New Frontiers in Chromatographyby Michael E. Swartz and Brian Murphy

High-performance liquid chromatogra-phy (HPLC) has proven to be the pre-dominant technology used in laborato-ries worldwide during the past 30-plusyears. One of the primary drivers for thegrowth of this technique has been theevolution of packing materials used toeffect the separation. The underlyingprinciples of this evolution are governedby the van Deemter equation, withwhich any student of chromatography isintimately familiar.1 The van Deemterequation is an empirical formula thatdescribes the relationship between lin-ear velocity (flow rate) and plate height(HETP or column efficiency). Sinceparticle size is one of the variables, a vanDeemter curve can be used to investi-gate chromatographic performance.

As illustrated in Figure 1, as the parti-cle size decreases to less than 2.5 µm,not only is there a significant gain inefficiency, but the efficiency does notdiminish at increased flow rates or lin-ear velocities. By using smaller parti-cles, speed and peak capacity (numberof peaks resolved per unit time) can beextended to new limits, termed UltraPerformance Liquid Chromatographyor UPLC™ (Waters Corp., Milford,MA). Using UPLC, it is possible totake full advantage of chromato-graphic principles to run separationsusing shorter columns and/or higherflow rates for increased speed withsuperior resolution and sensitivity. Fig-ures 2 and 3 further illustrate UPLC inaction. With UPLC, compromises areno longer necessary; in Figure 2 a sepa-ration of eight diuretics is accom-plished in under 1.6 min. The sameseparation on a 2.1 × 100 mm, 5-µmC18 HPLC column yields almost iden-tical resolution, but takes 10 min. Forsome analyses, however, speed is ofsecondary importance; peak capacityand resolution take center stage. Fig-ure 3 shows a peptide map in whichthe desired goal is to maximize thenumber of peaks. In this application,the increased peak capacity (numberof peaks resolved per unit time) ofUPLC dramatically improves the qual-

ity of the data, resulting ina more definitive map.

Chemistry of smallparticlesThe design and develop-ment of sub-2-µm particlesis a significant challenge,and researchers have beenactive in this area for sometime to capitalize on theiradvantages.2,3 Althoughhigh-efficiency, nonporous1.5-µm particles are com-mercially available, they suf-fer from poor loading capac-ity and retention due to lowsurface area. Silica-based particles havegood mechanical strength, but can sufferfrom a number of disadvantages, whichinclude a limited pH range and tailing ofbasic analytes. Polymeric columns canovercome pH limitations, but they havetheir own issues, including low efficien-cies and limited capacities.

XTerra® (Waters Corp.), a first-generation hybrid chemistry that tookadvantage of the best of both the silica

and polymeric column worlds, wasintroduced in 2000. XTerra columnsare mechanically strong, with highefficiency, and operate over anextended pH range. They are pro-duced using a classical sol-gel synthesisthat incorporates carbon in the form ofmethyl groups. In order to provide thekind of enhanced mechanical stabilityUPLC required, however, a second-generation bridged ethane hybrid(BEH) technology was developed:

Figure 1 van Deemter plot illustrating the evolution of particle sizes over the last three decades.

Figure 2 UPLC separation of eight diuretics. Column:2.1 × 30 mm, 1.7-µm ACQUITY UPLC BEH C18 at 35°C. A 9–45%B linear gradient over 0.8 min, at a flow rate of0.86 mL/min, was used. Mobile phase A was 0.1% formicacid; B was acetonitrile. UV detection was at 273 nm. Peaksare in order: acetazolamide, hydrochlorothiazide, impurity,hydroflumethiazide, clopamide, trichlormethiazide, inda-pamide, bendroflumethiazide, and spironolactone; 0.1 mg/mLeach in water.

Reprinted from American Laboratory February 2005

ACQUITY BEH (Waters Corp.).ACQUITY BEH 1.7-µm particlesderive their enhanced mechanical sta-bility by bridging the methyl groups inthe silica matrix, as shown in Figure 4.

Packing a 1.7-µm particle in repro-ducible and rugged columns was also achallenge that needed to be overcome.Requirements include a smoother inte-rior surface of the column hardware andredesigning the end frits to retain thesmall particles and resist clogging.Packed-bed uniformity is also critical,especially if shorter columns are tomaintain resolution while accomplish-ing the goal of faster separations. AllACQUITY BEH columns also includeeCord™ microchip technology, whichcaptures the manufacturing informationfor each column, including the qualitycontrol tests and Certificates of Analy-sis. When used in the ACQUITYUPLC™ System (Waters Corp.), theeCord database can also be updatedwith real-time method information suchas the number of injections, or withpressure information, to maintain acomplete column history.

Capitalizing on smallerparticlesInstrument technology also had to keeppace to truly take advantage of the

increased speed, superior resolution, andsensitivity afforded by smaller particles.Standard HPLC technology simply doesnot have the horsepower to take fulladvantage of sub-2-µm particles. A newsystem design with advanced technol-ogy in the pump, autosampler, detector,data system, and service diagnostics wasrequired. The ACQUITY UPLC Sys-tem has been holistically designed forlow system and dwell volume to take fulladvantage of low-dispersion and small-particle technology.

Achieving small-particle, high-peak-capacity separations requires a greaterpressure range than that achievable bytoday’s HPLC instrumentation. Thecalculated pressure drop at the opti-mum flow rate for maximum efficiencyacross a 15-cm-long column packedwith 1.7-µm particles is approx. 15,000psi. Therefore, a pump capable ofdelivering solvent smoothly and repro-ducibly at these pressures that cancompensate for solvent compressibilityand operate in both the gradient andisocratic separation modes is required.

Sample introduction is also critical.Conventional injection valves, eitherautomated or manual, are notdesigned and hardened to work atextreme pressure. To protect the col-umn from experiencing extreme pres-

Figure 3 Synthesis and chemistry of ACQUITY BEH 1.7-µm particles for UPLC.

sure fluctuations, the injection processmust be relatively pulse free. Theswept volume of the device also needsto be minimal to reduce potentialband spreading. A fast injection cycletime is needed to fully capitalize onthe speed afforded by UPLC, which inturn requires a high sample capacity.Low-volume injections with minimalcarryover are also required to realizethe increased sensitivity benefits.

With 1.7-µm particles, half-heightpeak widths of less than 1 sec areobtained, posing significant challengesfor the detector. In order to accuratelyand reproducibly integrate an analytepeak, the detector sampling rate mustbe high enough to capture enoughdata points across the peak. In addi-

Figure 4 HPLC vs UPLC peak capacity. In this gradient peptide map separa-tion, the HPLC separation (top) (on a 5-µm C18 column) yields 70 peaks, or apeak capacity of 143, while the UPLC separation (bottom) run under identicalconditions yields 168 peaks, or a peak capacity of 360, a 2.5× increase.

Figure 5 ACQUITY UPLC System.

tion, the detector cell must have mini-mal dispersion (volume) to preserveseparation efficiency. Conceptually,the sensitivity increase for UPLCdetection should be 2–3 times higherthan HPLC separations, depending onthe detection technique. MS detec-tion is significantly enhanced byUPLC; increased peak concentrationswith reduced chromatographic disper-sion at lower flow rates (no flow split-ting) promote increased source ioniza-tion efficiencies.

Shown in Figure 5, the ACQUITYUPLC System consists of a binary sol-vent manager, sample manager (includ-ing the column heater), detector, andoptional sample organizer. The binarysolvent manager uses two individualserial flow pumps to deliver a parallelbinary gradient. There are built-in sol-vent select valves that let the userchoose from up to four solvents. Thereis a 15,000-psi pressure limit (approx.1000 bar) to take advantage of the sub-2-µm particle in the linear velocity perthe van Deemter curve. The samplemanager also incorporates several tech-nology advancements. Low dispersionis maintained through the injectionprocess using pressure assist sampleintroduction, and a series of pressuretransducers facilitate self monitoring

and diagnostics. Needle-in-needle sam-pling improves ruggedness, and a nee-dle calibration sensor increases accu-racy. Injection cycle time is 25 secwithout a wash and 60 sec with a dualwash used to further decrease carryover.A variety of multiple-well plate formats(deep-well, mid-height, or vials) canalso be accommodated in a thermostati-cally controlled environment. Usingthe optional sample organizer, the sam-ple manager can inject from up to 22multiple-well plates. The sample man-ager also controls the column heater.Column temperatures up to 65 °C canbe attained. A “pivot out” design pro-vides versatility to allow the columnoutlet to be placed in closer proximityto the source inlet of an MS detector tominimize sample dispersion.

The ACQUITY UPLC Tunable UV(TUV) Detector (Waters Corp.)includes new electronics and firmwareto support Ethernet communications atthe high data rates necessary for UPLCdetection. Conventional absorbance-based optical detectors are concentra-tion sensitive; for UPLC use, the flowcell volume would have to be reduced instandard UV-VIS detectors to maintainconcentration and signal. Smaller-vol-

ume conventional flow cells would alsoreduce the pathlength upon which thesignal strength depends (recall Beer’sLaw). Worse, a reduction in cross-sec-tion means the light path is reduced andtransmission drops, increasing noise.Therefore, if a conventional HPLC flowcell is used, UPLC sensitivity would becompromised. The ACQUITY UPLCTUV detector cell consists of a light-guided flow cell equivalent to an opticalfiber. Light is efficiently transferreddown the flow cell in an internalreflectance mode that still maintains a10-mm flow cell pathlength with a vol-ume of only 500 nL. Tubing and con-nections in the system are efficientlyrouted to maintain low dispersion andto take advantage of leak detectors thatinteract with the software to alert theuser to potential problems.

ApplicationsChromatographers are used to mak-ing compromises, and one of themost common scenarios involves sac-rificing resolution for speed. In addi-tion, for complex samples such asnatural product extracts, added reso-lution can provide more informationin the form of additional peaks.

Figure 6 Comparison of HPLC and UPLC for the separation of aginger root extract. HPLC conditions—Column: 2.1 × 100 mm, 5.0-µmprototype BEH C18 at 28 °C. A 25–96%B linear gradient over 10 min,at a flow rate of 1.0 mL/min, was used. Mobile phase A was water; B wasacetonitrile. UV detection was at 230 nm, 10-µL injection. UPLC condi-tions—column: 2.1 × 100 mm, 1.7-µm ACQUITY BEH C18 at 28 °C.A 50–100%B linear gradient from 1.4 to 3.7 min followed by a hold until6.0 min, at a flow rate of 0.3 mL/min, was used. Mobile phase A waswater; B was acetonitrile. UV detection was at 230 nm, 5-µL injection.

Figure 7 UPLC separation of seven coumarins illustrating fast methoddevelopment. Column: 2.1 × 30 mm, 1.7-µm ACQUITY UPLC BEHC18 at 35 °C. A 20–40%B linear gradient over 1.0 min, at a flow rate of0.86 mL/min, was used. Mobile phase A was 0.1% formic acid; B was ace-tonitrile. UV detection was at 254 nm and 40 pts/sec. Peaks are in order: 7-hydroxycoumarin-glucuronide, 7-hydroxycoumarin, 4-hydroxycoumarin,coumarin, 7-methoxycoumarin, 7-ethoxycoumarin, and 4-ethoxycoumarin.

under 1 hr, including UPLC scoutingruns for gradient optimization andindividual runs for elution order iden-tification. These runs were performedin a fraction of the time that would benecessary with conventional HPLC,resulting in significant time savings inthe method development laboratory.

ConclusionAt a time when many scientists havereached separation barriers pushing thelimits of conventional HPLC, UPLCpresents the possibility to extend andexpand the utility of this widely usedseparation science. New ACQUITYUPLC technology in chemistry andinstrumentation provides more infor-mation per unit of work as UPLCbegins to fulfill the promise of increasedspeed, resolution, and sensitivity pre-dicted for liquid chromatography.

References1. van Deemter JJ, Zuiderweg FJ, Klinken-

berg A. Chem Eng Sci 1956; 5:271.2. Jerkovitch AD, Mellors JS, Jorgenson JW.

LC·GC North America 2003; 21:7.3. Wu N, Lippert JA, Lee MA. J Chromo-

togr 2001; 911:1.

Dr. Swartz is Principal Consulting Scientist, andMr. Murphy is Manager, Corporate Communica-tions, Waters Corp., 34 Maple St., Milford, MA01757, U.S.A.; tel.: 508-482-2742; fax: 508-482-3085; e-mail: michael_swartz@waters.com.The authors would like to acknowledge the contri-butions of the ACQUITY program team atWaters, particularly Eric Grumbach, PatMcDonald, Michael Jones, and Marianna Kele,for their contributions to this manuscript.

Figure 6 shows an HPLC versusUPLC separation comparison of aginger root extract sample whereboth speed and resolution areimproved, as well as an increase insensitivity. DryLab software (Rheo-dyne, Rohnert Park, CA) was used tomodel and redevelop the separationand transfer it to the ACQUITYUPLC System and BEH chemistry.

Faster separations can lead to higherthroughput and time savings whenrunning multiple samples. However, asignificant amount of time can also beconsumed in developing the methodin the first place. Faster, higher-resolution UPLC separations can cutmethod development time from daysto hours or even minutes. Figure 7 isan example of a UPLC separation ofseveral closely related coumarins anda metabolite that was developed in

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