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Journal of Chromatography A, 1217 (2010) 4561–4567

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

nstrumental considerations for the effective operation of short, highly efficientused-core columns. Investigation of performance at high flow rates and elevatedemperatures

avid V. McCalley ∗

entre for Research in Biomedicine, University of the West of England, Frenchay, Bristol BS16 1QY, UK

r t i c l e i n f o

rticle history:eceived 29 January 2010eceived in revised form 16 April 2010ccepted 23 April 2010vailable online 29 April 2010

a b s t r a c t

Fused core or superficially porous columns offer the advantages of higher efficiency compared with totallyporous columns of the same particle size, but similar operating pressures. However, their performancemay be adversely affected by extra-column effects that become more significant as the column efficiencyincreases, and as the diameter of the column is reduced. In this study, we show that 10 cm × 0.46 cmfused-core columns can be used on modified conventional instruments (“microbore systems”) without

eywords:used-core columnsuperficially porous columnsfficiencyead volume

serious loss in performance, and this approach is at present likely to yield superior results compared withuse of 0.21 cm columns (of identical efficiency) on current UHPLC instruments that have minimised extra-column volume. Furthermore, the true efficiency of commercial narrow-bore fused-core columns appearsto be reduced compared with those of conventional bore, which may be due to packing difficulties forthe former type. The fused-core columns in general gave excellent performance, showing no evidenceof an upturn in the Knox plots at high flow velocities and elevated temperatures. Careful control of

is nec

experimental conditions

. Introduction

Superficially porous or fused-core columns are a recent devel-pment of the original pellicular particles used in HPLC. In thesearticles, a porous outer layer surrounds an impervious core.he original pellicular particles typically consisted of porous lay-rs of 1–2 �m thickness, surrounding a solid non-porous core,ith overall particle diameter ∼30 �m [1]. Such particles show

aster mass transfer, due to the smaller diffusion distance throughhe porous layer, compared with (large) porous particles of theame diameter, but suffered from rather poor sample capacity,s less than 20% of the particle was porous. Later, particles ofuch smaller overall diameter (5 �m) with a 0.25 �m thick porous

ayer containing 30 nm pores were developed for the separationf macromolecules, to take advantage of the smaller diffusionistances for these low diffusivity solutes [2]. The efficiency of18 columns for small molecules was reported as similar to thatf totally porous phases. Newer C18 particles of overall diam-

ter 2.7 �m, with porous layer 0.5 �m thick, showed minimumeduced plate heights (hmin) as low as 1.5, a result replicated forhe equivalent bare silica hydrophilic interaction chromatographyHILIC) phase [3,4]. In comparison, totally porous particles gener-

∗ Tel.: +44 117 3282469; fax: +44 117 3282904.E-mail address: David.Mccalley@uwe.ac.uk.

021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2010.04.070

essary to ensure accurate data for these plots.© 2010 Elsevier B.V. All rights reserved.

ally show 2.0 < hmin < 2.5. For the new particles, the porous layerrepresents 75% of the volume of a totally porous particle, thusthe loading capacity is expected to be only slightly reduced. Thisresult was confirmed experimentally for the HILIC column [4]. Itwas proposed that the high efficiency of these particles mightbe due to their lower internal porosity (smaller B term) in vanDeemter/Knox type equations and narrower particle size distribu-tion and better packing (smaller eddy diffusion or A term), ratherthan the smaller diffusion distance and improved mass transfer(smaller C term) as originally suggested, at least for small molecules[3,5–7].

An apparent disadvantage of these columns was the unusualrate of increase of the plate height at high linear velocities, particu-larly at elevated temperature (up to 50 ◦C) as demonstrated for thetest solute naphtho[2,3-a]pyrene [5]. Comparison of the results foran excluded protein (bovine serum albumin) suggested that thisbehaviour was not related to mass transfer kinetics in the station-ary phase, but to some unexpected variation of the eddy dispersionwith the linear velocity at high temperatures, that might be con-nected with the roughness of the external surface of the fused-coreparticles. The authors suggested therefore that if the separation

of low molecular weight compounds is performed at high veloci-ties, the benefit of using such columns might vanish. However, ourown study of similar bare silica fused-core HILIC columns did notshow any evidence of this effect at high flow rate, using similartemperatures [4].

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562 D.V. McCalley / J. Chroma

The examination of column efficiency as a function of flow rate islearly of much importance in the characterisation of new columnaterials. However, the production of accurate van Deemter/Knox

urves is fraught with potential difficulties. These include:

a) The variable effect of instrumental dead volume on the mea-sured efficiencies. The detrimental effect will increase as theefficiency of the column increases (at a given retention factor,k) and with decreasing k, as both decrease the volume contain-ing the peak. The influence of the dead volume also increaseswith decreasing column internal diameter.

b) The data collection rate may be insufficient to assess accuratelythe shape of fast peaks that emerge at high flow rates.

(c) The column may be damaged by the higher pressures and flowsnecessary to construct the plots.

d) The column influent temperature may vary at high flow rate dueto insufficient pre-heating of the mobile phase. A colder influentreaches the oven temperature at the walls more quickly than atthe centre, giving a radial temperature gradient that can causepeak broadening-an effect that was first described more than25 years ago [8–11].

e) Column frictional heating may lead to axial and radial temper-ature gradients [5,12]. Radial gradients (with the column corehotter than the column walls) cause the mobile phase to travelfaster in the centre than at the walls, leading to band broad-ening effects. Such effects depend on many factors, includingthe type of column thermostat (e.g. still air, forced air, or waterbath), but can occur even for columns packed with relativelylarge (5 �m) particles [13].

Dorsey and co-workers stressed that consideration of suchffects was essential to the production of meaningful plate heights flow curves [14]. In the light of these potential problems, andhe important consequences of the conclusions of the previousnvestigation [5], we investigated further the behaviour of the sameused-core column under similar experimental conditions, takingote as far as possible of the potential difficulties discussed above.e were also interested in examining the effect of extra-column

andspreading and the performance of columns of conventional4.6 mm) and smaller internal diameter (2.1 mm). We used both a

odified conventional HPLC instrument (“microbore HPLC” sys-em) and an ultra-high pressure system (UHPLC), with the aimf determining the influence of extra-column effects on theseigh performance columns. We employed broadly similar con-itions to the previous study [5], using naphtho [2,3-a] pyreneelected apparently for its relatively low diffusion coefficient andigh hydrophobicity, enabling the attainment of high values of theeduced mobile phase velocity. However, it should be noted thatsing high concentrations of organic modifier does not inevitablyesult in high reduced velocities, as the effect of decreased viscosityf the mobile phase (allowing higher velocity) may be counteractedy the increase in the solute diffusion coefficient.

. Experimental

The microbore HPLC was a model 1100 binary high pressureixing HPLC system (Agilent, Waldbronn, Germany) with Chem-

tation, UV detector (1 �l flow cell), and Rheodyne 7725 valve5 �l injections). Connections were made with minimum lengthsf 0.012 cm I.D. tubing to minimise extra-column band spreading.

emperature was regulated using the Agilent column thermostat.he fastest data collection rate available on this instrument (14 Hz,esponse time <0.12 s) was invariably used. The ultra-high-pressureiquid chromatograph (UHPLC) was a UPLC® system from WatersMilford, MA, USA) including Empower data handling, binary sol-

1217 (2010) 4561–4567

vent manager, photodiode array (UV) detector (500 nl flow cell),and sample manager/injector valve (1 �l injections from partiallyfilled 5 �l loop with needle overfill) and was used for all experi-ments with 0.21 cm I.D. columns. The use of 1 �l injections withthe 0.21 cm column and 5 �l injections from the 0.46 cm columnshould result in similar detection sensitivity and column load-ing properties. Data collection rates were either 80 Hz (fast filter,0.0125 s) or 20 Hz (normal filter, 0.2 s) as specified. Duplicate sam-ple injections were made. For construction of van Deemter/Knoxplots, one data set was obtained increasing the flow rate graduallyto the maximum value, followed by a repeat data set as the flowrate was decreased back to its minimum value. Such an approachallows a check to be made for column damage resulting from useat the highest flow and pressure. The columns used were Halo C18(particle diameter 2.7 �m, pore size 80 Å, 10 cm × 0.46 cm I.D. and10 cm × 0.21 cm I.D.), from AMT (Wilmington, USA) and DiscoveryC18 (particle diameter 5 �m, pore diameter 180 Å, 15 cm × 0.46 cmI.D.) from Supelco (Bellafonte, USA).

Acetonitrile (far UV grade) was obtained from Fisher Scien-tific (Loughborough, UK). Naphtho [2,3-a] pyrene and uracil wereobtained from Sigma–Aldrich (Poole, U.K.).

System bandspreading was measured by replacing the columnwith a zero dead volume connector. � was calculated from mea-surements of the peak width at 4.4% of peak height (5� peak width)to take account of peak tailing typically shown for such injections.

The diffusion coefficients of naphtho[2,3-a]pyrene, as esti-mated from the Wilke–Chang equation, were taken as 1.29 × 10−5,1.23 × 10−5 and 1.52 × 10−5 cm2/s in pure ACN at 25 ◦C, ACN–water(90:10, v/v) at 37.5 ◦C, and ACN–water (85:15, v/v) at 50 ◦C, respec-tively [5]. Due to the low flow rates and low proportion of waterused with columns of 0.21 cm diameter, solvents were pre-mixedwhen using the UHPLC system to avoid the delivery of very smallvolumes by one of the pumps. For example, a flow of 0.05 ml/min ofacetonitrile–water (90:10, v/v) requires one pump to deliver accu-rately only 5 �l/min, which we found experimentally to be beyondthe capabilities of our system.

3. Results and discussion

3.1. Determination of bandspreading of microbore and UHPLCsystems

The experimentally measured variance of a chromatographicpeak is the sum of the column and extra-column variance, the latterin turn being the sum of the variance of the injector, detector, con-necting tubing and time-based events such as sampling rate anddetector time constant [15,16].

�2exptl. = �2

col. + �2excol.

Extra-column dispersion can be calculated in any convenientunits of time, volume or column length. We prefer the measure-ment in volume units as employed by many other workers [17–19].Clearly, it is best to use the actual compound employed for the vanDeemter/Knox curve measurements to probe extra-column effects,but we obtained additional data with uracil. There is no particularreason to choose uracil, commonly used as an unretained marker inRP separations, as no column is in place during the measurements.However, it is a compound often found in analytical laboratories,and serves therefore as a useful basis for comparative studies.

At the optimum flow of the 0.21 cm fused-core column (around

0.3–0.35 ml/min, see below) Fig. 1(a) shows the bandspreading ofthe UHPLC system was 3–3.5 �l2 for naphthopyrene, with slightlyhigher values for uracil, based on an injection volume of 1 �l. Allvalues were measured using acetonitrile–water (90:10, v/v) and37.5 ◦C, these being the median values of these parameters used in

D.V. McCalley / J. Chromatogr. A 1217 (2010) 4561–4567 4563

F obes/( r thet

tcgtttttbfirecGiNtt

ig. 1. System bandspreading measured for the two instruments using different prb) Effect of data gathering parameters on the bandspreading for naphthopyrene fohe microbore system.

he subsequent van Deemter/Knox plots (see below). The results areomparable with those reported by Neue and co-workers, who haveiven a detailed description of the complex factors that contributeo bandspreading, concluding that the value of the individual con-ributions is difficult to predict [18]. These factors were reportedo include the dispersion in the injector and detector, the contribu-ion of bandspreading in the tubing (which can be estimated usinghe Taylor–Aris equation), and the time-based contribution to peakroadening originating in the detector data collection rate and inlters used. The tubing used between the injector and detector isather short in both HPLC and UHPLC applications. The Taylor–Arisquation breaks down at low solute residence times. The authorsonsidered also an alternative treatment on the basis of work by

olay, finally proposing a mathematical description of broadening

n the tubing using a random walk computer model. According toeue [18], the typical maximum as in Fig. 1(a) can be attributed

o the onset of secondary flow or turbulence, possibly caused byhe roughness of the internal surface of the tubing. Such an effect

conditions. (a) Effect of different mobile phases and solutes for the UHPLC system.UHPLC system. (c) Comparison of bandspreading for naphthopyrene and uracil on

would cause mixing of the individual flow streams, limiting theeffects of bandspreading. As the values of bandspreading depend onthe diffusion coefficients of the solutes, there should also be vari-ation according to the mobile phase composition, particularly atlow flow rates where the Taylor–Aris treatment holds [18]. Indeed,Fig. 1(a) shows larger values of the bandspreading for uracil on theUHPLC system when measured in 10% ACN rather than 90% ACN.These results emphasise the need for estimating the system band-spreading under the same conditions as used for the subsequentanalysis.

Fig. 1(b) shows the effect of different detector data collectionparameters 80 Hz (0.0125 s filter) and 20 Hz (0.2 s filter) on �2 fornaphthopyrene, using the UHPLC system. As the flow rate increases

above 0.5 ml/min, the values of �2 are increasingly divergent, with�2 increasing almost linearly with flow rate for slower data collec-tion compared with the plateau achieved for fast collection. Notethat 20 Hz itself is a faster data collection rate than that of themicrobore system, however, the filter rate was 0.2 s compared with

4564 D.V. McCalley / J. Chromatogr. A

Fig. 2. Estimation of efficiency loss due to extra-column bandspreading for fused-core columns of different dimensions, on the two HPLC systems, as a function ofrbac

<ptsti

om2(1rcFoafiff

3n

lc0cI

etention factor. AG = microbore system; AQ = UHPLC system. Calculations based onandspreading values determined for naphthopyrene in ACN–water (90:10, v/v)t 37.5 ◦C. Upper diagram 10 cm columns with 25000 plates. Lower diagram 5cmolumns with 12500 plates.

0.12 s used on the microbore system. Indeed, 20 Hz and 0.05 s filterroduced considerably reduced bandspreading on the UHPLC sys-em. As the volumetric bandspreading contribution of the UHPLCystem is smaller than that of the microbore system, the detectorime-based contributions are likely to be more critical in the formernstrument.

Fig. 1(c) shows the bandspreading measured for naphthopyrenen the microbore system at 1.5 ml/min (in the region of its opti-um flow rate – determined subsequently, see below), was around

5 �l2 using 5 �l injections. As before, a slightly higher value27.5 �l2) was obtained for uracil. Bandspreading was reduced if�l injections were made, but the larger injections were used to

ender the results compatible in terms of absolute sensitivity andolumn loading with 1 �l injections on the 0.21 cm I.D. column.ig. 1(c) shows more or less constant values of �2 as a functionf flow rate between about 0.25 and 2 ml/min. However, at flowsbove 2 ml/min, �2 increased, which can be attributed to the insuf-cient data collection rate of the microbore system (14 Hz), that

ails to describe accurately the increasingly fast peaks emergingrom the column at high flow rate.

.2. Predicted effect of system bandspreading on efficiency ofaphthopyrene as a function of flow

Fig. 2 shows the predicted % loss in efficiency of columns of

ength 10 and 5 cm and true efficiency (in the absence of extra-olumn effects) 25,000 plates and 12,500 plates respectively, for a.46 cm column using the microbore HPLC, and 0.21 and 0.46 cmolumns on the UHPLC system, as a function of retention factor.t is likely in practice that columns will be operated in the region

1217 (2010) 4561–4567

of their optimum flow rate, generating the maximum efficiency. Atthis point, the effects of extra-column band broadening are likely tobe the most serious. The spreadsheet calculations were performedusing �2 = 25 �l2 for the microbore system and 3.5 �l2 for theUHPLC system, being the values corresponding to the bandspread-ing at the optimum flows for the 0.46 and 0.21 cm column respec-tively using naphthopyrene. Note that while �2 increases some-what above 25 �l2 for the microbore system above 2.5 ml/min, thetrue (theoretical) column efficiency declines due to mass transferband broadening processes, giving a concomitant reduced effectof extra-column effects on performance. Calculations showed thatthese effects of increased instrument bandspreading but reduced(true) column efficiency virtually cancelled each other out at highflow rate on this system, verifying the approach used. ConsideringFig. 2(a) for the 10 cm column, the curve for the 0.46 cm col-umn/microbore instrument indicates considerably less loss in effi-ciency than for the 0.21 cm column/UHPLC instrument. For exam-ple, the loss in efficiency at k = 2 is only 10% for the 0.46 cm columnbut above 20% for the 0.21 cm column. At k > 2, losses in efficiencydecrease, becoming very small for both systems at k = 5. However,at k = 1, the losses are quite serious at around 20% and almost 40% foreach system, respectively. For the 5 cm columns, the losses are moreserious as the peak volumes are considerably reduced and there-fore extra-column volumes become more significant. For exampleat k = 1, the losses in efficiency were around 33% and 55% for the 0.46and 0.21 cm columns, respectively. Clearly, the losses in efficiencyare minimised (although still not negligible for low k) using the0.46 cm column operated on the UHPLC instrument (calculations inthis case based again on �2 = 3.5 �l2 for a 1 �l injection). However,this column/instrument combination could not be used to obtaindata at high flow rate, due to the maximum 2 ml/min available onthe UHPLC (Waters Acquity) used in these experiments.

These results have important consequences for the experimen-tally measured performance of columns as a function of flow rate.Clearly, in all experimental measurements of column efficiency andplate height, the experimental value is always that of the columnplus the effect of the instrument (extra-column effects). It is pos-sible of course to correct for the extra-column contribution, andclearly such data treatment is necessary for a theoretical under-standing of column performance, especially when the instrumentalcontribution is significant, as with the 0.21 cm fused-core columns(see below). However, such an approach conceals the results thatare achievable practically with current instruments. A further dif-ficulty is that simple measurements of extra-column dispersioncannot easily account for effects such as column frictional heating,so these corrections may not be entirely accurate. To reduce theeffects of dead volume on results that are achievable practically,longer columns are preferable, although the maximum pressureavailable, and frictional heating are both factors to consider aswell as other factors such as the desired analysis time. Use ofexperimental conditions to generate high solute k are also recom-mended. For these relatively short columns, high k at least doesnot necessarily indicate prohibitively long analysis times. However,clearly extra-column effects are detrimental to fast analysis whena lower k might be preferred. System bandspreading values canbe greater for different solutes, and as shown in Section 3.1 arealso likely to be higher in mobile phases with a higher aqueouscontent than (the unusually low) values used in these experi-ments, giving more serious losses in efficiency than suggested byFig. 2.

The results have more general significance beyond fused-core

columns e.g. for the operation of sub-2 �m particle columns, whichgenerate similar efficiency to fused-core columns. These smallerparticle columns are often used in shorter lengths due to the highback pressures they generate, in which case instrumental band-spreading will be more significant.

D.V. McCalley / J. Chromatogr. A

Fig. 3. Effect of flow on h for fused-core columns of different dimensions on differentinstruments. The data for the 0.46 cm column was uncorrected for the (negligi-btflr

3f

uratflbtbaiciige

0T

h

a

�

wtooo1bptprT

le) extra-column bandspreading. Mobile phase ACN–water (90:10, v/v), columnemperature 37.5 ◦C. The reduced interstitial velocities correspond to volumetricow rates of 0.05–0.8 and 0.25–4.0 ml/min for the 0.21 and 0.46 cm I.D. columns,espectively.

.3. Measurement of Knox plots for 0.46 cm and 0.21 cmused-core columns on different systems

Table 1 shows the total instrument backpressure and the net col-mn pressure for the 10 cm × 0.46 cm fused-core column at a flowate of 1 ml/min for the various mobile phases used in the presentnd previous studies [5]. The net column backpressure corrected forhe extra-column contributions is about 60 bar in each case at thisow rate. Extrapolation of this figure would indicate a net columnackpressure of 360 bar for a 15 cm column operated at 4 ml/min,he highest flow employed in the previous study. For our micro-ore system, a 15 cm column operated at 4 ml/min would generatetotal system backpressure of around 480 bar, in excess of the max-

mum operating pressure of our system. In any case, operating theolumn at such high pressures could lead to greater frictional heat-ng effects. With these considerations in mind, and the small lossesn efficiency calculated for a 10 cm × 0.46 cm column at the high kiven by naphthopyrene, we decided to use 10 cm columns for allxperiments.

Fig. 3 shows Knox plots for naphthopyrene using 0.46 and.21 cm fused-core columns on the microbore and UHPLC systems.he plots are of the reduced plate height h

= H

dp(1)

gainst the reduced interstitial velocity �red,int

red,int = Fdp

�r2εextDm(2)

here F is the volumetric flow rate, dp the particle diameter, rhe column radius, εext the external porosity (determined previ-usly as 0.43 for this column [5]) and Dm the diffusion coefficientf the solute in the mobile phase. Highly symmetrical peaks werebtained for naphthopyrene, with asymmetry factors very close to.0. Fig. 3 shows the curve for the 0.46 cm column on the micro-ore system was the most favourable, with values of the reduced

late height as low as 1.5, similar to those obtained in HILIC [4] forhe bare silica version of this phase. (h = 1.5 corresponds to 25,000lates on the 10 cm column). The retention factor of naphthopy-ene was 5.4 in the mobile phase used (90% ACN at 37.5 ◦C, seeable 2) and Fig. 2 shows that the loss of efficiency for a column

1217 (2010) 4561–4567 4565

with 25,000 plates is only around 2.5% at such high retention, a neg-ligible figure. Fig. 3 shows also a partial Knox curve for the 0.46 cmcolumn on the UHPLC, corresponding to volumetric flow rates of0.5–2.0 cm3/min, the maximum flow rate that can be used withour instrument. Surprisingly, the curve lies mostly slightly abovethat for the same column on the microbore instrument, despitethe smaller bandspreading of the UHPLC, especially at the higherflow rates. This result might be attributable to some small incom-patibility in the temperature of the influent to that of the columntemperature at high flows on this instrument, due to less effectivesolvent pre-heating. In contrast to the UHPLC system, the microboresystem uses a serpentine capillary embedded in a massive heat-ing block; this pre-heating system is reasonably effective, althoughsome cooling can take place at high set temperatures in the con-necting capillary between the heating block and the column, dueto cold spots in the oven [13]. Apparently, the very small advantageof the lower instrumental bandspreading of the UHPLC system hasno significance at this high value of k (see Fig. 2). However, forpeaks of lower k, use of the 0.46 cm column on the UHPLC sys-tem is likely to generate the highest efficiency, subject to the pumpbeing able to generate sufficient flow to achieve the minimum ofthe Knox curve. The curve for the 0.21 cm fused-core column on theUHPLC instrument lies clearly above that for the 0.46 cm columnon the microbore system. The predicted loss of efficiency for sucha column with 25,000 plates on this instrument is still only around6%, due to the high k of the solute. Correcting the reduced plateheight for the effects of the system bandspreading still shows lessfavourable results for this 0.21 cm column (a maximum of around20,900 plates for the true efficiency) compared with the 0.46 cmcolumn. This result could be attributed to the greater difficulties inpacking of the smaller diameter fused-core column [20]. A similarreduction in efficiency of narrower bore compared with 0.46 cmfused-core columns (after correction for instrumental effects) wasalso shown for those from a different manufacturer (Kinetex, Phe-nomenex, Torrance, USA, results not shown [21]). Table 2 showsfits of these plots to the Knox equation:

h = A�0.333 + B

�+ C� (3)

The fitting coefficients reported for the 0.21 cm fused-core col-umn were calculated from the data corrected for the extra-column(instrumental) contribution to plate height, whereas for the 0.46 cmcolumns, the data were uncorrected as the extra-column contribu-tions are extremely small. The plot for the 0.21 cm column showsA = 0.50, considerably higher than for the 0.46 cm column (A = 0.34),indicating that differences in the packing could be responsible. Nev-ertheless, there is clearly some convergence of the curves for the0.46 and 0.21 cm columns in the C term region of the plots at highreduced velocity (see Fig. 3). Table 2 also indicates C = 0.034 forthe 0.21 cm column compared with 0.046 for the 0.46 cm fused-core column. It is possible that this result could be explainedby greater frictional heating effects in the larger bore column,as there is a greater difference in temperature between the coreand the walls in such columns, as heat is less easily conductedaway.

In conclusion, the combination of the 10 cm × 0.46 cm col-umn and the microbore system gave the optimum results.In combination with the high k for naphthopyrene, this

combination also yields values of column efficiency that donot require correction for extra-column dispersion. Clearlyhowever, the contribution of extra-column effects needscareful consideration for each individual experimental situa-tion.

4566 D.V. McCalley / J. Chromatogr. A 1217 (2010) 4561–4567

Table 1Backpressure measurements using different mobile phases at a flow of 1 cm3/min for a 10 cm × 0.46 cm fused core and 15 cm × 0.46 cm totally porous columns, microboresystem.

Column Mobile phase Total system pressure (bar) Extra-column pressure (bar) Column pressure (bar)

10 cm × 0.46 cmfusedcore

100% ACN, 25 ◦C 78 19 5990% ACN, 37.5 ◦C 82 22 6085% ACN, 50 ◦C 81 23 58

15 cm × 0.46 cm totally porous 90% ACN, 37.5 ◦C 40 22 18

Table 2Fits of plots of reduced plate height for naphtho[2,3-a]pyrene against reduced interstitial velocity to the Knox equation.

Column/dimensions Instrument Mobile phase k A B C

10 cm × 0.46 cmfusedcore2.7 �m

Microbore 100% ACN, 25 ◦C 3.2 0.33 2.9 0.06790% ACN, 37.5 ◦C 5.4 0.34 3.8 0.04685% ACN, 50 ◦C 5.7 0.38 3.7 0.041

10 cm × 0.21 cm fused core 2.7 �m UHPLC 90% ACN, 37.5 ◦C 4.5 0.50 4.2 0.03415 cm × 0.46 cm totally porous 5 �m Microbore 90% ACN, 37.5 ◦C 3.3 0.41 7.2 0.038

The data for the 0.21 cm I.D. column was corrected for extra-column effects, whereas the data for the 0.46 cm columns is uncorrected, due to the very small contribution ofextra-column effects under the experimental conditions.

Fs

3a

atrrpattelAtaphirAF

ig. 4. Effect of flow on h for 10 cm × 0.46 cm fused-core column with the microboreystem and different mobile phases/temperatures.

.4. Comparison of Knox plots for the 0.46 cm fused-core columnt different temperatures

Fig. 4 shows the Knox plots for the 0.46 cm column at 25, 37.5nd 50 ◦C, as used previously [5] with 100, 90 and 85% ACN, respec-ively. The ACN concentration was reduced as the temperature wasaised presumably to keep k approximately constant. Use of theeduced (interstitial) velocity means that curves for different tem-eratures should overlay exactly, in the absence of unusual effects,s the variation of the diffusion coefficient with temperature isaken into account. Within experimental error, this indeed appearso be the case. Small errors could be caused by inaccuracies in thestimation of Dm using the Wilke–Chang equation, especially in theight of reports that this is indeed likely in mobile phases of highCN content [22]. Variations in the temperature of the oven from

he set temperature, and variable frictional heating effects shouldlso be considered [13]. The results however, contrast with thosereviously reported [5]. No strong upturn is shown in the curve at

igh mobile phase velocity, and good performance at high flow rate

s indicated. For example, values of h exceeding 5 were previouslyeported for a reduced interstitial velocity of ∼20 at 37.5 ◦C in 90%CN, whereas a value barely above 2 is shown in the present work.ig. 3 also shows no upturn in the curve for the 0.21 cm column, and

Fig. 5. Effect of flow on h for 15 cm × 0.46 cm totally porous (5 �m) particle column.Mobile phase ACN–water (90:10, v/v), column temperature 37.5 ◦C. The reducedinterstitial velocities correspond to volumetric flow rates of 0.25–4.0 ml/min.

neither was the upturn noted for a bare silica fused-core column inHILIC [4]. A recent report of efficiency measurements with fused-core columns in which the instrumental variance was subtractedfrom the observed peak variance also showed quite flat plots of H vsflow [23]. Nevertheless, we did not study higher molecular weightsolutes, whose performance might be affected by partial exclusionfrom the relatively small pores of the fused-core column used.

3.5. Comparison of Knox plots for a totally porous particle column

Fig. 5 shows a similar Knox plot for a totally porous 5 �m pack-ing column run on the microbore instrument. The column showsexcellent efficiency at the optimum flow velocity for a packingof this type, yielding a minimum value of h = 1.95, correspond-ing to >100,000 plates/m. Table 2 indicates a slightly higher valueof the C coefficient on the 0.46 cm fused-core column run underthe same conditions (37.5 ◦C, 90% ACN) compared with the porous

5 �m phase (C = 0.046 and 0.038, respectively). Nevertheless, thisresult could be attributed entirely to frictional heating effects in the0.46 cm fused-core phase, as the value of C for the 0.21 mm fused-core column is only 0.034. Clearly the backpressure on the 5 �mcolumn was much lower (see Table 1) despite its longer length,

togr. A

iftcssetmoioma

�

wupct(b

4

tcsHeetmn

[[

[[[[[[

[

[[20] J.J. Kirkland, AMT Technologies, personal communication, 2009.[21] D.V. McCalley, unpublished observations.

D.V. McCalley / J. Chroma

ndicating much lower frictional heating. Considering the 0.46 cmused-core column, values of the A and B coefficients were lowerhan the totally porous column. These results agree with previousonclusions that the good packing properties and narrow particleize distribution of the fused-core material lead to low eddy diffu-ion, while the low internal porosity of the packing leads to smallerffective diffusivity of the sample [5,6]. For small molecules at least,hese considerations appear to be more important than improved

ass transfer in the stationary phase. However, over-interpretationf these fitting coefficients for different stationary phase materialss inadvisable using our approach, as the coefficients clearly dependn consistent measurement of particle size. Nevertheless, measure-ents of the backpressure of the totally porous column indicatedparticle size of 5.2 �m, according to the equation

P = 1000F�L

�r2d2p

(4)

here F is the volumetric flow rate, � is the viscosity, L the col-mn length, r the internal radius and dp the particle size of theacking [19]. However, this expression is only approximate, andould lead to errors in the estimated particle size. More impor-antly, the equations used to describe the dependence of h on flowthe Knox treatment) may be too simplistic; for example, there areetter ways to calculate the B contribution [5,24].

. Conclusions

A microbore LC and a UHPLC system were carefully charac-erised as to their effect on the determination of van Deemter/Knoxurves for highly efficient fused-core columns. Instrumental band-preading effects can have serious effects on the measurement ofETP. However, for the high k solute used in these studies, the

ffects were minimised and negligible when using a large diam-ter 0.46 cm column together with a microbore LC and smallerhan for a 0.21 cm column used with a UHPLC system. Further-

ore, it appears that at present it may not be possible to packarrower bore fused-core columns of as high (true) efficiency as

[[[

1217 (2010) 4561–4567 4567

conventional bore columns. These results are unfortunate, in thatthe use of narrower bore columns limits the detrimental effects offrictional heat generation, and reduces solvent consumption. Weexamined the performance of the fused-core columns at high flowrate and elevated temperature, considering carefully the problemsthat may be encountered in generating accurate van Deemter/Knoxcurves. No unusual features of the van Deemter/Knox plots werereported, indicating that these columns are eminently suitable forfast analysis.

References

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