As published in LPI- June 2004 •

During the past 30 or so years, high perform-

ance liquid chromatography (HPLC) has

proven to be the predominant technology

used in laboratories worldwide. One of the

primary drivers for the growth of this tech-

nique has been the evolution of the packing

materials used to effect the separation. The

underlying principles of this evolution are

governed by the van Deemter equation, which

any student of chromatography is familiar

with [1]. The van Deemter equation is an

empirical formula that describes the relation-

ship between linear velocity (flow rate) and

plate height (HETP or column efficiency).

Since particle size is one of the variables, a van

Deemter curve can be used to investigate

chromatographic performance, as illustrated

in Figure 1.

Figure 1 demonstrates that as the particle size

decreases to less than 2.5 µm, not only is there

a significant gain in efficiency, but the effi-

ciency does not diminish at increased flow

rates or linear velocities. By using smaller par-

ticles, speed and peak capacity (number of

peaks resolved per unit time) can be extended

to new limits, termed Ultra Performance

Liquid Chromatography, or UPLC. Using

UPLC, it is now possible to take full advantage

of chromatographic principles to run separa-

tions using shorter columns, and/or higher

flow rates for increased speed, with superior

resolution and sensitivity. UPLC is further

illustrated in Figure 2, which compares the

separation of a five component sample mix-

ture using HPLC and UPLC.

Chemistry of small particlesThe design and development of

sub-2 µm particles is a significant chal-

lenge, and researchers have been very active

in this area to capitalise on their advantages

[2, 3]. Although high efficiency non-porous

1.5 µm particles are commercially available,

they suffer from low surface area, leading to

poor loading capacity and retention. To

maintain retention and capacity similar to

HPLC, UPLC must use a novel porous par-

ticle that can withstand high pressures.

Silica based particles have good mechanical

strength, but suffer from a number of dis-

advantages. These include tailing of basic

analytes and a limited pH range. Another

alternative, polymeric columns, can over-

come pH limitations, but they have their

own issues, including low efficiencies and

limited capacities.

In 2000, Waters introduced a first generation

hybrid chemistry, called XTerra, which com-

bines the advantageous properties of both sil-

ica and polymeric columns - they are

mechanically strong, with high efficiency, and

operate over an extended pH range. XTerra

columns are produced using a classical sol-gel

Ultra performance liquid chromatography:tomorrow's HPLC technology today

In recent years, significant technological advances havebeen made in particle chemistry performance, systemoptimisation, detector design, and data processing andcontrol. When brought together, the individual achieve-ments in each discipline have created a step-functionimprovement in chromatographic performance. Ultraperformance liquid chromatography (UPLC) is a newcategory of analytical separation science that retainsthe practicality and principles of HPLC, while increasingthe overall interlaced attributes of speed, sensitivity andresolution.

by Dr. Michael E. Swartz and Brian J. Murphy

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

NOVEL PHASES FOR HPLC

As published in LPI- June 2004 •

synthesis that incorporates carbon in the

form of methyl groups. However, in order to

provide the kind of enhanced mechanical sta-

bility UPLC requires, a second generation

hybrid technology was developed [4], called

ACQUITY UPLC. ACQUITY 1.7 µm particles

bridge the methyl groups in the silica matrix,

as shown in Figure 3, which enhances their

mechanical stability.

Packing a 1.7µm particle in reproducible and

rugged columns was also a challenge that

needed to be overcome. The column hardware

required a smoother interior surface and the

end frits were re-designed to retain the small

particles and resist clogging. Packed bed uni-

formity is also critical, especially if shorter

columns are to maintain resolution while

accomplishing the goal of faster separations.

All ACQUITY columns also include the eCord

microchip technology that captures the man-

ufacturing information for each column,

including the quality control tests and certifi-

cates of analysis. When used in the Waters

ACQUITY UPLC system, the eCord database

can also be updated with real time method

information, such as the number of injec-

tions, or pressure information, to maintain a

complete column history.

Capitalising on smaller particlesTo truly take advantage of the increased

speed, superior resolution and sensitivity

afforded by small particles, instrument tech-

nology also had to keep pace. A completely

new system design with advanced technology

in the pump, autosampler, detector, data sys-

tem, and service diagnostics was required. The

ACQUITY UPLC system has been holistically

designed for low system and dwell volume to

take full advantage of low dispersion and

small particle technology.

Achieving small particle, high peak capacity

separations requires a greater pressure range

than that achievable by today's HPLC instru-

mentation. The calculated pressure drop at

the optimum flow rate for maximum efficien-

cy across a 15 cm long column packed with

1.7 µm particles is about 15,000 psi. Therefore

a pump capable of delivering solvent smooth-

ly and reproducibly at these pressures, which

can compensate for solvent compressibility

and operate in both the gradient and isocrat-

ic separation modes, is required.

Sample introduction is also critical.

Conventional injection valves, either auto-

mated or manual, are not designed and hard-

ened to work at extreme pressure. To protect

the column from experiencing extreme pres-

sure fluctuations, the injection process must

be relatively pulse-free. The swept volume of

the device also needs to be minimal to reduce

potential band spreading. A fast injection

cycle time is needed to fully capitalise on the

speed afforded by UPLC, which in turn

requires a high sample capacity. Low volume

injections with minimal carryover are also

required to realise the increased sensitivity

benefits.

With 1.7 µm particles, half-height peak

widths of less than one second are obtained,

posing significant challenges for the detector.

In order to accurately and reproducibly inte-

grate an analyte peak, the detector sampling

rate must be high enough to capture enough

data points across the peak. In addition, the

detector cell must have minimal dispersion

(volume) to preserve separation efficiency.

Conceptually, the sensitivity increase for

UPLC detection should be 2-3 times higher

than HPLC separations, depending on the

detection technique. MS detection is signifi-

cantly enhanced by UPLC; increased peak

concentrations with reduced chromatograph-

ic dispersion at lower flow rates (no flow split-

ting) promotes increased source ionisation

efficiencies.

Figure 2. The top chromatogram is an overlay of both conventional (3 µm) HPLC and1.7 µm UPLC for a five component sample mixture. The bottom is an expansion of thefirst 0.6 minutes of the overlay to show the increased speed of UPLC, while resolution isstill maintained. Solvent use is also greatly reduced with UPLC.

NOVEL PHASES FOR HPLC

Figure 3. Synthesis and chemistry ofACQUITY 1.7µm particles for UPLC.

As published in LPI- June 2004 •

ApplicationsChromatographers are used to making compromises, and one of the

most common scenarios involves sacrificing resolution for speed. UPLC

overcomes such problems, as illustrated in Figure 4, which shows a sep-

aration of eight diuretics in under 1.6 minutes. The same separation on

a 2.1 by 100mm 5µm C18 HPLC column yields almost identical resolu-

tion, but takes ten minutes. For some analyses, however, speed is of a sec-

ondary importance, and peak capacity and resolution are the priority.

Figure 5 shows a peptide map where the desired goal is to maximise the

number of peaks. In this application, the increased peak capacity (num-

ber of peaks resolved per unit time) of UPLC dramatically improves the

quality of the data, resulting in a more definitive map.

ConclusionAt a time when any scientists have reached separation barriers pushing

the limits of conventional HPLC, UPLC presents the possibility to

extend and expand the utility of this widely used separation science.

The new ACQUITY technology in chemistry and instrumentation

provides more information per unit of work as UPLC begins to fulfill

the promise of increased speed, resolution and sensitivity predicted for

liquid chromatography.

References

1. van Deemter JJ, Zuiderweg FJ, and Klinkenberg A. Chem. Eng. Sci.

1956; 5: 271.

2. Jerkovitch AD, Mellors JS, and Jorgenson JW. LCGC North America.

2003; 21: 7.

3. Wu N, Lippert JA, and Lee ML. J. Chromotogr. 2001; 911: 1.

4. Wyndham KD, O’Gara JE, et al. Anal. Chem. 2003; 75: 6781-6788.

The authors

Michael E. Swartz, Ph.D., Principal Consulting Scientist,

Brian J. Murphy, Manager, Corporate Communications

Waters Corporation

34 Maple Street

Milford, MA 01757 USA

Tel.: +1 508 482 2614

Fax: +1 508 482 3443

Email: brian_j_murphy@waters.com

Figure 4. UPLC separation of eight diuretics. Column: 2.1 by 30mm 1.7 µm ACQUITY UPLC C18 at 35°C. A 9-45% B linear gra-dient over 0.8 minutes, at a flow rate of 0.86 mL/min was used.Mobile phase A was 0.1% formic acid, B was acetonitrile. UVdetection at 273nm. Peaks are in order: acetazolamide,hydrochlorothiazide, impurity, hydroflumethiazide, clopamide,trichlormethiazide, indapamide, bendroflumethiazide, andspironolactone, 0.1mg/mL of each in water.

Figure 5. HPLC v’s UPLC peak capacity. In this gradient peptidemap separation, the HPLC (top) separation (on a 5µm C18 col-umn) yields 70 peaks, or a peak capacity of 143, while the UPLCseparation (bottom) run under identical conditions yields 168peaks, or a peak capacity of 360, a 2.5 x increase.

NOVEL PHASES FOR HPLC