Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems

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Article

Reference

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems

FEKETE, Szabolcs, et al.

Abstract

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates.

FEKETE, Szabolcs, et al . Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems. Journal of Chromatography. A , 2021, vol. 1650, p.

462258

DOI : 10.1016/j.chroma.2021.462258 PMID : 34058594

Available at:

http://archive-ouverte.unige.ch/unige:152868

Disclaimer: layout of this document may differ from the published version.

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ContentslistsavailableatScienceDirect

Journal of Chromatography A

journalhomepage:www.elsevier.com/locate/chroma

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems

Szabolcs Fekete

^a^,^b^,^∗

, Amarande Murisier

^a^,^b

, Gioacchino Luca Losacco

^a^,^b

, Jason Lawhorn

^c

, Justin M. Godinho

^c

, Harry Ritchie

^c

, Barry E. Boyes

^c

, Davy Guillarme

^a^,^b

aSchool of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland

bInstitute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland

cAdvanced Materials Technology, 3521 Silverside road, Suite 1-K, DE 19810, Wilmington, United States

a rt i c l e i nf o

Article history:

Received 30 March 2021 Revised 11 May 2021 Accepted 14 May 2021 Available online 18 May 2021 Keywords:

Narrow-bore column Micro-bore column Eﬃciency loss System dispersion Superﬁcially porous particles

a b s t r a c t

Thisarticledescribestheuseofanewprototypecolumnhardwaremadewith1.5mminternaldiameter (i.d.)anddemonstratessomebenefitsoverthe1.0mmi.d.micro-borecolumn.Theperformanceof2.1,1.5 and1.0mmi.d.columnsweresystematicallycompared.Withthe1.5mmi.d.column,thelossofapparent column efficiencycanbesignificantlyreducedcomparedto1.0mmi.d.columns inbothisocratic and gradientelutionmodes.Intheend,the1.5mmi.d.columnisalmostcomparableto2.1mmi.d.column fromapeakbroadeningpointofview.Theadvantagesofthe1.5mmi.d.hardwarevs2.1mmi.d.narrow- borecolumnsarethelowersampleandsolventconsumption,andreducedfrictionalheatingeffectsdue todecreasedoperatingflowrates.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction

Analyticalscale liquidchromatographic(LC)columnsare com- mercially available in 4.6, 3.9, 3.0, 2.1, 2.0 and 1.0 mm internal diameters (i.d.). Terms such as standard-bore (4.6, 3.9 and 3.0 mm i.d.), narrow-bore (2.1 and 2.0 mm i.d.) and micro-bore (1.0 mm i.d.),are often usedto describe anddistinguish the dif- ferentcolumnformatsandtherequiredoperatingﬂowrate[1,2,3].

Columns based on3.0 – 4.6mm havehistorically dominatedthe ﬁeld ofchromatographyhowever,therehasbeen asigniﬁcant increase inusing2.1 mm i.d. columns,largely dueto theadoption ofultra-highpressureliquidchromatographic(UHPLC)technology andUHPLC–MSsystems[3].Conversely,theuseof1.0mmi.d.col- umnformatisstillnotwidelyadopted.

As possible advantages, smaller column diameters result in lower solventconsumption,thusreducingthecostofanalysisand offeringagreenersolution[4].Onsmalldiametercolumns,theop- timal ﬂow rate is lower, therefore frictional heat effects become lessimportant,andgive risetomoreeﬃcientdesolvation forLC–

MSanalyses,resultinginhighersensitivity.Finally,thesamplecon-

∗Corresponding author at: Waters Corp., School of Pharmaceutical Sciences, CMU - Rue Michel Servet, 1, 1211 Geneva, Switzerland.

E-mail address: szabolcs.fekete@unige.ch (S. Fekete).

sumptionisalsoreducedwithsmallercolumns,sinceinjectedvol- umehastobescaledindirectproportionwiththecolumnvolume, tomaintainthesamesensitivity.

On the other hand, the disadvantages of micro-bore columns may lie in limited loading capacity and decreased apparent effi- ciencyduetoextra-columnband broadening.The limitedloading capacityisoftennotverycritical,butthelossofapparentcolumn efficiencycanbeserious.Lestremauandco-workerscomparedthe apparentefficiencyof1.0×100 mmand2.1×100 mmcolumns packedwiththesamematerial-usingamodernUHPLC system– andonlyabout67%oftheefficiencywasobtainedonthe1.0mm i.d.columncomparedtothe2.1mmi.d.one,inisocraticmode[4]. The apparentefficiency couldbe improved byincreasing the col- umnlength and thereforethe ratioof columnvolume to system volume. In gradient mode, the contribution of the extra-column bandspreading was significantly reduced and a peak capacity of about80%oftheirequivalent2.1mmi.d.columnscanbeobtained on the 1.0 mm i.d. column [4]. Wu and Bradley reported about 60%dropinplatenumbers(N)whencomparing2.1×50mmand 1.0× 50 mm columnspacked with1.8μm particles andoperat- ingthemunderisocraticconditions(N=9010platesvs.3580)[5]. Theyconcludedthattheefficiencylossduetoextra-columnband broadeningincreases asthecolumn diameterandcolumnlength decrease.Thiseffectwaseven morepronounced forearly eluting components. Another study also reported that the extra-column

https://doi.org/10.1016/j.chroma.2021.462258

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S. Fekete, A. Murisier, G.L. Losacco et al. Journal of Chromatography A 1650 (2021) 462258

dispersion ofagivenLCsystemcandramaticallydecreasetheap- parentperformanceofhighlyeﬃcientnarrow-borecolumns[6].To properly operate2.1×50 mmcolumns,an LCsystempossessing systemdispersionaslowas

σ

²≤10μL²isrequiredtomaintainat least55%ofintrinsiccolumneﬃciency.Whencouplingmicro-bore columnstoMS,thetubingusedtointerfacetheUHPLC systemto the MS device is particularly critical inboth isocratic andgradi- ent modes, because this tubing is located after the column outlet,wherethebandcompressioneffectsthatcompensateforband broadeningdonot occur [7].Standardcommercial UHPLC–MSin- struments(unmodiﬁed)exhibit

σ

²^values^ranging^from²⁰^to^more

than100μL².However,byminimizingthevolumeoftheinterfac- ing tube, the extra-columnvariance can be reduced to

σ

² ≤ 20 μL² foranytype of MS detector(please note that routinely, very long interfacingtubesare usedinpractice,such as50– 100cm).

WithanoptimizedUHPLC–MSconﬁguration,thelossineﬃciency witha 2.1mmI.D. columnwasnegligible atretentionfactors(k) higher than 7,while the 1mm I.D. columnwas hardly compati- ble withcurrent instrumentation,even atk > 20. The impact of the extra-columnband broadeningonthechromatographicpeaks ingradientmodewassubtle,thoughstillunacceptablewithmicro- borecolumns[7].

The lower efficiencyof 1.0mm i.d.columns isnot exclusively dueto extra-columndispersionbutcan alsobe causedby poorly packedbeds,fritdispersionandaxialbedheterogeneity.Grittiand Wahab reportedthat thepackedbednearthewallofthecolumn isdenserthanthatofthebulkpacking,whichresultsindifferences inboth soluteandmobilephase velocitythroughthecolumn[8]. Fora1.0mmi.d.column,thewallregionvolume(denser)tobulk region volume (lessdense) ratioiscloseto 1 whichisthe worst case.Forlarger-diametercolumns,thewallregionvolumebecomes less significant and - forsmaller column internal diameter -the bulkregionbecomeslesssignificant.Thus,the1.0mmi.d.isoften considered to be the worst-casefrom bedheterogeneity point of view.

Despite the expectedly high eﬃciency loss with 1.0 mm i.d.

columns, these columns can be used by utilizing carefully opti- mizedsystems,optimalcolumnhardwaredesign,verylowinjected volume andby beneﬁting fromband focusing effects usingweak injectionsolvent[9,10].Schoorsandco-workersreportedthesuc- cessfulusageof1.0mmi.d.columnsfortheanalysisofmonoamine neurotransmitters[11].Theydemonstratedasigniﬁcantincreasein sensitivityusing1.0mmi.d.columnascomparedtoa2.1mmi.d.

column. In addition, weak solvent injection helped focusing the sampleatthecolumninlet.

It seems today that despite the sensitivity increase and reduced solvent consumption, the adoption of micro-borecolumns isstill limited.Theeffortsneededtocompensateforsystemband broadening seem to be a strong barrierfor mostusers. The aim of this study was to evaluate a compromise between 1.0 and 2.1 mmcolumns.Thus, a prototype1.5mm i.d.columnwaspre- paredandsystematicallycomparedtocommercial2.1and1.0mm i.d. columnspackedwithahighlyefficientcolumnpackingmate- rial (2.7μmsuperficiallyporous 90 ˚AC18). Columnefficiencyob- served inbothisocraticandgradientmodeswerecomparedusing averylowdispersion(AcquityI-Class)andastandardUHPLC(Ac- quityH–Class)systems.Inaddition,thesensitivityofMSdetection wasalsostudied.

2. Experimental

2.1. Chemicalsandsamples

Acetonitrile (AcN), methanol (MeOH), ethanol (EtOH), water and formic acid were purchased from Fisher Scientiﬁc (Reinach,Switzerland).Uracil,methylparaben,ethylparaben,propy-

lparaben, butyl–paraben, cannabidivarine (CBDV), cannabigerolic acid (CBGA), tetrahydrocannabivarin (THCV), cannabichromene (CBC), delta9-tetrahydrocannabinolic acid (THCA-A) and hu- man serum albumin (HSA), were purchased from Sigma–

Aldrich.Cannabidiolic acid(CBDA), cannabigerol(CBG), cannabid- iol (CBD), cannabinol (CBN), (-)-delta9-THC (d9-THC) and (-)- delta8-THC(d8-THC)werepurchasedfromLipomedAG(Arlesheim, Switzerland).Terbutaline,fenﬂuramine,norfentanyl,atenolol,ben- zoylecgonine, probenecid, hydrochlorothiazide, etacrynic acid, furosemide, chlorthalidone, bumetanide and bendroﬂumethiazide solutionat1mg/mLinMeOHwerekindlyprovidedby theSwiss LaboratoryforDopingAnalyses(Epalinges,Switzerland).

2.2. Chromatographicsystem

ForUHPLC-UV measurements,two UHPLC systems were used.

One wasa very low-dispersion system, namelya Waters Acquity UPLCI-Class (Waters, Milford, MA, USA) equippedwith a binary solventdelivery pump, anautosamplerandUV detector.The sys- temincludedaflowthroughneedle(FTN)injectionsystemwith15 μLneedleanda0.5μLUV flow-cell.Theextra-columnvolumeof thesystemwasmeasuredasVec=7.5μL,whilethegradientdelay volume wasV_d = 98μL.The other systemwasa WatersAcquity UPLCH–Classequippedwithaquaternarysolventdelivery pump, an autosampler and UV detector. The system includeda FTNin- jectionsystem with15 μLneedle anda 0.5μL UV flow-cell. The extra-columnvolume ofthe system wasmeasured asV_ec = 11.5 μL,whilethegradientdelayvolumewasV_d=370μL.

ForUHPLC-MS/MSmeasurements,athirdWatersAcquityUPLC I-Class, composed of a binary solvent delivery pump andan autosampler(loop oﬄine), washyphenated to a WatersTQD Triple Quadrupolemassspectrometer,ﬁttedwithaZ-sprayESIsource.A capillaryvoltageof±1.5kV,sourcetemperatureof150°C,desolva- tiontemperatureat450°C,desolvationandconegassetat750L/h and0L/h wereappliedtoallanalyses.Nitrogen (N₂)wasusedas bothdesolvationandconegas,whileargon(Ar)wasemployedas the collision gas.Multiple reaction monitoring (MRM)mode was usedduringUHPLC-MSanalyses.TheUHPLCsystemwasconnected totheMSvia65μmx50cmPEEKtube.

Data acquisition and instrument control for UHPLC-UV measurements were performed by Empower Pro 3 software (Waters, Milford, MA, USA), while for UHPLC-MS analyses, MassLynx v4.1 (Waters, Milford, MA, USA) wasused. Data was treatedin Excel (Microsoft)forUHPLC-UVanalyses,whileTargetLynxv4.1(Waters, Milford,MA,USA)wasusedforUHPLC-MSmeasurements.

2.3. Columns

Anewprototype1.5×100mmcolumnpackedwith2.7μmsu- perﬁciallyporous90 ˚AC18particlesandcommercial1.0×100mm and 2.1 × 100 mm columns packed with the same material wereprovidedbyAdvancedMaterialsTechnology(Wilmington,DE, USA).

2.4. Sampleandmobilephasepreparation

Amixsolution containinguracil,methylparaben, ethylparaben, propylparaben and butylparaben wasprepared in 10:90 v/v acetonitrile:waterat50μg/mL.Uracilandparabenswereelutedinthe mobilephase,namely35:65v/vacetonitrile:water.

Amixsolutioncontainingelevencannabinoids(i.e.CBDV,CBGA, THCV, CBC, THCA-A,CBDA, CBG, CBD, CBN, d9-THC andd8-THC) was prepared from individual stock solutions diluted in solvent having the same composition as mobile phase “A” at 45 μg/mL.

The individual stocksolutions were preparedin eithermethanol, acetonitrileorethanoldependingontheir solubility.Cannabinoids 2

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Fig. 1. Apparent plate numbers ( N app) as function of solute retention factor ( k ) observed on Acquity I-Class system at high (A) and low ﬂow rates (B) and on H-class system at high (C) and low (D) ﬂow rates with 1.0, 1.5 and 2.1 mm i.d columns. Sample: methyl-, ethyl-, propyl- and butyl–paraben.

were separated in gradient mode. Mobile phase “A” was 0.1%

formicacidinwater,whilemobilephase“B” was0.1%formicacid inacetonitrile.Alineargradientof60– 95%Bwasappliedattwo different gradient steepness (corresponding to gradient times of t_G1 = 10 min andt_G2 = 20 min), since peak width dependson gradientsteepness.

For UHPLC-MS analyses, two mixtures of doping agents were used.Bothmixtureswerepreparedin5:95v/vmethanol:waterat a final concentration of 200 pg/mL. Analyses were performed in gradient mode. Mobile phase “A” was 0.1% formic acid in water, whilemobilephase“B” was0.1%formicacidinacetonitrile.Alin- ear gradient from 5% to95% of mobile phase “B” wasapplied at two differentgradient steepness (t_G1 = 5min forall analyses at high-flowrateson eachcolumn, t_G2 =3.3minforallanalyses at low-flowrates).

2.5. Comparisonofeﬃciency

The linear mobile phase velocity (u₀) and the total column porosity(

ε

T)weredeterminedfromthefollowingequation:

u₀= L t₀ = 4F

ε

Td_c²

π

⁽¹⁾

whereListhenominalcolumnlength,t₀isthecolumndeadtime (corrected forsystemresidencetime),Fis themobile phaseﬂow rate, anddc thenominalcolumndiameter.Thecolumndeadtime wasmeasuredbyinjectingnon-retainedcompound(uracil).

When comparing column eﬃciency, two mobile phase velocities were set (low and high level). Linear velocities u₀ = 15 and 25 cm/min were considered, corresponding to F ~ 0.3 and 0.5 mL/min on the 2.1 mm i.d., F ~ 0.15 and 0.26 mL/min on the 1.5 mm i.d. and F ~ 0.07 and 0.11 mL/min on the 1.0 mm

i.d. column. Mobile phase temperature wasset to 30°C. In both isocraticandgradientmodes, threeinjectionvolumesweretested (V_inj =0.1, 0.5and1μL)since injectedvolume mightimpact both systemandcolumnband-broadening,especiallyforsmall volume columns.

In isocratic mode, theapparent platenumbers (Napp, not cor- rectedforsystem dispersion)obtainedwith theparaben mixture werecompared.PlotsofNappversusretentionfactor(k)wereplot- tedandlogarithmictrendswereﬁtted.Theretentionfactorsofthe parabens were comprised betweenk ~ 1and 11,which is repre- sentative of common practice. Since in isocratic mode, both the pre-andpostcolumndispersionsimpact thetotalband broadening,twoUHPLCsystemswereusedtoseethedifferencesofappar- enteﬃciency.

Ingradientmode,thepeakwidthsofcannabinoidsmeasuredat halfheight(w_1/2) werecomparedandplottedasafunctionofap- parent retention factor (k_app, based on observed retention time).

The gradient measurements were performed only on the low- dispersionsystem. Please notethat ingradientmode,itismostly thepostcolumnvolumethatimpactstheoverallpeakbroadening, since the pre-column dispersion is compensatedby the gradient band focusing effect.The postcolumnvolume ofthelow dispersion(AcquityI-Class)andastandardUHPLC(AcquityH–Class)systems were the same (0.5 μL UV cell and postcolumn connector tubingof65μmx30cm), thuswhyonlyone systemwastested here.

2.6. Comparisonofsensitivity

Peakintegrationandsignal-to-noise(S/N) measurementswere performed via the TargetLynx tool available in MassLynx v4.1.

Smoothingprocess wasapplied to all chromatogramsprior toall

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Fig. 2. Comparative chromatograms, obtained with 1.0 ×100 mm, 1.5 ×100 mm and 2.1 ×100 mm columns - packed with superﬁcially porous C18 material - in isocratic mode (standard UHPLC, Acquity H –Class). Mobile phase 35:65 v/v acetonitrile:water, mobile phase velocity u 0= 25 cm/min. Sample: methyl-paraben (1), ethyl-paraben (2), propyl-paraben (3) and butyl–paraben (4).

calculations, using aSavitzky-Golay methodwitha smoothingit- eration value of 3 and a smoothing width of 2. The “Peak-to- Peak” methodwasappliedforsignal-to-noisecalculation,measur- ing peak signal level from the baseline andby ﬁxing a constant noisesignalwindowof1.0minforallchromatograms.

3. Resultsanddiscussion

In the ﬁrstinstance, the columnvolumesandporosities were determined. Column volumes, V₀,were equal to173, 100 and49 μL, whileporosities

ε

T were equal to 0.50, 0.57 and0.63forthe 2.1,1.5and1.0mmi.d.columns,respectively.

Regarding V₀,it isimportanttokeep inmind thegeneral10%

rule of thumb forextra-column band broadening andassociated eﬃciencyloss[12]:theextra-columnvolumeshould notbemore than 10%ofthecolumn’spackedbedvolume(to limitthecontri- butionofsystemdispersion).Theextra-columnvolumeofourvery low dispersion UHPLC system isVec = 7.5μL,which corresponds to 4, 7 and 15%of the 2.1, 1.5 and1.0 mm i.d. columns volume (100mmlength),respectively.Ontheotherhand,thesystemvol- umeofourstandardUHPLCsystemisVec =11.5 μL,whichcorre- spondsto7,11and23%ofthe2.1,1.5and1.0mmi.d.columnsvol- ume(100mmlength),respectively.Thesevaluessuggest thatthe 1.5× 100 mmcolumncan be operatedona verylow dispersion

systemwithoutsignificantefficiencyloss,whileonastandardUH- PLC,lowerapparentefficiencyisexpected,buttoamuchlesserex- tentthanona1.0×100mmcolumn.Tooperatea2.1×100mm i.d. column (packed with superficially porous particles) without significant efficiency loss,a system volume of V_ec ≤ 17μL is re- quired.Fora1.5and1.0× 100mmcolumn, Vec ≤10μL,andVec

≤5μLarerecommended,respectively.Thislattercriterion(Vec ≤ 5μL)isproblematic,since commercialUHPLC systemsallpossess Vec >6–7μL(typicallybetween6and20μL)[6,7,12].

Itisalsoworthmentioningthat theobservedcolumnporosity increaseswhendecreasing thecolumndiameter.Thisobservation islogicalandcanprobablybeexplainedbythefollowingreasons:

Theapparentporosityofsmallcolumnsincreaseswhendecreasing column diameteror length, due to the higher ratio ofextra-bed volume (V_eb) to packedbed volumeanddue tosome differences inpacking quality (density) too [13,14]. The extra-bedvolume of a column hardware was recently described as the total volume ofcolumn hardwareflow distributor, flow collector,frits, andin- let/outlet connections [13,14]. To have an idea about its value, a recentstudyreportedextra-bedvolumeofV_eb ~ 4μLfora2.1mm i.d.columnhardware[14].Therefore,forverylowvolumecolumns (suchasshort columnsof 1.0, 1.5and2.1 mmi.d.),not only the extra-columnsystemvolume, butalsothe extra-bedcolumnvol- umeneedtobeconsideredaspossiblesourceofefficiencyloss.

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Fig. 3. Peak widths ( w 1/2) as function of solute apparent retention factor ( k app) observed at b = 0.025 (A), 0.04 (B), 0.05 (C) and 0.08 (D) gradient steepnesses with 1.0, 1.5 and 2.1 mm i.d columns. Sample: cannabinoinds standard mixture. The widths of only the well-resolved peaks are considered. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.)

3.1. Apparenteﬃciencyinisocraticmode

Fig. 1showsthemeasured platenumbers (Napp) asafunction ofsoluteretentionfactor(k).Figs.1AandBcorrespondtothevery low dispersion system.At highﬂowrate, Napp =15,000– 19,000 plates wereobtainedonthe 2.1mmi.d. column.The 1.5mmi.d.

column resulted in N_app = 12,500 - 19,000 and the 1.0 mm i.d.

column provided Napp = 9500 – 16,000. In average, the 1.5 and 1.0 mm i.d. columns performed ~93% and ~76% efficiency compared to the 2.1 mm i.d. column, respectively. At low flow rate, Napp = 19,500 – 21,000 (2.1 mm i.d.), Napp = 16,500 – 21,000 (1.5 mm i.d.)andNapp = 11,500 – 16,700(1.0 mm i.d.)were observed. Inaverage,we observed~94%and69%efficiencywiththe 1.5and1.0mmi.d.columns,respectively,comparedtothe2.1mm i.d.column.

It can alsobe seen that the less retained peaksare moreaf- fectedbysystemdispersion.Thisisobviouslyduetothefactthat columnpeakvariance(

σ

col²)-andthereforepeakwidth-depends onsoluteretention:

σ

col² = V₀²

N_col

(

¹+k

)

² ⁽²⁾

where N_col is the column intrinsic efficiency (plate number un- affected by system dispersion). Therefore, at low k, the ratio of system dispersion to columndispersion increases dueto the de- creaseofcolumndispersion.Whencomparingtheefficiencycorre- spondingtopoorly(k~ 1)andhighlyretained(k~ 10)compounds, theefficiencyobtainedforthepoorlyretainedcompounddropsby about20,35and40%onthe2.1,1.5and1.0mmi.d.columns,respectively, atlow flowrate. Similarly, at highflow rate, thiseffi- ciencylosscorresponds to7%(2.1mmi.d.),17%(1.5mmi.d.)and 31%(1.0mmi.d.).

Figs.1CandDshowtheapparenteﬃciencyobtainedonastan- dard UHPLC system. At high ﬂow rate, Napp = 13,000 – 16,000 plateswereobservedwiththe2.1mmi.d.column.The1.5mmi.d.

columnprovidedinNapp=10,500– 16,000,whilethe1.0mmi.d.

columnperformedNapp= 8500– 15,000.Inaverage,the1.5mm i.d. column performed ~92% while the 1.0 mm i.d. column performed ~88% efficiency compared to the 2.1 mm i.d. column. At lowflowrate,Napp=17,500– 19,000(2.1mmi.d.),Napp=14,200 – 18,500 (1.5 mm i.d.) and Napp = 8200 – 16,000 (1.0 mm i.d.) were observed. In average, we obtained ~90%and 80% efficiency withthe1.5mmand1.0mmi.d.columns,respectively,compared tothe2.1mmi.d.column.Betweenpoorlyandhighlyretainedso- lutes,athighflowrates,wesaw19,34and43%differenceinplate numberson the2.1, 1.5and1.0mm i.d.columns,respectively. At low flow rate, we observed 8% (2.1mm), 23% (1.5 mm)and49%

(1.0mm)differencesinplatenumbersbetweenk~ 1and10.

Asanexample,Fig.2showscorrespondingchromatogramsob- tained with the standard UHPLC system operating at high ﬂow rate.Itisworthmentioningthatnotonlyplatenumbers,butpeak symmetryisalsoaffectedbythecolumndiameter.The poorlyre- tained compounds elute in more asymmetrical peaks on smaller borecolumns.

3.2. Apparenteﬃciencyingradientmode

Ingradientelutionmode,theapparent eﬃciencyisexpectedly less affected by system dispersion, since most of the dispersion occurring in the pre-column volumes are compensated by band focusing taking place at the top of the column. Therefore, pre- columndispersionisalmostnegligible,whileapparenteﬃciencyis mostlyaffected by post-column dispersion [15]. The post-column systemvolumeofmostUHPLCsystemsrangesbetween1and3μL (typicallydetector cell of0.5to 2 μL andconnectingtube of <1 μL).

Fig. 3 shows the peak widths measured at different gradientsteepness.Themeasurementswereperformedattwogradient times(t_G) andtwo ﬂow ratescorresponding tofour differentin-

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Fig. 4. Comparative chromatograms, obtained with 1.0 ×100 mm, 1.5 ×100 mm and 2.1 ×100 mm columns - packed with superﬁcially porous C18 material - in gradient mode (standard UHPLC, Acquity H –Class). Mobile phase “A” is 0.1% formic acid in water, mobile phase “B” is 0.1% formic acid in acetonitrile. Linear gradient of 60 – 95% B at t G= 10 min and at u 0= 15 cm/min. Sample: CBDV (1), CBDA (2), CBGA (3), CBG (4), CBD (5), THCV (6), CBN (7), d9-THC (8), d8-THC (9), CBC (10) and THCA-A (11).

trinsicgradientsteepness(b):

b=S·

ϕ

^·^t_t⁰

G

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where Sis solute dependentparameter and

ϕ

^is ^the^difference

betweentheinitialandﬁnalmobile phasecompositionexpressed involumefraction.Peakcapacity(n_c)wasdeterminedaccordingto thefollowingequation:

nc=1+ tG−t0

1.699w1/2

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At b = 0.025 (correspondingto highflow rateand longgradient time, Fig. 3A),the peak widthranged betweenw_1/2 = 0.032 and 0.072min,correspondingtoan averagepeakcapacityofnc =223 on the 2.1mm i.d.column. On the1.5mm i.d. columnwe mea- suredw_1/2=0.036 and0.076min(averagenc =205),whilewith the 1.0mm i.d.columnwe obtainedw_1/2 =0.045and0.089 min (average n_c = 178). At b = 0.04(corresponding to low flow rate andlonggradienttime,Fig.3B),averagepeakcapacityof185,163 and145wereobtainedwiththe2.1, 1.5and1.0mmi.d. columns, respectively.Atb=0.05(correspondingtohighflowrateandshort gradient time,Fig. 3C),we measured peakcapacities asnc =154 (2.1 mm), 141 (1.5 mm)and 119(1.0 mm). And finally, withthe steepest gradient (b = 0.08, corresponding to low flow rate and shortgradienttime,Fig.3D)weobservednc =130(2.1 mm),115 (1.5mm)and95(1.0mm).

Comparedtothe2.1mmi.d. column,the1.5mmcolumnper- formedabout7–11%lesspeakcapacity,whilethe1.0mmi.d.col- umnprovided 20–27%lower peak capacity. The largesteﬃciency loss was observed with the steepest gradient, while the shal- low gradients resulted in somewhat less signiﬁcant post-column dispersion. This observation is in agreement with theory, since the gradient band compression effect is more pronounced with steep gradients and therefore thinner peaks are expected [14]. Fig.4showsacomparativeexampleofcannabinoidsmixturesep- arationsperformedon2.1,1.5and1mmi.d.columnsatagradient steepnessofb=0.08.

3.3. StudyingLC-MSsensitivity

Besidesthe impactofreducing columndiameteroneﬃciency, we have alsoevaluated its effecton MS sensitivity.For thispart of the work, various doping agents were analysed in both ESI+ and ESI- conditions, at two different ﬂow rates (low and high).

The ﬂowrates andinjected volumeswere geometrically adjusted between the columns of 2.1, 1.5 and 1 mm i.d. In addition, ESI conditions were optimized depending on the mobile phase ﬂow rate. Fig. 5 highlights the behavior of one of the doping agents, namelychlorthalidone.Similarly,towhatwaspreviouslydescribed with UV detection, peak widths remain comparablebetween 2.1 and1.5 mm i.d.,but peaksare clearly broaderon the 1 mm i.d.

column. Due to the additional band broadening observed on the 6

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Fig. 5. LC-ESI-/MS/MS chromatograms obtained for chlorthalidone on 2.1, 1.5 and 1.0 mm i.d. at two different ﬂow rates. Here, the ﬂow rates and injected volumes were geometrically scaled with the column volumes.

1.5vs.2.1mmi.d.column, thepeak heightwasreducedbyupto afactor2atthelowestflowrate.Importantly,thesignallosswas much morepronounced onthe1 mmi.d. column(uptoa factor 7atlow flowrate).Forthe12dopingagentstestedhere,S/Nwas inaveragedecreasedby afactor3.9and1.6atlowandhighflow rate,respectively,whenmovingfroma2.1toa1.5mmi.d.column.

Whenmodifyingcolumni.dfrom2.1to1mmi.d.,theaverageS/N values were reducedby 10.3and 6.9forhighandlow ﬂow rate, respectively. Based onthisdata,it isprobablethat S/N wouldbe less negatively impacted if a higherﬂow ratewould be selected on the2.1mmi.d.columnandgeometricallytransferred toother columndimensions.

Contrary to our expectations, the sensitivity decreased when using asmallercolumni.d. (injectedvolumeandﬂow rateswere adjustedindirectproportiontothecolumnvolume).Theonlyad- vantagewhenusing1.5mmi.d.inLC-MSistoconsumelesssam- ple (which can be important whenanalysing precious sample of limitedvolume),whilehavingalimitedimpactonpeakwidths.

It is ﬁnally well known that theimpact ofmobile phase ﬂow rateonESI/MS sensitivitycanbevery differentdependingonthe geometry ofthe ionization source(and the MS brand). Evenifit has not beentested, itis possiblethat an improvementof sensi- tivitywouldbeobservedwiththe1.5vs.2.1mmi.d.columnfora differentMSdevice.

4. Conclusion

Ourpurposewastoﬁndacompromisebetween1.0and2.1mm i.d. columns.Therefore, aprototype 1.5mmi.d. columnwaspre- paredandcomparedto2.1and1.0mmi.d. columnspackedwith thesamematerial(2.7μmsuperﬁciallyporous90 ˚AC18).

Inisocraticmode,thelossofaverageapparenteﬃciency(plate numbers) was6– 10%and12– 31% withthe1.5and1.0mmi.d.

columns,respectively,comparedtothe2.1mmi.d.column.

Ingradientmode,theloss ofaverage peakcapacitywasabout 7 – 11% withthe 1.5 mm i.d. columnand about 20– 27% with the1.0mmi.d.columnwhencomparedtothe2.1mmi.d.column.

Thelargesteﬃciencylosswasobservedwiththesteepestgradient, whilethe shallowgradientsresulted insomewhat lesssigniﬁcant post-columndispersion.

When comparing the average S/N for the studied 12 doping agents,itdecreased by afactor 3.9and1.6atlow andhighﬂow rate, respectively, on the 1.5 mm i.d. column compared to the 2.1mmone.Whenreplacingthe2.1mmi.d.columnbythe1mm i.d.one, theaverageS/N valueswerereducedby 10.3and6.9for highandlowﬂowrate,respectively.

Based on the resultsobserved in thisstudy, the new1.5 mm i.d. columnhardwareseems to ﬁtmuch better tocurrentUHPLC instrumentation thanthe 1.0mm i.d. column hardware. The loss inapparent eﬃciency andS/N ratiois much lessimportantwith the 1.5mm id.columnthan withthe 1.0mm i.d. column. How- ever, decreasing the extra-column volume of current instrumen- tationwould resolve most ofthe problems related to theuse of 1.0mmi.d.columns.

Other benefits of the 1.5 mm i.d. hardware compared to the 2.1mm i.d.columns arethe lowersampleandsolvent consump- tions.Incaseofproperlyscaledflowrates,theeluentconsumption isaboutthehalf(factor0.51)onthe1.5mmi.d.columncompared toa2.1mmi.d. column.Inaddition,probablylessimportantfric- tionalheatingeffectsareexpectedduetothedecreasedoperating flowrate.

DeclarationofCompetingInterest

Theauthorsdeclarethattheyhavenoknowncompetingﬁnan- cialinterestsorpersonalrelationshipsthatcouldhaveappearedto inﬂuencetheworkreportedinthispaper.

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S. Fekete, A. Murisier, G.L. Losacco et al. Journal of Chromatography A 1650 (2021) 462258 CRediTauthorshipcontributionstatement

Szabolcs Fekete: Writing - original draft, Methodology, Inves- tigation, Validation. AmarandeMurisier: Investigation,Validation, Writing - review & editing. GioacchinoLuca Losacco: Investiga- tion,Validation, Writing-review &editing.JasonLawhorn:Con- ceptualization, Resources. JustinM. Godinho: Conceptualization, Resources, Writing - review & editing. Harry Ritchie: Conceptu- alization, Resources. BarryE. Boyes: Conceptualization, Writing - review&editing.DavyGuillarme:Supervision,Writing-review&

editing.

Acknowledgement

TheauthorswishtothankCedricSchellingfromtheUniversity of Geneva for preparing and providing the cannabinoid samples andfordiscussions,andMr.BenLibertatAMTforpreparationand testingofprototypecolumns.

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