Dc

MD

a

ARRAA

KASPERI

1

gprafotfaLtural

aitaa

0h

Journal of Chromatography A, 1312 (2013) 124– 133

Contents lists available at ScienceDirect

Journal of Chromatography A

j our nal homep age: www.elsev ier .com/ locate /chroma

etermination of adsorption isotherms in supercritical fluidhromatography

artin Enmark, Patrik Forssén, Jörgen Samuelsson, Torgny Fornstedt ∗

epartment of Engineering and Chemical Sciences, Karlstad University, SE-651 88 Karlstad, Sweden

r t i c l e i n f o

rticle history:eceived 19 July 2013eceived in revised form 31 August 2013ccepted 2 September 2013vailable online 7 September 2013

eywords:

a b s t r a c t

In this study we will demonstrate the potential of modern integrated commercial analytical SFC-systemsfor rapid and reliable acquisition of thermodynamic data. This will be done by transferring the follow-ing adsorption isotherm determination methods from liquid chromatography (LC) to supercritical fluidchromatography (SFC): Elution by Characteristic Points (ECP), the Retention Time Method (RTM), theInverse Method (IM) and the Perturbation Peak (PP) method. In order to transfer these methods to SFCin a reliable, reproducible way we will demonstrate that careful system verification using external sen-

dsorption isothermsupercritical fluid chromatographyerturbation Peak methodlution by Characteristic Pointsetention Time Method

sors of mass flow, temperature and pressure are needed first. The adsorption isotherm data generatedby the different methods were analyzed and compared and the adsorption isotherms ability to predictnew experimental elution profiles was verified by comparing experiments with simulations. It was foundthat adsorption isotherm data determined based on elution profiles, i.e., ECP, IM and RTM, were able toaccurately predict overloaded experimental elution profiles while the more tedious and time-consuming

ll inje

nverse Method PP method, based on sma

. Introduction

The interest in supercritical fluid chromatography (SFC) hasrown rapidly during the last years and many process units in theharmaceutical industry have started to replace liquid chromatog-aphy (LC) with SFC. The main reasons are that the production ratesre generally higher and the separations are more environmentallyriendly as SFC uses CO2 instead of the harmful organic solventsften used in LC. SFC also has a large economic potential: althoughhe investment cost, and the cost for educating personnel, is higheror SFC than for LC, SFC’s increased productivity should make itdvantageous to use in many situations. SFC is more complex thanC due to of the compressibility of the mobile phase which leadso large variations in density and viscosity and makes fundamentalnderstanding and modeling more complicated [1]. SFC thereforeequires more research and investigations, both in the academiand in the industry, in order for the technology to reach the sameevel of maturity as LC.

In order to predict optimal separation conditions in SFC, as wells in LC, it is crucial to have accurate determination of adsorptionsotherms over a broad concentration range. From LC we know

hat, if properly processed, this adsorption data gives informationbout the degree of heterogeneity, the energy of interactionsnd monolayer capacities of each individual type of adsorption

∗ Corresponding author. Tel.: +46 76 774 31 58.E-mail address: Torgny.Fornstedt@kau.se (T. Fornstedt).

021-9673/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.chroma.2013.09.007

ctions on concentration plateaus, failed in doing so.© 2013 Elsevier B.V. All rights reserved.

site. From this one can determine the types of interactions, e.g.,dipole–dipole, van der Waals etc. [2,3].

Frontal analysis (FA) is more or less considered to be a refer-ence method for adsorption isotherm determination in LC. It isusually carried out by injecting a series of increasing concentra-tion pulses or by injecting a concentration “staircase” [4,5]. FA canbe used for any type of adsorption isotherm and are not affected byslow or concentration dependent kinetics. However, the methodsfor adsorption isotherm determination that were intend to trans-fer from LC to SFC, and then validate, are the Perturbation Peak(PP) method, the Elution by Characteristic Points method (ECP), theRetention Time Method (RTM) and the Inverse Method (IM). PP isbased on the retention time of small disturbance peaks on concen-tration plateaus. The PP method has been validated for LC and foundto be as accurate as FA in the single component case [6] and betterthan FA in the multi-component case [7], therefore the PP methodwas selected instead of FA in this study. ECP, RTM and IM are allbased on overloaded elution profiles, possibly combined with theretention time of small analytical peaks. ECP use the diffuse partof an overloaded elution profile, RTM use the retention times ofthe sharp overloaded elution profiles breakthrough fronts and theretention time of an analytic peak and IM uses whole overloadedelution profiles.

All the methods we are going to transfer have successfully been

used to determine adsorption isotherms in LC [8,9] and they all havetheir advantages and drawbacks which are well known from LC. PPand ECP will give raw adsorption isotherm data which make themsuitable for conducting fundamental adsorption studies because

dx.doi.org/10.1016/j.chroma.2013.09.007http://www.sciencedirect.com/science/journal/00219673http://www.elsevier.com/locate/chromahttp://crossmark.crossref.org/dialog/?doi=10.1016/j.chroma.2013.09.007&domain=pdfmailto:Torgny.Fornstedt@kau.sedx.doi.org/10.1016/j.chroma.2013.09.007

atogr

wwmhuonPatfilRgeaI

omatifim

•

•

•

•

•

irtaeaptvacikt

column. From the mobile phase density and total mass flow, theeffective volumetric flow through the column, FV,total, can be calcu-

M. Enmark et al. / J. Chrom

e do not need to assume adsorption isotherm models in advancehich is required for RTM and IM. IM also requires a columnodel which is not needed by any other method in this study;

owever this might also be an advantage because it can then besed for system with very slow kinetics. PP, ECP and RTM on thether hand are all derived using the ideal model, that assumes infi-ite efficiency, and therefore requires moderate to high efficiency.P and IM can be used to estimate multicomponent competitivedsorption isotherms from mixtures while ECP only can be usedo determine single component adsorption isotherm and there-ore requires pure components. The strength of PP in LC is thatt gives accurate adsorption isotherm data; however, it consumesarge quantities of pure solutes and is time consuming. ECP andTM are fast and convenient methods that requires only a few sin-le component elution profiles and are simple to process. IM canstimate multicomponent competitive adsorption isotherms from

few multicomponent elution profiles and this advantage makesM a viable method for process optimization.

In SFC, only a handful papers deals with the determinationf adsorption isotherms [1] and in even fewer cases the esti-ated adsorption isotherms are verified by comparing simulated

nd experimental overloaded elution profiles [10–12]. We suspecthree reasons for this: (i) the lack of fundamental SFC understand-ng, (ii) the lack of commercial SFC systems properly evaluated forundamental studies and (iii) the previous relatively low interestn the SFC area. Among the few studies done the following can be

entioned,

Depta et al. [13] used the FA method to determine quadraticadsorption isotherms of cis- and trans-phytol on a LiChrosphersilica column with 2-propanol as modifier. A density decrease of3% was taken into account by using the average density.Lübbert et al. [14] used the PP method to determine cubicadsorption isotherms of tocopherol enantiomers on differentunmodified silica columns with 2-propanol as modifier. The den-sity of pure CO2 was used to approximate the density of theCO2/2-propanol mixture.Ottiger et al. [10] used IM to determine bi-Langmuir adsorptionisotherms of 1-phenyl-1-propanol enantiomers on a Chiralcel-OD column with different amounts of MeOH as modifier. Thedensity drop was in all cases less than 0.2%.Wenda and Rajendran [11] used IM to determine Langmuir andbi-Langmuir adsorption isotherms of flurbiprofen enantiomerson a Chiralpak AD-H column with different amounts of MeOH asmodifier. The density variation never exceeded 1.63%.Bao et al. [12] used ECP to determine the Langmuir adsorptionisotherms of paroxol enantiomers on a Chiralpak AD-H columnwith different alcohols as modifiers. Although investigating theeffect of pressure, no detailed discussion of possible variations involumetric flow rate due to density gradients was given.

Easily reproducible fundamental studies in LC can be done usingntegrated commercially available system provided that the accu-acy of delivered volumetric flow, the temperature control andhe system void volumes are properly verified [8,15]. However, in

recent study it was shown that, in order to have reproduciblexperiments using commercially available system SFC systems,dditional sensors are needed to measure the mass flow and theressure at the column inlet and outlet [9]. These measurementsogether with temperature can be used to calculate the effectiveolumetric flow rate and density at each point in the column whichre needed in order to reproduce fundamental SFC studies. In this

ontext its worth mentioning that for CO2/MeOH eluent mixturest is also possible to calculate the density of the mobile phase bynowing the pressure and temperature, this approach has beenaken by several authors [9–11,16]. The error in such calculation

. A 1312 (2013) 124– 133 125

was recently estimated by Tarafder et al. to be less than 1.75% for amolar composition of 88/12 CO2/MeOH at 40 ◦C and at 150 bar [16].

The first aim of this study is to determine the conditions in thecolumn by conducting system verification on a commercially avail-able SFC-system. This is going to be accomplished by measuringtemperature, mass-flow and pressure with extra sensors to ensurethat all calculations are based on the “true” conditions and not oninstrumental settings. The second aim is to determine adsorptionisotherms on the verified system using RTM, ECP, PP and IM. Theadsorption isotherms are then going to be compared and used topredict elution profiles. As model system antipyrine separated ona silica column is going to be used with CO2/MeOH as eluent.

2. Theory

In this study it is assumed that the separation conditions overthe whole column are close to isopycnic (constant density) andisothermal conditions and this is verified using the theory in Section2.1. For simulations the equilibrium dispersive model and the idealmodel will be used and these are described in Section 2.2. Adsorp-tion isotherms can be determined using several different methods.In this study we will use four different methods that are describedin Sections 2.3–2.6: the Perturbation Peak method (PP), the Elutionby Characteristic Points method (ECP), the Retention Time Method(RTM) and the Inverse Method (IM).

2.1. Mobile phase density and effective volumetric flow

To verify isothermal conditions, the surface temperature of thecolumn at different points can be measured, but measuring densityis a little more complicated. One way to check isopycnic conditionsis to calculate the density of the MeOH/CO2 mixture using an appro-priate Equation of State (EOS). The EOS calculation requires thatthe pressure, temperature and fluid composition (mass or mole-fraction of CO2 and MeOH) is known. In this study we use animplementation of the Kunz and Wagner model [17] to calculatemixture densities. Pressure can be measured as easily as temper-ature, but to get the fluid composition we also need to know themass flow of each component in the separation. The total inlinemass flow, ṁtotal, and the mass flow of MeOH, ṁMeOH, can be mea-sured and from this the mass flow of CO2, ṁCO2 , can be calculatedusing,

ṁtotal = ṁCO2 + ṁMeOH. (1)

Now the molar fraction xMeOH and xCO2 can be calculated using,

xCO2 =ṁCO2 /MCO2

ṁMeOH/MMeOH + ṁCO2 /MCO2, xMeOH = 1 − xCO2 , (2)

where M* is the molecular weight. Using the fluid compositiontogether with pressure and temperature readings the density ofthe mixture can be calculated using an EOS. Isopycnic conditionsmeans that the density of the mobile phase at the column inlet isvery close to the density at the outlet, i.e.,

�(Pinlet, Tinlet) ≈ �(Poutlet, Toutlet). (3)

In reality, the density at the inlet will always be slightly largerthan at the outlet, otherwise there will be no flow through the

lated using,

FV,total =ṁtotal

�. (4)

1 atogr

2

msaa

⎧⎪⎪⎪⎪⎨⎪⎪⎪⎪⎩

wptaflb0epts

jnsfiTSlrud

m

l

a(tfiLwce

q

wb

2

fLia

26 M. Enmark et al. / J. Chrom

.2. Column mass balance

Several different column models have been used to describe theigration of a solute through the column in LC and SFC [4]. In this

tudy the Equilibrium Dispersive (ED) model and the ideal modelre going to be used. The ED model is valid for the relatively fastdsorption kinetics that is often observed in SFC and can be written,

∂C(x, t)∂t

+ F ∂q(C(x, t))∂t

+ u ∂C(x, t)∂x

= Da ∂2C(x, t)

∂x2,

0 ≤ x ≤ L, t≥0,C(x, 0) = C0,

C(0, t) = ϕ(t),

(5)

here C and q are the concentration, in the mobile and stationaryhase respectively, of the solute at time t and column position x. F ishe phase ratio (ratio between the volume of the stationary phasend the mobile phase, here calculated from the lumped mass trans-er and dispersion coefficient), u is the linear velocity and Da is theumped mass transfer and dispersion coefficient (here estimatedy fitting a Gaussian function to an analytical elution profile). C(x,) = C0 is the initial condition, i.e., the concentration of solute in theluent, is usually 0 but can also be an established concentrationlateau prior to injection; see the PP method in Section 2.3. C(0,) is the boundary condition, i.e., the injection profile, and in thistudy a rectangular profile is used.

The difference between the ideal model and the ED model isust that Da = 0 in Eq. (5), i.e., the efficiency is infinite. In real lifeo separation system have infinite efficiency, but for separationystems with high column loads and efficiencies the elution pro-les calculated using the ideal and ED are more or less identical.o decide if the two models give more or less identical results thehirazi-number can be used [18]. The Shirazi-number, m, is a unit-ess measurement that is calculated from the column load, Lf (theatio between moles adsorbed and moles at saturation), the col-mn efficiency, N, and the retention factor for the solute at infiniteilution, k0,

= NLf(

k01 + k0

)2. (6)

If m is larger than 50 the ideal and ED model will give more oress the same results [4,18].

The stationary phase concentration is described using andsorption isotherm which relates the concentrations in the mobileC) and stationary phases (q), respectively. The Langmuir model ishe simplest homogenous model and describes a homogenous sur-ace with a single type of adsorption site and the bi-Langmuir models the simplest heterogeneous two-site model. In this study the bi-angmuir model will be used, see Sections 2.4 and 4.4 for the reasonhy this adsorption model was chosen. The bi-Langmuir model

ontains the sum of two Langmuir terms to the right side of thequality sign and can be written [4],

(C) = a1C1 + b1C

+ a2C1 + b2C

, (7)

here a* is the distribution coefficient, or the Henry constant, and* is the association equilibrium constant.

.3. The Perturbation Peak method

The Perturbation Peak method (PP) is a well-established and

requently used method for adsorption isotherm determination inC [4,7,19–22]. The adsorption isotherm is determined by inject-ng a small excess or deficiency of the solute into a column thatre already equilibrated with an eluent containing identical solute

. A 1312 (2013) 124– 133

molecules, i.e., a concentration plateau. The perturbation peak willbe a single peak in the chromatogram and the retention time ofthis peak is related to the slope of the adsorption isotherm, see Eq.(8). By repeating this experiment on several different concentra-tion plateaus the whole adsorption isotherm can be determined.The prerequisites for using the PP method are (i) moderate to highcolumn efficiency and (ii) the possibility of generating small sym-metric and detectable concentration disturbances on establishedconcentration plateaus. The retention time of the perturbation peakcan be calculated from [4],

tR(C0) −tinj2

= tM(

1 + F dqdC0

), (8)

where tR(C0) is the retention time of the perturbation peak at anestablished concentration plateau with concentration C0, tinj is theinjection time, tM is the column hold up time and dq/dC0 is the slopeof the adsorption isotherm. Eq. (8) is derived from the ideal modelof chromatography and assumes constant volumetric flow.

2.4. Elution by Characteristic Points

The Elution by Characteristic Points (ECP) method generatesadsorption isotherm data from the diffuse part(s) of an elution pro-file [4,23,24] and the theory is derived using the ideal model thatassumes infinite column efficiency. However, the efficiency of a realcolumn is finite [4] and this will lead to some error in the adsorptionisotherm determination but this error decreases with increasingcolumn efficiency [25,26]. In SFC we usually have higher efficiencythan in LC so this source of error is usually of minor concern, butthis needs to be verified prior to calculations. The ECP also needconcentration data, i.e., conversion of response to concentration isnecessary.

Another source of error is that the ECP-theory assumes rect-angular injection profiles [27], but experimental injection profilesare not rectangular [28,29] and this could lead to large errors. Thiserror increases with increasing injection volume [27] and unfortu-nately it is nearly always necessary to inject very large volumes tobe able to measure adsorption isotherms over a wide concentra-tion range. This error was recently more or less eliminated fromLC by a technical improvement called the ECP-CUT method wherea sharp rectangular slice was made experimentally from the rearof the injection zone [27]. When injections are conducted in theeluent stream in SFC, only small injection volumes as comparedto the column volume can be used; if larger injections are madethe injection solvent will strongly affect the elution profiles. Smallinjections will reduce the error due to non-rectangular injectionprofiles, but the drawback is that we will not be able to span a wideconcentration range due to the lower column load.

A systematic error can be caused by the selection of the inte-gration starting point at concentration equal to 0 and this errorwas reduced in a recent study by replacing the classical integrationwith the use of raw slope data [24]. This approach allows excludingextreme low concentration data, which are more affected by theseparation systems limited efficiency. With this modification it isno longer required that the elution profiles contain continuous dif-fuse parts and adsorption isotherms with inflection points can alsobe determined [23]. This approach was used here and the adsorp-tion data was determined using the following relationship for typeI adsorption isotherms (convex, e.g., the bi-Langmuir model),

dq(C) tR(C) − tinj − tM

dC

=tMF

, (9)

where tR is the elution time corresponding to the mobile phaseconcentration C.

atogr. A 1312 (2013) 124– 133 127

2

fl

k

I

k

t([

loeip

m

wtaaa(msoi

2

oomciumupcwp

edii

3

3

oH(

Fig. 1. Schematic representation of the external measurements of temperature,pressure and mass flow in the SFC system where the arrows indicate the flow ofmobile phase. In (a) it is shown how inlet and outlet pressure and temperature(using surface attached probes) were measured. During these measurements the

M. Enmark et al. / J. Chrom

.5. The Retention Time Method

In the Retention Time Method (RTM) [4,30–32] the retentionactor, k0, for a small analytic injection is measured first. The fol-owing relationship holds for this retention factor,

0 = F · limC→0

dq

dC ·(10)

In this study the bi-Langmuir adsorption isotherm model is used.nserting Eq. (7) into Eq. (10) we have that,

0 = F(a1 + a2) ⇔ a1 =k0F

− a2 (11)

Using the ideal model of chromatography one can calculate elu-ion profiles for any type I (convex, e.g., bi-Langmuir) or type IIIconcave) adsorption isotherm when one have infinite efficiency33].

The ideal model will have an infinite sharp front for an over-oaded injection, with retention time tF, when using the ideal modelf chromatography. Front retention times can then be used tostimate adsorption isotherm parameters from overloaded exper-mental elution profiles by solving the following least squaresroblem,

in∑n

i=1(t̃F − tF)2, (12)

here t̃F is experimental elution profiles front retention time, tF ishe calculated retention time of the front using the ideal modelnd n is the number of experimental chromatograms. By usingn analytic injection and Eq. (11) one can reduce the number ofdsorption isotherm parameters that needs to be estimated in Eq.12), e.g., three adsorption isotherm parameters needs to be esti-

ated in Eq. (12) for a bi-Langmuir adsorption isotherm. Here onehould use at least as many measured front retention times, fromverloaded experimental elution profiles, as there are adsorptionsotherm parameters that are going to be estimated in Eq. (12).

.6. The Inverse Method

The Inverse Method (IM) is a fast method that has been devel-ped for obtaining adsorption isotherm parameters directly fromverloaded elution profiles and has been successfully applied forany years [34–36]. IM is based on solving a column model’s so

alled inverse problem, here the equilibrium dispersive model’snverse problem, see Eq. (5). The inverse problem is to estimatenknown model parameters from known solutions to the columnodel; the direct problem is to calculate the solution to the col-

mn model from known model parameters (i.e., to simulate elutionrofiles). In this inverse problem experimental elution profiles areonsidered to be known solutions to the column model and wante want to estimate the unknown adsorption isotherm modelarameters.

The inverse problem is here solved by estimating the param-ters, in some adsorption isotherm model, that minimizes theifference between measured elution profiles and simulated ones

n a least squares sense. In this study the bi-Langmuir adsorptionsotherm model, Eq. (7), will be used.

. Experimental and procedures

.1. Materials

Antipyrine (1,2-dihydro-1,5-dimethyl-2-phenyl-3H-pyrazol-3-ne) analytical standard (Sigma–Aldrich, Steinheim, Germany),PLC grade MeOH (Fischer Scientific, Loughborough, UK) and CO2

>99.995%, AGA Gas AB, Sweden) were used in this study.

column was mounted in the SFC column oven. In (b) it is shown how mass flowwas measured outside the column oven by connecting the column inlet and outletcapillaries to the Coriolis mass flow meter.

The SFC system used in this study was a Waters UPC2

system (Waters Corporation, Milford, MA, USA) in its defaultconfiguration with a 2-column oven and PDA detector. Watersproprietary Empower software was used to control the systemand collect data. All separations were conducted on a Kromasilsilica 100 mm × 4.6 mm column, packed with 5 �m nominal par-ticle size, 100 Å pore size (AkzoNobel Eka, Bohus, Sweden). Thecolumn temperature was measured with two PT-100, 4-wire,resistance temperature detectors with a flat measuring surfaceof 2 mm × 2 mm from Pentronic AB, Gunnebo, Sweden, see theschematic representation in Fig. 1. On the column surface, aboutone cm from the column inlet and outlet, PT-100 elements withaccuracy ±0.2 ◦C were permanently attached using a silver basedepoxy adhesive (Arctic Silver, CA, USA). The signals were loggedusing a data logger from Pico Technology (Cambridgeshire, UnitedKingdom). The pressure at the column inlet and the outlet weremeasured using two absolute pressure transmitters (EJX530A,Yokogawa Electric Corporation, Tokyo, Japan) with a read accuracyof ±1 bar, see Fig. 1. They were connected via reducing unions to a1 m 1/16′′ OD steel impulses capillary which were connected inlineusing low dead volume union tees. The total mass flow was mea-sured using a Bronkhorst mini CORI-FLOW model M12 (BronkhorstHigh-Tech B.V., Ruurlo, Netherlands) with an accuracy of ±0.2%of the mass flow rate, see Fig. 1. The MeOH mass flow was con-ducted simply by weighing the amount of MeOH pumped over timeat room temperature using Mettler-Toledo B3002 scale (Mettler-Toledo GmbH, Switzerland).

3.2. Procedures

All experiments were conducted at a set flow rate of 1 mL/min

using 90/10% (v/v) CO2/MeOH as eluent at a back pressure of 150 barand a column temperature of 35 ◦C. Observe that these are instru-mental settings and not necessarily the true conditions. The settingswere verified by additional measurements of mass flow, pressure

1 atogr. A 1312 (2013) 124– 133

am

tMuv

u7Ail

4

ttn4biSu

4

mucpsSca(e

tcrtamwse3oatapvt

Fig. 2. In (a) the measured column inlet (solid black) and outlet (solid gray) tem-perature is shown over a period of 1 h when running at a set method of 1 mL/min,90/10 (v/v) CO2/MeOH at 35 ◦C and the automated back pressure regulator set to

TTD

28 M. Enmark et al. / J. Chrom

nd temperature to ensure all adsorption isotherm determinationethods used correct conditions (see Section 2.1).The dead volume of the system capillaries, and connections from

he injector to the detector, was estimated by using injections ofeOH with and without the column. Without the column, the col-

mn inlet and outlet capillaries were connected using a low deadolume union.

A 250 g/L stock solution of antipyrine dissolved in MeOH wassed throughout the entire study. Solutions of 10, 20, 30, 40, 50,0, 100, 130, and 150 g/L were prepared from the stock solution.

50 �L fixed loop was used for all injections, full loop or partialnjections. The elution profiles obtained, with fixed loop and partialoop injections, were nearly identical for the same injected volume.

. Results and discussion

Due to the compressibility of the mobile phase it is importanthat the SFC instrument is first verified so that one knows thathe results are based on actual conditions inside the column andot just on the operational system settings, this is done in Section.1. This step is also important because the experiments can thene reproduced on any SFC system. In Sections 4.2–4.6 adsorption

sotherms were estimated using different methods and finally, inection 4.7 the estimated adsorption isotherms were compared andsed to simulate elution profiles.

.1. Mass flow, temperature and pressure

To accurately determine adsorption isotherms using dynamicethods, as we do in this study, it is important to know the vol-

metric flow of the mobile phase in the column [4,5,9,37]. Toalculate the volumetric flow, see Eq. (4), we need the total mobilehase mass flow (which can be measured) as well as the den-ity of the mobile phase (which needs to be calculated). Here theFC-system were set to deliver a flow of 1 mL/min using an eluentomposition of 90/10 (v/v) CO2/MeOH and operated at a temper-ture of 35 ± 0.5 ◦C, with the Automated Back Pressure RegulatorABPR) set at 150 ± 1 bar. These are the set conditions used in thentire study.

Pressure and temperature was measured in-line according tohe schematic representation in Fig. 1(a). Impulse capillaries wereonnected from low volume tees to the pressure transmitters usingeducing unions and the pressure was continuously read fromhe transmitter. Using the set method, a stable reading of 161 bart the column inlet and 156 bar at the outlet could be noted forore than 2 h. The union tee in itself was measured to contributeith a pressure drop of about 1 bar. The temperature was mea-

ured at the column inlet and outlet using surface mounted PT-100lements (RTD); see Fig. 1(a). The column inlet temperature was4.5 ◦C and the outlet temperature was 34.2 ◦C for a period ofver 2 h, see Fig. 2(a). Temperature was continuously monitored inll experiments and there was no measurable difference betweenhe temperatures when pumping pure eluent or when pumping

ntipyrine at different plateau levels in the bulk eluent for theerturbation experiments. A slight transient positive temperatureariation, never exceeding 0.2 ◦C at the column inlet and 0.1 ◦C athe column outlet, was observed for each injection of 50 �L.

able 1emperature (T), pressure (P), effective volumetric flow (utotal) and density (�) at the coluensity is calculated using REFPROP [36].

T [◦C] P [bar] utotal [mL/min]

Column inlet 34.5 161 1.07 Column outlet 34.2 156 1.07

Average 34.4 158.5 1.07

150 bar, the dashed lines are average values. In (b) the measured total in-line massflow (solid gray) is shown when running the system at the same set conditions for∼2 h, the dashed line are average value.

To measure the total inline mass flow the column was removedand the system default inlet and outlet capillaries were connectedto the inlet and outlet of the Coriolis flow meter using reducingunions see Fig. 1(b). Then, the system was set to run at the ordi-nary settings of 1 mL/min with a set composition of 90/10 (v/v)CO2/MeOH. The temperature was set to 35 ± 0.5 ◦C and the ABPRset to 150 ± 0.5 bar. The measurement was run for more than 1 hand the signal was stable at 0.92 g/min, see Fig. 2(b).

The mass flow of MeOH was measured by weighing the amountof fluid pumped for about 4 h. The mass flow was estimated to be0.11 g/min. At the measured room temperature of 22 ◦C the den-sity of pure MeOH is 0.789 g/mL and the volumetric flow is thus0.13 mL/min. Subtracting the mass flow of MeOH from the totalmass flow gives the mass flow of neat CO2, see Eq. (1). Now themolar composition of the mobile phase can be calculated to be0.84/0.16 CO2/MeOH according to Eq. (2).

Using an average pressure of 158.5 bar and an average tempera-ture of 34.4 ◦C the average density was calculated to be 0.859 g/mLusing the NIST Reference Fluid Thermodynamic and TransportProperties Database version 9.1 (REFROP) [38]. Using the boundaryvalues of pressure and temperature, the inlet density was calcu-lated to be 0.860 g/mL and the outlet density was calculated to be0.858 g/mL. To conclude: the average density is, considering theuncertainty of the calculations and the measurements (the accu-racy of these calculations was discussed in detail in [16]), the sameas the inlet and outlet density; this means the column is operatingunder iso-pycnic conditions. For a molar composition of 0.84/0.16,at a pressure of 158.5 bar and at 40 ◦C, the relative error made by

REFPROP is estimated to be less than 1.75%. Because data at exactly35 ◦C is not available in literature, our best estimate is that the dif-ference between calculated and actual density of such compositionis small. Using the average density and the total mass flow, the

mn inlet and outlet together with mass flow of CO2 (ṁCO2 ) and of MeOH (ṁMeOH).

� [g/mL] ṁCO2 [g/min] ṁMeOH [g/min]

0.860 0.81 0.110.858

0.859 0.81 0.11

M. Enmark et al. / J. Chromatogr. A 1312 (2013) 124– 133 129

F ntipyro f ∼5 g∼

vcspi

4

i(RAtmtnv

dtiaanim0WrHvaa

perturbations in Fig. 3(b)–(d) are distinctly negative and occur at7.3, 6.3 and 5.7 min for injections on 1.3, 6.4 and 19.2 g/L plateaus,respectively. The adsorption data were fitted to bi-Langmuir modeland the adsorption isotherms are presented Table 2 and Fig. 4. The

Table 2Summary of adsorption isotherm parameters obtained using the perturbation peakmethod (PP), the Elution by Characteristic Points method (ECP), the Retention TimeMethod (RTM) and the Inverse Method (IM).

Method a1 b1 [L/g] a2 b2 [L/g]

ig. 3. Perturbation peak results: in (a) an overlay of four 3 �L injections of ∼1 g/L af 1 g/L antipyrine on a plateau of ∼1 g/L, in (c) an overlay of four 3 �L injections o13 g/L antipyrine on a plateau of ∼13 g/L.

olumetric flow rate was estimated to be 1.07 mL/min. In con-lusion, the chromatographic system does not deliver the exactet volumetric flow and volumetric composition of the mobilehase. The measured and calculated parameters are summarized

n Table 1.

.2. Adsorption isotherm determination

In the following sections the adsorption isotherm for antipyrines going to be determined using (i) the Perturbation Peak methodPP), (ii) the Elution by Characteristic Points method (ECP), (iii) theetention Time Method (RTM) and (iv) the Inverse Method (IM).ll these are dynamic adsorption acquisition methods and require

hat the all measurements are carried out at isopycnic, isother-al conditions and that the volumetric flow is constant; all of

hese requirements were verified in Section 4.1. However, we alsoeed to determine the column holdup volume and the system voidolume.

The measured hold up volume is actually the sum of the systemead volume and the columns void volume. System dead time athe calculated flow rate of 1.07 mL/min was determined by replac-ng the column with a low volume union, equilibrating the systemnd then making several 1 �L injections of highly diluted antipyrinend the system dead time was estimated to be 0.055 min (RSD 1%,

= 7) or about 60 �L. To determine the column void volume, 1 �Lnjections of MeOH and injections of diluted toluene in MeOH were

ade. Both methods gave an average time of about 1.24 min (RSD.55%, n = 5), which corresponds to a void volume of about 1.30 mL.e are well aware that error in the holdup volume estimation could

esult in errors in the determined adsorption isotherm [39,40].

owever, a mechanistic investigation elucidating suitable holdupolume markers remains to be performed in SFC and until then wessume that void markers “suitable” for normal phase applicationslso works well for SFC.

ine in pure MeOH in a stream of CO2/MeOH, in (b) an overlay of four 3 �L injections/L antipyrine on a plateau of ∼5 g/L and in (d) an overlay of four 3 �L injections of

4.3. The Perturbation Peak method

The Perturbation Peak method (PP) was performed using a sin-gle modifier pump and an injection loop of 50 �L. After thoroughequilibration of the column, using pure CO2/MeOH eluent, an injec-tion of 3 �L sample of 1 g/L antipyrine was performed, followed bythree replicates. The repeatability was excellent, see Fig. 3(a), anda retention time of 7.7 min could by recorded. Plateaus were thenestablished by pumping stock solutions of 10, 20, 30, 40, 50, 70, 100,130, and 150 g/L through the column for 3 h before the first injectionwas made. On each plateau four perturbations were generated byinjecting 3 �L of the corresponding stock solution diluted 10 times;by this approach it was assured that the perturbation was as smallas possible to reduce errors [22]. Because the measured volumet-ric fraction MeOH was 0.13, these perturbations always resultedin negative disturbances. Fig. 3(b)–(d) has several similarities anddissimilarities with what typically is observed in LC. Several distur-bances are present around and after the column void time. Thesedisturbances are typically observed in SFC and their magnitude alsodepends on the choice of wavelength (not shown), they have oftenbeen used to estimate the column holdup volume [6]. The actual

PP 10.89 ∼0 6.77 0.080ECP 15.56 0.023 3.16 0.468RTM 16.81 0.029 0.971 0.266IM 17.18 0.031 0.915 39.2

130 M. Enmark et al. / J. Chromatogr. A 1312 (2013) 124– 133

0 2 4 6 8 100

20

40

60

80

100

120

140

PPE CPRTMIM

0 5 10 15 2011

14

17

PP data

FitR aw

F d blacM showa

ifi

4

fwcducibttotstftmc

ig. 4. Adsorption isotherms obtained by the Perturbation Peak method (PP, soliethod (RTM, dash-dotted black) and the Inverse Method (IM, solid gray). The inset

dsorption isotherm (black line).

nset in Fig. 4 shows the raw perturbation data and the slope of thetted bi-Langmuir adsorption isotherm.

.4. Elution by Characteristic Points

The Elution by Characteristic Points (ECP) method was per-ormed using the diffuse rear of four replicated elution profilesith a 50 �L injection of 250 g/L antipyrine. Chromatograms were

onverted from response to concentration using the approachescribed earlier [10]. A two-step procedure, involving a combinedse of Scatchard plots and adsorption energy distribution (AED) cal-ulations, was used prior to the adsorption isotherm model fittingn order to determine the degree of heterogeneity of the models toe tested [2,3]. The Scatchard plots (not shown) were made fromhe raw data; these were concave which indicates that the adsorp-ion cannot be described with a Langmuir adsorption isotherm andne should use a heterogeneous model, like bi-Langmuir. Adsorp-ion energy distributions were calculated from the ECP data (nothown), but because the column load in this study were too lowhe calculations did not converge and no conclusions can be drawn

rom these calculations [39,41]. The bi-Langmuir model was fittedo the data; see Eq. (7), Table 2 and Fig. 4. To validate if the ECP

ethod is accurate in this case the Shirazi-number, see Eq. (6), wasalculated to be more than 300 which is considerably larger than 50

k), Elution by Characteristic Points method (ECP, dashed black), Retention Times the perturbation peak raw slope data (symbols) and the best fit of the Bi-Langmuir

[4] that is required to be able to accurately describe the separationwith the ideal model [18].

4.5. The Retention Time Method

The Retention Time Method (RTM) was used to estimate thebi-Langmuir adsorption isotherm parameters from 6 experimentalelution profiles: (i) 2 �L injection of a 1 g/L sample with tR = 7.7 min,(ii) 10 �L injection of a 250 g/L sample with t̃F = 6.30 min, (iii) 20 �Linjection of a 250 g/L sample with t̃F = 5.78 min, (iv) 30 �L injec-tion of a 250 g/L sample with t̃F = 5.44 min, (v) 40 �L injection ofa 250 g/L sample with t̃F = 5.15 min and (vi) 50 �L injection of a250 g/L sample with t̃F = 4.93 min.

The adsorption isotherm estimated was estimated using Eq.(12) where the ideal model’s simulated front retention times werecalculated using the algorithm in [33]. The estimated adsorptionisotherm parameters are presented in Table 2 and plotted in Fig. 4.

4.6. The Inverse Method

The Inverse Method (IM) was used to estimate bi-Langmuiradsorption isotherm parameters from three experimental elutionprofiles: (i) 2 �L injection of a 1 g/L sample, (ii) 30 �L injection of a250 g/L sample and (iii) 50 �L injection of a 250 g/L sample, i.e., we

M. Enmark et al. / J. Chromatogr. A 1312 (2013) 124– 133 131

0

5

10

15

20

C [g

/L]

PP

(a) ExperimentSimulation

7 7. 5 8 8. 50

2

4

6

8x 10

−3

0

2

4

6

8

10

12

ECP

(b)

7 7. 5 8 8. 50

2

4

6

8x 10

−3

5 6 7 80

2

4

6

8

10

12

Time [min.]

C [g

/L]

RTM

(c)

7 7. 5 8 8. 50

2

4

6

8x 10

−3

5 6 7 80

2

4

6

8

10

12

Time [min.]

IM

(d)

7 7. 5 8 8. 50

2

4

6

8x 10

−3

Fig. 5. Experimental (solid lines) 10–50 �L injection of 250 g/L antipyrine in the main figures and an analytical 2 �L injection of a 1 g/L antipyrine in the insets. The corre-s , mainl a) the(

utt

uut1epwtitipo

4

oPt1ieSwi

ponding overloaded elution profiles simulated using the ideal model (dashed linesines, insets), are overlaid for each adsorption isotherm determination method: in (ECP), in (c) the Retention Time Method (RTM) and in (d) the Inverse Method (IM).

sed an analytical peak and two overloaded elution profile. Noticehat we here only used 3 experimental elution profiles, instead forhe 6 that was used for RTM in Section 4.5.

The solution of the equilibrium dispersive model, i.e., the sim-lated elution profiles, see Eq. (5) and Section 2.6, was calculatedsing the Rouchon algorithm [42]. This algorithm has been showno give accurate solutions in most cases if the efficiency exceeds000 theoretical plates [4,43] and in this case the efficiency wasstimated to be 5415 theoretical plates. When solving the inverseroblem the used elution profiles were assigned equal weighthen solving the least squares problem and the estimated adsorp-

ion isotherm parameters are presented in Table 2 and plottedn Fig. 4. But one can instead choose to assign different weightso the used elution profiles, e.g., proportional to the correspond-ng injected amount and the result will then be biased towardredicting the larger injection more accurately than the smallnes.

.7. Prediction of elution profiles

As can be seen from Fig. 4 the shape of the adsorption isotherms,btained using ECP, RTM and IM are nearly identical, while theP results differ significantly. We used the determined adsorp-ion isotherm parameters to simulate the elution profile for a0, 20, 30, 40 and 50 �L injection of 250 g/L antipyrine using the

deal model, these simulations, together with the corresponding

xperimental elution profiles, is plotted in Fig. 5(a)–(d). As thehirazi-numbers are considerably larger than 50, see Section 4.4,e choose to use the ideal model as it is suitable for predict-

ng elution profiles in this case [4,33]. The insets in Fig. 5(a)–(d)

figures), or the equilibrium dispersive model with 5415 theoretical plates (dashed perturbation peak method (PP), in (b) the Elution by Characteristic Points method

show an analytic 2 �L injection of 1 g/L antipyrine, here we usedthe equilibrium dispersive model with 5415 theoretical plates forsimulation.

From Fig. 5(a) it is obvious that the adsorption isotherm param-eters obtained using the PP method, in contrast to the parametersfrom the other methods, see Fig. 5(b)–(d), could not predict the cor-rect elution profile for any volume except for the analytic injection.The discrepancy between simulation and experiment decreaseswith decreasing injection volume. The fronts of the simulatedelution profiles are constantly predicted to occur later than theexperimental profiles; only the very final part of the tail is predictedaccurately. The adsorption isotherm data from ECP, RTM and IM cansuccessfully be used to predict experimental elution profiles, seeFig. 5(b)–(d). This is not surprising as these methods directly use theexperimental elution profiles to determine the adsorption isothermparameters. IM is based on minimizing the difference between sim-ulated and experimental profiles and is balanced between havingaccurate prediction of the front’s position and the correct shape ofthe tail. RTM uses only the position of the fronts, and the retentiontime of an analytical peak, and is therefore biased toward accu-rate prediction of the front’s position and ECP only use the tail ofan overload elution profile and is therefore biased toward givingthe correct shape of the tail. Here it can be noted that ECP predictsan analytical peak at 8 min, while the other methods successfullypredicts it at 7.7 min. This can be expected as the other methodsactually use the position of an analytic peak. To conclude, RTM,

ECP and IM can accurately predict the shape of the elution pro-file, within the injection concentration range used to determinethe adsorption isotherms, also for other injections than the onesused for determination.

132 M. Enmark et al. / J. Chromatogr

Fig. 6. Comparison of the diffuse rear of overloaded 50 �L injections of 250 g/Lat(

totaocwilF

ti%omtit5tTftptrEatis

oewectp

ntipyrine injection (solid lines) and the determined perturbation peak retentionimes on different plateaus (symbols): in (a) for 12.5% (v/v) MeOH and in (b) for 10%v/v) MeOH.

Why the PP method fails to predict any elution profile needso be investigated. Plotting the position of the perturbation peaksn the diffuse rear of an overloaded injection clearly shows thathe perturbation theory is not directly applicable in this cases it predicts that the perturbation peaks positions should ben the diffuse rear; see Fig. 6(a). To verify if this discrepancyould be reproduced for different conditions the PP experimentas repeated using the same settings except 10% (v/v) MeOH

nstead of 12.5% (v/v). The perturbation peaks still does not over-ap with the diffuse rear of the equivalent overloaded injection, seeig. 6(b).

A possible explanation to this phenomenon lies in the injec-ion plug. If pure MeOH is injected in the system, it will eluten the void, i.e., it has no retention when using 87.5/12.5 (v/v)

CO2/MeOH. If the system is equilibrated using 100% MeOH, anverloaded injection of antipyrine will also elute in the void. Thiseans that antipyrine has no retention at all in pure MeOH and

hat there is a considerable mismatch of elution strength in thenjection plug compared to the eluent. The injection volumes ofhe overloaded injections used in this study varied from 10 to0 �L; in comparison, the PP method obtains data from 3 �L injec-ions, i.e., 3–16 times less volume than the overloaded injections.his means that the mismatch zone will be smaller when per-orming PP analysis than when injecting large volumes. Becausehe overlap of the simulated elution profile and the experimentalrofile increases with decreasing injection volume, this explana-ion is plausible. However, the effect is contraindicated by otheresults. For example, the adsorption isotherm determined usingCP is made using the largest 50 �L injection. If this profile wasffected by an elution strength mismatch zone one would expecthat predictions made for smaller volumes would be increas-ngly worse, however this is only true for the analytic injection,ee Fig. 5(b).

Another possible explanation is that the volumetric fractionf MeOH is different for each perturbation point. At the high-st concentration of antipyrine, the volume fraction of MeOHill be less than at low concentration of antipyrine. This would

xplain why the deviation increases with increasing plateau con-entration. To conclude, further studies are required to investigatehe true origins of the failure of PP data to predict elutionrofiles.

. A 1312 (2013) 124– 133

5. Conclusions

To transfer dynamic LC adsorption isotherm acquisition meth-ods [4,5,37] to SFC one must ensure that the column is operatedat constant volumetric flow rate under isopycnic and isothermalconditions. Furthermore, it is also necessary to have accurate mea-surements of the actual mass flow, pressure and temperature inorder for SFC experiments to be reproducible. We have shownhere how a commercially available SFC system can be verified byusing external pressure, mass flow and temperature sensors. Thesesensors, and the corresponding measurements, are necessary inorder to have reliable, reproducible results, as the instrumentalsettings might not reflect the true conditions inside the column.They are also necessary in order to ensure that we have the isopy-cnic/isothermal conditions, and the constant volumetric flow rate,that is needed to transfer LC adsorption isotherm acquisition meth-ods to SFC.

On a SFC system, verified according to the above, we estimatedadsorption isotherms for antipyrine using the Perturbation Peakmethod (PP), the Elution by Characteristic Points method (ECP),the Retention Time Method (RTM) and the Inverse Method (IM).The adsorption isotherms determined using IM, RTM and ECP aresimilar, but the one determined by PP differed significantly. As thecalculated Shirazi-number was high, SFC usually has much higherefficiency than LC, the ideal model could be used to describe thesystem [33] and this model was used to simulate elution profileswith the determined adsorption isotherms. These simulations werecompared with experimental elution profiles, the simulations usingthe adsorption isotherms determined by IM, RTM and ECP gavegood predictions of the overloaded elution profiles; but simula-tions using the adsorption isotherm determined using PP failed inpredicting the overloaded experimental results.

The reason why the PP adsorption isotherm differs, and couldnot be used to predict overloaded elution, is unclear. Here it shouldbe noted that PP use small injections on concentration plateausand all the other methods uses large injections and overloaded elu-tion profiles. Therefore one possible explanation might be solventstrength mismatch of the injection plug which would increase withincreasing injection volume, another explanation would be increas-ing differences in MeOH volume fraction on the different plateaus;however this remains to be investigated.

To conclude we have demonstrated that system verification,using external sensors, is necessary in order have to reliable, repro-ducible results and to ensure that we have SFC conditions thatenables us to transfer LC adsorption isotherm acquisition methodsto SFC. We have also shown that the IM, RTM and ECP methods canbe used in SFC to determine adsorption isotherms that can accu-rately predict overloaded elution profiles. Finally we observed thatthe PP method adsorption isotherm differs from the other methodsand could not be used to predict overloaded elution. The reason forthis interesting result is unclear and remains to be investigated.

Acknowledgements

This work was supported by the Swedish Research Council(VR) in the project “Fundamental studies on molecular interactionsaimed at preparative separations and biospecific measurements”Grant # 621-2012-3978. The work was also supported by theSwedish Knowledge Foundation in the KK HÖG 2011 project“Improved Purification Processes to Satisfy Modern Drug QualityAssurance and Environmental Criteria” and by the Research Coun-

cil for Environment, Agricultural Sciences and Spatial Planning inthe project “High-Value Compounds from Agricultural and ForestryWaste by Sustainable Methods – an Interdisciplinary Approach forBioresource Utilization”.

atogr

R

[[[[[[

[

[

[[

[

[[

[[

[[[[

[

[[[

[[[[

[[

[

[[41] B.J. Stanley, J. Krance, J. Chromatogr. A 1011 (2003) 11.

M. Enmark et al. / J. Chrom

eferences

[1] G. Guiochon, A. Tarafder, J. Chromatogr. A 1218 (2011) 1037.[2] T. Fornstedt, J. Chromatogr. A 1217 (2010) 792.[3] X. Zhang, J. Samuelsson, J.-C. Janson, C. Wang, Z. Su, M. Gu, T. Fornstedt, J.

Chromatogr. A 1217 (2010) 1916.[4] G. Guiochon, D.G. Shirazi, A. Felinger, A.M. Katti, Fundamentals of Preparative

and Nonlinear Chromatography, 2nd ed., Academic Press, Boston, MA, 2006.[5] A. Seidel-Morgenstern, J. Chromatogr. A 1037 (2004) 255.[6] J. Lindholm, P. Forssén, T. Fornstedt, Anal. Chem. 76 (2004) 4856.[7] J. Lindholm, P. Forssén, T. Fornstedt, Anal. Chem. 76 (2004) 5472.[8] F. Gritti, G. Guiochon, J. Chromatogr. A 1217 (2010) 7677.[9] A. Tarafder, G. Guiochon, J. Chromatogr. A 1285 (2013) 148.10] S. Ottiger, J. Kluge, A. Rajendran, M. Mazzotti, J. Chromatogr. A 1162 (2007) 74.11] C. Wenda, A. Rajendran, J. Chromatogr. A 1216 (2009) 8750.12] Z. Bao, B. Su, H. Xing, Y. Yang, Q. Ren, J. Sep. Sci. 33 (2010) 3256.13] A. Depta, T. Giese, M. Johannsen, G. Brunner, J. Chromatogr. A 865 (1999) 175.14] M. Lübbert, G. Brunner, M. Johannsen, J. Supercrit. Fluids 42 (2007) 180.15] J. Samuelsson, M. Enmark, P. Forssén, T. Fornstedt, Chem. Eng. Technol. 35

(2012) 149.16] A. Tarafder, K. Kaczmarski, D.P. Poe, G. Guiochon, J. Chromatogr. A 1258 (2012)

136.17] O. Kunz, R. Klimeck, W. Wagner, M. Jaeschke, The GERG-2004 Wide-Range

Equation of State for Natural Gases and Other Mixtures, Fortschr.-Ber. VDI,VDI-Verlag, Düsseldorf, 2007.

18] S. Golshan-Shirazi, G. Guiochon, J. Chromatogr. 506 (1990) 495.19] M. Enmark, J. Samuelsson, T. Undin, T. Fornstedt, J. Chromatogr. A 1218 (2011)

6688.

20] D. Åsberg, M. Leśko, M. Enmark, J. Samuelsson, K. Kaczmarski, T. Fornstedt, J.

Chromatogr. A 1299 (2013) 64.21] P. Forssén, J. Lindholm, T. Fornstedt, J. Chromatogr. A 991 (2003) 31.22] J. Samuelsson, R. Arnell, J.S. Diesen, J. Tibbelin, A. Paptchikhine, T. Fornstedt,

P.J.R. Sjoberg, Anal. Chem. 80 (2008) 2105.

[

[

. A 1312 (2013) 124– 133 133

23] J. Samuelsson, T. Undin, T. Fornstedt, J. Chromatogr. A 1218 (2011) 3737.24] J. Samuelsson, T. Undin, A. Törncrona, T. Fornstedt, J. Chromatogr. A 1217 (2010)

7215.25] H. Guan, B.J. Stanley, G. Guiochon, J. Chromatogr. A 659 (1994) 27.26] L. Ravald, T. Fornstedt, J. Chromatogr. A 908 (2001) 111.27] J. Samuelsson, T. Fornstedt, Anal. Chem. 80 (2008) 7887.28] J. Samuelsson, L. Edström, P. Forssén, T. Fornstedt, J. Chromatogr. A 1217 (2010)

4306.29] P. Forssén, L. Edström, J. Samuelsson, T. Fornstedt, J. Chromatogr. A 1218 (2011)

5794.30] S. Golshan-Shirazi, G. Guiochon, Anal. Chem. 61 (1989) 462.31] A. Rajendran, W. Chen, Sep. Purif. Technol. 67 (2009) 344.32] F. Gritti, G. Guiochon, Anal. Chem. (2013), http://dx.doi.org/10.1021/ac4015897

(in press).33] S. Golshan-Shirazi, G. Guiochon, J. Phys. Chem. 94 (1990) 495.34] P. Forssén, R. Arnell, T. Fornstedt, Comput. Chem. Eng. 30 (2006) 1381.35] D. Antos, W. Piątkowski, K. Kaczmarski, J. Chromatogr. A 874 (2000) 1.36] F. James, M. Sepúlveda, F. Charton, I. Quiñones, G. Guiochon, Chem. Eng. Sci. 54

(1999) 1677.37] J. Samuelsson, R. Arnell, T. Fornstedt, J. Sep. Sci. 32 (2009) 1491.38] E.W. Lemmon, M.L. Huber, M.O. McLinden, NIST Standard Reference Database

23: Reference Fluid Thermodynamic and Transport Properties-REFPROP, Ver-sion 9.1, National Institute of Standards and Technology, Standard ReferenceData Program, Gaithersburg, 2013.

39] J. Samuelsson, J. Zang, A. Murunga, T. Fornstedt, P. Sajonz, J. Chromatogr. A 1194(2008) 205.

40] J. Samuelsson, P. Sajonz, T. Fornstedt, J. Chromatogr. A 1189 (2008) 19.

42] P. Rouchon, M. Schonauer, P. Valentin, G. Guiochon, Sep. Sci. Technol. 22 (1987)1793.

43] M. Enmark, R. Arnell, P. Forssén, J. Samuelsson, K. Kaczmarski, T. Fornstedt, J.Chromatogr. A 1218 (2011) 662.

http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0005http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0010http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0015http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0020http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0025http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0030http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0035http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0040http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0040http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0040http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0040http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0040http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0040http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0040http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0040http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0040http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0040http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0045http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0045http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0045http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0045http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0045http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0045http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0045http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0045http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0045http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0045http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0050http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0055http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0055http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0055http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0055http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0055http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0055http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0055http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0055http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0055http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0055http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0060http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0065http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0070http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0075http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0080http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0085http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0090http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0090http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0090http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0090http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0090http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0090http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0090http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0090http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0090http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0095http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0100http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0105http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0110http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0115http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0120http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0125http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0130http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0130http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0130http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0130http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0130http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0130http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0130http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0130http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0130http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0130http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0135http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0135http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0135http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0135http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0135http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0135http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0135http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0135http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0135http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0140http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0145http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0150http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0150http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0150http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0150http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0150http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0150http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0150http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0150http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0150http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0155http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0155http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0155http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0155http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0155http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0155http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0155http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0155http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0155http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0155dx.doi.org/10.1021/ac4015897http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0165http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0165http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0165http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0165http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0165http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0165http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0165http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0165http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0165http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0165http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0170http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0175http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0180http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0185http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0190http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0195http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0200http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0205http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0205http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0205http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0205http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0205http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0205http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0205http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0205http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0205http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0205http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0210http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref0215http://refhub.elsevier.com/S0021-9673(13)01417-9/sbref02