High temperature to increase throughput in liquid chromatography and liquid chromatography–mass spectrometry with a porous graphitic carbon stationary phase

J. Sep. Sci. 2007, 30, 1115 –1124 L. Pereira et al. 1115

Luisa PereiraStephen AspeyHarald Ritchie

Thermo Fisher Scientific,Runcorn, UK

Original Paper

High temperature to increase throughput in liquidchromatography and liquid chromatography–massspectrometry with a porous graphitic carbonstationary phase

High separation temperatures in liquid chromatography and liquid chromatogra-phy–mass spectrometry with a porous graphitic carbon column were investigated.Separation temperature was varied up to 2008C, and the effect on retention, analysistime, and sensitivity was measured. Analysis times were reduced more than six-fold,whilst baseline resolution was maintained. The impact of the separation tempera-ture on signal-to-noise ratio with atmospheric pressure chemical ionisation or elec-trospray mass spectrometric detection was also investigated. The potential of usingsuperheated water for the analysis of some very polar compounds is illustrated.Monitoring of column stability detected no loss of performance, due to the highlystable nature of the 100% carbon stationary phase.

Keywords: High-temperature liquid chromatography / Speed / Sensitivity /

Received: December 14, 2006; revised: January 12, 2007; accepted: February 8, 2007

DOI 10.1002/jssc.200600521

1 Introduction

The overwhelming majority of HPLC separations are runat temperatures between 25 and 408C, and although theadvantages of using higher temperatures have beenunderstood for many years [1, 2], until recently this hasnot been fully exploited [3–6]. The main benefits of ele-vated temperature are shorter analysis times withoutloss of efficiency, reduced pressure drop across the col-umn, and the need for reduced organic modifier in themobile phase. Additionally, temperature can be used toimprove separation selectivity and temperature pro-gramming [7, 8] can replace or be combined with solventgradients.

Concerns regarding analyte and column thermal stabi-lity, and the availability of column ovens capable ofworking at higher temperature have stalled the use oftemperature as another method development tool. How-ever, the trend towards miniaturisation in column inter-nal diameter and particle size, and the constant need toincrease sample throughput, have been driving forces

behind recent developments in chromatographic hard-ware that allow for the use of high separation tempera-tures and temperature programming. When high tem-peratures are used analyses are faster and solute resi-dence times in the column are shorter, which meansthat even complex molecules and solutes that are rela-tively unstable can be analysed without degradation,which has been attributed [9] to the solvated state of thecomplex molecules. However, the stability of silica-basedstationary phases is an issue; therefore, other packingmaterials which are stable at high temperatures must beexplored. For instance, porous graphitic carbon is a sta-tionary phase which is not affected by physical or chemi-cal degradation at high temperature regardless of themobile phase used.

The use of high temperatures in reversed-phase liquidchromatography has several practical advantages.Mobile phase viscosity is reduced at higher temperatures[10] which enhances the mass transfer of the solutebetween the mobile and stationary phase, resulting inbetter chromatographic performance. It has beendemonstrated [11] for a range of chromatographic sup-ports that an increase in column temperature results inhigher column efficiency (optimum reduced plate heightdecreases with temperature) and also improves peakasymmetry due to elimination of secondary interactions.There are two major benefits to obtaining more efficientand symmetrical peaks: increased resolution and peakheight, which in turn improves signal-to-noise ratios and

Correspondence: Dr. Luisa Pereira, Thermo Fisher Scientific,112 Chadwick Road, Runcorn, Cheshire WA7 1PR, UKE-mail: [email protected]: +44 (0) 1928 588106

Abbreviations: APCI, atmospheric pressure chemical ionisation;DVB, divinylbenzene; FID, flame ionisation detector; PBD, poly-butadiene; PGC, porous graphitic carbon

i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

1116 L. Pereira et al. J. Sep. Sci. 2007, 30, 1115 – 1124

the sensitivity of the analysis. Another consequence ofreduced solvent viscosity at high temperatures isreduced backpressure, allowing for the use of higherflow rates to increase the speed of analysis, without los-ing efficiency. In fact, the optimum linear velocityincreases proportionally to T/g (temperature/eluent visc-osity) [11], and therefore the flow rate for optimum effi-ciency is shifted to a higher value at higher tempera-tures. As a consequence, the analysis speed can beimproved 5 to 15-fold, when temperature is increasedfrom ambient to 2008C. Reduced backpressure at hightemperature also allows longer columns packed withsmall particles to be used to facilitate the resolution ofcomplex samples.

Another interesting aspect of high-temperature liquidchromatography (HT-LC) is the possibility of using high-temperature water [12, 13] as the mobile phase (other ter-minologies such as superheated water and subcriticalwater are also found in the literature). At room tempera-ture water is too weak as a solvent to elute all except themost polar analytes but as the temperature of the water isincreased, particularly above its boiling point, its dielec-tric constant (which is a measure of polarity) decreases[14], thereby increasing its elution strength for reversed-phase liquid chromatography (RP-LC). The dielectric con-stant of water at 2258C is comparable to that of acetoni-trile and, therefore, water can potentially be used as areversed-phase eluent and its elution strength adjustedby changing temperature. At high temperatures, thehydrogen bond donor strength of water decreases signifi-cantly, whereas the hydrogen acceptor strengthincreases; these changes may indicate a disruption of thehydrogen-bond network in the water, which increasesthe solubility of non-polar solutes [15]. It has been demon-strated that the selectivity changes as a function of tem-perature are different from those obtained as a functionof the mobile phase composition in conventional RP-LC,which makes these two approaches complementary [16].The advantages of using high-temperature water are thereduction or elimination of hazardous organic solvents,thereby providing a more environmentally friendly andcheaper LC solution, and also the possibility of couplingwith flame ionisation detection (FID) [17], which is a verysensitive, universal detection technique.

HT-LC is expected to show advantages when coupledwith mass spectrometry. In LC–MS with ESI the columneffluent is nebulised in the ion source by a high voltageapplied to the electrospray needle and a nebulising gas,followed by droplet desolvation with high temperature.In atmospheric pressure chemical ionisation (APCI) thecolumn effluent is vaporised by a combination of hightemperature applied to the probe and a nebulising gas,followed by ionisation in the gas phase. When HT-LC isused in combination with ESI and APCI, the mobile phasereaches the ion source at elevated temperature which

may aid the vaporisation and desolvation processes, thusincreasing the ionisation efficiency and consequentlythe sensitivity of the analysis.

Routine HT-LC requires a thermally stable column.However, columns packed with alkyl-modified silicas,which are generally used in RP-LC, should not be usedabove 60–708C, a limit that is dependent upon the silica,ligand, and mobile phase composition (water content,pH). At these temperatures hydrolysis of the organosi-lane bond or dissolution of the silica may occur leadingto column failure. Some silica-based phases use bondingtechnologies in which the siloxane bond is protectedfrom hydrolysis, imparting stability up to 90–1008C [8].Polymeric columns have traditionally been used in gelpermeation chromatography at high temperatures andtherefore it is already known that this type of materialpossesses good thermal stability. Recently, poly(styrene-divinylbenzene) (PS-DVB) columns have been provenstable up to temperatures of 1508C under a range ofmobile phase conditions [8, 18], although efficiencies ofsuch columns are lower than those of equivalent silica-based columns. Zirconia-based phases have good thermalstability and it has been shown that polybutadiene (PBD)-modified zirconia is stable for 1300 column volumes at2008C [19]. However, in temperature gradient HT-LC, zir-conia-based phases show a significant rise in the baselinesignal in UV detection [8], possibly caused by loss of thePBD polymer. Porous graphitic carbon (Hypercarb) isstable at a relatively very high temperature for LC regard-less of the mobile phase used, provided there are no PEEKcomponents in the column hardware. Hypercarb is 100%carbon with no bonded chemistry, and is therefore che-mically very stable. It has been demonstrated [8, 20] thatHypercarb can be used routinely up to 2008C under iso-thermal or temperature gradient conditions, withoutany loss in performance. Moreover, because Hypercarbhas no bonded phase, column bleed does not occur,which means that this column packing can be used inHT-LC with detection techniques such as mass spectrome-try, evaporative light scattering detection and FID, whichare sensitive to column bleed.

Ovens and column heaters specifically designed forHT-LC are now commercially available. These includetemperature programming, mobile phase preheating,and mobile phase pre-detector cooling. Mobile phase pre-heating is an important requirement at column tempera-tures above 808C to prevent band dispersion caused bythermal mismatch across the diameter of the column[21]. When using UV detection it is also necessary to coolthe mobile phase to prevent damage of the flow cell; themobile phase pre-detector cooler is bypassed when FID orMS detection are used. Another system requirement inHT-LC is that a backpressure regulator is used to main-tain the mobile phase in the liquid state, especially if thetemperature is close to or above the boiling point.


J. Sep. Sci. 2007, 30, 1115 –1124 Liquid Chromatography 1117

In the work presented in this paper, examples of theeffect of separation temperature on retention, speed ofanalysis and sensitivity (ESI and APCI signal intensityand signal-to-noise ratios) are shown using a porous gra-phitic carbon (PGC) column. The separation of purinesand pyrimidines using a mobile phase of 100% water atvery high temperatures illustrates the potential for thismethodology as a “green” LC option for the analysis ofpolar compounds. These examples illustrate the poten-tial of high temperature as a complementary methoddevelopment parameter in LC and LC-MS. Data on col-umn stability is also presented.

2 Experimental

2.1 Columns and instrumentation

All the columns used were Hypercarb 5 lm, manufac-tured by Thermo Scientific (Bellefonte, PA), in the follow-ing dimensions: 10064.6 mm, 10062.1 mm,5062.1 mm.

The instrumentation used to perform the UV workconsisted of a HPLC system with a quaternary pump,degasser, autosampler and variable wavelength UV detec-tor (Agilent 1100 series, Agilent Technologies, Wald-bronn, Germany) and a programmable oven with mobilephase preheating, Polaratherm Series 9000 (SelerityTechnologies, Salt Lake City, UT). The mobile phase pre-heater consists of a small diameter stainless steel lineinside a cartridge, that is heated independently from thecolumn compartment, and which heats the mobilephase just before it enters the column. The autosampleris connected to the mobile phase preheater with stainlesssteel tubing 0.178mm in internal diameter. The oven wasoperated either with a temperature gradient or isother-mally; in both cases the effluent cooler was set to 258C.The data acquisition was performed using ChromQuestversion 4.0 (Thermo Scientific, San Jose, CA). To preventevaporation of the mobile phase in the column at hightemperatures, extra backpressure was introduced in thesystem, downstream from the column, using 5-cm lengthof 50-lm internal diameter tubing. The instrumentationused to perform the LC–MS work consisted of the Finni-gan Surveyor (pump with degasser and autosampler) andFinnigan Surveyor MSQ (Thermo Scientific, San Jose, CA)fitted with Polaratherm Series 9000. The outlet of theautosampler was connected to the column placed in theoven and the outlet of the column was connected to theMS inlet using stainless steel tubing. In the LC–MS sys-tem setup the effluent cooler was bypassed. In the experi-ments carried out to measure the signal intensity as afunction of the mobile phase temperature the instru-mental conditions were as follows: APCI, probe tempera-ture 6008C, corona discharge 5 lA, entrance cone voltage60 V, full scan from m/z 250 to 280 in 0.2 s, positive ion

polarity; ESI, probe temperature 4508C, electrospray nee-dle voltage 3.5 kV, entrance cone voltage 60 V, full scanfrom m/z 250 to 280 in 0.2 s, positive ion polarity. TheLC–APCI-MS work used a probe temperature of 5508C,corona discharge 5 lA, entrance cone voltage 60 V, fullscan from m/z 200 to 300 in 0.2 s, positive ion polarity.The LC–ESI-MS work used a probe temperature of 5008C,electrospray needle voltage 2.5 kV, entrance cone voltage70 V, full scan from m/z 200 to 300 in 0.2 s, positive ionpolarity. Additional methodology is described in figurecaptions.

2.2 Chemicals

Water, methanol, and acetonitrile were HPLC grade, pur-chased from Fisher Scientific (Loughborough, UK) orRathburn Chemicals (Walkeburn, UK); formic acid, tri-fluoroacetic acid and ammonium acetate were HPLCgrade purchased from Fisher Scientific (Loughborough,UK), Riedel –de Ha�n (Sigma–Aldrich, LaborchemikalienGmbH, Seelze, Germany) and Fluka Chemie GmbH (Stein-heim, Germany) respectively. All the chemicals used inthe test mixtures were purchased from Sigma (Sigma–Aldrich Co, St. Louis, MO) or Riedel –de Ha�n (Sigma–Aldrich, Laborchemikalien GmbH, Seelze, Germany).Test solutions were prepared in water, methanol, orwater/methanol (1:1) at a concentration of 1 mg/mL andfurther diluted to the working concentrations.

3 Results and discussion

3.1 Effect of temperature on retention on porousgraphitic carbon

Generally, in reversed-phase separations an increase intemperature will produce a reduction in retention times.The effect of varying separation temperature on theretention factors of several solutes is demonstrated inFig. 1 with a plot of log k versus 1/T (Van't Hoff plot [22]),for the temperature range between 30 and 1808C. A quad-ratic relationship provides a better fit of the experimen-tal data than a linear relationship, with correlation coef-ficients R2 A0.998. Linear Van't Hoff plots are usuallyreported for neutral, low molecular mass solutes inhydro-organic mobile phases [23, 24] with temperaturesup to 908C. In a recent report [11] log k versus 1/T, fromambient to 2008C, were plotted for several mobile andstationary phases, and both linear and non-linear Van'tHoff relationships were observed, depending on the sta-tionary phase and mobile phase combinations. A non-classical curvilinear Van't Hoff relationship has beenattributed to a change of heat capacity of the systemdependent on temperature [25], and is likely to be consis-tent with a change in the retention mechanism as tem-perature is increased.



3.2 High temperature to reduce analysis time

Figure 2 illustrates the gain in speed of analysis that canbe obtained by using temperature programming. Sevenherbicides and three metabolites of atrazine were sepa-rated with a solvent gradient of water and acetonitrile,at conventional temperature (Fig. 2a). These compoundshave a wide range of hydrophobicity, log P = 0.32 for atra-zine-desethyl-1-desisopropyl and log P = 3.07 for propa-nil. Porous graphitic carbon [26] allows for good reten-tion of the polar metabolites but also strongly retainshydrophobic solutes such as propanil. Under thesemobile phase and temperature conditions propanil doesnot elute in 45 min; a stronger solvent such as isopropa-nol or tetrahydrofuran [27] instead would be required toelute this solute. In Fig. 2b, the solvent gradient (5 to100% organic) was replaced with a temperature gradientfrom 140 to 2008C at 208C/min and an isocratic mobilephase (50:50 v/v, water/acetonitrile); analysis time isreduced from 28 to 9 min, with elution of the 10 analytesand still exhibiting full baseline resolution. Because athigh temperatures the flow rate for optimum perform-ance is higher, on Fig. 2c the flow rate was increased to2 mL/min (all other conditions kept unchanged), whichfurther reduced the analysis time to just over 5 min. Theobserved resolution between any pair of solutes is A1.5in all of these runs (refer to Table 1). Comparison of theresolution of later eluting peaks (after peak 4) run at 1and 2 mL/min reveals that for the latter resolution isincreased because peaks are narrower (data not shown).This effect can be attributed to the reduced longitudinaldiffusion experienced by solutes which have a shorterresidence time in the column through increased flowrate.

A further practical advantage of using a temperaturegradient in replacement of a solvent gradient is that col-umn re-equilibration at the end of the gradient is faster,allowing for shorter cycle times. Typically ten columnvolumes are used for re-equilibration and therefore the10064.6 mm column running at 1 mL/min requiredapproximately 10 min to re-equilibrate. In the exampleshown in Figs. 2b and 2c the time required between tem-perature gradient runs to cool the oven down to the start-ing temperature (1408C) was around 3 min.

3.3 LC separations with high-temperature(superheated) water

Purines and pyrimidines are very polar solutes and canbe difficult to retain under reversed-phase conditionsunless an ion pair is used in the mobile phase or the sta-


Figure 1. Effect of temperature(1/T) on retention (log k) for (g)uracil, mobile phase H2O/MeCN(95:5), (f) phenol, (0) resorcinol,(h) phloroglucinol, mobile phaseH2O+0.1% formic acid/ACN(80:20, v/v). Column: Hypercarb5 lm, 10064.6 mm. Tempera-ture was varied between 30 and2008C. Regression coefficients(R2) are for a quadratic fitting.

Table 1. Comparison of resolution (USP) for chromatogramsin Figs. 2a, 2b, and 2c.

Resolution

Peak pair Solventgradient(Fig. 2a)

T gradient1 mL/min(Fig. 2b)

T gradient2 mL/min(Fig. 2c)

2, 1 18.68 4.24 3.513, 2 6.70 2.18 1.954, 3 3.45 2.72 2.595, 4 7.96 3.70 3.866, 5 2.55 1.87 1.897, 6 8.65 1.71 2.068, 7 2.44 3.11 3.409, 8 8.85 4.64 5.20

10, 9 – 6.08 7.04


tionary phase has some polar character that enables anadditional interaction mechanism with the polar groupsin the analyte. On porous graphitic carbon these com-pounds are well retained without the need for an ionpair reagent, as demonstrated on Fig. 3a; retention andseparation are obtained with a mobile phase of water,acetonitrile and formic acid, at 508C. The mechanism ofretention of polar solutes on porous graphitic carbon isthrough dipole-dipole interaction: the polar solute has apermanent dipole, which induces a dipole on the polari-sable surface of the graphite as it approaches it [28]. Theformic acid in the mobile phase behaves as an electronicmodifier that competes with the solutes for the surface

of the graphite thereby reducing retention. By removingthe organic component (acetonitrile) and the electronicmodifer (formic acid) from the mobile phase, the elutionstrength is greatly reduced, and at conventional tem-peratures this is too weak to elute the solutes from thePGC column. However, if the temperature of water isincreased its elution strength increases due to adecreased dielectric constant. In Fig. 3b, the purines andpyrimidines are separated with a mobile phase of purewater at 1908C. Although the elution order is main-tained, the separation selectivity obtained with 100%water at 1908C is different and overall peak symmetry isimproved at this relatively high temperature. Asymme-


Figure 2. Separation of herbicides and metabolites at con-stant temperature (408C) or with a temperature gradient. Col-umn: Hypercarb 5 lm, 10064.6 mm; mobile phase: H2O/ACN; detection: UV at 215 nm. (a) Gradient: 5 to 100% ACNin 15 min; temperature: 408C; flow rate: 1 mL/min. (b) Iso-cratic (50:50, v/v); temperature: 1408C to 2008C at 208C/min;flow rate: 1 mL/min. (c) Isocratic (50:50, v/v); temperature:1408C to 2008C at 208C/min; flow rate: 2 mL/min. Analytes:1) atrazine-desethyl-1-desisopropyl; 2) atrazine-desethyl; 3)atrazine-desisopropyl; 4) propazine; 5) prometryn; 6) atra-zine; 7) ametryn; 8) simazine; 9) symetryn; 10) propanil.


try values for peaks 3 and 4 (at 10% height) fall from 1.37and 1.56 under conventional conditions to 1.22 and 1.19respectively at 1908C.

Temperature programming is particularly usefulwhen developing a method using a mobile phase con-taining only water. Parameters that can be changed arestarting temperature, final temperature, and heatingrate (8C/min). The effect of these parameters on selectiv-

ity, run time, and resolution are illustrated on Fig. 4 forthe separation of the same mixture of purines and pyri-midines. This type of separation combines the ability ofthe PGC material to retain very polar molecules inreversed-phase LC with the ability to change the elutionstrength of water with temperature programming.

3.4 HT-LC–MS

The sensitivity of a LC–MS method is generally measuredas the signal-to-noise ratio (S/N) of the chromatographicpeak for each solute and is affected by method param-eters such as the flow rate, the composition and pH ofthe mobile phase, and the ratio of organic-to-aqueous inthe mobile phase. Mobile phases with high organic con-tent give better sensitivity in ESI because of lower surfacetension and therefore better spraying. The effect of thetemperature of the mobile phase is not generally consid-ered as most LC–MS methods are developed at conven-tional temperatures. It is expected that if the mobilephase reaches the ionisation source at high temperaturethen the desolvation of the solutes will be improved.Additionally, the process of ionisation of the solute inthe mobile phase in ESI and the process of solute vapori-


Figure 3. Comparison of the separation of purines and pyri-midines under conventional conditions (a) and with high-tem-perature water (b). Column: Hypercarb 5 lm, 10064.6 mm;detection: UV at 254 nm. (a) Mobile phase: H2O + 0.1% for-mic acid/ACN (85:15, v/v); flow rate: 0.8 mL/min. Tempera-ture: 508C. (b) Mobile phase: H2O; flow rate: 2.0 mL/min.temperature: 1908C. Analytes: 1) cytosine; 2) uracil; 3) thy-mine; 4) hypoxanthine; 5) guanine; 6) xanthine.

Figure 4. Temperature programming in LC with superheatedwater. Column: Hypercarb 5 lm, 10064.6 mm; mobilephase: H2O; flow rate: 1.5 mL/min; detection: UV at 254 nm.Temperature program: (a) 100 to 2008C, at 108C/min; (b)100 to 2008C, at 158C/min; (c) 150 to 2008C, at 158C/min.Analytes: 1) cytosine; 2) uracil; 3) thymine; 4) hypoxanthine;5) guanine; 6) xanthine.


sation in APCI will change as the mobile phase is heatedto higher temperatures. Therefore, when working withhigh separation temperatures in LC–MS it is necessary toconsider the changes in the chromatography and alsoionisation in the detector source.

To measure the changes in ionisation efficiency withincreased temperatures, the mobile phase temperaturewas varied between 30 and 1908C and a basic compound(sulfamerazine) was injected into the LC flow (with nocolumn). The mobile phase composition and flow ratesused are typical of LC–MS with ESI or APCI. The detectorsignal was measured as peak area and peak height of theextracted mass chromatograms for [M+H]+ (m/z 265) andplotted as a function of the mobile phase temperature(Figs. 5a and 5b, respectively). It was observed that athigher temperatures there is an improvement in the sig-nal intensity for both ESI and APCI, as can be seen in

Fig. 5a: peak area increases as the temperature of themobile phase increases, with an optimum at approxi-mately 1508C. These improvements are believed to be aresult of better analyte desolvation at higher tempera-tures and changes in solution chemistry in ESI orchanges in gas phase chemistry in APCI, which enhancethe ionisation process. However, increased separationtemperature also affects chemical noise, particularly inESI, therefore the measure of S/N is a better probe of thesensitivity of the method in LC–MS. For this reason peakheight was also measured and the observation (Fig. 5b) isthat there is a 4-fold increase in APCI and a 1.7–foldincrease in ESI when the temperature of the mobilephase is increased from 30 to 1508C.

The effect of separation temperature on the S/N of foursulfonamides was measured in LC–APCI-MS and LC–ESI-MS (Figs. 6a and 6b, respectively). The experimental con-


Figure 5. Effect of the temperature of the mobile phase on the ESI and APCI signal, measured as peak height (a) and peak area(b) of sulfamerazine injected into the LC flow (no column). Mobile phase: H2O/ACN (50:50, v/v) + 0.1% formic acid for ESI, H2O/ACN (1:1) for APCI; flow rate: 0.2 mL/min for ESI, 0.5 mL/min for APCI.


ditions are described in the figure caption; for each set ofexperiments temperature was varied between 30 and1808C. The change in peak height has a chromatographiccontribution, and a detection contribution, as discussedabove. As the column temperature increases, retention isreduced and therefore the chromatographic peakbecomes narrower and taller. The detection contributionis demonstrated in Fig. 6b. In APCI the separation tem-perature for the best S/N is 1808C, except for sulfagua-nine, which exhibits an optimum at 908C, and sulfathia-zole, which exhibits an optimum at 1208C. In ESI, how-ever, above separation temperatures of 90 to 1208C, noiseincreases more rapidly than the signal intensity, there-fore, S/N drops as temperature is increased. The S/N curveprofile for sulfamonomethoxine is different from the

other sulfonamides; at 308C this solute is stronglyretained and elutes with 100% organic mobile phase.However, when the temperature is raised it elutes with a

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com

Figure 6. Impact of the separation temperature on the sig-nal-to-noise ratio (S/N) in APCI (a) and ESI (b) (solutes –sulfonamides). Experimental conditions for APCI: Column:Hypercarb 5 lm, 10062.1 mm; mobile phase: ammoniumacetate 10 mmol/L pH 9/ACN (65:35, v/v); flow rate: 0.2 mL/min; Detection: +APCI. Experimental conditions for ESI: Col-umn: Hypercarb 5 lm, 5062.1 mm; mobile phase: A – H2O+ 0.1% formic acid, B – ACN + 0.1% formic acid, gradient:50 to 100% B in 5 min; flow rate: 0.2 mL/min; Detection:+ESI.

Figure 7. LC-MS of sulfonamides at high temperature: (a)APCI: Column: Hypercarb 5 lm, 10062.1 mm; mobilephase: ammonium acetate 10 mmol/L pH 9/ACN (65:35,v/v); flow rate: 1 mL/min; column temperature: 1808C; detec-tion: +APCI. (b) ESI: Column: Hypercarb 5 lm, 5062.1 mm;mobile phase: A – H2O + 0.1% formic acid, B – ACN + 0.1%formic acid, gradient: 50 to 100% B in 5 min; flow rate:0.2 mL/min (top chromatogram) 0.4 mL/min (bottom chroma-togram); column temperature: 308C (top chromatogram),908C (bottom chromatogram); detection: +ESI. Analytes: 1)sulfaguanidine; 2) sulfathiazole; 3) sulfamerazine; 4) sulfa-monomethoxine.


lower percentage of organic solvent; therefore, there arethree variables determining the signal intensity: reten-tion that affects peak width, separation temperature(temperature of the mobile phase), and percentage oforganic solvent in the mobile phase.

Examples of the separation of this group of com-pounds at high temperatures are shown in Fig. 7. InFig. 7a the separation of the four sulfonamides at 1808C,with APCI detection, is achieved in 0.7 min. Because ofthe reduced mobile phase viscosity at 1808C it is possibleto run the 2.1 mm id column at 1 mL/min without exces-sive backpressure, thereby further reducing analysistime. In ESI such a high flow rate would cause a decreasein sensitivity (optimum flow rate for ESI is approximately200 lL/min) and therefore the analysis (Fig. 7b) of thesame four sulfonamides is performed at 0.4 mL/min and908C (bottom chromatogram) and compared with thesame analysis at 0.2 mL/min and 308C (top chromato-gram). Under those conditions complete elution wasobserved in 2.5 min, versus 17 min at the lower tempera-ture and flow rate.

3.5 Column stability

Column stability and performance is an essential ele-ment of HT-LC. Hypercarb column performance wasmonitored by running the same test mixture both whenthe column was new and again after the column hadbeen used under high temperature conditions (up to2008C) for a period of six weeks. During this period,mobile phases used included water/methanol, water/acetonitrile and water/acetonitrile with 0.1% formic acidmixtures, with a total volume of approximately 6000 col-umn volumes. In Fig. 8 the two chromatograms obtainedare compared. Retention times are approximately thesame and the efficiency of the last eluting peak (3,5-xyle-nol) decreases by less than 10%.

4 Concluding remarks

The effects of separation temperature on retention,speed of analysis, mobile phase composition, and sensi-tivity in LC–MS with ESI and APCI were investigated,using a temperature stable stationary phase of porousgraphitic carbon. It was observed that an increase in thetemperature results in a decrease in the retention timesand that the relationship between capacity factor (log k)and absolute temperature (1/T) is quadratic between 30and 1808C. This non-linear relationship is indicative of achange in retention mechanism as temperature isincreased. When using high temperatures, six-fold gainsin speed of analysis were observed, and when solvent gra-dients are replaced with temperature gradients analysis

cycle time can be further improved due to faster systemre-equilibration after each run.

The potential of using 100% aqueous mobile phases inRP-LC and controlling elution strength by changing tem-perature was demonstrated with the separation of polarcompounds (purines and pyrimidines). This is a lessexpensive and more environmentally friendly way ofdoing LC.

The use of high separation temperatures in LC–MS hasan impact not only on the analysis time, but also on thesensitivity of the method. The signal-to-noise ratios inLC–MS were measured at temperatures between 30 and1808C and improvements of 1.7-fold for ESI at 908C and 4-fold for APCI at 1808C were observed. Temperatureshigher than 908C produce an increase in ESI signal butalso in noise, which may actually decrease S/N.

The availability of columns that are stable at high tem-peratures as well as specialised column ovens enables theuse of temperature as another method development toolin order to develop fast, sensitive, and economical meth-ods.

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Figure 8. Hypercarb column stability under high temperatureconditions. Before and after 6 weeks of use at high tempera-tures. Column: Hypercarb 5 lm, 10064.6 mm; mobilephase: MeOH/H2O (95:5, v/v); flow rate: 0.8 mL/min; detec-tion: UV @ 254 nm; analytes: 1) acetone; 2) phenol; 3) p-cre-sol; 4) 3,5-xylenol.


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High temperature to increase throughput in liquid chromatography and liquid chromatography–mass spectrometry with a porous graphitic carbon stationary phase