separate por-

phyrins. Today, the typical implementation of SFC uses carbon diox-

ide. Carbon dioxides enter the supercritical defined state at and be-

yond 304.12 K (~31 °C) and 74.5 bar.

There is a continuous ongoing debate about the name Supercritical

Fluid Chromatography which may be misleading for most current typ-

ical applications where liquid co-solvents are added to the carbon di-

oxide which means that a supercritical phase is never reached and

thus “Supercritical Fluid Chromatography” can be technically mis-

leading [7,9,10]. Most major manufacturers of chromatographic in-

struments today provide equipment built and optimized for utilizing

carbon dioxide [11]. There is little difference between HPLC or

UHPLC and SFC instrumentations in terms of equipment, besides a

modified pump to be able to pump chilled and compressed carbon

dioxide as well as a back-pressure regulator (BPR) to maintain a par-

ticular density of the mobile phase [11].

The reason why SFC is an important chromatographic technique is

related to the properties of the mobile phase [6,7]. Neat carbon diox-

ide or carbon dioxide with added co-solvent at sub- or supercritical

conditions has lower viscosity (µ), higher solute diffusion coefficients

(Dm) and higher compressibility than comparable liquids used for liq-

uid chromatography [6,7]. Some values of viscosities and adiabatic

compressibility’s are summarized in Table 1. Literature suggests so-

lute diffusion coefficients in neat carbon dioxide of a magnitude larger

than in liquids [7].

11

Table 1: Comparison of solvent properties; data adapted and modified from [9]

except for heptane [12]. Atmospheric pressure = 1.01 bar.

Solvent Tempera-

ture [°C]

Pressure

[bar]

Viscosity

[cP]

Adiabatic com-

pressibility

[10-5 bar-1]

CO2 20 138 0.09 37

CO2 40 138 0.06 82

CO2/MeOH

70/30 mol%

20 138 0.16 15

Water 20 1.01 1 4.6

Methanol 20 1.01 0.59 10

Acetonitrile 20 1.01 0.35 9.6

Heptane 20 1.01 0.41 11

The practical consequences of lower viscosity and higher solute diffu-

sion coefficients are the possibility of operating at higher linear veloci-

ties than liquid chromatography or utilizing longer columns to obtain

high efficiency. These consequences are beneficial for both analytical

and preparative SFC. Higher compressibility means that properties

such as density and temperature of the mobile phase can be altered by

changing the pressure, which in turn will affect the chromatographic

separation process. Furthermore, because there always exist a pres-

sure drop along the column, there will be gradients of these properties

along and across the column, something observed for both neat car-

bon dioxide or carbon dioxide with addition of methanol [13]. A sim-

ple schematic figure of the most important components in a SFC sys-

tem are summarized in Figure 1 together with the typical gradients

experienced in in SFC illustrated along the column.

12

Figure 1. Schematic figure of the major components in a SFC system. The occur-

rence and shape of typical gradients encountered in SFC are overlaid

along the column, from inlet to outlet, left to right in the figure. Gradients

of increasing volumetric flow, decreasing v% co-solvent, pressure, density

and temperature are also illustrated (not to scale).

The most common detector in SFC is UV but also evaporative light

scattering detectors, flame ionization detectors, polarimetric detectors

and mass spectrometry [3,4,6,7] are used.

1.1.2 Application overview

SFC using packed columns has historically been utilized for a wide

variety of applications. In general, SFC utilizes a less polar mobile

phase, either neat CO2 or CO2 modified with a more polar co-solvent,

classifying it as a normal phase separation technique [6]. The station-

ary phase is typically of porous silica type, for example bare silica,

diol, amino-propyl, 2-ethylpyridine or chiral stationary phases [3,5–

7], most of which are utilized in normal phase liquid chromatography.

Analytical SFC has been used for qualitative and quantitative chiral

and achiral pharmaceutical analysis, analysis of pesticides, fossil

fuels, polymers, peptides, natural products and more [4–7,14–16]. As

noted by Lesellier and West [4] the historically documented applica-

tions of SFC may not accurately represent how analytical SFC is used

today. Due to instrumental difficulties, for example detector noise due

13

to pumping and back-pressure regulation, SFC has had difficulties

achieving sufficient accuracy and precision for GMP qualification [3].

With the advent of new and improved instrumentation as well as col-

laboration between instrument manufacturers and industry, SFC can

and has been qualified for GMP operations [3,11]. However, detector

noise in SFC systems is still worse than in LC systems [4].

The analytical applications of SFC are small in comparison to the

dominating application; preparative chiral separations, see review

articles [3,4,6,7,17]. Since the first reported chiral separation using

SFC [18], it has grown into the dominating technique for obtaining

enantiomerically pure material in the discovery phase in the pharma-

ceutical industry [3,4,17,19]. Today, the pharmaceutical industry rou-

tinely uses SFC for high-throughput screening and purification of chi-

ral compounds [3,6,19]. Generally, g to kg amounts of compounds can

be obtained using instrumentation that delivers flows between 10

g/min to 1 kg/min, using 1-10 cm inner diameter columns [3,6,17]. In

almost all described applications of preparative chiral SFC it is used in

batch mode and typically by stacking injections [17]. Simulated Mov-

ing Bed (SMB) applications in SFC has been reported and investigated

by researchers but its complexity and cost of implementation has

made its use limited [17]. Typically UV detection is used for detection

and control of fraction collection but also MS.

The reason for the success of SFC in this field is because it can offer

advantages over normal phase liquid chromatography. This is mainly

due to the possibility of operating at higher linear velocities, i.e.

shorter cycle time and hence increased productivity (purified amount

per unit time). The cost benefit is not only related to decreased analy-

sis time but also the reduced cost of mobile phase, i.e. predominantly

carbon dioxide which can either be vented to the atmosphere or recy-

cled [3,6]. Furthermore, purified components can be collected in a

smaller volume, i.e. only the residual fraction of liquid co-solvent as

the depressurization of the mobile phase allows carbon dioxide to be

evaporated easily. This reduces the generation of liquid waste. Proper-

ties such as increased productivity and easier sample handling make

the large scale use of preparative SFC interesting [3,6].

14

The number of reported publications of large scale SFC separations

beyond the kilogram scale are limited especially because most such

applications are confidential operations in the pharmaceutical indus-

tries [3,19]. As far as the author knows, the largest currently as of

2015 actively used preparative SFC unit is a system built by Novasep

using a 20 cm inner diameter column by the company Johnson Mat-

they for a non-disclosed multi-component isomer separation problem

[20]. Even larger scale systems of 35 cm inner diameter column has

been reported but details remains unclear [3].

While the empirical evidence clearly has afforded SFC to become an

important chromatographic tool, the fundamental knowledge of the

technique is lacking. This was clearly laid out in the comprehensive

review of SFC by the now late Georges Guiochon and Abhijit Tarafder

[6] which was published around the same time the work on this thesis

was started. Many researchers of SFC had also described a lack of in-

terest in SFC by academy [4–6,11,21] with most applications taking

place in the pharmaceutical industries.

1.2 Aim of study

From decades of research in LC it is well know that being able to

quantify adsorption has been vital to the understanding and ad-

vancement of chromatography, hence Paper I-II were dedicated to

the topic of adsorption isotherm determination methods. Particular

focus was on the prerequisites for applying each method. While ad-

sorption isotherms provide a physical chemical description of the

separation process, a more rapid quantitative description in terms of

its retention, productivity or arbitrary response, can be achieved by

utilizing a Design of Experiments (DoE) approach. This was investi-

gated in Paper III. The fundamental aspects of sample injection in

SFC were investigated in Paper IV. Focus was put on quantitatively

describing peak distortions observed in SFC. Utilizing the knowledge

from Paper I-IV, the topic of scaling up SFC was investigated in Pa-

per V where a chiral separation system was scaled up from a 4.6 mm

inner diameter column to a 50 mm inner diameter column. The aim

was to investigate what was needed to achieve a predictable scale-up

in terms of maintaining the elution volume.

15

2. Theory and Methodology

2.1 Measurement of mass flow, pressure and temperature

Due to the compressibility of the mobile phase in SFC, the density and

hence the volumetric flow rate can vary considerably along the flow

path including the column in a SFC instrument [22,23]. If not meas-

ured, the volumetric flow can only be determined if the mass or molar

composition of carbon dioxide and co-solvent and pressure and tem-

perature are known. From these measurements the density of the

mobile phase can be calculated and hence the volumetric flow rate. It

must be emphasized that errors in either measurements or in the

Equation of State (EoS) used to calculate density still poses an uncer-

tainty in the calculation of the actual volumetric flow [22]. The basic

knowledge of volumetric flow, pressure and temperature were

deemed to be the most basic information needed in order to perform

reproducible fundamental research in SFC. This is in clear compari-

son to LC where any reproducible study using commercially available

instruments should be verified in terms of the accuracy of delivered

flow rate and system void volumes [24,25]. A more particular reason

would be because in order to utilize many dynamic methods of ad-

sorption isotherm determination, the volumetric flow rate needs to be

known and preferably constant. This can be verified by calculating the

volumetric flow rate at the inlet and outlet of the column.

Today, no commercial SFC instrument except for some preparative

instruments is equipped with mass flow regulation or read-out. Be-

cause of this it was decided to interface Coriolis mass flow meters

(CFM) which presents a reliable way of measuring fluid mass flow

[22,26]. The approach of measuring mass flow in using CFMs in SFC

was reported in the 1980’s by Schoenmakers and Uunk and was re-

vived in 2010’s by Tarafder et al. and later as well as by other authors

[22,27–29].

On the typical analytical commercial SFC instrument, there are no

more than a couple of pressure transducers, typically near the pump

and at the back-pressure regulator. Rajendran el al. showed how the

pressure drop was distributed in different sections of a custom built

SFC system [30,31], clearly demonstrating why more pressure trans-

16

ducers would be required to quantify pressure drop over the column,

but also the possibility of predicting the pressure drop at points not

measured.

Temperature control on commercially available SFC instrumentations

typically entails still or circulating air ovens with or without eluent

heat exchangers which can be set at a constant temperature. Due to

the adiabatic decompression of the mobile phase, the column can ex-

perience a significant temperature gradient with a lower temperature

at the outlet of the column. This was demonstrated by Poe et al.

[32,33] and later also by other authors [34,35]. Axial and radial tem-

perature gradients were also measured and modeled by Kaczmarski

and Poe [13]. While radial temperature gradients can lead to loss of

efficiency due to an uneven flow profile across the column, axial gra-

dients could change the local retention factor [36].

The SFC system used in Paper I-V was a Waters UPC2 system (Wa-

ters Corporation, Milford, MA, USA), which can be considered as the

third generation of commercially available SFC instruments [37,38].

The system was used in its basic configuration with a binary pump, a

2-column compartment, a Photodiode Array (PDA) detector and a

back-pressure regulator. It maintains a constant low temperature at

the pump which maintains carbon dioxide in its liquid state [7]. The

system has two pressure transducers, one at the pump and one at the

back-pressure regulator. For studies in Paper I-V except the metha-

nol mass flow in Paper I, total and methanol mass flow was meas-

ured using one (Paper I-II) or two (Paper III-V) Coriolis flow me-

ters. For all studies in Paper I-V the inlet and outlet pressure of col-

umn in use was measured using two absolute pressure transducers.

Surface inlet and outlet temperature of the column in use was moni-

tored with PT-100 resistance temperature detectors which were per-

manently attached to the column.

17

Figure 2. Schematic representation of how the external measurements of tem-

perature, pressure and mass flow were made in the SFC system. In (a) it is

shown how inlet and outlet pressure and temperature were measured. In

(b) it is shown how mass flow �� was measured outside the column using

the Coriolis mass flow meter. Adapted from Paper I.

18

2.2 Calculation of density and methanol volume fraction

To determine the volumetric flow rate at a specified point in the flow

path of an SFC instrument, measurements of mass flow, pressure and

temperature must be complemented with calculations of density. In

Paper I-V density was calculated using the Reference Fluid Thermo-

dynamic and Transport Properties Database (REFPROP) v 9.1 pro-

gram by the National Institute of Standards and Technologies [12].

REFPROP implements the EoS of Span and Wagner [39] and the mix-

ture density of carbon dioxide and methanol using the mixing rules of

Kunz et al. [40]. This approach has been reported by several authors

[13,41]. For a 88/12 carbon dioxide methanol molar mixture, the error

in density was estimated to less than 1.8 % at 40 °C and 150 bar [41].

In Paper I-V the average volumetric flow rate was used. This flow

rate corresponds to the average of the flow rate at the inlet and outlet

of the column. This approximation first assumes that the mass flow

and fractions are constant, which could be verified by continuous

measurements during experiments. It further requires that the pres-

sure and temperature gradient along the column is kept at a mini-

mum. This leads to the definition of near-isopycnic conditions, iso-

pycnic meaning constant density:

𝜌(𝑃𝑖𝑛𝑙𝑒𝑡 , 𝑇𝑖𝑛𝑙𝑒𝑡) ≈ 𝜌(𝑃𝑜𝑢𝑡𝑙𝑒𝑡 , 𝑇𝑜𝑢𝑡𝑙𝑒𝑡) ≈ 𝜌(𝑃𝑎𝑣𝑒𝑟𝑎𝑔𝑒, 𝑇𝑎𝑣𝑒𝑟𝑎𝑔𝑒)

(1)

Where 𝜌 is the density, P and T is the pressure and temperature at

the inlet and outlet of the column. From the average density, the aver-

age volumetric flow rate can be calculated.

19

Another important parameter is the volumetric percent methanol.

Because it is known to be the most important parameter controlling

retention it SFC [7,9,42–45] it was imperative that it was verified

properly. To calculate this we need to estimate the molar volume (V)

of carbon dioxide and methanol [46]:

𝑉 =𝑀

𝜌

𝑀 = 𝑥𝐶𝑂2𝑀𝐶𝑂2 + 𝑥𝑀𝑒𝑂𝐻𝑀𝑀𝑒𝑂𝐻

(2)

Where M is the molecular weight of the fluid, ρ is the density of the

fluid and x is the mole fraction. To estimate the volumetric fraction,

the partial molar volume (Vi) needs to be calculated:

𝑉𝐶𝑂2 = 𝑉 + 𝑥𝑀𝑒𝑂𝐻

𝜕𝑉

𝜕𝑥𝐶𝑂2

𝑉𝑀𝑒𝑂𝐻 = 𝑉 − 𝑥𝐶𝑂2𝜕𝑉

𝜕𝑥𝐶𝑂2

(3)

From the calculated molar volume and measured mass flows �� of

carbon dioxide and MeOH the volumetric fraction of MeOH can be

calculated:

𝑣%𝑀𝑒𝑂𝐻 =

��𝑀𝑒𝑂𝐻

𝑀𝑀𝑒𝑂𝐻𝑉𝑀𝑒𝑂𝐻

��𝑀𝑒𝑂𝐻

𝑀𝑀𝑒𝑂𝐻𝑉𝑀𝑒𝑂𝐻 +

��𝐶𝑂2

𝑀𝐶𝑂2𝑉𝐶𝑂2

∙ 100

(4)

The molar fractions were estimated using the measured methanol and

total mass flow. The partial derivatives ∂V/∂x were numerically esti-

mated [47].

20

2.3 Adsorption isotherms and their determination

One of the aims of the thesis was to investigate what methods of ad-

sorption isotherm determination could be transferred from liquid

chromatography. The adsorption isotherm refers to the quantitative

description of a solute’s adsorption equilibria between the moving

phase (mobile phase) and the stationary phase [48]. Adsorption iso-

therms are typically classified into different types. The type I adsorp-

tion models (Langmuir, Tóth, Jovanovicí) assumes homogenous or

heterogeneous adsorption energy distributions. The Langmuir ad-

sorption isotherm can be expressed as:

𝑞 = 𝑞𝑆𝐾𝐶

1 + 𝐾𝐶

(5)

Where q is the concentration of the solute in the stationary phase, qs

and K is the monolayer saturation capacity and association equilibri-

um constant and C the concentration of solute in the mobile phase.

The adsorption isotherm q is related to the retention time, tR of a di-

lute injection:

𝑡𝑅 = 𝑡0 (1 + 𝐹𝑑𝑞

𝑑𝐶|𝐶=0

)

(6)

Where t0 is the void time and F the phase ratio which is the ratio of

the stationary phase volume and the mobile phase volume. The ad-

sorption isotherm is a key property for fundamental understanding,

simulation and optimization of many chromatographic separation

processes [48–56].

The retention time is related to the retention factor (k) and selectivity

(α) as follows:

𝑘 =𝑡𝑅 − 𝑡0𝑡0

𝛼 =𝑘2𝑘1, 𝑘2 ≥ 𝑘1

(7)

21

An example of a bi-Langmuir adsorption isotherm (sum of two Lang-

muir terms) and the corresponding experimental and simulated elu-

tion profile is shown in Figure 3.

Figure 3. Figure illustrating the correlation between the elution profile (a) and

the adsorption isotherm (b) of a 400 µL injection of 30 g/L omeprazole on

an 250x4.6 10 µm amylose-based chiral stationary phase (CSP) in normal

phase LC. In (a) the black line is the experimental profile and grey a simu-

lated profile while in (b) the black line represents the adsorption isotherm

of the first eluting enantiomer of omeprazole while the grey represents the

later eluting. Modified from Paper VII.

Methods to determine adsorption isotherms in liquid chromatog-

raphy are well characterized and understood [48,57]. One method is

frontal analysis (FA) which is considered a reference method [48,57].

The FA method uses the breakthrough volumes of a number of plat-

eau reaching injections of increasing concentrations to determine

points on the adsorption isotherm. Another is the perturbation peak

method (PP) which has been shown to be as accurate or even more so

as FA [48,58–62].

The perturbation peak method exploits the theoretical relation be-

tween the retention time tR,i of a small injected deficiency or excess

concentration pulse on a concentration plateau Ci and the slope of the

adsorption isotherm for this plateau concentration dqi/dCi.

𝑡𝑅,𝑖 = 𝑡0 (1 + 𝐹𝑑𝑞𝑖𝑑𝐶𝑖

)

(8)

22

By establishing a number of plateaus and measuring the retention

times the adsorption isotherm q can be determined by regression.

The PP method can be used to determine single or multicomponent

competitive adsorption isotherms.

Figure 4. Illustration of the principles of the perturbation peak method (PP).

The top subplot shows the retention times of different perturbation peaks,

beginning at the zero plateau tR,1 where the column is equilibrated with

pure mobile phase. The bottom subplot shows the relation between the

slope of adsorption isotherm dqi/dCi and the retention time of the pertur-

bation peak tR,i on a plateau concentration Ci.

23

Other methods frequently utilized are the Elution by Characteristic

Points (ECP) Method [48,63,64], Retention Time Method [48,65–

67], Inverse Method (IM) [54,68,69]. The ECP, RTM and IM all ex-

tracts adsorption data from overloaded elution profiles. The ECP uses

the retention times of concentration zones of the diffuse part of elu-

tion profiles which correlates to the slope of the adsorption isotherm

for each concentration.

The RTM uses the retention factor and the retention time of the fronts

of elution profiles for injections of different volume and/or concentra-

tion. The difference between experimentally obtained front retention

times and calculated times using an adsorption isotherm model is

then minimized until valid adsorption isotherm parameters are

found.

Both the ECP and RTM methods are derived from the ideal model of

chromatography [48] why the error in their application will increase

with decreasing column efficiency. The IM is based on iteratively solv-

ing a column mass balance model, for example Equation 9, where the

elution profile of the solute(s) is described by an adsorption isotherm.

The parameters of the adsorption isotherm are changed such that

eventually the simulated and experimental chromatograms overlap.

The adsorption isotherm parameters for which this occurs is then said

to describe the separation system.

The number of publications concerning the determination of adsorp-

tion isotherms in SFC is still small but growing [70–75]. All adsorp-

tion isotherm determination methods require precise control and

knowledge of volumetric flow, temperature, system and column void

volumes [24,25,76].

24

2.4 Simulation of chromatographic processes

When the adsorption isotherm of a solute is known, it is possible to

model the separation process in terms of convection, dispersion and

adsorption and simulate the propagation of a solute through a column

[48]. One model that is often applied to small molecule separation

systems with sufficiently high column efficiency is the so called Equi-

librium-Dispersive (ED) model [6] often referred to as “the simplest

realistic model of chromatography” [6]. The choice of column model

must however be made not to oversimplify, or misunderstandings of

the process can be made [77]. The ED model can be formulated as fol-

lows.

𝜕𝐶𝑖(𝑥, 𝑡)

𝜕𝑡+ 𝐹

𝜕𝑞𝑖(𝑥, 𝑡)

𝜕𝑡+ 𝑢

𝜕𝐶𝑖(𝑥, 𝑡)

𝜕𝑥= 𝐷𝑎,𝑖

𝜕2𝐶𝑖(𝑥, 𝑡)

𝜕𝑥2

0 ≤ 𝑥 ≤ 𝐿, 𝑡 ≥ 0, 𝑖 = 1,2, … , 𝑛

𝐶𝑖(𝑥, 0) = 𝐶𝑖,0, 𝐶𝑖(0, 𝑡) = 𝜑𝑖(𝑡)

(9)

where Ci(x, t) and qi(x, t) are the concentrations of substance i at time

t and position x in the mobile and stationary phase, F is the phase

ratio, u is the mobile phase linear velocity and 𝐷𝑎,𝑖 =𝐿∙𝑢

𝑁𝑖 is the lumped

mass transfer and dispersion coefficient, Ni is the column efficiency.

Φi is the injection profile. The model can be solved numerically by

many different approaches [68,78,79] and [VI]. The number of publi-

cations in SFC using simulations in an effort to predict experimental

data limited [72,73,80] and [I-IV].

25

2.5 Design of Experiments

The concept of Design of Experiments, DoE, refers to a methodology

to design a minimal number of experiments to obtain a maximum

amount of information about the studied system [81]. Paper III and

V utilized a full factorial design with three factors; methanol level,

pressure and temperature. The response which was studied was the

retention factor (k), selectivity (α) and productivity (purified amount

per unit time). From literature it is known that the retention factor

has a quadratic relationship with the methanol content [42–45]. It is

also known that there are interactions between factors such as pres-

sure and temperature in how they affect retention. Based on this a two

level design would be insufficient [81]. A three level design which var-

ied the factors at a low, middle and high was selected. This means at

least 33 runs plus additional center point runs.

The regression model used to fit the responses is on the following

form:

𝑌 = 𝑝0 + 𝑝1𝐶 + 𝑝2𝑃 + 𝑝3𝑇 + 𝑝4𝐶𝑃 + 𝑝5𝐶𝑇 + 𝑝6𝑃𝑇 + 𝑝7𝐶2 + 𝑝8𝑃

2 + 𝑝9𝑇2 (8)

Where Y is the response (retention factor, selectivity or productivity),

p0-p9 constants, C the methanol concentration (v%), P pressure and T

temperature. Coefficients were estimated using multiple linear regres-

sion and the regression models were evaluated using analysis of vari-

ance (ANOVA). All calculations were performed using MODDE ver-

sion 7 (Umetrics, Umeå, Sweden).

26

3. Discussion of papers

3.1 Word analysis of Paper I-V

By analyzing the occurrence of words in Paper I-V, the superficial

reader can understand that the focus of this thesis has been on ad-

sorption and how to determine the isotherms. Methanol has exclu-

sively been used as co-solvent. Temperature and pressure has been

investigated.

Figure 5. Word analysis of Paper I-V presented as a word cloud where the size

of the font is correlated to the frequency of occurrence of certain words.

Bigger font means more frequent occurrence. Data generated using Word

Cloud Python (https://github.com/amueller/word_cloud, accessed Sep-

tember 2015)

27

3.2 Paper I

The motivation of this paper was the lack of knowledge about how to

characterize adsorption in SFC. The number of previous works [70–

73] was limited and in general lacked a thorough discussion about the

applicability of the methods used. Three main reasons were suspected

to be (i) the lack of fundamental SFC understanding, (ii) the lack of

commercial SFC systems properly evaluated for fundamental studies

and (iii) the previous relatively low interest in the SFC area. A few no-

table studies could be found. Depta et al., Lübbert et al., Ottiger et al.,

Wenda et al. and Bao et al. used the FA, PP, IM, IM and ECP method

respectively to determine adsorption isotherms of different solutes in

mixed carbon dioxide/co-solvent mobile phases [70–73,80]. Some

like Ottiger et al. and Wenda et al. had a discussion about density gra-

dients; others like Lübbert et al. approximated mixed phase densities

with that of neat carbon dioxide.

In the planning of the study it was decided to evaluate four different

methods, the Perturbation Peak method (PP) [48,58,61,82,83], the

Elution by Characteristic Points (ECP) [48,63,64], the Inverse Meth-

od (IM) [54,68,69] and the Retention Time Method (RTM)

[48,65,66,84]. As model system, the retention of 1,2-Dihydro-1,5-

dimethyl-2-phenyl-3H-pyrazol-3-one (antipyrine) on a bare silica

column was studied. The set experimental conditions were 90/10 v%

CO2/methanol at 150 bar and 35 °C. The set volumetric flow was 1

mL/min.

Particular focus was paid to the prerequisites for applying each meth-

od. The basic criterion of any experiment must of course be to know

the actual conditions. As was explained in Section 2.1, pressure, tem-

perature and mass fraction affects both density and volumetric com-

position of the eluent. Studies have demonstrated the importance of

verifying and estimating the pressure drop [30], the variations of

mass and volumetric flow of commercial systems [28] and combined

pressure, temperature and density drop in SFC [13].

28

Table 2. Temperature (T), pressure (P), average volumetric flow (utotal) and den-

sity (ρ) at the column inlet and outlet together with mass flow of CO2

(��𝐶𝑂2) and of MeOH(��𝑀𝑒𝑂𝐻). Reproduced and reprinted with permission

of Elsevier from Paper I.

T

[°C]

P

[bar]

utotal

[mL/min.]

ρ

[g/mL]

mCO2

[g/min.]

��𝑀𝑒𝑂𝐻

[g/min.]

Column

inlet

34.5 161 1.07 0.860

0.81 0.11 Column

outlet 34.2 156 1.07 0.858

Average 34.4 158.5 1.07 0.859 0.81 0.11

The Waters UPC2 system was modified by interfacing pressure trans-

ducers and temperature probes on the column inlet and outlet. Car-

bon dioxide mass flow was measured using a Coriolis mass flow me-

ter. Methanol mass flow was measured by weighing the amount

pumped over time.

In Table 2 the measured and calculated conditions are presented.

Based on this data it was concluded that experiments conducted could

be considered near isothermal, isobaric and isopycnic. The conse-

quence of this is that retention will not vary along the column and

both the PP, RTM, ECP and IM can be applied directly. Perturbation

peak experiments were performed by pumping 10 stock solutions be-

tween 10 and 150 g/L in the 10 v% co-solvent line and perturbing the

system with stock solution diluted 10 times. Perturbations on zero to

19.2 g/L plateaus are presented in Figure 6.

29

Figure 6. Perturbation peak results: in (a) an overlay of four 3 µL injections of 1

g/L antipyrine in neat MeOH in a stream of CO2/MeOH, in (b) an overlay

of four 3 µL injections of 1 g/L antipyrine on a plateau of 1.3 g/L, in (c) an

overlay of four 3 µL injections of 5 g/L antipyrine on a plateau of 6.4 g/L

and in (d) an overlay of four 3 µL injections of 13 g/L antipyrine on a

plateau of 19.2 g/L. Reproduced and reprinted with permission of Elsevier

from Paper I.

ECP experiments were performed by injecting 50 µL of 250 g/L anti-

pyrine while RTM and IM experiments by injecting different volumes

of varying concentration.

The corresponding bi-Langmuir adsorption isotherms are presented

in Figure 7 where it is apparent that the data obtained by ECP, RTM

and IM are very similar and that PP data is different. Using the ob-

tained data to simulate overloaded elution profiles shows that either

ECP, RTM or IM can be used to predict this data if using the Equilib-

rium-Dispersive model of chromatography. Different reasons to why

the data obtained using the PP data could not be used to predict elu-

tion profiles within the investigated concentration range were dis-

cussed. One likely explanation was that the methanol volume fraction

30

differs at each plateau. The conclusion of the study was therefore that

the PP method is not a suitable method for SFC. Instead, methods us-

ing the elution profiles should be preferred.

Figure 7. Adsorption isotherms obtained by the Perturbation Peak method (PP,

solid black), Elution by Characteristic Point method (ECP, dashed black),

Retention Time Method (RTM, dash-dotted black) and the Inverse Method

(IM, solid gray). The inset shows the Perturbation Peak raw slope data

(symbols) and the best fit of the Bi-Langmuir adsorption isotherm (black

line). Reproduced and reprinted with permission of Elsevier from Paper

I.

31

3.3 Paper II

As a consequence of the observations in Paper I about the Perturba-

tion Peak method (PP) it was decided to perform an in depth study of

why adsorption data obtained using the PP method could not describe

elution profiles as well as data obtained using ECP, RTM or the IM.

The main hypothesis was that since the instrument pumps co-solvent

at a constant rate, the actual methanol content will decrease as con-

centration of antipyrine increases. Hence, each perturbation peak cor-

responds to a point on the adsorption isotherm for a particular meth-

anol volume fraction. The actual volume fraction methanol can be cal-

culated according to Equation 9 where mtot is the weight of a volume

Vtot containing mantipyrine dissolved in methanol with density ρMeOH. So-

lutions of concentrations of up to 100 g/L antipyrine were prepared

and their methanol volume fraction calculated, see Table 3.

𝑣% =𝑚𝑡𝑜𝑡 −𝑚𝑎𝑛𝑡𝑖𝑝𝑦𝑟𝑖𝑛𝑒

𝜌𝑀𝑒𝑂𝐻𝑉𝑡𝑜𝑡∙ 100

(9)

The prepared concentrations were chosen in such a way that the devi-

ation could be compensated by the pump which has a minimum

change of 0.1 v%. For example, if 10 v% methanol plateau is sought

and the concentration of antipyrine in the co-solvent is about 60 g/L

the volume fraction methanol is about 95 v% and the system needs to

pump 10.5 v% to maintain 10 v% methanol volume fraction.

Table 3 Experimentally obtained values of the MeOH volume fraction in samples

with different concentrations of antipyrine at 22 °C. Reproduced and re-

printed with permission of Elsevier from Paper II.

Antipyrine

[g/L]

0 13.52 25.59 37.43 49.05 60.46 71.67 82.67 93.47

MeOH volume

fraction [-]

1 0.990 0.981 0.971 0.963 0.954 0.945 0.937 0.928

When plotting the retention times of each perturbation peak in theory

they should overlap on the rear slope of an overloaded elution profile

at the same concentration. In Figure 8 it is apparent that the pertur-

32

bation peaks obtained when pumping the same percentage co-solvent

irrespectively of the antipyrine concentration does not overlap but

instead predicts longer retention of the equivalent concentration. The

deviation does however increase with increasing antipyrine concen-

tration on each plateau. When maintaining the same methanol vol-

ume fraction on each plateau by compensating by changing the pump

flow, the correspondence is better but still not exactly following the

diffuse rear. The conclusion was therefore that the initial hypothesis

could not be confirmed. What was clarified using the ECP method was

the strong dependence of antipyrine retention to the volume fraction

methanol, see Figure 9.

Figure 8. In (a) experimental elution profiles for injections of 250 g/L antipy-

rine at different volumes using an instrumental setting of 10 v% MeOH.

The symbols are the perturbation peak retention times obtained from per-

turbation injections on established antipyrine plateaus. The circles using

the “standard” approach with set modifier fraction of 10 v% MeOH and

the squares using the “compensated” approac. In (b) ECP adsorption iso-

therm slopes (solid line), symbols are experimental data from perturba-

tion peaks at an instrumental setting of 10 v% MeOH (circles) or using

compensated MeOH fractions (squares) with fitted bi-Langmuir model

(dashed and dotted line). In (c) the adsorption isotherms obtained using

the ECP (solid grey), PP uncorrected (dotted), PP corrected (dashed) and

FA (circles) methods. Reproduced and reprinted with permission of Else-

vier from Paper II.

33

Figure 9. In (a) the natural logarithm of the retention factor for antipyrine (k)

is plotted for 8, 8.5, 9.1, 9.6, 10, 10.5, 11, 11.5 and 12 v% MeOH in the eluent

(circles) and the line is the best fit of the data to Eq. (8). In (b) the adsorp-

tion energy distributions calculated for adsorption data determined using

the ECP method for the same MeOH compositions as in figure (a). In (c)

the bi-Langmuir model fit to the ECP adsorption data at the same eluent

compositions as for figure (a). Reproduced and reprinted with permission

of Elsevier from Paper II.

34

3.4 Paper III

Understanding retention in SFC is vital to the design and optimiza-

tion of separation systems and to evaluate method to characterize ad-

sorption and retention and adsorption was therefore the purpose of

Paper I-II. The purpose of Paper III was to propose and evaluate a

methodology to study the convoluted effects of pressure [7,85], tem-

perature [6,43,86,87] and methanol content [7,9,42–45] on the sepa-

ration of two small neutral racemic compounds on a chiral column

(Kromasil Cellucoat). This is important aspect besides studying how

the stationary phase effects retention in SFC [86,88–93]. An explora-

tory Design of Experiments (DoE) approach was used to study the

main factors and their possible interactions. Based on the observa-

tions from Paper I-II, particular attention was directed to account

for the discrepancies between system set and actual values inside the

column as well as maintaining close to isobaric, isothermal and iso-

pycnic conditions. To calculate the retention factor and selectivity, the

void volume (void time multiplied by the volumetric flow) of the col-

umn [94] must be determined.

Figure 10. The void volume determinations using pycnometry (dashed line),

and injections of nitrous oxide, 1,3,5-tri-tert-butyl-benzene (TTBB) and

MeOH (bars), are presented for 2.5, 5.0 and 7.5 v% MeOH. The volumetric

flow rate was set to 0.7 mL/min but the elution volume was calculated

from the actual estimated volumetric flow rate for each experiment from

the measured mass flow and density of the mobile phase. The back pres-

sure was set to 160 bar and the temperature was set to 30°C. Reproduced

and reprinted with permission of Elsevier from Paper III.

35

Based on a limited number of investigations dedicated to this, three

dynamic and one static method were evaluated [95,96]. In Figure 10

it is apparent that the retention volume of nitrous oxide [95] is con-

stant between 2.5 to 7.5 v% methanol while the same volume for both

methanol and 1,3,5-tri-tert-butyl-benzene (TTBB) [97,98] varies.

Based on this observation, the void volume derived from the elution

time of nitrous oxide was used in calculating the retention factor.

A three-level full factorial design was used in order to accurately mod-

el the response within the boundaries of the design, also with the an-

ticipation of both quadratic and interaction terms. The design was

spanned by 120, 160 and 200 bar, 24, 30 and 36°C, and 2.5, 5 and 7.5

v% methanol for trans-1,2-diphenyloxirane, (TSO) or 15, 20 and 25

v% methanol for (1,1'-binaphthalene)-2,2'-diol (BINOL). Note that

these values correspond to instrument set values. Retention factors

were calculated for each point in the design region. Analysis of data

showed that methanol was the most important factor controlling re-

tention of both enantiomers of BINOL and TSO as is seen by the nega-

tive coefficient bars in Figure 11. Additionally, a quadratic methanol

term was found to be the third most important factor. The second

most important factor for both enantiomers of TSO and BINOL was

pressure, also indicated by negative bars in Figure 11. The selectivity

for TSO was found to be most affected by methanol content, while for

BINOL temperature. Both pressure and temperature had opposite

effect on the selectivity for BINOL and TSO.

At each point in the design space, injections of 12-22 µL of 40 g/L

TSO were made. The maximum injection volume fulfilling 100 % yield

and purity was used to calculate the productivity [48,99] for the col-

lection of either enantiomer in so called stacked injection mode. Anal-

ysis of variance revealed that the most important factor was the meth-

anol content followed by temperature, for both an increase gave in-

creased productivity, see Figure. 12.

36

Figure 11. Centered and normalized coefficients from the model fit for the first

and second retention factor, respectively, for: a) TSO and b) BINOL. The

error bars represent the 95% confidence interval of the coefficients. Re-

produced and reprinted with permission of Elsevier from Paper III.

Figure 12. a) Centered and normalized coefficients with 95 % confidence inter-

val from the model fit to the productivity for the optimum touching-band

chiral separation of TSO is plotted. In b) the productivity is plotted as a

function of amount of modifier in the eluent and the temperature. Repro-

duced and reprinted with permission of Elsevier from Paper III.

In conclusion, the study presented important insights into how to

properly study analysis of variance for important factors in SFC. The

exploratory results obtained can be used for method optimization or

as foundation to fundamental studies investigating the underlying

mechanisms.

37

3.5 Paper IV

All column based chromatography is based on the application or in-

jection of sample into the mobile phase stream. To minimize distor-

tions of elution peaks the injection solvent in LC should be identical to

the eluent or peak deformations can follow [100–102]. This includes

properties such as solvent composition and pH. In SFC, the current

practice is to inject in mixed stream. This means that different diluent

will always be injected, either with dissimilar or similar elution

strength as the mobile phase. The purpose of the paper was to investi-

gate and quantify the origins of peak deformations caused by the in-

jection, a well know phenomena albeit investigated by few authors in

SFC [103–105].

In SFC there are at least two principally different injection methods

[17,106]. One is the mixed stream and the other the modifier stream

injection, see Figure 13. For both methods, a sample loop is filled with

sample and eventually transferred onto the column. Mixed stream

injection is similar or identical to how sample is injected in liquid

chromatography, the total flow of carbon dioxide and co-solvent

transports the sample plug onto the column, see Figure 13(a). For

modifier stream, it is the stream of co-solvent which transfers the

sample from the loop, to the mixing vessel and finally onto the col-

umn.

Figure 13. Schematic figure illustrating instrumental plumbing for (a) mixed stream

injection and (b) modifier stream injection. In all experiments the mixer corre-

sponds to a 250 µL passive mixer in the Waters UPC2 system. Reproduced and

reprinted with permission of Elsevier from Paper IV.

38

Initial experiments were performed to qualitatively investigate the

shape of the elution profiles of injections performed in mixed and

modifier stream injections. For this purpose, two neutral probes were

studied. 0.5, 30 and 75 µL of 0.2 and 100 g/L antipyrine and 0.5 and

20 g/L 2-Hydroxy-N-phenylbenzamide (salicylanilide) were injected

separately in mixed and modifier stream injections. Studying antipy-

rine in Figure 14(a) it is apparent that severe peak distortion takes

place for injection volumes over 5 µL. Only when injecting 5 µL in

mixed and modifier stream mode is the elution profile identical. It is

apparent that elution profiles obtained in mixed stream mode shifts

the center of mass to shorter retention. The same phenomena are ob-

served for injections of more concentrated solutions, but more sharp-

ening of the front is observed.

Figure 14. Comparisons between mixed (solid black line) and modifier stream

(solid grey line) injections of antipyrine and salicylanilide. In the top row

5, 30 and 75 µL injections of antipyrine. In (a) 0.25 g/L and in (b) 100

g/L. In the bottom row 5, 30 and 75 µL injections of salicylanilide. In (c)

0.5 g/L and in (b) 100 g/L. Reproduced and reprinted with permission of

Elsevier from Paper IV.

39

To investigate the correlation of distortion with the diluent, 0.2 g/L

antipyrine was prepared in methanol, ethanol and toluene. 75 µL in-

jections of the sample were made and it could be noted that using

methanol as diluent gave the most severe peak distortion, followed by

ethanol and toluene, see Figure 15(a). A similar experiment was per-

formed in NPLC, where the same column was used but antipyrine was

eluted using 15/85 v% ethanol/heptane. 0.2 g/L antipyrine was pre-

pared in the eluent, ethanol and isopropanol. Injections showed that

the least peak distortion was observed using the eluent, followed by

isopropanol and ethanol. The important conclusion from these exper-

iments are that the peak-distortion phenomena likely are similar in

SFC and NPLC and related to the eluent strength of the injection sol-

vent compared to that of the mobile phase

Figure 15. Observations of the peak distortion of antipyrine and salicylani-

lide when injected in SFC and NPLC mode. In (a) experiments conducted

in SFC mode with 75 µL ca 0.2 g/L antipyrine injected in toluene (grey),

ethanol (dashed grey) and methanol (black). Running conditions were

90/10 v% CO2/MeOH. In (b) experiments conducted in NPLC mode with

75 µL of ca 0.2 g/L antipyrine injected in 2-propanol (dashed-grey), etha-

nol (black) and 85/15 v% heptane/EtOH (gray). Running conditions 85/15

v% heptane/EtOH. In (c) and (d) the equivalent injections of 0.2 g/L salic-

ylanilide. Reproduced and reprinted with permission of Elsevier from

Paper IV.

40

If the elution strength that is the most important factor contributing

to the peak distortion, the distortion should be able to be simulated

using the Equilibrium-Dispersive model of chromatography. This also

requires that the adsorption isotherm has a quantifiable dependency

on the methanol content in the eluent. This dependency was investi-

gated by injecting 5 µL of 300 g/L antipyrine at different methanol

levels from 7.2 to 100 % and then extracting the adsorption data by

using the ECP method. While the monolayer saturation capacity de-

creases with increasing methanol content, the association equilibrium

constant has a more complex relationship. To quantitatively describe

the dependency, a cubic polynomial was used to describe the relation-

ship of both the monolayer capacity and the association equilibrium

constant.

Using the determined adsorption isotherm and its dependency of the

methanol content as well as the broadening of the injected methanol

plug, it was possible to quantify the observed peak distortion for dif-

ferent injection volumes at low concentration of analytes, see Figure

16. Simulations did not predict the experimental outcome exactly

which was attributed to the possibility of occurrence of viscous finger-

ing [107–110], a phenomena well know from liquid chromatography.

Liquid chromatography experiments mimicking the viscosity ratio

observed in SFC was undertaken, strengthening this hypothesis.

41

Figure 16. Experimental (a) and simulated (b, c) elution profiles of antipyrine is

plotted. In (a) experimental elution profiles for 2, 5, 10, 20, 30, 60 and 75

µL 0.25 g/L antipyrine in eluent containing 7.2 v% MeOH are plotted. In

(b) the corresponding simulated injections when the methanol plug is not

retained and (c) same as in (b) but now the methanol is retained. Repro-

duced and reprinted with permission of Elsevier from Paper IV.

42

3.6 Paper V

One of the most important applications of SFC today is preparative

chiral chromatography where g to kg amounts of compounds needs to

be purified [3,6,17,19]. Liquid chromatography has been the de facto

standard to obtain enantiomerically pure material in early pharma-

ceutical development [99,111]. Chromatography is a very scalable

technology which has been proven to reliably scale up a million times

[99]. This merit of chromatography means that method development

and optimization can be carried out in small laboratory scale where

the consumption of materials and energy is minimized and subse-

quently scaled up to a scale which satisfies the needed throughput.

Although preparative SFC is such an important application, it remains

relatively little investigated, in particular concerning the topic of scal-

ing up [112]. Because of the compressibility of the mobile phase and

the known correlation between retention shifts and the gradients

caused by this compressibility any difference in column length or par-

ticle size between the small and large scale system will hinder reliable

scale-up. Tarafder et al. has suggested the concept of average density

matching to compensate for such differences [112]. Furthermore, as

there are different manufacturers of small and large scale systems,

their design in terms of flow regulation, mixing of carbon dioxide and

co-solvent and system pressure contribution, the situation becomes

complicated.

The topic of Paper V was to investigate if a reliable i.e. preserved elu-

tion volume scale-up could be made from one small scale system (Wa-

ters UPC2) to one preparative system (Novasep SuperSep 600) by us-

ing a 250x4.6 mm column scaled to a 250x50 mm packed with the

same chiral stationary phase. An overview of how this approach was

made is presented in Figure 17.

43

Figure 17. Schematic overview of how the scale-up from 4.6 mm inner diameter

column used in the analytical Waters UPC2 to the 50 mm column in the

preparative NovaSep SuperSep 600 system was performed. The first step

includes setting the desired conditions on the UPC2 system followed by

measuring its performance. Mass flow is then set according to the scale-

up factor (50/4.6)2 on the SuperSep 600 as well as other parameters

measured on the UPC2 system. The preparative system was then tuned to

obtain the identical performance as the UPC2 system.

A separation system consisting of the chiral resolution of trans-1,2-

diphenylethylene oxide (TSO) was chosen as model system. The chiral

stationary phase was chosen based on what column was available at

AstraZeneca R&D Mölndal [19] and at the same time could be packed

in a smaller diameter column using the same batch. The first step was

to investigate which parameter had the greatest impact on the reten-

tion factors and selectivity of the enantiomers of TSO, using the DoE

approach of Paper III. The methanol level was found to be the most

44

important parameter for both retention factors, followed by pressure

and temperature. Selectivity was most affected by temperature, fol-

lowed by methanol level and pressure. The conclusion was that a

scale-up must preserve all parameters, but most importantly metha-

nol content. Because the retention factor only corresponds to the ini-

tial slope of the adsorption isotherm, it was decided to verify the con-

clusion using high concentration and high volume injections on the

50 mm inner diameter column. The experiments could well confirm

the DoE conclusion, see Figure 18.

Figure 18. Figure showing large scale elution profiles at a 20 % positive or

negative change of methanol fraction, pressure and temperature after the

injection of 4 mL of 40 g/L TSO. The solid gray line is the reference chro-

matogram acquired at 8.5 wt.% methanol, a BPR of 132 bar and tempera-

ture at 32 °C. In (a) the methanol content is increased 10.2 wt.% (dotted

line) or decreased to 6.8 wt.% (dashed line). In (b) the BPR pressure is in-

creased to 158 bar (dotted line) or decreased to 106 bar (dashed line). In

(c) the temperature is changed to 38.4 °C (dotted line) or to 25.6 °C

(dashed line). The preparative SuperSep 600 instrument system was used.

Reproduced and reprinted from Paper V.

45

The core of the approach of scaling-up was to transfer the exact condi-

tions in terms of flow, methanol content, pressure and temperature

between the 4.6 and 50 mm column. Because the SuperSep 600 is

controlled using built in Coriolis mass flow meters, which also acts as

an automatic control system of the pumps, the mass flow on the UPC2

system must be determined. This was done as described in Paper I-

IV. The determined mass flow and methanol mass fraction on the

UPC2 system could then directly be scaled by a factor of 118 to the Su-

perSep 600 system. This is the column volume increase going from

inner diameter 4.6 mm to 50 mm. To match the pressure drop along

the column in both systems, inlet and outlet pressures were moni-

tored and the back-pressure on both systems adjusted to a value for

which the pressure gradient matched. The same approach was made

using the surface temperature measurements.

Differences in system void volume were accounted for by matching

the retention time of TTBB on both systems. Having matched the col-

umn length, packing material, mass flow and fraction methanol, pres-

sure and temperature gradient on both systems, injections of identical

load were made. Their analytical retention volumes were found to

match well, se Figure 19. However, distinct fronting of the overloaded

elution profiles obtained on the 50 mm column was observed. At pre-

sent, no confirmed explanation to this observation exists. Neverthe-

less, the conclusion from Paper V demonstrated that scale-up in SFC

can be made more reliable, albeit not completely predictable.

46

Figure 19. The results from the matched scale-up at different eluent methanol

level in wt.% units: (a) 4.2 (b) 8.5 and (c) 12.8. The black lines in all fig-

ures/insets correspond to injections on the analytical (UPC2) unit with the

250 x 4.6 mm column and the gray lines correspond to injections on pre-

parative (SuperSep) unit with the 250 x 50 mm column. The insets corre-

spond to analytical injections using the analytical UPC2 respective the

preparative SuperSep unit, respectively. The main figure corresponds to

different injected volumes of 40 g/L TSO on the analytical UPC2 and on

the preparative SuperSep unit. Corrections were made for system void

volumes by matching the elution time of the TTBB marker. Reproduced

and reprinted from Paper V.

To illustrate the complexity of SFC separations, a seemingly simple

experiment was devised on the analytical system. Elution profiles for

equal injections of TSO were recorded at 1 and 4 mL/min. In order to

match the elution volumes of the chromatograms obtained at the dif-

ferent flows, both the column average pressure and the methanol vol-

ume fraction delivered at each flow had to be adjusted using both cal-

culations of density and methanol content.

47

4. Concluding remarks

The expression “two steps forward one step back” rather precisely

summarizes the work undertaken in this thesis. Everything began

with the basic question if reproducible research could be performed

using the latest generation commercial SFC instrumentations. Based

on previous work by several authors, some of whom built their own

SFC systems, the answer was clearly no. The compressibility of the

mobile phase made user set parameters such as volumetric flow, pres-

sure and even modifier content ambiguous. At which point in the flow

path were these parameters valid, at the pump, at the inlet of the col-

umn or at the back pressure regulator?

The approach of interfacing Coriolis mass flow meters was the first

attempt of making the research more reproducible because mass flow

can be reported unambiguously. By coupling pressure transducers

and temperature probes at the inlet and outlet of the column, experi-

mental conditions can be even more unambiguously reported. To-

gether, these measurements also allow for calculation of the fluid den-

sity, which in turn can be used to calculate the volumetric flow rate.

Because the calculation of density relies on the accuracy of the Equa-

tion of State, this parameter must still be used with caution, as its er-

ror is only known for certain conditions. An investigation of the accu-

racy of the available Equation of States with mixing rules should be

investigated further. Additionally, density can only be calculated for a

limited amount of mixtures, why more basic research is needed.

Nevertheless, the additional measurements allowed us to find so

called near-isopycnic (also near isothermal) where methods to deter-

mine adsorption isotherms typically used in LC could be directly

transferred to SFC. The results were interesting but at the same time

discouraging, as it revealed that the most accurate methods in LC are

very complicated or maybe impossible to use in SFC. To use the per-

turbation peak methods, solute dissolved in the liquid modifier will

displace the liquid and if no adjustment is made, each increased con-

centration plateau will decrease modifier level. The adsorption data is

likely valid but each obtained point represents a unique point on an

adsorption isotherm for a particular modifier content. This data can-

48

not be used to simulate overloaded injections in isocratic elution

mode. Even if it was shown that adsorption isotherm determination

methods which rely on the use of overloaded elution profiles could be

used to predict elution profiles of varying injection volume, Paper IV

made it clear that the possibility of elution profile distortion must al-

ways be taken into account. Furthermore, Paper I-II also relies on

the assumption that co-solvent adsorption is implicitly accounted for

in the adsorption isotherm. This may or may not be valid over differ-

ent ranges of co-solvent levels and/or stationary phases and remains

to be investigated. Further research into methods to determine ad-

sorption isotherms in SFC is necessary to gain deeper insight into the

separation process, as has been shown numerous times in LC.

Due to the findings of Paper I-II, Paper III was devoted to a more

descriptive approach, i.e. the use of Design of Experiments to quantify

the performance of the separation process. The attempt was success-

ful and could be used to model the response within the design space.

This is an important observation since it would allow experimenters

to use the predictive power within the design space to for example op-

timize the conditions for a preparative separation as was demonstrat-

ed in the paper. As one can fit any response given a sufficient number

of parameters, caution must of course be taken when extrapolating

beyond the design space.

The observant reader may have noticed that all experiments in Paper

I-IV were performed at low flow rates, which may seem odd given

that that one of the best aspects of SFC is its possibility to operate at

very high flow rates. The reason for this is that methods of adsorption

isotherm determination otherwise would need to take into account

gradients in density, flow rate, temperature. This would be very chal-

lenging but the recent work by Kaczmarski et al. shows that it not im-

possible.

Paper V utilized SFC at higher volumetric flows and managed to pre-

sent a viable approach for scaling-up separation systems in SFC. By

scaling up SFC using the same length of column, no calculation of

mobile phase density is required if mass composition and pressure

drop are preserved during the scale-up.

49

To summarize I would like to quote Stellan Hjertén “No doubt the fi-

nal success is, in most cases a consequence of a series of consecutive

advances and improvements and, therefore, not concentrated in one

or a few days”

SFC is a usable technique that has found, and will continue to find

suitable applications where it in some aspects will outperform liquid

chromatography. Its ultimate success will require a continued re-

search effort, which today actually seems promising.

50

6. Swedish Summary

Kromatografi är en kemisk separationsteknik som baserar sig på att

de ämnen, eller molekyler, som skall separeras ifrån varandra fördelar

sig på olika sätt mellan en rörlig (mobil) fas och en stillastående (stat-

ionär) fas. Vid vätskekromatografi är den rörliga fasen en vätska, ofta

vattenbaserade lösningar innehållande salter eller organiska lönings-

medel med definierat pH. Den stationära fasen består ofta av små po-

rösa kisel-dioxidpartiklar (små pärlor) som modifierats kemiskt på

olika sätt. Den stationära fasen nedpackas i en stålcylinder med en

diameter på cirka 3-5 mm och en längd på 50 – 150 mm. Den rörliga

fasen pumpas vid mer eller mindre högt tryck alltifrån cirka 50 bar till

nästan 1000 bar.

Separationen inleds med att en provlösning sugs upp från en prov-

behållare och sedan injiceras in i mobila fasen – via en högtrycksven-

til – under det att den mobila fasen pumpas genom kolonnen varvid

provblandningen introduceras till kolonntoppen och dess olika ingå-

ende ämnen transporteras vidare genom kolonnen. Det kemiska

ämne som adsorberas starkast till de kemiskt modifierade pärlorna

ytor rör sig långsammast genom kolonnen. En detektor kopplad till en

dator registrerar hur de separerade molekylerna rör sig ut ur kolon-

nen. Om man kan registrera en normalfördelad (klockformad) topp

och väl separerad för varje substans som ska separeras så är separat-

ionen lyckad. Då kan de ingående substanserna haltbestämmas, vilket

görs vid analytisk kromatografi. Eller så kan ämnena isoleras, och

samlas upp i fraktionsrör, vilket görs vid preparativ kromatografi.

Vid preparativ kromatografi är syftet renframställning istället för

kvantitativ information, och stora mängder substans introduceras vid

provinjektionen. De höga koncentrationerna medför att substanser-

nas kromatografiska toppar blir överladdade; den högre koncentrat-

ionen som finns i toppen strävar efter att komma ut snabbare vilket

medför att toppens framsida blir skarp medan baksidan tar formen av

en skidbacke. Fenomenet kallas ”svansning” och beror på att antalet

adsorptionsställen – ställen som molekylerna kan fastna på – är be-

gränsade hos den stationära fasen i kolonnen. Om två sådana över-

laddade toppar vandrar nära varandra bildas blandzoner med under-

51

liga topputseenden till följd vilket medför att det är svårt att veta när

man ska börja och sluta fraktionera för att få rena substanser. Därför

är preparativ kromatografi mycket svårare att optimera än analytisk.

Men med kunskap om ämnenas adsorptionsisotermer, som visar

sambanden vid konstant temperatur mellan koncentrationen av äm-

net i mobila fasen och den på stationära fasen, kan man förutsäga de

överladdade topputseendena.

Denna avhandling har fokuserat på en utökad förståelse för s.k. över-

kritisk fluid kromatografi (eng. förkortning SFC) där man istället för

vätska eller gas som mobil fas använder ett lösningsmedel i ett till-

stånd mitt emellan gas och vätska. Många lösningsmedel kan under

vissa speciella tryck- och temperaturförhållanden uppnå ett sådant

definierat tillstånd, redan vid lågt tryck och temperatur för koldioxid.

Denna form av kromatografi använder nästan uteslutande koldioxid

som huvudkomponent av den mobila fasen vilket innebär att man

slipper använda miljö- och hälsovådliga organiska lösningsmedel.

Metoden ger snabbare separationer då överkritisk koldioxid ger

mindre pumpmotstånd, men är samtidigt komplicerad att använda

och modellera.

Enbart koldioxid är ett dåligt lösningsmedel för många polära före-

ningar varför man idag i stor utsträckning blandar in mindre mängder

polärt organiskt lösningsmedel som metanol eller etanol i den mobila

fasen. Det faktum att det kromatografiska experimentet kan utföras

snabbare innebär att analyser kan genomföras på kortare tid och att

produktiviteten, d.v.s. mängd substans renad per tidsenhet kan ökas.

Koldioxid som mobil fas ger också enklare uppsamling av de värde-

fulla fraktionerna rent ämne, i huvudsak eftersom all koldioxid helt

enkelt förgasas vid atmosfärstryck eller återvinns.

Trots att det finns mycket empirisk kunskap kring SFC och att läke-

medelsindustrin i stor utsträckning nyligen implementerat SFC för

deras reningsprocesser av läkemedel, så saknas en djupare kunskap

om SFC. Många decenniers forskning kring vätskekromatografi har

resulterat i en god förståelse för de underliggande fysikaliska förlop-

pen vilket har inneburit att man med matematiska modeller kan si-

mulera och förutse hur de flesta förändringar av styrparametrar på-

52

verkar systemet. Med hjälp av numeriska simuleringar kan man op-

timera det vätskekromatografiska systemet till att prestera optimalt. I

SFC kan man i många fall inte ens sätta upp en enkel modell som kan

beskriva hur systemet förändras då man ändrar pumpflödet. Kun-

skapsbristen inom SFC beror framförallt på att mobila fasen i överkri-

tiskt tillstånd är komprimerbar till skillnad från en vätska. SFC är som

en slags elastisk gummivariant av vätskekromatografi. I vanlig väts-

kekromatografi vandrar lösningsmedlet genom kolonnen med jämn

hastighet men i SFC får man gradienter när det gäller tryck, densitet,

temperatur och hastighet. Tillsammans påverkar dessa gradienter se-

parationsprocessen på ett mycket invecklat sätt.

Syftet med mitt avhandlingsarbete har varit att undersöka flera

grundläggande aspekter av SFC. Förutsättningen för detta har varit

att noggrant mäta och/eller beräkna parametrar såsom tryck, tempe-

ratur, flöden, densiteter och olika kompositioner av mobila fasen i ett

kommersiellt SFC instrument. Den senaste generationens SFC system

ger en begränsad information om hur dessa parametrar varierar och

ger därför dåliga förutsättningar för reproducerbara studier. Resulta-

ten av mina forskningsrön redovisades i två vetenskapliga publikat-

ioner (Artikel I och II). I huvudsak visade det sig att de metoder för

bestämning av adsorptionsisotermer som ger högst noggrannhet i

vätskekromatografi, så kallade platåmetoder, fungerar dåligt för SFC.

Istället rekommenderas metoder som normalt har sämre noggrannhet

i vätskekromatografin som baseras sig på data från överladdade kro-

matogram.

Därefter sattes statistiskt baserad försöksplanering upp (”Experimen-

tell design”) för att ta reda på den relativa betydelsen av de olika expe-

rimentella parametrarna och inställningarna på instrumentet för

bästa tänkbara separation, såväl analytisk som preparativt. Resultaten

kunde bekräfta tidigare observationer såsom att metanolhalten följt

av trycket visade sig vara de relativt viktigaste parametrarna för att

styra retentionen i två modellsystem. Temperaturen var viktigast för

selektiviteten. För att öka produktiviteten var en större andel metanol

och högre temperatur viktigt (Artikel III).

53

Artikel IV fokuserade på bakomliggande orsaker till toppdeforme-

ring i SFC, vilket allvarligt begränsar hur stor volym man kan injicera

av provet vid SFC, till skillnad mot vätskekromatografi. Den domine-

rade metoden att injicera prov i SFC är i vätskeform, precis som vid

vätskekromatografi. Detta innebär att man injicerar en plugg av 100

% polärt organiskt lösningsmedel (i vätskeform) in i en mobilfas som

huvudsakligen består av koldioxid, i överkritiskt tillstånd. Denna

stora skillnad leder till att molekylerna i provlösningen upplever en

kraftig jämviktstörning mellan mobil och stationärfas vilket man tidi-

gare visat ge kraftiga toppdeformationer. Studien kunde både kvalita-

tivt och kvantitativt genom simuleringar förklara uppkomsten av

toppdeformeringarna. Denna information är speciellt viktig för pre-

parativ SFC eller då större volymer måste injiceras.

En av vätskekromatografins fördelar jämfört med SFC, är att det är

mycket enkelt att skala upp ett analytiskt småskaligt experiment till

preparativ skala. Det är precis tvärtom vid SFC och jag ville försöka

komma tillrätta med detta problem i Artikel V. Jag använde ett en-

kelt separationssystem som modell och studerade vad som krävdes

för att skala upp detta från en kolonn med 4.6 mm inre diameter till

en motsvarande 50 mm kolonn. Studien genomfördes på ett analy-

tiskt system på universitetet och ett storskaligt instrument på Astra-

Zeneca i Mölndal. En nyckel till att lyckas med denna metodöverfö-

ring var att först noggrant karaktärisera flöden, tryck och temperatu-

rer i det analytiska systemet och sedan överföra dessa i enheter som

användes av det preparativa systemet.

54

5. Acknowledgement

This thesis would not have been possible without the inspiration, am-

bition and persistent help of many others. I would especially like to

thank the following:

Dr. Torgny Fornstedt for inspiring me to endeavor in the field of

chromatography. You have showed me that “good enough” is not good

enough when it comes to research.

Dr. Jörgen Samuelsson for your endless knowledge in anything relat-

ed (or not) to chromatography. Thanks for pushing me to continuous-

ly adopt a more thorough approach to my studies, and of course, for

converting me from Ubuntu to Arch Linux.

Dr. Patrik Forssén for help with calculations and excellent eye for

detail in the writing of our joint papers.

Dennis Åsberg for all help and great contributions to my studies. Also,

for a very nice time during our stay in Uppsala.

All colleagues at Karlstad University and Uppsala University. Torgny

Undin and Lena Edström for a nice collaboration during my first year

as a PhD student. Mikael Andersén for always helping me with course

work preparations.

Magnus Klarqvist, Hanna Leek, Kristina Öhlén for excellent support

and contributions to the scale-up study during my stay at AstraZene-

ca, Mölndal.

Dr. Olle Gyllenhaal for giving me my first experience with SFC and for

introducing me to the SFC community at the SFC conference in Brus-

sels 2012.

Dr. Joakim Högblom for helping me start up the SFC research using

the “should be placed in the attic and forgotten” instrument.

55

Dr. Arvind Rajendran for continuous feedback on my research during

our meetings in Sweden, Europe and the U.S. Your early work in SFC

guided me in the right direction.

Dr. Terry A. Berger for letting me know that I was on the right track in

my research.

Dr. Robert Arnell for showing me how large scale chromatography

works and how fundamental research can be applied to improve such

processes.

Dr. Krzysztof Kaczmarski for quick, thoughtful and always helpful

support. Of course also for traditional polish food and oil field sight-

seeing in southern Poland.

Dr. Abhijit Tarafder for a lot of helpful feedback to my research. Also

thank you for the facility tour in Milford.

Dr. Fabrice Gritti for always asking the difficult but necessary ques-

tions during my “lecture series “on SFC in Boston 2013 and 2014.

Dr. Georges Guiochon for some interesting and rewarding discussions

on my research. The opportunity to read the proof of an early version

of your seminal SFC review made me realize what fun lay ahead.

Personnel at Waters, Paris, France, HICHROM Ltd, Reading United

Kingdom, Waters, Milford United States (especially Michael D Jones

for supplying you know what). Victor Abrahamsson at Lund Universi-

ty.

The Swedish Research Council and the Swedish Knowledge Founda-

tion for generous grants supporting the research effort.

My family, friends for all your support. The foundation for this jour-

ney was laid long before the start of my PhD.

Finally, Sanne, even though we now both live on new continents, I

love you more than ever.

56

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Fundamental Investigations of Supercritical Fluid Chromatography

Martin Enmark

Martin E

nmark | Fundam

ental Investigations of Supercritical Fluid Chrom

atography | 2015:45

Fundamental Investigations of Supercritical Fluid Chromatography

This thesis aims at a deeper understanding of Supercritical Fluid Chromatography (SFC). Although preparative SFC has started to replace Liquid Chromatography (LC) in the pharmaceutical industry - because of its advantages in speed and its less environmental impact - fundamental understanding is still lacking. Therefore there is no rigid framework to characterize adsorption or to understand the impact of changes in operational conditions.

In Paper I-II it was demonstrated why most methods applied to determine adsorption isotherms in LC could not be applied directly for SFC. Methods based on extracting data from overloaded profiles should be preferred.

In Paper III a Design of Experiments approach was successfully used to quantitatively describe the behavior of several solutes in a separation system. This approach can be used to optimize SFC separations or to provide information about the separation system.

In Paper IV severe peak distortion effects often observed in SFC were carefully investigated and explained using experiments and simulations.

Finally, in Paper V, the prerequisites for performing reliable and predictable scale-up of SFC were investigated by small and large scale experiments.

DISSERTATION | Karlstad University Studies | 2015:45 DISSERTATION | Karlstad University Studies | 2015:45

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