Effect of parallel segmented flow chromatography on the height equivalent to a theoretical plate II...

Accepted Manuscript

Title: Effect of parallel segmented flow chromatography onthe height equivalent to a theoretical plate II- Performances of4.6 × 30 mm columns packed with 2.6 μm Accucore-C18

superficially porous particles

Author: Fabrice Gritti Georges Guiochon

PII: S0021-9673(13)01312-5DOI: http://dx.doi.org/doi:10.1016/j.chroma.2013.08.060Reference: CHROMA 354654

To appear in: Journal of Chromatography A

Received date: 13-5-2013Revised date: 18-7-2013Accepted date: 5-8-2013

Please cite this article as: Fabrice Gritti, Georges Guiochon, Effect of parallel segmentedflow chromatography on the height equivalent to a theoretical plate II- Performances of4.6 × 30 mm columns packed with 2.6 μm Accucore-C18 superficially porous particles,Journal of Chromatography A (2013), http://dx.doi.org/10.1016/j.chroma.2013.08.060

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

http://dx.doi.org/doi:10.1016/j.chroma.2013.08.060

http://dx.doi.org/10.1016/j.chroma.2013.08.060

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- Parallel segmented flow chromatography was applied to columns packed with shell particles

- Short 4.6 x30 mm columns packed with sub-3 um particles are not radially equilibrated

- PSFC at the column outlet improves the overall efficiency by a factor of nearly two.

- The sensitivity of weakly retained compounds increase by 25% when applying PSFC.

- Post-column band broadening should be minimized for best gradient elution performance

*Highlights (for review)

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Effect of parallel segmented flow chromatography on1

the height equivalent to a theoretical plate2

II- Performances of 4.6 × 30 mm columns packed with3

2.6 µm Accucore-C18 superficially porous particles4

Fabrice Gritti and Georges Guiochon ∗,

Department of Chemistry, University of Tennessee

Knoxville, TN 37996–1600, USA

5

Abstract6

The reduced trans-column (or long-range eddy dispersion) height equivalent to a theo-7

retical plate (HETP) of short and wide 4.6 × 30 mm columns packed with 2.6 µm Accucore-C188

superficially porous particles was measured under conventional (no split flow) and parallel seg-9

mented (outlet and inlet) flow chromatography. The overall reduced HETP was derived from10

the true moments of the recorded concentration profiles. The longitudinal diffusion HETP term11

was measured at a very small flow rate (0.05 mL/min). The solid-liquid mass transfer resistance12

was derived from the shell diffusivity, using the composite Garnett-Torquato model of effective13

diffusion in a heterogeneous system made of a dense packing of core-shell particles immersed14

in a continuous matrix (the eluent). The trans-channel and short-range interchannel eddy15

dispersion HETP terms were assumed to be equal to the calculated h data after solving the16

Navier-Stokes equation and simulating the advection-diffusion transport process.17

Experimental results confirmed that the optimum efficiency of these short columns was18

increased by a factor of about two. The ratio of the detection sensitivities on the PSFC stream19

and on a regular stream increased from 1 to 1.45 when the retention factor decreases from20

∗Corresponding author: (e-mail) [email protected]; (fax) 865 974–2667.

*Manuscript

http://ees.elsevier.com/chroma/viewRCResults.aspx?pdf=1&docID=29151&rev=1&fileID=1218905&msid=81752507-ACF1-476D-8C0A-0C5A40350E4D

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about 10 to 0.5. These phenomena are due to a strong reduction of the trans-column eddy21

dispersion HETP term. The system loses about 60% of the sample mass when only outlet22

skimming is carried out when the flow rate ratio of 55% is applied, as was done in this work. It23

loses about 50% of the sample when inlet/outlet segmentation is applied. In gradient elution,24

the peak capacity is increased by only 15%, due to post-column band spreading, which should25

imperatively be minimized when the outlet flow is split.26

Keywords: Column efficiency; inlet and outlet segmented flow chromatography; eddy diffusion;27

trans-column eddy dispersion; Accucore-C18, naphthalene.28

* Fax: 865-974-2667; email address: [email protected]

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1 Introduction30

Parallel segmented flow chromatography (PSFC) was recently designed to increase the column effi-31

ciency, to lower the detection limits, to perform simultaneously various complementary detections,32

and to match the optimum flow rates applied in HPLC and for LC-MS applications 1–5. The main33

motivation behind PSFC was to minimize the negative effects caused by the wall and border re-34

gions of a chromatographic column. The properties of the packed bed in the wall region differ from35

those in the bulk central region of the column 6. This was initially observed in preparative chro-36

matography with wide I.D. columns using local detection of eluted band profiles across the whole37

column diameter 7–12. The same center-to-wall column heterogeneity was also revealed in analytical38

columns (4.6 mm I.D.) irrespective of the nature of the particles used to pack the chromatographic39

column 13–31.40

The implementation of PSFC relies on the fabrication of new column frits and endfittings that41

allow the inlet and/or the outlet stream of the column to be split into two parallel streams. The42

first stream of eluent percolates through the column along the central zone while the second one43

circulates along the peripheral region of the column. The flow rate ratio of the streams is optimized44

empirically. It was shown that a ratio between 50 and 60% gives the optimum column efficiency45

5. From a physical viewpoint, segmenting the outlet flow prevents from detecting the sample46

molecules that are retarded in the peripheral region of the column. It allows improvement of the47

column performance by detecting only the sample molecules that are eluted along the central zone of48

the column, where axial dispersion is minimum and rather uniform. The peak tailing observed with49

traditional short and classical 4.6 × 30 mm columns packed with 3.0 µm fully porous Hypersil-C1850

particles is virtually eliminated when they are operated in PSFC. Columns operated under PSFC51

appear to be more efficient than if they were used under standard conditions of detection (no outlet52

split flow) 1,2, 32. The column efficiency corrected for extra-column band spreading increased by53

nearly a factor two at a maximum flow rate of 4 mL/min. The signal intensity increased by 25 to54

50% when the sample volume is injected only in the central region of the column (inlet split flow).55

In gradient elution, the relative gain of peak capacity was measured at about +15%, only, because56

of the nefarious post-column bandspreading.57

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In summary, it was previously demonstrated that the effect of using PSFC on the van Deemter58

curve of columns was to significantly reduce the long range trans-column eddy dispersion HETP59

term. This result was not unexpected because the overall eddy dispersion HETP term accounts60

for at least 80% of the total band broadening in today’s HPLC columns 6. The question remains61

whether similar improvements were observed for columns packed with superficially porous particles.62

Traditional 4.6 mm I.D. and 10 to 15 cm long columns packed with sub-3 µm particles show63

exceptionally low minimum reduced plate heights, around 1.4 instead of 2.0 for the columns of64

the size but packed with fully porous particles 16–18,22,23,33–37. For small molecules at low reduced65

velocities, the advantage of columns packed with core-shell particles is due to the decrease of66

the longitudinal diffusion HETP term (' -30 %) and of the trans-column eddy dispersion HETP67

term (- 40 %). However, local electrochemical detection across the column diameter has shown that68

these columns are still heterogeneous from the center to the wall 17,38. Additionally, experiments69

and the theory of eddy dispersion support the fact that the trans-column flow heterogeneity and70

the dispersion caused by the frit/endfitting assemblies contribute significantly to the column plate71

height 6. In theory, the incompressible minimum reduced HETP of a non-retained compound is72

about 0.6 for a bed porosity of 40% 39. In practice, the minimum reduced plate height73

observed for large aspect ratio columns (column-to-particle diameter) was measured74

at 1.2 for a 2.4 × 16.6 cm column packed with 200-220 µm glass beads having an75

external porosity of 38% 40. It was later measured at 1.0 for a particular column 4.6 × 15076

mm column packed with 2.6 µm core-shell particles and for an external porosity of 39.5 % 35. This77

column came from a lot of columns set apart for having an efficiency more than ± 2 standard78

deviations above the mean efficiency of a large batch of columns. Similarly low reduced plate79

heights were measured by a few authors who used columns with aspect ratios smaller than 8,80

for which there is no clear distinction between the structures of the center and the wall regions of81

the column 41,42. Therefore, there is still room to further increase the efficiency of classical 4.6 mm82

I.D. columns packed with core-shell particles. Applying the concept of PFSC to columns packed83

with sub-3 µm core-shell particles is an attractive option.84

The goal of this work was to study the effect of PSFC on the performance of short 4.6 × 30 mm85

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columns packed with 2.6 µm Accucore-C18 core-shell particles. The segmentation flow ratio was set86

at 55% (peripheral port). The analyte was the small molecule naphthalene for which adjusting the87

volume fraction of acetonitrile in water at 42.5, 62.0 and 90.0% provided three different retention88

factors. The potential advantage of PSFC on these columns operated in gradient elution is finally89

discussed.90

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2 THEORY91

2.1 Definitions92

The total, external and internal porosities of the packed bed are εt, εe, and εp, respectively. ρ is the93

core-to-particle diameter ratio for superficially porous particles. Dm is the diffusion coefficient of94

the analyte in the bulk mobile phase. The apparent analyte diffusivity through the porous region of95

the particles is Dp = ΩDm, with Ω being the dimensionless ratio of the sample diffusivity through96

the porous region of the particles to its bulk diffusion coefficient. The effective diffusion coefficient97

along the heterogeneous packed bed made of superficially porous particles in the external eluent98

is Deff . Ka is the equilibrium constant between the stationary phase and the eluent99

phase.100

The zone retention factor, k1, is defined as 43:101

k1 =1− εeεe

[εp + (1− εp)Ka] (1− ρ3) (1)

This retention factor, k1, refers to the elution time of the compound with respect to the time spent102

by the analyte in the interstitial column volume (εeπr2c ). In contrast, the conventional retention103

factor, k′, refers to the same elution time but with respect to the total accessible pore volume104

(εtπr2c ).105

The reduced interstitial linear velocity, ν, is written 44:106

ν =udpDm

(2)

where u in the average interstitial linear velocity along the column given by:107

u =Fvεeπr2c

(3)

where Fv is the flow rate and rc is the inner column radius.108

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2.2 Reduced HETP equation109

In the absence of significant effects due to frictional heating (which is the general case with beds of110

core-shell particles, even at high heat friction power 45,46), the overall reduced plate height is the111

sum of the longitudinal diffusion term, the trans-particle mass transfer resistance term, and the112

eddy dispersion term or 32:113

h =B

ν+A(ν) + Cpν (4)

By definition, the A(ν) HETP term includes all the sources of sample band broadening along114

the column that are not caused by the apparent longitudinal diffusion along the column and by115

the mass transfer resistance across the stationary phase volume. Next, we provide the explicit116

equations used in this work to express the B, A(ν), and the Cp term descriptions of each of these117

three HETP terms. For more detailed information on the derivation of these equations, the reader118

is referred to the following references 43,45,47–50.119

2.2.1 The reduced longitudinal diffusion HETP term120

The longitudinal diffusion term is derived from the effective diffusion coefficient of the analyte in121

the heterogeneous bed of packing material containing the external eluent, the porous shells, and122

the impermeable solid cores. A physically relevant model of effective diffusion in random packed123

beds with superficially porous particles is given by the Garnett-Torquato composite model 48,51,52,124

which is written 53,54:125

Deff =1

εe(1 + k1)

[1 + 2(1− εe)β − 2εeξ2β

2

1− (1− εe)β − 2εeξ2β2

]Dm (5)

with126

β =

1−ρ3

1+ ρ3

2

Ω− 1

1−ρ3

1+ ρ3

2

Ω + 2(6)

In these equations, ξ2 is an adjustable parameter estimated from the experimental external ob-127

struction factor for Ω = 0 and k1 = 0 (non-porous particles). From Eq. 5, the general expression128

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of γe =DeffDm

as a function of εe and ξ2 is:129

γe =2(1− ξ2

2 )

3− εe(1 + ξ2)(7)

The obstruction factor of a 4.6 × 100 mm column packed with 3.3 µm non-porous silica cores was130

found to be 0.633 for an external porosity of 0.415 55. Therefore, the value of ξ2 given by Eq. 7131

and used for columns packed with 2.6 µm Accucore-C18 superficially porous particles will be 0.493.132

The reduced B coefficient in Eq. 4 is then 53:133

B = 2(1 + k1)Deff

Dm(8)

2.2.2 The reduced solid-liquid mass transfer resistance HETP term134

The general expression of the solid-liquid mass transfer resistance coefficient (Cp) is 44,47,56:135

Cp =1

30

εe1− εe

(k1

1 + k1

)2 1 + 2ρ+ 3ρ2 − ρ3 − 5ρ4

(1 + ρ+ ρ2)21

Ω(9)

In this equation, the value of Ω is obtained by applying the model of effective diffusion (Eqs 5 and136

6) to the best experimental value of Deff estimated at the lowest reduced velocity applied (ν <137

0.3).138

2.2.3 The reduced eddy dipersion HETP term139

The general expression of the eddy dispersion HETP term, A(ν), is given in details in 32.140

2.3 Transverse dispersion coefficient across beds packed with core-shell parti-141

cles142

Estimates of the asymptotic transverse dispersion coefficient were recently refined for true packed143

beds (wide particle size distribution, non porous particles, no retention k1=0) by solving numeri-144

cally the Navier-Stokes equation and simulating the advection-diffusion transport process 27. The145

best fit between the simulated asymptotic transverse reduced plate height data and the general146

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mathematical expression (power law) of the transverse dispersion coefficient for reduced velocities147

in the range from 0.5 to 500 is 27 :148

Dt

Dm= γe + 0.133ν0.782 (10)

After generalization for any packed beds made of fully and/or superficially porous particles and149

taking into account the zone retention factor k1 of any retained analyte, the effective transverse150

dispersion coefficient is then written 6,32:151

Dt

Dm=Deff

Dm+

0.133

1 + k1ν0.782 (11)

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3 Experimental152

3.1 Chemicals153

The mobile phases were mixtures of acetonitrile and water (42.5/57.5, 62/38, and 90/10, v/v). All154

these solvents were HPLC grade from Fisher Scientific (Fair Lawn, NJ, USA). Acetonitrile was155

filtered before use on a surfactant-free cellulose acetate filter membrane, 0.20 µm pore size (Suwan-156

nee, GA, USA). Eleven polystyrene standards (MW=590, 1100, 3680, 6400, 13200, 31600, 90000,157

171000, 560900, 900000, and 1870000) were purchased from Phenomenex (Torrance, CA, USA) in158

order to perform inverse size-exclusion chromatography (ISEC) measurements on the standard Ac-159

cucore column. The RPLC checkout sample (1 mL) was purchased from Agilent Technologies (Little160

Fall, DE, USA). This mixture contains 100.3 µg/mL (± 0.5 %) of acetophenone, propiophenone,161

butyrophenone, valerophenone, hexanophenone, heptanophenone, octanophenone, benzophenone,162

and acetanilide dissolved in water-acetonitrile (65/35, v/v). Naphthalene was purchased from163

Fisher Scientific, with a minimum purity of 99%.164

3.2 Apparatus165

The same configurations of the 1290 Infinity system as those applied in reference 32 were used. For166

the sake of clarity and the visualization of the different flow rates in and out of the column, the167

schematics of the standard (S), parallel segmented outlet flow (O) and parallel segmented inlet and168

outlet flow (IO) configurations are shown in Figure 1 of reference 32.169

3.3 Columns170

Three different 4.6 × 30 mm prototype columns packed with the same batch of 2.6 µm Accucore-171

C18 superficially porous particles (the diameter of the solid silica core is 1.9 µm so the structural172

parameter is ρ=0.73) were generously given by the column manufacturer (ThermoFisher, Lough-173

borough, UK). One column was assembled with a standard endfitting at both the inlet and174

outlet. A second column was assembled with a special outlet endfitting that segments the outlet175

flow into two parallel streams. The wall fraction or peripheral region of the outlet stream is directly176

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sent to the waste bottle while the central fraction flows to the detector. The third column was as-177

sembled with one of these special endfittings at each end. The inlet flow stream that comes from178

the pump is split into two parallel streams, allowing one part to be sent directly into the column,179

into its peripheral region, bypassing the injection valve while the second stream is sent through180

the injection valve into the central region of the column. So the sample is locally injected into the181

central region of the column. For more detailed information on these prototype column endfittings,182

the reader is referred to references 1–5 or to the manufacturer (ThermoFisher, Loughborough, UK).183

The external, total, and internal (shell porosity) porosities of the standard 4.6 × 30 mm column184

were measured by ISEC at 0.429, 0.538, and 0.313, respectively. The external porosity of this short185

column packed with 2.6 µm fully porous particles is significantly larger than those usually measured186

for longer columns (10-15 cm long) packed with similar core-shell particles (0.39 < εe < 0.41) 19–21.187

This suggests that the inlet and outlet frits used in this short and wide column contribute somehow188

to the column hold-up volume. Commercially available stainless steel 0.5 µm and 2.0 µm frits have189

the same diameter of 4.78 mm and thickness of 1.88 mm. Their void fractions are 19.2 and 23%,190

respectively (data obtained from a web commercial catalogue). So, they generate an extra flow191

path volume of around 7 µL each. After corrections for these additional extra- column packing192

volumes, the true external and total porosities of the chromatographic beds were estimated to be193

εe=0.413 and εt=0.525. The average mesopore size of the packing material after derivatization was194

assessed at about 120 A from the intersection of the exclusion and retention ISEC branches.195

3.4 Inverse size-exclusion chromatography (ISEC)196

ISEC measurements were made only on the standard 4.6 × 30 mm Accucore-C18 column. Neat197

THF was used as the eluent. Eleven polystyrene standards with molecular weights between 500198

and 2 millions Dalton were used as probe molecules. They cover a wide range of molecular sizes,199

between 10 and 950 A. The average mesopore size before binding the core-shell Accucore particles200

with C18 ligands was 125 A according to the manufacturer. After this derivatization followed by201

trimethylsilane (TMS) endcapping, the average pore size was decreased to about 120 A 57–59. The202

flow rate was set at 0.50 mL/min and the sample size set at 2 µL. The detection wavelength was set203

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at 254 nm with a bandwidth of 4 nm. The external porosities (εe) were derived by extrapolation204

to a zero molecular weight of the exclusion branch of the ISEC plot of the elution volumes of the205

polymers versus their hydrodynamic molecular radius. The accuracy of the ISEC experimental206

protocol lies within 1%. The total porosity (εt) was measured from the elution time of toluene in207

pure tetrahydrofuran. The internal porosity of the porous shell (εp) was derived from:208

εp =εt − εe

(1− εe)(1− ρ3)(12)

3.5 Diffusion coefficients of naphthalene209

The bulk molecular diffusivities, Dm, of naphthalene were measured at 1.08 × 10−5, 1.48 × 10−5,210

and 2.60 × 10−5 cm2/s for volume fractions of acetonitrile in water of 0.425, 0.620, and 0.900,211

respectively 32.212

3.6 HETP measurements213

The same sequence of flow rates was applied to all three column configurations and mobile phase214

compositions. The flow rate was increased successively from 0.05 to 0.10, 0.25, 0.50, 0.75, 1.00,215

1.50, 2.00, 3.00 and to 4.0 mL/min. Depending on the flow rate, the data sampling frequency216

was adjusted between 2.5 and 160 Hz in order to record at least 120 data points for the whole217

band profile of naphthalene. For the extra-column band profiles, the sampling frequency was fixed218

at 160 Hz for all flow rates. Samples of 1 µL of the naphthalene solution (concentration < 0.5219

g/L in the respective mobile phase) were injected into the three columns and the chromatograms220

were recorded at a wavelength of 290 nm. A constant UV bandwidth of 4 nm was selected. The221

temperature was set by the laboratory air-conditioning system at 24.1 ± 0.5 oC.222

For the same sequence of flow rates, the extra-column contributions of the system (standard223

1290 Infinity) to the retention times and time variances were measured in the above three system224

configurations (S, O, and IO). Due to the split flow (in the O and IO configurations) and the225

subsequent reduction of the flow rate from the column outlet to the detector cell, the system first226

and second time moments are larger than those measured under the standard configuration S.227

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Examples of band profiles recorded in the presence and absence of the chromatographic column are228

shown in Figures 3, 4, and 5 at the maximum flow rate of 4 mL/min. Without exception, all band229

profiles appear smooth when connecting two successive data points with a straight line.230

The first and second central moments were all measured using a well-defined numerical analysis.231

All the details for the measurement of the HETP data and their accuracy are given in 60–62. In order232

to increase the accuracy of the measurements, the peak area was determined with the Simpson233

integration method instead of the standard trapezoidal method. The first and second central234

moments of the eluted band profiles were derived from the discreet Simpson sums given in235

32. The first (left cut) and the last (right cut) elution times in these sums were unambiguously236

determined from the two elution times recorded when the sample concentration began to increase237

(left) then decrease (right) to 0.1% of the peak height after linear baseline correction. This method238

has the advantage of clearly and systematically adjusting the integration window, regardless of the239

degrees of peak fronting and peak tailing that may fluctuate in various ways depending on the240

nature of the sample injected and on the flow rate applied. It is highly sensitive to any packing241

heterogeneity or any source of flow heterogeneity in the column which would not be perceptible by242

the classical half-height peak width method. Therefore, this method is highly suitable for assessing243

the accurate effect of PSFC on the peak moments.244

The intrinsic (corrected for the extra-column volume contributions) reduced plate height h is245

then measured according to classical relationships given in 32. The accuracy of the h246

values is around 10 and 4% under non-retained and retained conditions, respectively. In addition,247

let us recall that the measurement of the corrected reduced plate height, h, assumes that the extra-248

column contributions (µ1,ex and µ′2,ex) measured by replacing the column with the zero dead249

volume (ZDV) union connector are the true ones. It should be kept in mind that the conditions250

of pressure (upstream the column) and of sample concentration (downstream the column) are251

necessarily different in the presence and in the absence of the chromatographic column. This252

contribute to a certain extent to lower the accuracy of the h data, especially with short columns.253

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4 Results and Discussion254

The first part of this work shows evidence of a pre-asymptotic dispersion regime across the diameter255

(4.6 mm) of short (3 cm) columns packed with 2.6 µm Accucore-C18 core-shell particles, on the basis256

of the best transverse dispersion coefficient calculated for packing materials made of particles with a257

narrow particle size distribution 27. In the second part, the effect of PSFC on the corrected reduced258

plate height is assessed for 4.6 × 30 mm columns packed with 2.6 µm Accucore-C18 particles. The259

same three system configurations (S for standard, O for outlet split flow, and IO for inlet and outlet260

split flow) as those applied in reference 32 are also considered in this work. In the last part, the261

three different system configurations are tested and compared in gradient elution for the separation262

and resolution of a sample of nine small molecules.263

4.1 Evidence for the pre-asymtotic dispersion regime264

The sample concentration profile across the column diameter is considered to be uniform when265

the injected (z=0) sample molecules have sampled at least once the whole column inner diameter266

(dc=0.46 cm) during their migration along the column until they reach the column outlet (z=L).267

This defined the complete asymptotic dispersion regime 63. The use of short and wide columns268

does not favor the establishment of an asymptotic transverse and axial dispersion regime because269

the residence times of the analytes are too short to allow the complete radial equilibrium across270

the 4.6 mm I.D. columns.271

The asymptotic radial (or transverse) dispersion coefficient of the analyte is best given by Eq.272

11. The retention time tR(ν) and the standard deviation of the radial excursion distance r(ν)273

spread by sample molecules from the inlet to the outlet of the column at a reduced velocity ν are274

given by :275

tR(ν) = (1 + k1)L

u= (1 + k1)

LdpνDm

(13)

and276

r(ν) =√

4Dt(ν)tR(ν) = 2

√Ldpν

(B

2+ 0.133ν0.782

)(14)

The reduced longitudinal B coefficient was measured for the three different retention factors.277

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For k1=14.5, 3.3, and 1.0 the values of the B coefficient measured were 8.40, 3.25, and 1.73,278

respectively. Figure 1 shows the plots of the radial distance r(ν) as a function of the applied flow279

rate, which increases from 0.05 to 4.00 mL/min. In this graph, the blue, thick, solid horizontal line280

represents the inner radius of the 3 cm long column. It is noteworthy that the standard deviation281

of the radial dispersion distance of a sample band injected in the center of the column is always282

smaller than the column diameter. Clearly, the column is not equilibrated radially, irrespective of283

the mobile phase composition and of the flow rate applied. For small molecules, the stream-284

splitting distances are naturally small and the diffusion coefficient of small molecules285

in the mobile phase is too small. Remarkably, r(ν) decreases with increasing flow rate because286

the residence times in the column decrease and, so, the radial convective dispersion term287

is marginal for 2.6 µm spherical particles.288

In conclusion, the overall axial dispersion coefficient is measured at the column outlet in a289

transient dispersion regime because the column is far from being radially equilibrated. The kinetic290

performance of a short 3 cm long column is sensitive to the initial distribution of the sample291

molecules at the column inlet and to their final collection at the column outlet 6. Accordingly, in292

theory, PSFC should definitely affect the apparent axial dispersion coefficients of 3 cm long and293

4.6 mm wide columns packed with fine sub- 3 µm particles. The purpose of the next sections is to294

report measurements that verify this theoretical prediction.295

4.2 Column performance under isocratic conditions296

The purpose of this section is to assess the advantages of PSFC (system configurations O and IO)297

on the intrinsic kinetic performance of a short standard 4.6 × 30 mm Accucore-C18 column under298

isocratic conditions. The corrected reduced plate heights of naphthalene were measured for three299

different retention factors (k′= 11.4, 2.5, and 2.6). Figure 2A-C show the corresponding corrected300

reduced h versus ν plots. Note that the different values of Dm narrow the experimental301

range of reduced velocities at increasing acetonitrile content in the mobile phase from302

Figure 2A to 2B and to 2C. It is important to recall that the corrected HETP data were303

measured according to the numerical integration of the whole peak profile. For such304

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short and wide 4.6 mm × 30 mm columns, the border and wall effects are maximum305

and the minimum reduced plate height does not match that of longer 4.6 × 100 mm306

columns. As a result, peaks are often tailing at their base. Additionally, the contribu-307

tion of the extra-column contributions is large for such short column and the possible308

underestimation of the true peak variance when replacing the chromatographic column309

with the ZDV union connector can lead to an overestimation of the corrected reduced310

plate height. Therefore, for both these reasons, the minimum reduced HETP can be311

much larger than 2.0 for fully porous particles 32 and 1.5 for core-shell particles (this312

work). Again, the goal of this work is not to assess the efficiency of these standard 3313

cm long columns but to report on the efficiency gain obtained when segmenting the314

whole inlet and outlet flow into a central and a wall streams.315

4.2.1 Strong retention factor : k′> 10316

For k′=11.4, the reduced longitudinal diffusion B coefficient is 8.40 (Figure 2A). This high value317

is explained by the occurrence of surface diffusion 64 in the RPLC mode. This justifies the rapid318

decrease of the h plots with increasing reduced velocity. The minimum reduced HETP of the319

standard column is hmin=3.6. This value is larger than the usual optimum value of h, 1.5 when320

measured for 10 or 15 cm long columns packed with core-shell particles 18–21. The wall and border321

effects are clearly enhanced in short and wide columns such as those used in this work. Additionally,322

the possible errors made in the measurement of the extra-column peak variance by replacing the323

column with a ZDV union connector could explain this high minimum reduced plate height.324

The kinetic performance of the PSFC columns (O and IO configurations) are significantly better325

than that of the standard column (no split flow). The optimum reduced velocity remains constant326

at νopt=11 whereas the hmin decreases from 3.6 (S configuration) to 2.0 (O configuration) and327

1.9 (IO configuration). Remarkably, the larger the flow rate, the better the performance of the328

PSFC than that of the classical columns. This is explained by the fact that the distance from329

radial equilibrium increases with increasing flow rate as shown in Figure 1. The collected sample330

molecules have mostly been axially dispersed through the central homogeneous region of the column.331

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Their residence time is too short to allow a significant dispersion across the column inner diameter.332

The efficiency gain is then maximum (+80%) for the largest flow rate beyond 1 mL/min. This333

also means that border effects dominate wall effects in such short columns.334

Clearly, the elimination of the wall region stream of eluent exiting the column is advantageous.335

Figure 3 shows the corresponding peak profiles at 4 mL/min and compares them to the extra-column336

peak profiles recorded by replacing the chromatographic column with the appropriate ZDV union337

connectors. The main visible effect of using PSFC columns is the almost total elimination of the338

tailing at the base of the peak for strongly retained compounds. The second striking observation is339

how the relative peak heights contrast with those of the extra-column peaks. For strongly retained340

compounds, the extra-column effects on the peak variance are small (< 0.6, 3.3, and 6.2% for341

the S, O, and IO column configurations). Results show that the detection sensitivity in the IO342

configuration is only 5% larger than in the standard configuration. Finally, the fraction of the343

sample injected that is actually detected can be estimated from the outlet flow rate through the344

detector, which is equal to only 45% of the total flow rate. While, obviously, 100% are detected for345

the standard column (no outlet split flow), only 41% and 47% are detected for the O and IO PSFC346

column configurations, respectively. Roughly, half the mass injected is lost.347

4.2.2 Moderate retention factor : k′= 2.5348

The reduced h versus ν plots are shown in Figure 2B. The B coefficient is now lower, at 3.25.349

As a result, the optimum reduced plate height hmin decreases from 3.6 to 3.3 for the standard350

column. Again, the PSFC column generate smaller hmin values at 2.2 (IO configuration) and351

1.9 (O configuration). In contrast to what was reported in the previous section, the IO column352

configuration is not as efficient as the O column configuration for moderate retention factors. This353

demonstrates experimentally that the local inlet injection of the sample (56.3 % of the total eluent354

stream flows directly through the peripheral port of the column and bypasses the injection valve)355

causing a harmful bias in the center-to-wall velocity bias compared to that in the classical injection356

mode. Figure 4 shows the corresponding band profiles in the presence (right graph) and in the357

absence (left graph) of the column at the same flow rate of 4 mL/min. Remarkably, the intrinsic358

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efficiencies of columns operated under PSFC mode is larger than that of standard columns but is359

not useful for analysts because the extra-column band broadening contributions to the total peak360

variance become large (8, 24, and 32 % for the S, O, and IO column configurations). Eventually,361

the gain in sensitivity remains moderate, around 10%.362

4.2.3 Weak retention factor : k′< 0.6363

The reduced h versus ν plots are shown in Figure 2C. The B coefficient is minimum at 1.73.364

The relative contribution of the extra-column volume to the total peak variance measured become365

large, at 21, 59, and 57% for the S, O, and IO column configurations. The optimum corrected366

reduced plate height hmin is 4.2 for the standard column. The use of PSFC configuration decreases367

the corrected hmin value to 3.6 (IO configuration) and 2.2 (O configuration). Again, the gain368

in column efficiency provided by the IO configuration is not as large as the one observed with369

the O configuration. This is consistent with the previous result reported for moderate retention370

factors. The peak profiles are shown in Figure 5. The gain in sensitivity is now obvious at about371

+45% for the O configuration. Remarkably, the front part of the peak profile is sharper for the O372

configuration than for the S column. whereas the peak tailing is virtually unchanged due to the373

large extra-column volume contribution.374

As it was shown in section 4.2 where a pre-asymptotic dispersion regime is expected for all375

retention factors, the advantage of PSFC at the column outlet is virtually the same for all retention376

factors.377

4.2.4 Analysis of the trans-column eddy dispersion HETP term378

Figure 6A-C summarizes the advantages of using PSFC configuration regarding the reduction of379

the reduced transient trans-column eddy dipersion HETP term hTC . These plots were generated380

after subtracting the Bν HETP terms (B=8.40, 3.25, and 1.73), the trans-channel, the short-range381

inter-channel reduced HETP terms (hTS and hIC), the solid-liquid mass transfer resistance reduced382

HETP term Cpν (Ω=5.1, 1.0, and 0.2 so Cp=0.0019, 0.0064, and 0.012) from the total h data. The383

largest diminution of hTC are observed for the PSFC O configuration, for the smallest retention384

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factor, and in almost the whole range of flow rates applied.385

4.3 Gradient application386

The standard and the two PSFC column configurations (O and IO) were tested in gradient elution387

at a constant flow rate of 2 mL/min. The test sample was the Agilent RPLC check-out sample388

which contains nine small molecules. The mobile phase composition was linearly increased from389

ϕ=0.40 to ϕ=0.95. The gradient volume VG was set at six times the hold-up column volume (VG=6390

× 0.268 cm3= 1.608 cm3). The volume gradient slope was βv=0.280 cm−3. The expected peak391

capacities were estimated from the peak standard deviation σi variance of seven fully resolved peaks392

i=1, 2, 3, 6, 7, 8, and 9 (the peaks #4 and #5 are not fully resolved for all the system contributions)393

:394

Pc = 1 + 7t9 − t14∑

i σi(15)

Figure 7 shows the corresponding gradient chromatograms recorded under the S (red color), O395

(blue color), and IO (green color) system configurations. As previously observed with 4.6 × 30 mm396

columns packed with 3.0 µm fully porous Hypersil-C18 particles, the IO configuration does not seem397

to be well adapted for gradient elution. Peaks are both fronting and tailing and a poor resolution398

is achieved with a peak capacity of only 27. Fronting is possibly due to the asynchronous399

distribution of the gradient eluent at the inlet wall and central ports. This could400

mean that the dwell time from the pump mixer to the inlet wall region is shorter401

than that to the inlet central region. A fraction of the molecules injected in the wall402

region hits first the central outlet first due to transverse dispersion. Similarly, the403

peak tailing is due to the late eluted molecules injected in the central region of the404

column. A fraction of these molecules remains in the central region of the column405

during migration and are eluted last. Despite the local injection of the sample volume in406

the central region of the column, larger detection limits are observed in the IO than in either the407

O or the S system configurations. The peak heights are relatively low and a large peak tailing is408

observed in the green chromatograms in Figures 4 and 5 for small retention factors. In contrast,409

the gradient performance of the column in the PSFC configuration O is clearly better than that of410

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the standard column. The peak capacity increases from 38 to 43 when the outlet stream is split (a411

+ 12% gain).412

It is worth noting that for a flow rate of 2 mL/min, the relative gains in intrinsic column413

efficiency are +75% (for k′=11.4 in Figure 2A), +97% (for k

′=2.5 in Figure 2B) and +104% (for414

k′=0.6 in Figure 2C). Therefore, on the average, if the extra-column contributions to the peak415

variance were negligible in gradient elution, the relative gain in peak capacity would be around416

35-40%. In fact, however, this gain is only around +12%. Indeed, the average peak variance (over417

the seven resolved peaks) measured in gradient elution are 0.105 s2 (S configuration) and 0.083 s2418

(O configuration). We may estimate the post-column extra-column peak variances from half the419

total extra-column peak variances (ideal column inlet focusing). At a flow rate of 2 mL/min, it is420

0.008 s2 (S configuration) and 0.019 s2 (O configuration). After correction for these extra-column421

contributions, the peak variance decreases to 0.097 s2 and to 0.064 s2. The peak capacities would422

then increase to 39 and 51, respectively, in better agreement with the intrinsic kinetic performances423

of the columns.424

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5 Conclusion425

Parallel segmented flow chromatography (PSFC) was applied to study its effects on the kinetic426

performance of short (3 cm) and wide (0.46 cm I.D.) columns packed with 2.6 µm Accucore-C18427

core-shell particles. A segmentation flow ratio of 55% along the wall region was set. The minimum428

reduced HETP of such columns packed with these particles (hmin=4.1) was found to be larger than429

one (hmin=1.4) usually observed for longer (10-15 cm) columns. This confirms that the wall and430

border effects of the column are more important for short than for long columns. Additionally, the431

impact of the extra-column band broadening is important for such short columns and the possible432

underestimation of its measurement leads to an overestimate of the corrected reduced plate height.433

PSFC set at the outlet of short columns has the same effects whether they are packed with 2.6434

µm core-shell particles or with 3.0 µm fully porous particles. This lowers the trans-column eddy435

dispersion HETP by at least a factor 2 for all retention factors. The minimum reduced HETP436

decreased from 4.1 to only 1.9. Thus, the intrinsic column efficiency is doubled over most of range437

of flow rates applied to 4.6 mm I.D. columns (from 1 to 4 mL/min). For large retention factors,438

the baseline peak tailing observed for the standard column is eliminated. The small fraction of439

sample molecules retarded in the wall region of the column is discarded and sent directly to the440

waste bottle instead of being detected. For small retention factors and for these short columns, this441

significant advantage is partially counter-balanced by the relative increase of the extra-column time442

peak variance from the needle seat to the column inlet (inlet split flow) and/or from the column443

outlet to the detection cell (outlet split flow). In gradient elution, the peak capacity are increased444

by only 15%, due to the decrease of the flow rate and to the post-column dispersion between the445

column outlet and the detector cell. The inlet split flow was not successful, most probably because446

the compositions of the eluent flowing through the peripheral and the central inlet regions of the447

column were slightly asynchronous. This could explain the systematic peak fronting observed for448

retained analytes.449

The detection sensitivities under PSFC and standard flow conditions were found to be similar for450

large retention factors (the reduction of the trans-column eddy dispersion HETP term is roughly451

compensated by the larger extra-column time peak variance). The detection limit decreased by 10452

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to 45% when the retention factor decreases from about 2.5 to 0.5 (the reduction of the trans-column453

eddy dispersion HETP term is not entirely cancelled out by the larger extra-column time peak454

variance). Overall, the segmentation of the flow rate eliminates about 50% of the total number of455

injected molecules.456

In conclusion, PSFC does significantly improve the kinetic performance of short and wide HPLC457

columns, irrespective of the nature of the particles. The influence of the column length on this458

improvement should be investigated for a series of columns packed with the same material but of459

different lengths (5, 7.5, and 10 cm long). Increasing the column length should favor the relaxation460

of the radial concentration gradients across the column and should a priori maximize and minimize461

the impacts of the wall and border, effects, respectively, on the observed efficiency of standard462

HPLC columns. Additionally, the impact of the diffusion coefficient of the analyte on the efficiency463

gain will be analyzed from both a theoretical and experimental vioewpoints. The corrected reduced464

HETPs will be compared for small (Dm ' 10−5 cm2/s) and high-molecular-weight (Dm ' 10−6465

cm2/s) compounds.466

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6 Acknowledgements467

This work was supported in part by the cooperative agreement between the University of Tennessee468

and the Oak Ridge National Laboratory. We thank Andrew Shalliker and Harald Ritchie (Ther-469

moFisher, Loughborough, UK) for the generous gift of the Accucore-C18 columns and the special470

fittings and for useful advice and discussions.471

References472

[1] Camenzulli, M.; Ritchie, H. J.; Ladine, J. R.; Shalliker, R. J. Chromatogr. A 2012, 1232, 47.473

[2] Shalliker, R.; Camenzulli, M.; Pereira, L.; Ritchie, H. J. J. Chromatogr. A 2012, 1262, 64.474

[3] Camenzulli, M.; Ritchie, H. J.; Shalliker, R. J. Chromatogr. A 2012, 1270, 204.475

[4] Camenzulli, M.; Ritchie, H. J.; Ladine, J. R.; Shalliker, R. Analyst 2011, 136, 5127.476

[5] Camenzulli, M.; Goodie, T. A.; Bassanese, D. N.; Francis, P.; barnett, N.; Ritchie, H. J.;477

Ladine, J. R.; Shalliker, R. Rapid Commun. mass Spectrom. 2012, 1232, 47.478

[6] Gritti, F.; Guiochon, G. Anal. Chem. 2013, 85, 3017.479

[7] Farkas, T.; Guiochon, G. Anal. Chem. 1997, 69, 4592.480

[8] Farkas, T.; Sepaniak, M. J.; Guiochon, G. AIChE J. 1997, 43, 1964.481

[9] Yun, T.; Smith, M. S.; Guiochon, G. J. Chromatogr. A 1998, 828, 19.482

[10] Yew, B. G.; Ureta, J.; Shalliker, R. A.; Drumm, E. C.; Guiochon, G. AIChE J. 2003, 49,483

642–664.484

[11] Shalliker, R. A.; Wong, V.; Broyles, B. S.; Guiochon, G. J. Chromatogr. A 2002, 977, 213.485

[12] Mriziq, K. S.; Abia, J. A.; Lee, Y.; Guiochon, G. J. Chromatogr. A 2008, 1193, 97.486

[13] Knox, J. H.; Laird, G. R.; Raven, P. A. J. Chromatogr. 1976, 122, 129.487

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[14] Eon, C. J. Chromatogr. 1978, 149, 29–42.488

[15] Gritti, F.; Guiochon, G. J. Chromatogr. A 2011, 1218, 1592.489

[16] Gritti, F.; Leonardis, I.; Shock, D.; Stevenson, P.; Shalliker, A.; Guiochon, G. J. Chromatogr.490

A 2010, 1217, 1589.491

[17] Gritti, F.; Leonardis, I.; Abia, J.; Guiochon, G. J. Chromatogr. A 2010, 1217, 3219.492

[18] Guiochon, G.; Gritti, F. J. Chromatogr. A 2011, 1218, 1915.493




[22] Gritti, F.; Guiochon, G. LC-GC North Am. 2012, 30, 586.497

[23] Gritti, F. Chromatography Today 2012, 3, 4.498

[24] Gritti, F.; Horvath, K.; Guiochon, G. J. Chromatogr. A 2012, 1263, 84.499

[25] Khirevich, K.; Holtzel, A.; Seidel-Morgenstern, A.; Tallarek, U. J. Chromatogr. A 2012, 1262,500

77.501

[26] Daneyko, A.; Khirevich, S.; Holtzel, A.; Seidel-Morgenstern, A.; Tallarek, U. J. Chromatogr.502

A 2011, 1218, 8231.503

[27] Daneyko, A.; Hlushkou, D.; Khirevich, S.; Tallarek, U. J. Chromatogr. A 2012, 1257, 98.504

[28] Bruns, S.; Tallarek, U. J. Chromatogr. A 2011, 1218, 1849.505

[29] Hormann, K.; Muellner, T.; Bruns, S.; Holtzel, A.; Tallarek, U. J. Chromatogr. A 2012, 1222,506

46.507



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[35] Gritti, F.; Guiochon, G. Chem. Eng. Sci. 2010, 65, 6327.513



[38] Abia, J.; Mriziq, K.; Guiochon, G. J. Chromatogr. A 2009, 1216, 3185.516

[39] Khirevich, S.; Holtzel, A.; Seidel-Morgenstern, A.; Tallarek, U. Anal. Chem. 2009, 81, 7057–517

7066.518

[40] Magnico, P.; Martin, M. J. Chromatogr. 1990, 517, 31.519

[41] Kennedy, R.; Jorgenson, J. Anal. Chem. 1989, 61, 1128.520

[42] Patel, K. D.; Jerkovich, A. D.; Link, J. C.; Jorgenson, J. W. Anal. Chem. 2004, 76, 5777.521


[44] Guiochon, G.; Felinger, A.; Katti, A.; Shirazi, D. Fundamentals of Preparative and Nonlinear523

Chromatography, 2nd ed.; Academic Press, Boston, MA, 2006.524



[47] Giddings, J. Dynamics of Chromatography; Marcel Dekker, New York, NY, 1965.527

[48] Torquato, S. J. Appl. Phys. 1985, 58, 3790.528


[50] Khirevich, S.; Daneyko, A.; Holtzel, A.; A., S.-M.; Tallarek, U. J. Chromatogr. A 2010, 1217,530

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[51] Garnett, J. C. M. Philos. Trans. R. Soc. London, Ser. B 1904, 203, 385.532

[52] Torquato, S. Random Heterogeneous Materials. Microstructure and Macroscopic Properties;533

Springer: New York, 2002.534




[56] Kaczmarski, K.; Guiochon, G. Anal. Chem. 2008, 79, 4648.538

[57] Rustamov, I. Farcas, T.; Ahmed, F.; Chan, F.; LoBrutto, R.; McNair, H.; Kazakevich, Y. J.539

Chromatogr. A 2001, 913, 49.540


[59] Gritti, F.; Kazakhevich, Y.; Guiochon, G. J. Chromatogr. A 2007, 1169, 111.542


[61] Stevenson, P.; Gritti, F.; Guiochon, G. J. Chromatogr. A 2011, 1218, 8255.544

[62] Stevenson, P.; Gao, H.; Gritti, F.; Guiochon, G. J. Sep. Sci. 2013, 36, 279.545

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[64] Miyabe, K.; Guiochon, G. J. Chromatogr. A 2010, 1217, 1713.548

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7 List of symbols549

Roman letters550

A(ν) Reduced eddy dispersion term551

B Reduced longitudinal diffusion coefficient with reference to the interstitial linear velocity552

Ci Sample concentration at time ti (s)553

Cp Reduced solid-liquid mass transfer resistance coefficient554

dp Average particle diameter (m)555

Deff Effective diffusion coefficient of the analyte in the packed column (m2/s)556

Dm Bulk molecular diffusion coefficient (m2/s)557

Dt Transverse dispersion coefficient of the analyte across the packed column (m2/s)558

Fv Flow rate (m3/s)559

h Reduced plate height560

hTS Reduced trans-channel eddy dispersion plate height561

hIC Reduced short-range eddy dispersion plate height562

hTC Reduced trans-column plate height563

k′

Retention factor564

k1 Zone retention factor.565

Ka Equilibrium Henry’s constant for the sample adsorption-desorption between the solid566

phase in the porous volume of the particle and the liquid eluent phase.567

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L Column length (m).568

rc Column inner radius (m)569

ti Discretized elution time (s)570

u Interstitial linear velocity (m/s)571

VG Gradient volume (m3)572

Greek letters573

β Parameter in Torquato’s model of effective diffusion in packed beds defined by Eq. 6574

βv Volume gradient slope (m−3)575

εe External column porosity576

εp Particle porosity577

εt Total column porosity578

γe Obstruction factor caused by randomly packed non-porous particles to the diffusion in the579

external bulk mobile phase580

µ1 First moment (s)581

µ′2 Second central moment (s2)582

µ1,ex First moment recorded in absence of the chromatographic column (s)583

µ′2,ex Second central moment recorded in absence of the chromatographic column (s)584

ν Reduced interstitial linear velocity585

Ω Ratio of the effective diffusivity of the sample in the porous region of the particle to its586

bulk diffusion coefficient587

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ω1 Diffusion eddy dispersion coefficient related to trans-channel velocity bias588

λ1 Flow eddy dispersion coefficient related to trans-channel velocity bias589

ω2 Diffusion eddy dispersion coefficient related to short-range inter-channel velocity bias in590

columns packed with non-porous particles591

λ2 Flow eddy dispersion coefficient related to short-range inter-channel trans-column velocity592

bias in columns packed with non-porous particles593

ϕCH3CN Volume fraction of acetonitrile in water594

ξ2 Adjustable parameter in Torquato’s model of effective diffusion Eq. 5595

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0 1 2 3 40.0

0.1

0.2

rC=0.23 cm

Rad

ial d

ispe

rsio

n [c

m]

Flow rate [mL/min]

k'=11.4 k'=2.5 k'=0.6

Figure 1: Plot of the average radial dispersion spread of injected analyte molecules at the columnoutlet as a function of the applied flow rate for three different retention factors as indicated in thegraph. Column length : 3 cm. Packing material : 2.6 µm Accucore-C18 core-shell particles.

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0 10 200

10

20

30CH3CN=0.425

h=H

/dp

Reduced velocity

Standard column Oulet split flow Inlet-Outlet split flow

0 10 200

10

20

30

Reduced velocity

h=H

/dp


CH3CN=0.62

0 10 200

10

20

30 CH3CN=0.90


Reduced velocity

h=H

/dp

Figure 2: Plots of corrected reduced plate height plots, h versus ν, measured for three differentPSFC configurations as indicated in the legend. The volume fraction of acetonitrile in the mobilephase increases from 42.5 (A) to 62.0 (B) and 90.0 % (C). The retention factors decrease fromk=11.4 to 2.4 and 0.6. Column: 4.6 × 30 mm Accucore-C18. Room temperature.

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0.0 0.5 1.0 1.50

400

800CH3CN=0.425

Abs

orba

nce

[mA

U]

Time [s]

Standard column Outlet split flow Inlet-Outlet split flow

45 48 51 540

50

100

CH3CN=0.425

Abs

orba

nce

[mA

U]

Time [s]


Figure 3: Band profiles of naphthalene eluted through the ZDV connector and the extra-columnvolume (left) and through the column (right) recorded for three different configurations of the 1290Infinity system as indicated in the legend. The volume fraction of acetonitrile in the mobile phasewas 42.5 %. The retention factors was k=11.4. Column: 4.6 × 30 mm Accucore-C18. Roomtemperature. Flow rate : 4 mL/min. Injection volume : 1 µL.

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0.0 0.5 1.0 1.50

400

800CH3CN=0.62

Abs

orba

nce

[mA

U]

Time [s]


14 160

100

200 CH3CN=0.62A

bsor

banc

e [m

AU

]

Time [s]


Figure 4: Band profiles of naphthalene eluted through the ZDV connector and the extra-columnvolume (left) and through the column (right) recorded for three different configurations of the1290 Infinity system as indicated in the legend. The volume fraction of acetonitrile in the mobilephase was 62.0 %. The retention factors was k=2.4. Column: 4.6 × 30 mm Accucore-C18. Roomtemperature. Flow rate : 4 mL/min. Injection volume : 1 µL.

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0.0 0.5 1.0 1.50

400

800

CH3CN=0.90

Abs

orba

nce

[mA

U]

Time [s]


6 70

100

200

300

400

CH3CN=0.90A

bsor

banc

e [m

AU

]

Time [s]


Figure 5: Band profiles of naphthalene eluted through the ZDV connector and the extra-columnvolume (left) and through the column (right) recorded for three different configurations of the1290 Infinity system as indicated in the legend. The volume fraction of acetonitrile in the mobilephase was 90.0 %. The retention factors was k=0.6. Column: 4.6 × 30 mm Accucore-C18. Roomtemperature. Flow rate : 4 mL/min. Injection volume : 1 µL.

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0 10 20 300

2

4

6

Reduced velocity

hTC Standard column Oulet split flow Inlet-Outlet split flow

CH3CN=0.425

0 10 200

2

4

6

hTC

Reduced velocity

CH3CN=0.62 Standard column Oulet split flow Inlet-Outlet split flow

0 10 20 300

2

4

6

hTC


CH3CN=0.90

Reduced velocity

Figure 6: Plots of the reduced transcolumn eddy dispersion plate height, hTC versus ν, measuredfor the three different PSFC configurations of the 1290 Infinity system as indicated in the legend.The volume fraction of acetonitrile in the mobile phase was increased from 42.5 (A) to 62.0 (B)and 90.0 % (C). The retention factors decreased from k=11.4 to 2.4 and to 0.6. Column: 4.6 × 30mm Accucore-C18. Room temperature.

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Figure 7: Gradient chromatograms recorded with the three different system configurations (see thelegend and the three different colors in the graph) presented in Figure 1 of reference 32. Column:4.6 × 30 mm Accucore-C18. Room temperature. Flow rate : 4 mL/min. Injection volume : 1 µL.Analytes : Agilent RPLC check-out samples (see experimental section for more details).

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Effect of parallel segmented flow chromatography on the height equivalent to a theoretical plate II...

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