Accepted Manuscript

Title: Current and future trends in UHPLC

Author: Szabolcs Fekete, Julie Schappler, Jean-Luc Veuthey, Davy Guillarme

PII: S0165-9936(14)00200-3

DOI: http://dx.doi.org/doi: 10.1016/j.trac.2014.08.007

Reference: TRAC 14320

To appear in: Trends in Analytical Chemistry

Please cite this article as: Szabolcs Fekete, Julie Schappler, Jean-Luc Veuthey, Davy Guillarme,

Current and future trends in UHPLC, Trends in Analytical Chemistry (2014), http://dx.doi.org/doi:

10.1016/j.trac.2014.08.007.

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1

Current and future trends in UHPLC

Szabolcs Fekete, Julie Schappler, Jean-Luc Veuthey, Davy Guillarme * School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d’Yvoy 20, 1211

Geneva 4, Switzerland

HIGHLIGHTS

Ultra-high-performance liquid chromatography UHPLC is the standard LC platform

Columns packed with sub-2µm particles come for chiral-LC, SEC, IEX, HILIC and SFC

Detailed characterization of biopharmaceuticals is an important UHPLC application

Particle-size reduction and system-pressure increase are technologically difficult

Superficially porous particles directly improve the kinetic performance of UHPLC

ABSTRACT

Since its commercial introduction in 2004, there has been a considerable interest in ultra-high-

performance (pressure) liquid chromatography (UHPLC), which dramatically increases the

throughput of regular HPLC methods. Although the ability to achieve fast separations and

good resolution are the main drivers for the increasing use of UHPLC, we describe a number

of other trends in this review, such as:

(1) use of UHPLC technology to perform high-resolution analysis of complex samples;

(2) development of columns packed with sub-2-µm particles to achieve different

chromatographic modes (i.e. chiral LC, SEC, IEX, HILIC, and SFC);

(3) evaluation of higher pressure drop, higher temperature, smaller particle sizes and sub-2-

µm core-shell particles, to further improve kinetic performance;

(4) use of UHPLC for enhancing the characterization of biopharmaceuticals; and,

(5) development of UHPLC-MS for applications, including bioanalysis, multi-residue

screening, and metabolomics.

Keywords:

Bioanalysis

Biopharmaceutical

Fast analysis

Fully porous particle

High-resolution analysis

HILIC

Metabolomics

Superficially porous particle

UHPLC

UHPLC-MS

* Corresponding author. Tel.: +41 22 379 34 63; Fax: +41 22 379 68 08.

E-mail address: davy.guillarme@unige.ch (D. Guillarme)

1. Introduction

The term UHPLC was originally coined by Jorgenson in 1997 and stands for ultra-high-

pressure liquid chromatography. This group was the first to describe the use of nano-columns

packed with non-porous 1.0–1.5-µm silica-based particles on a prototype system compatible

with very high pressure {up to 4100 bar in 1997 [1] and 7200 bar in 2003 [2]}. Next to the

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work of Jorgenson, Lee et al. also confirmed the potential of UHPLC for pressures up to 3600

bar [3]. Based on these proofs of concept, the first chromatographic system compatible with a

pressure of 1000 bar was commercialized by Waters in 2004 under the trademark ultra-

performance liquid chromatography (UPLC), together with narrow-bore columns packed with

1.7-µm fully porous particles (FPPs) [4,5]. The term UHPLC is preferred for the name of the

technology and stands for “ultra-high-performance liquid chromatography” or “ultra-high-

pressure liquid chromatography”. As an alternative, the term very high-pressure liquid

chromatography (VHPLC) has been reported in a few papers.

In the past 10 years, most of the chromatographic providers (at least 10 suppliers) have

commercialized their own UHPLC systems (more than 20 different systems) compatible with

pressures in the range 600–1400 bar [6]. The design of numerous detector devices improved

and all of them can be coupled to UHPLC, including ultraviolet (UV), UV-diode-array

detector (UV-DAD), evaporative light-scattering detector (ELSD), corona aerosol-discharge

(CAD), refractive index (RI), fluorescence detector (FD) and mass spectrometry (MS).

The number of columns packed with fully porous sub-2-µm particles has also grown, with

more than 100 supports and >10 different phase chemistries accessible from at least 15

providers. All these stationary phases are not equivalent in terms of particle size (1.5–2 µm),

pressure tolerance (600–1400 bar), and pH and temperature ranges. As a consequence of these

important technological achievements, the number of applications has grown exponentially,

as shown in Fig. 1.

In the early days of its commercialization, UHPLC was mostly used to achieve fast

separations. The use of a UHPLC column of 50 x 2.1 mm I.D., 1.7 µm instead of a regular

HPLC column of 150 x 4.6 mm I.D., 5 µm, enables a reduction in analysis time by a factor 9,

with equivalent kinetic performance [7]. This high-throughput feature is attractive in

environmental, food, and chemical analysis, where the productivity has to be enhanced, due to

the large number of samples. Another driving force for fast separations is the pharmaceutical

field. Enhanced productivity and reduced costs are particularly needed during the drug-

discovery and development process, in applications, such as quality control,

pharmacokinetics, and drug metabolism. In addition to high-speed separations, there are

several other benefits related to the use of UHPLC.

The goal of this review is to discuss the current and future trends of this technique,

illustrated with various examples.

2. Technical constraints and recent evolution of UHPLC instrumentation

Several instrumental characteristics need to be considered for successful operation of

UHPLC, as outlined in Fig. 2:

(i) system upper pressure limit and flow rate capability;

(ii) type of column temperature-control module [8];

(iii) instrument dead volume and associated extra-column band broadening;

(iv) gradient delay volume (also named dwell volume); and,

(v) data-acquisition rate of the detector [6].

For several years, the system upper pressure limit was considered the most important

attribute of UHPLC systems. The first generation of instruments provided pressure limits

fixed at 1000 bar, up to 1 mL/min. When increasing the flow rate to 2 mL/min, the pressure

was linearly decreased from 1000 bar to 600 bar. Today, providers offer powerful dedicated

UHPLC systems with an extended pressure range (up to 1200–1400 bar) and a flow rate in the

upper range of 2–5 mL/min. However, it is important to consider that a reasonable amount of

system pressure is consumed through use of narrow connecting tubing. As a low-cost

alternative, HPLC/UHPLC hybrid systems can be found and are able to operate at pressures

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and flow rates of up to 600–800 bar and 5 mL/min, respectively [6]. Regarding stationary

phases, the number of UHPLC columns compatible with pressures of up to 1000 bar is

continually increasing, although the choice is limited above 1200 bar.

Another important, and often underestimated, feature of UHPLC systems is the instrument

dead volume and associated extra-column dispersion (σ²ext). This source of peak broadening is

particularly critical using narrow bore and short columns (e.g., 50 x 2.1 mm I.D.) [9]. In order

to maintain reasonable band broadening, the tubing dimensions of UHPLC instruments were

optimized (shortest tubing of 0.005-inch I.D.) and zero dead-volume fittings employed.

UHPLC autosamplers allowing fast, repeatable injection volumes of only 0.1–2 µL were also

developed. Finally, the UV detector cell volumes were minimized (Vcell 0.5–2 µL) without

altering sensitivity (due to a path length remaining equal to 10 mm). With these improved

characteristics, the dispersion obtained with UHPLC systems is 6–30 µL², while it is equal to

70–200 µL² with regular HPLC devices [6,10]. The recently launched Waters Acquity I-Class

system, plumbed with 0.003-inch I.D. tubing (generating an elevated residual system

pressure) and mounted with an active mobile-phase pre-heater, offers system dispersion of

about 2 µL², rendering the system potentially compatible with 1-mm I.D. UHPLC columns

[11]. To reduce system dead volume further, it would be of interest to have a UHPLC system

where it would be possible to attach the column inlet directly at the injection valve and the

outlet at the detector, without the need for any connection tubing.

The gradient dwell volume, which represents the volume from the mixing point of solvents

to the head of the analytical column, also needs to be taken into account, particularly with fast

UHPLC gradient analysis [12]. Because of this gradient dwell volume, the sample is

subjected to an undesired additional isocratic migration under the initial mobile-phase

composition. This increases the time required to elute the compounds of interest from the

column and reduces the throughput. In addition, it generates problems for transfer to other

instruments. To maintain a reasonable dwell volume (Vd 100–300 µL), the first generations of

UHPLC instruments were exclusively commercialized with high-pressure mixing systems

(where each solvent is delivered to the mixer by a dedicated pump). To further reduce dwell

volume while preserving appropriate mixing of the mobile phases and to reduce background

noise on the baseline, microfluidic mixing devices were developed on at least two high-

pressure mixing instruments, namely the Shimadzu Nexera and the Agilent 1290 Infinity.

With these systems, the volume of the mixing chamber was reduced down to only 20 µL and

35 µL, respectively. In parallel with the improvement in high-pressure mixing instruments, all

UHPLC providers developed low-pressure mixing systems (where the mobile-phase

components are blended before they reach the pump). In this configuration, only one pump is

required, reducing the cost and the maintenance needed. It also offers more flexibility for

method development (with the possibility of working with ternary and quaternary mobile

phases), but the dwell volume is larger (300–900 µL), reducing the throughput [6]. Today, the

number of UHPLC models with high-pressure devices is equivalent to those with low-

pressure mixing devices.

A strong commercial argument in the early days of UHPLC was the high data-acquisition

rate of UV detectors. Due to the narrow peaks produced by state-of-the-art stationary phases

packed with sub-2-µm particles (down to only 1 s in some cases), the need for fast detectors

in UHPLC has emerged rapidly, to get at least 10–15 acquisition points per peak for

acceptable quantitative performance [13]. The latest generations of spectroscopic detectors

(UV, UV-DAD, and FD) available for UHPLC systems offer data-acquisition rates of 80–200

Hz, which is more than sufficient for any type of application. The achievement of high

acquisition rates with MS devices is more difficult and only the latest generation of

instruments meets these requirements [14]. Today, the fastest triple-quadrupole device

(Shimadzu LC-MS 8050) is able to perform one selected reaction monitoring (SRM)

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experiment in less than 1 ms. The fastest QqTOF/MS instruments (AB Sciex TripleTOF

5600+) is able to acquire m/z ratios in a wide range in only 10 ms with a resolution of ~40,000

(full width at half maximum, FWHM).

Because the stationary phases employed in UHPLC are increasingly efficient, extra-

column dispersion of UHPLC instruments has to be further reduced (below 1 µL²) to limit

efficiency loss. Also, the data-acquisition rate of the slowest detectors still needs to be

increased while maintaining reasonable sensitivity. As example, only a limited number of MS

devices are sufficiently fast for proper UHPLC operation.

3. Future trends of UHPLC instrumentation and columns

Below, we critically discuss various technical solutions that can be implemented to

improve the current performance of UHPLC technology, such as:

(1) increase the system-pressure drop beyond 1400 bar;

(2) elevation of the mobile-phase temperature (>60°C);

(3) reduction of the particle size (<1.5 µm) or modification of the particle-size distribution;

and,

(4) use of sub-2 µm superficially porous particles (SPPs).

According to kinetic plot methodology, it is possible to calculate the time required to

achieve a given plate count or the efficiency that can be reached for a given analysis time, at a

fixed pressure drop. As example, an efficiency of 10,000 plates can be attained for a column

packed with fully porous 1.7-µm particles in 1.9 min at a pressure of 400 bar, while it requires

only 0.7 min and 0.6 min at 1000 bar and 1200 bar, respectively [6]. In other words, analysis

time is inversely proportional to the ΔPmax, and elevated system pressure may be beneficial for

increasing throughput and maximizing kinetic performance, provided that the deleterious

effects of frictional heating remain reasonable [4].

Similarly, for high-resolution separations (i.e., 100,000 plates), analysis time can be

reduced by three-fold between systems possessing ΔPmax of 400 bar and 1200 bar. In this case,

it corresponds to a significant reduction in analysis time, from around 3 h to 1 h, using

columns packed with 1.7-µm particles [6]. These observations confirm that working with a

chromatographic system possessing an elevated upper pressure limit is also relevant for high-

resolution analysis.

To improve further the kinetic performance of current UHPLC technology, it could be

beneficial to have a system able to work at pressure beyond the limit of 1200–1400 bar.

However, such a system may be difficult to build, and pumping and injector technologies able

to work under such extreme conditions are still not available. In addition, and as discussed in

the next section, frictional heating may become detrimental at pressures beyond 1400 bar, and

micro or capillary columns would certainly be required instead of narrow-bore columns [15].

Increasing mobile-phase temperature is an attractive way to improve kinetic performance

in UHPLC, as demonstrated elsewhere [16]. At elevated temperatures (>60°C), the mobile-

phase viscosity is reduced (up to 13 times between 20°C and 180°C for a 60:40 MeOH-water

mixture), leading to increased analyte-diffusion coefficients and low column-pressure drop

[17]. Consequently, the flow rate can be increased to improve throughput, or the column

length can be extended to increase efficiency and resolution. Although the performance

improvement is very important using temperatures in the range 150–200°C, they may be

extremely critical for stability of the sample and the column [18], and the UV cell. A

temperature of 60–100°C appears a good compromise in UHPLC, as it is still beneficial in

terms of kinetic performance without issues related to compound and column degradation

[16]. However, the diversity of stationary phases compatible with such a temperature range is

still limited, so new column chemistries are needed before this temperature range can be

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routinely and reliably used. At elevated temperature, the mobile phase should also be

preheated before it enters the column, involving a passive or active preheating device added

between the injection valve and the column inlet. Active preheater technology is much more

beneficial since the required tubing length for efficient preheating is greatly reduced and

limits peak dispersion.

It is well known in LC that chromatographic efficiency is inversely proportional to particle

diameter. The particles currently employed in the UHPLC technology are 1.5–2.0 µm. If the

particle diameter was reduced below 1.5 µm, the kinetic performance could be significantly

improved. However, the price to pay would be high, since the backpressure is inversely

proportional to the square of the particle size, and even to the cube of the particle size at the

optimal linear velocity. In other words, the pressure drop increases much faster than the

chromatographic efficiency. As example, between columns packed with 1.7 µm and 1.0 µm

particles, the expected efficiency improvement is ~70%, while the backpressure is five-fold

higher under optimal flow-rate conditions. Again, the upper pressure limit of current UHPLC

instrumentation needs to be greatly increased to expect some benefits from particle-size

reduction. Modification of the particle-size distribution is another way to improve kinetic

performance in UHPLC, with bimodal or narrow particle-size distribution, or absence of fine

particles [19,20]. However, it was recently demonstrated using simulations that these

modifications were not always beneficial [21].

The morphology of the sub-2-µm FPPs is another characteristic that can be modified to

improve performance in UHPLC further. SPPs (also named fused-core, or core-shell) are

particles made of a solid, non-porous core surrounded by a shell of a porous material, and

they possess properties similar to those of the fully porous materials employed in HPLC and

UHPLC. Due to some significant improvements of eddy diffusion (A term of the van Deemter

equation), moderate decrease of longitudinal diffusion (B term of the Van Deemter equation),

and limited improvement of mass-transfer resistance (C term of the van Deemter equation), it

has been experimentally demonstrated that chromatographic efficiency could be increased by

~30–50% between columns packed with FPPs and SPPs of the same dimensions [22–24],

while the permeability and the associated pressure drop remain similar between both

technologies. The sub-2-µm SPP columns were recently evaluated [25–28] and it was

demonstrated that the 1.6-µm SPP phase was the most promising one for current UHPLC

instrumentation {for a column of 50 x 2.1 mm I.D., 1.6 µm provides about 19,000 plates

[25]}, taking into account the upper pressure limit of the system and the permeability of the

stationary phase. Columns packed with 1.3-µm SPPs were very powerful (a column of 50 x

2.1 mm I.D., 1.3 µm provides about 25,000 plates) but it was difficult to work under optimal

conditions due to their low permeability and the strong contribution of the UHPLC system to

band broadening [26]. As example, Fig. 3 shows the separations of a cashew-nut extract

performed with columns packed with SPPs of different sizes (2.6 µm, 1.7 µm and 1.3 µm).

Due to its important benefits, SPPs will probably tend to replace FPPs progressively in

UHPLC, while waiting for the UHPLC upper pressure to be increased and the system

dispersion to be reduced. Today, there are only two providers that offer columns packed with

sub-2-µm SPPs, but there will certainly be substantial growth in the near future.

4. UHPLC for very high-resolution analysis

It has been theoretically demonstrated that the use of columns packed with sub-2-µm

particles under very high pressure is not the best approach to reach ultra-high efficiency

(>100,000 plates) without considering any time limit. However, highly permeable columns

(packed with 5-µm particles) of longer length can achieve higher efficiencies due to the lower

pressure imposition from their larger particle size and the lower optimum linear velocity (it is

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important to keep in mind that a plate count beyond 100,000 is often not relevant for real-

world applications). This statement is graphically demonstrated in Fig. 4 using the kinetic plot

methodology, which allows visualization of the speed-efficiency limits for a given

chromatographic condition [29,30]. For intermediate efficiency (in the range 20,000–

100,000), it is obvious that long columns packed with sub-2-µm particles and very high-

pressure conditions represent the best strategy. It is predictable that efficiency in isocratic

mode can be increased by three-fold in UHPLC (1.7-µm particles) versus HPLC (5-µm

particles) using identical column lengths and similar analysis times. The calculations in

gradient mode are much more complex but UHPLC has proved to be a viable strategy to

reach high peak capacity and high resolution [31,32]. For these reasons, UHPLC was

employed in recent years to attain high efficiency and high resolution, and up to 35,000 plates

can be reached with a UHPLC column of 150 mm. Some separations involving longer

UHPLC columns were also reported in the literature for profiling complex natural-product

extracts or for analyzing tryptic digests of protein mixtures [33,34], although optimal flow-

rate conditions were difficult to attain, due to the important backpressure generated by long

columns packed with sub-2-µm particles.

In order to allow compatibility of long UHPLC columns with existing instruments, the

mobile-phase temperature can be increased to 60–90°C [35]. Under such conditions, the

mobile-phase viscosity is reduced, as does the pressure drop. The excellent performance of

UHPLC using long columns and elevated temperature (up to 90°C) was demonstrated for

small molecules and peptides [36]. A maximum peak capacity (number of peaks theoretically

separated with a resolution of 1) of ~1100 was achieved in about 100 min [36].

To improve the resolving power of UHPLC further, beyond a peak capacity of 1000,

comprehensive on-line two-dimensional LC (LC x LC) set-ups can be used. If the goal is to

achieve ultra-high peak capacity within a reasonable analysis time (3 h maximum), an ultra-

fast gradient run can be employed in the second dimension involving the use of UHPLC

columns at elevated temperature (90°C). Based on a recent study [37], it appears that an

RPLC x RPLC system, with C18 material in both dimensions, was the most attractive

combination. Surprisingly, the HILIC x RPLC combination was found to be less interesting,

due to the incompatibility of the dissolution solvent between the two dimensions. The highest

degree of orthogonality was achieved in RPLC x RPLC using ammonium acetate at pH 6.8

and formic acid at pH 2.7 in the first and second dimensions, respectively, providing a pH

modification relevant for ionizable compounds or peptides. The practical peak capacity was

very elevated (>4000), due to the use of a short UHPLC column under extreme conditions

(ultra-fast gradient, highest possible flow rate, pressure, and temperature) in the second

dimension. Fig. 5 shows the current limits of 1D-LC for the separation of peptides and

demonstrates the potential of 2D-LC, using UHPLC technology in the second dimension [38].

From these results, it is clear that UHPLC is a promising strategy for improving the

performance of comprehensive LC and will certainly be increasingly used in the near future.

However, quantitation and data treatment are still the main bottlenecks of comprehensive LC

and need to be tackled before such a strategy becomes widespread.

5. Potential solutions to limit frictional heating in UHPLC

As discussed above, the use of columns packed with very small particles at high mobile-

phase velocities generates very high pressures. Consequently, the friction of the mobile phase

percolating through the column bed induces heat. This phenomenon is known as frictional

heating and may be detrimental to separation performance. It was largely described in

preliminary works of Jorgenson [39] and Lee [3], and, since the commercial introduction of

UHPLC, there have been more studies performed by Guiochon and Sandra [40–42].

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The generated heat dissipates across and along the chromatographic column, inducing the

formation of radial and longitudinal temperature gradients, respectively. The temperature

variations between the inlet and the outlet of the column at a fixed pressure of 1000 bar were

experimentally determined in the range 13–20°C and 3–16°C for various lengths of 2.1-mm

and 1-mm I.D. columns, respectively. The radial gradients are mostly observed under

isothermal conditions (i.e., the column is placed in a forced-air oven or in a thermostatted

water bath) and impact column efficiency. The longitudinal gradients occur when the column-

wall temperature is not kept constant (adiabatic case, i.e., the column is placed in a still-air

oven). They impact retention and selectivity due to changes in the average column

temperature, while the effect on column efficiency is negligible. This behavior is illustrated in

Fig. 6, showing the impact of mobile-phase temperature (Fig. 6A) and system pressure (Fig.

6B) on retention and selectivity of a chromatographic separation. The first series was

performed at a constant pressure of 100 bar as temperature increased between 30°C and 50°C.

The second series of chromatograms was realized at a constant temperature of 30°C as

pressure increased between 100 bar and 1000 bar. This study confirmed that the pressure

increase has the same effect on chromatographic retention and selectivity as a temperature

increase. In order to limit the frictional heating phenomenon, various solutions can be

envisaged.

The first solution involves reducing the inner diameter of the column since the dissipation

power is directly proportional to the mobile-phase flow rate. This is why Jorgenson employed

nano-columns of 30–50-µm I.D. exclusively to perform experiments at pressures in the range

4000–7000 bar [1–3]. However, UHPLC instrumentations is currently incompatible with

column I.D. less than 1 mm, due to the strong contribution of extra-column variance to peak

broadening, but it could be possible to employ commercial nano-UHPLC systems also

compatible with pressures up to 1000 bar. In addition, it is important to keep in mind that

columns of 0.5–1 mm I.D. are more difficult to pack than narrow-bore or capillary columns,

due to wall effects [43,44].

The reduction of the pressure drop inside the column could be considered to alleviate

frictional heating effects, since the dissipation power is also directly proportional to the

pressure drop. However, with the goal to improve further kinetic performance in UHPLC, it is

not advisable to reduce the pressure drop, particularly when using columns packed with sub-

2-µm FPPs (except if interstitial porosity can be slightly increased, which might be a good

point to minimize thermal effects).

The coupling of several short columns (instead of a single long column) is another

approach described in the literature to reduce frictional heating, since the effect of

longitudinal temperature gradient decreases as the number of coupled columns increases.

With this set-up, active cooling is needed to dissipate the viscous heat on the capillaries

connecting the short columns [45]. The primary trade-off of this approach is the substantial

cost increase related to the use of multiple columns. Also, this solution cannot be

implemented if the original separation is performed on a 50-mm column length because

shorter columns packed with sub-2-µm particles are still not available.

The nature of the solid inner core of sub-2-µm SPPs can also be modified to reduce

frictional heating. The use of high heat-conductivity materials, such as alumina (thermal

conductivity, 40 W/m/K) or gold (thermal conductivity, 320 W/m/K) dramatically reduces the

influence of the heat effects on column efficiency [46]. With these materials, the amplitude of

the radial temperature gradients decreases and the heat dissipates, as for columns made of

silica-based materials, but at a much faster rate, due to the larger thermal conductivities

compared to neat silica (1.4 W/m/K). This promising solution is compatible with only SPP

technology, and currently appears technologically difficult and expensive.

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Finally, because the type of oven directly influences the effects of the frictional heating on

chromatographic behavior, it should be theoretically possible to develop a UHPLC oven

allowing a reduction or even elimination of frictional heating effects. Such an oven is not yet

available, but the idea would be to insulate the column perfectly, to limit as much as possible

the longitudinal and radial temperature gradients.

6. Towards better integration of UHPLC technology in the pharmaceutical

regulatory environment (QC laboratories)

Due to the obvious benefits of the UHPLC technology (high-throughput separation and

improved resolution), methods can be successfully developed with adequate performance.

The high-throughput feature of UHPLC allows fast screening of stationary phases, mobile-

phase pH, and organic modifiers, finally to select the best combination for a given mixture of

compounds. It was recently demonstrated that 24 generic gradient conditions, including four

short UHPLC columns, three pH values, and two organic modifiers, can be screened in a few

hours, with the help of software (e.g., Fusion) able to interact with the UHPLC instrument.

The software automatically creates the sample set and all UHPLC methods. It also evaluates

the resulting chromatograms and counts the number of separated peaks, allowing easy

selection of the best combination of conditions [47]. Once the best combination of column,

pH and organic modifier is selected, the method can easily be improved by properly adjusting

the gradient profile and the mobile-phase temperature. Again, this task can be easily and

quickly performed using well-known HPLC modelling software packages (e.g., DryLab,

Chromsword, or Osiris). The robustness of the final method can also be assessed in an

automated way [48]. If the resolution remains inadequate after proper adjustment of retention

and selectivity, the chromatographic efficiency can finally be enhanced by extending the

UHPLC column length to meet the expected critical resolution criteria. Fig. 7 shows this

generic method-development procedure.

In the last stage, the UHPLC method has to be validated according to existing guidelines

and protocols (evaluation of trueness, intermediate precision, and accuracy) [49]. Numerous

examples of successful UHPLC method validation were published in recent years [50,51],

demonstrating that this technology can be considered mature for routine use in QC

laboratories. Despite these recognized achievements, UHPLC remains rarely used in routine

QC laboratories, mostly because they are not equipped with this recent technology, and also

because there is no need for a QC laboratory to have UHPLC equipment if there are no

UHPLC methods approved for the products that it is releasing.

After a relatively fast method development and validation under UHPLC conditions, the

optimal method is frequently transferred back to HPLC conditions, considering similar

stationary-phase chemistry, pH, organic modifier, and temperature. In this case, the gradient

profile, mobile-phase flow rate, and injected volume have to be adjusted by applying

geometrical transfer rules and taking into account the difference in dwell volumes between

UHPLC and HPLC instruments [52,53]. Because the development and the validation of a

method under UHPLC conditions and its routine use under HPLC conditions are not allowed

by the European Pharmacopeia (EP) and the United States Pharmacopeia (USP), the

transferred method has to be validated again under HPLC conditions. This additional step can

be circumvented if the method presents the same kinetic performance (plate count or peak

capacity) within the ranges of adjustments provided by EP or USP. These ranges are very

restrictive [54], and they are clearly unsuitable for a transfer between UHPLC and HPLC (too

many changes in column dimensions).

In conclusion, analysts face an issue caused by the lack of flexibility of EP and USP in the

framework of method transfer from UHPLC to HPLC and HPLC to UHPLC. Rather than

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giving fixed limits for the modification of chromatographic parameters, a possible solution,

suggested by Neue et al. [55], could be a performance-based approach. In this case, the same

or better performance (identical or higher ratio L/dp, which defines the performance of a

column) should not require a complete method revalidation.

7. Extension of UHPLC technology to alternative chromatographic modes

For several years, columns packed with sub-2-µm particles were employed under RPLC

conditions exclusively. Today, there is a trend towards use of this technology in other modes

of separation, including:

(1) chiral separation;

(2) size-exclusion chromatography (SEC);

(3) ion-exchange chromatography (IEX);

(4) hydrophilic interaction chromatography (HILIC); and,

(5) supercritical fluid chromatography (SFC).

Chiral separations are often performed on columns packed with 5-µm particles, but particle

size can be reduced to improve kinetic performance, similarly to RPLC. Some providers have

commercialized chiral stationary phases (CSPs) packed with 3-µm particles, allowing benefits

in terms of throughput and efficiency [56]. Gasparrini et al. recently developed prototypes of

brush-type Pirkle CSPs with particles of 4.3 µm, 2.6 µm and 1.9 µm. Analysis times of 15–40

s were attained for the separation of various enantiomeric pairs with sub-2-µm particles [57].

Today, only one company is offering sub-2-μm coated polysaccharide CSPs [58], but this

market is expected to grow in the future. It was demonstrated that chiral separations could be

performed under achiral RP-UHPLC conditions after a chemical derivatization, avoiding the

need for a dedicated CSP [59].

In SEC, the analysis time is mostly determined by the mobile-phase flow rate and the

column dead volume, since all analytes of interest are eluted before the total void time of the

column. To shorten the separation time in SEC, the ratio of the column-void volume to the

flow rate needs to be decreased. Then, reduction of the column size and increase of the flow

rate are straightforward ways to perform fast SEC analysis, even if the plate count is also

reduced under these conditions. The most common column dimensions in SEC are 30 cm

column length, 4.6–8-mm I.D., 5–10-µm particles [60]. These provide an analysis time of

generally 15–50 min.

Recently, a new sub-2-µm SEC material was introduced, enabling very fast SEC

separations [61]. As expected, the SEC column packed with 1.7-µm particles offered 2–4-fold

faster separation than columns packed with 5-µm or 3-µm particles, although the very high

pressure drop generated by small particles caused artefacts (on-column generation of

additional aggregates) circumventing the quantitation of protein aggregates [62].

Today, the main application of IEX is the separation of protein variants with non-porous

particles. Because IEX is a kinetically slow process compared to partitioning [63], the use of

small particle sizes should improve mass transfer [64]. As the longitudinal diffusion is

negligible with large molecules, only the Eddy’s dispersion and mass-transfer processes

contribute to band broadening. However, because non-porous materials are conventionally

used in IEX, mass transfer is negligible, so the kinetic performance is not dramatically

improved with the reduction of the particle size, explaining why there are only a few IEX

phases packed with sub-2-µm particles.

HILIC has gained importance for the analysis of hydrophilic and/or ionizable compounds.

It is characterized by a polar stationary phase and a mixture of aqueous buffer and organic

aprotic solvent (generally acetonitrile in high percentage, > 60%) commonly used as mobile

phase [65]. The HILIC retention mechanism is multimodal and involves hydrophilic

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partitioning, adsorption, and ion exchange [66]. Columns packed with sub-2-µm particles are

available for HILIC operation, in the form of bare silica or silica bonded with various polar

groups [67]. As previously discussed, the main limitation of the RP-UHPLC approach relates

to the sigbnificant column pressure drop that could limit accessible throughput or resolution

and generate frictional heating. The pressure drop under HILIC conditions is more reasonable,

due to the low viscosity of the mobile phase containing a high proportion of acetonitrile [i.e.,

60–95% (v/v)], so the main limitations of UHPLC technology are less critical in HILIC

conditions. Numerous papers have demonstrated that HILIC-UHPLC was a powerful strategy

[68–70]. The range of stationary-phase chemistries available for HILIC-UHPLC should

therefore be extended in the future.

SFC is a well-known technique that recently regained strong interest in it, due to the

commercialization of a new generation of instruments, with improved performance, reliability

and robustness [71]. These systems are largely based on the recent developments in UHPLC

instruments, including reduced system dispersion and higher upper pressure limits. They also

exhibit good compatibility with modern column dimensions (short, narrow-bore columns

packed with sub-2-µm particles). As demonstrated in a recent study, high kinetic performance

(>20,000 plates for 100-mm column length) obtained with sub-2-µm particles in supercritical

conditions are comparable to those achieved under UHPLC on similar columns [72]. In

addition to its excellent kinetic performance, SFC can be considered a green analytical

strategy and the versatility of SFC in terms of analyzed compounds opens up numerous fields

of application {e.g., this approach was successfully employed for the analysis of relatively

polar and ionizable compounds [73,74], but also for lipids and liposoluble vitamins [75]}. We

expect that, in the near future, an increasing number of laboratories will be equipped with

modern SFC instruments, able to work with columns packed with sub-2-µm particles.

Surprisingly, there is no paper dealing with the possibility of performing NPLC with

columns packed with sub-2-µm particles. This field will certainly be covered in the near

future, since NPLC columns with polar moieties are available and various suppliers offer

dedicated pumping kits able to deal with NPLC solvents.

8. UHPLC for the characterization of biopharmaceuticals

Nowadays, there is a trend towards the production of new therapeutic drugs from

biological sources (i.e., biological systems or biological molecules). These products of

pharmaceutical biotechnology are known as biopharmaceuticals and include recombinant

peptides, proteins, monoclonal antibodies (mAbs), and antibody-drug conjugates (ADCs)

[76]. In 2004, only 1-in-10 drugs that generated the highest revenue was a biopharmaceutical.

In 2010, this proportion increased to 7-in-10, confirming the trend in the pharmaceutical

market.

Because the development of biopharmaceuticals is quite complex, regulatory authorities

such as the FDA (Food and Drug Administration) and EMA (European Medicines Agency)

require demonstration of the drug-substance characterization (e.g., verifying primary structure

and appropriate post-translational modifications), lot-to-lot and batch-to-batch comparisons,

stability studies, impurity profiling, and glycoprofiling [77]. For this purpose, a single

analytical technique is not sufficient, and a variety of spectrophotometric, chromatographic,

electrophoretic, and MS methods have to be employed to describe fully such a complex

sample. Chromatography plays a pivotal role in the characterization of biopharmaceuticals

because of its high speed, elevated resolution power, significant sensitivity and

reproducibility. In addition to the historical SEC or IEX reference techniques, the interest in

RPLC is growing since it can be easily coupled to MS.

The analysis of large biomolecules in RPLC involves numerous issues, such as:

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(1) protein adsorption;

(2) secondary interactions;

(3) protein-diffusion coefficients; and,

(4) poor kinetic performance [78].

We therefore recommend using a stationary phase with limited access to residual silanols

and a sufficiently large pore size. Because the diffusion coefficient of large biomolecules is

relatively small, UHPLC columns packed with sub-2-µm particles should be considered to

reduce mass-transfer resistance, in addition to the non-negligible gain in efficiency (inversely

proportional to the particle-size reduction).

Wide-pore C3, C4, C8, and C18 stationary phases are now available from several providers

and successful separations of very large molecules were reported, as illustrated in Fig. 8 for

the separation of intact IgG2 of 150 kDa or mAb fragments of 25–50 kDa [79]. The mobile-

phase conditions should also be optimized and contain an ion-pairing reagent to limit

secondary ionic interactions (e.g., 0.1% TFA). The temperature should be set in the range 60–

90°C to improve peak shape, reduce adsorption, and further improve kinetic performance.

In 2008, Everley et al. were the first to employ a combination of columns packed with sub-

2-µm particles at very high-pressure, high temperatures (up to 65°C), and a strong organic

modifier (i.e., isopropanol) to enhance resolution and sensitivity, and throughput (about three-

fold) [80]. A large number of works are currently studying the characterization of

biopharmaceuticals under UHPLC conditions [81,82] and this number will certainly continue

to grow in the near future.

9. Recent developments in UHPLC-MS

The coupling of LC and MS is considered the gold standard for many applications. As

soon as UHPLC technology was commercialized, the first attempts were made to combine it

with several types of MS analyzer.

As discussed in Section 2, the primary constraint of fast-LC approaches is the detector

data-acquisition rate. This is particularly true for MS detector devices where a compromise is

often made between speed and sensitivity. Highly sensitive, fast MS instruments were not

available until MS providers integrated into their development that chromatographic peaks

were thinner in UHPLC technology. They now provide MS instruments, such as time-of-

flight (TOF) and quadrupole-based devices, which can work very rapidly without

compromising sensitivity. Only very high-resolution MS devices, such as Orbitrap and

Fourier-transform ion-cyclotron resonance MS (FT-ICR/MS) are rarely combined with

UHPLC technology, due to their slow acquisition rate [83].

A recent innovation that could also be applied to UHPLC-MS is the commercialization of a

simple MS detector built around the needs of chromatographers. The single-quadrupole

analyzer has the size of an optical detector and can be used without any special training or

expertise [84]. This type of detector will probably contribute to the expansion of MS in

laboratories with strong chromatographic activity, as it can enhance the analytical value and

productivity of each analysis.

Today, most UHPLC-MS applications (>60% of the published papers, as shown in Fig. 1)

are in the fields of bioanalysis, drug-metabolism studies, multi-residue screening,

metabolomics, and protein characterization, as reported in recent reviews [13,14,83,85]. We

report below two relevant applications in the doping and metabolomics fields to emphasize

how UHPLC-MS is a recognized reference technique to increase throughput and resolution.

Doping-control laboratories mostly employ LC-MS methods for routine, high-throughput

screening and confirmation assays. UHPLC-MS with low-MS resolution is particularly

beneficial due to its high throughput. The possibility to shorten analysis time is valuable due

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to the increasing demands for sample turn-around, but also the ability to separate more

analytes efficiently within commonly accepted chromatographic run times [86].

Metabolomics is a biological approach, which involves analyzing and modeling the metabolic

response to biological interventions and disease processes [87]. This field has rapidly

expanded, despite the complexity of the approach that requires comprehensive identification

and quantitation of all metabolites in a complex biological system. Hence, there is a need for

analytical systems with very high resolution and increased sensitivity for separation and

detection, such as those achieved with UHPLC-TOF/MS and UHPLC-QqTOF/MS platforms.

Recently, a UHPLC-MS protocol for the global metabolic profiling of urine was proposed by

Want et al. [88]. In addition to throughput and resolution benefits, the UHPLC strategy was

found more beneficial than conventional HPLC, with improved retention time, reproducibility

and signal-to-noise ratios.

10. Conclusions

UHPLC instrumentation has become the standard LC platform adopted by most

manufacturers and practitioners. As illustrated in this review, UHPLC technology has been

applied successfully since its commercial introduction in 2004 to a wide range of samples and

conditions, in combination with spectroscopy and MS. The main advantage of UHPLC is the

possibility to achieve ultra-fast and/or high-resolution separations, with reduced solvent

consumption, using columns packed with sub-2-µm particles and chromatographic systems

compatible with pressures up to 1400 bar. Because the benefits of using small particles can be

extended to chromatographic techniques other than RPLC, there is a trend towards using

UHPLC technology in several modes, including chiral LC, SEC, IEX, HILIC, and SFC.

Among the numerous application fields of UHPLC, the detailed characterization of

biopharmaceuticals appears of prime importance, since these molecules represent the driving

force of the pharmaceutical market.

Various directions are possible for UHPLC developments. The reduction of particle size

and the increase in the system-pressure limit are technologically difficult, due to safety

problems, packing issues, and frictional heating effects, but they will probably be proposed by

providers. However, the use of SPPs instead of sub-2-µm FPPs is certainly a straightforward

way to improve kinetic performance of UHPLC and will probably continue its expansion.

References

[1] J.E. MacNair, K.C. Lewis, J.W. Jorgenson, Ultrahigh-Pressure Reversed-Phase Liquid Chromatography in Packed Capillary Columns, Anal. Chem. 69 (1997) 983-989. [2] A.D. Jerkovich, J.S. Mellors, J.W. Jorgenson, The use of micron-sized particles in ultrahigh-pressure liquid chromatography, LC-GC Europe 16 (2003), 20–23. [3] J.A. Lippert, B. Xin, N. Wu, M.L. Lee, Fast ultrahigh-pressure liquid chromatography: On-column UV and time-of-flight mass spectrometric detection, J. Microcol. Sep. 11 (1999) 631-643. [4] J.R. Mazzeo, U.D. Neue, M. Kele, R.S. Plumb, Advancing LC Performance with Smaller Particles and Higher Pressure, Anal. Chem. 77 (2005) 460A-467A. [5] R.E. Majors, Fast and Ultrafast HPLC on Sub-2-µm Porous Particles — Where Do We Go from Here?, LCGC North Am. 23 (2005) 1248-1255. [6] S. Fekete, I. Kohler, S. Rudaz, D. Guillarme, Importance of instrumentation for fast liquid chromatography in pharmaceutical analysis, J. Pharm. Biomed. Anal. 87 (2014) 105-119. [7] D.T.T. Nguyen, D. Guillarme, S. Rudaz, J.L. Veuthey, Fast analysis in liquid chromatography using small particle size and high pressure, J. Sep. Sci. 29 (2006) 1836-1848.

Page 12 of 17

13

[8] M. M. Fallas, M. R. Hadley, D. V. McCalley, Practical assessment of frictional heating effects and thermostat design on the performance of conventional (3 lm and 5 μm) columns in reversed-phase high-performance liquid chromatography, J. Chromatogr. A, 1216 (2009) 3961-3969. [9] F. Gritti, C.A. Sanchez, T. Farkas, G. Guiochon, Achieving the full performance of highly efficient columns by optimizing conventional benchmark high-performance liquid chromatography instruments, J. Chromatogr. A 1217 (2010) 3000-3012. [10] S. Fekete, J. Fekete, The impact of extra-column band broadening on the chromatographic efficiency of 5 cm long narrow-bore very efficient columns, J. Chromatogr. A 1218 (2011) 5286-5291. [11] S. Fekete, J.L. Veuthey, D.V. McCalley, D. Guillarme, The effect of pressure and mobile phase velocity on the retention properties of small analytes and large biomolecules in ultra-high pressure liquid chromatography, J. Chromatogr. A 1270 (2012) 127-138. [12] J.W. Dolan, Dwell volume revisited, LCGC North Am. 24 (2006) 458-466. [13] D. Guillarme, J. Schappler, S. Rudaz, J.L. Veuthey, Coupling ultra-high-pressure liquid chromatography with mass spectrometry, Trends Anal. Chem. 29 (2010) 15-27. [14] M. Rodriguez-Aller, R. Gurny, J.L. Veuthey, D. Guillarme, Coupling ultra high-pressure liquid chromatography with mass spectrometry: Constraints and possible applications, J. Chromatogr. A 1292 (2013) 2-18. [15] N. Wu, J.A. Lippert, M.L. Lee, Practical aspects of ultrahigh pressure capillary liquid chromatography, J. Chromatogr. A 911 (2001) 1-12. [16] D.T.T. Nguyen, D. Guillarme, S. Heinisch, M.P. Barrioulet, J.L. Rocca, S. Rudaz, J.L. Veuthey, High throughput liquid chromatography with sub-2µm particles at high pressure and high temperature, J. Chromatogr. A 1167 (2007) 76-84. [17] S. Heinisch, J.L. Rocca, Sense and nonsense of high-temperature liquid chromatography, J. Chromatogr. A 1216 (2009) 642-658. [18] H.G. Gika, G. Theodoridis, J. Extance, A.M. Edge, I.D. Wilson, High temperature-ultra performance liquid chromatography–mass spectrometry for the metabonomic analysis of Zucker rat urine, J. Chromatogr. B 871 (2008) 279-287. [19] J. Billen, D. Guillarme, S. Rudaz, J.L. Veuthey, H. Ritchie, B. Grady, G. Desmet, Relation between the particle size distribution and the kinetic performance of packed columns: Application to a commercial sub-2µm particle material, J. Chromatogr. A 1161 (2007) 224-233. [20] http://www.sigmaaldrich.com/analytical-chromatography/hplc/columns/titan/reporter-titan-intro.html - consulted in February 13, 2014. n o o ltzel, S. Khirevich, U. Tallarek, Influence of the Particle Size Distribution on Hydraulic Permeability and Eddy Dispersion in Bulk Packings, Anal Chem. 83 (2011) 3903-3910. [22] D.V. McCalley, Some practical comparisons of the efficiency and overloading behaviour of sub-2 μm porous nd sub-3 μm sh ll p rticl s in r v rs d-phase liquid chromatography, J. Chromatogr. A 1218 (2011) 2887-2897. [23] J. Ruta, D. Zurlino, C. Grivel, S. Heinisch, J.L. Veuthey, D. Guillarme, Evaluation of columns packed with shell particles with compounds of pharmaceutical interest, J. Chromatogr. A 1228 (2012) 221-231. [24] S. Fekete, J. Fekete, K. Ganzler, Shell and small particles; Evaluation of new column technology, J. Pharm. Biomed. Anal. 49 (2009) 64-71. [25] B. Bobaly, D. Guillarme, S. Fekete, Systematic comparison of a new generation of columns packed with sub-2 µm superficially porous particles, J. Sep. Sci. 37 (2014) 189-197. [26] A.C. Sanchez, G. Friedlander, S. Fekete, J. Anspach, D. Guillarme, M. Chitty, T. Farkas, Pushing the performance limits of reversed-phase ultra high performance liquid chromatography with 1.3 μm cor –shell particles, J. Chromatogr. A 1311 (2013) 90-97. [27] S. Fekete, D. Guillarme, Kinetic evaluation of new generation of column packed with 1.3 µm core–shell particles, J. Chromatogr. A 1308 (2013) 104-113. [28] S. Fekete, D. Guillarme, Possibilities of new generation columns packed with 1.3 µm core–shell particles in gradient elution mode, J. Chromatogr. A 1320 (2013) 86-95.

Page 13 of 17

14

[29] G. Desmet, P. Gzil, D. Clicq, Kinetic Plots to Directly Compare the Performance of LC Supports, LCGC Eur. 18 (2005) 403-409. [30] G. Desmet, Comparison techniques for HPLC column performance, LCGC North Am. 21 (2008) 310-320. [31] D. Guillarme, E. Grata, G. Glauser, J.L. Wolfender, J.L. Veuthey, S. Rudaz, Some solutions to obtain very efficient separations in isocratic and gradient modes using small particles size and ultra-high pressure, J. Chromatogr. A 1216 (2009) 3232-3243. [32] Y. Zhang, X. Wang, P. Mukherjee, P. Petersson, Critical comparison of performances of superficially porous particles and sub-2μm p rticl s und r optimized ultra-high pressure conditions, J. Chromatogr. A 1216 (2009) 4597-4605. [33] E. Grata, D. Guillarme, G. Glauser, J. Boccard, P.A. Carrupt, J.L. Veuthey, S. Rudaz, J.L. Wolfender, Metabolite profiling of plant extracts by ultra-high-pressure liquid chromatography at elevated temperature coupled to time-of-flight mass spectrometry, J. Chromatogr. A 1216 (2009) 5660-5668. [34] E. Grata, J. Boccard, D. Guillarme, G. Glauser, P.A. Carrupt, E.E. Farmer, J.L. Wolfender, S. Rudaz, UPLC–TOF-MS for plant metabolomics: A sequential approach for wound marker analysis in Arabidopsis thaliana, J. Chromatogr. B 871 (2008) 261-270. [35] J. Ruta, D. Guillarme, S. Rudaz, J.L. Veuthey, Comparison of columns packed with porous sub-2 µm particles and superficially porous sub-3 µm particles for peptide analysis at ambient and high temperature, J. Sep. Sci. 33 (2010) 2465-2477. [36] P.J. Eugster, D. Biass, D. Guillarme, P. Favreau, R. Stöcklin, J.L. Wolfender, Peak capacity optimisation for high resolution peptide profiling in complex mixtures by liquid chromatography coupled to time-of-flight mass spectrometry: Application to the Conus consors cone snail venom, J. Chromatogr. A 1259 (2012) 187-199. [37] ’ ttom C Griv l S inisch On-line comprehensive two-dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part I: Orthogonality and practical peak capacity considerations, J. Chromatogr. A 1262 (2012) 148-159. [38] ’ ttom S inisch On-line comprehensive two dimensional separations of charged compounds using reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography. Part II: Application to the separation of peptides, J. Chromatogr. A, 1306 (2013) 27-36. [39] J.E. MacNair, K.D. Patel, J.W. Jorgenson, Ultrahigh-Pressure Reversed-Phase Capillary Liquid Chromatography:  Isocratic and Gradient Elution Using Columns Packed with 1.0-μm Particles, Anal. Chem. 71 (1999) 700-708. [40] M. M. Fallas, U. D. Neue, M. R. Hadley, D. V. McCalley, Further investigations of the effect of pressure on retention in ultra-high-pressure liquid chromatography, J. Chromatogr. A, 1217 (2010) 276-284. [41] A. de Villiers, H. Lauer, R. Szucs, S. Goodall, P. Sandra, Influence of frictional heating on temperature gradients in ultra-high-pressure liquid chromatography on 2.1 mm I.D. columns, J. Chromatogr. A 1113 (2006) 84-91. [42] F. Gritti, G. Guiochon, Effects of the thermal heterogeneity of the column on chromatographic results, J. Chromatogr. A 1131 (2006) 151-165. [43] F. Gritti, G. Guiochon, Theoretical and experimental impact of the bed aspect ratio on the axial dispersion coefficient of columns packed with 2.5 μm p rticl s, J. Chromatogr. A, 1262 (2012) 107- 121. [44] F. Gritti, G. Guiochon, Kinetic investigation of the relationship between the efficiency of columns and their diameter, J. Chromatogr. A 1218 (2011) 1592-1602. [45] K. Broeckhoven, J. Billen, M. Verstraeten, K. Choikhet, M. Dittmann, G. Rozing, G. Desmet, Towards a solution for viscous heating in ultra-high pressure liquid chromatography using intermediate cooling, J. Chromatogr. A 1217 (2010) 2022-2031. [46] F. Gritti, G. Guiochon, The current revolution in column technology: How it began, where is it going?, J. Chromatogr. A 1228 (2012) 2-19.

Comment [DG1]: Add reference

Page 14 of 17

15

[47] B. Debrus, D. Guillarme, S. Rudaz, Improved quality-by-design compliant methodology for method development in reversed-phase liquid chromatography, J. Pharm. Biomed. Anal. 84 (2013) 215-223. [48] R. Kormány, I. Molnár J. Fekete, D. Guillarme, S. Fekete, Robust UHPLC Separation Method Development for Multi-API Product Amlodipine and Bisoprolol: The Impact of Column Selection, Chromatographia (2014) DOI 10.1007/s10337-014-2633-9. [49] R. Kormány, J. Fekete, D. Guillarme, S. Fekete, Reliability of simulated robustness testing in fast liquid chromatography, using state-of-the-art column technology, instrumentation and modelling software, J. Pharm. Biomed. Anal. 89 (2014) 67-75. [50] I. Baranowska, S. Magiera, Development and validation of a UHPLC method for the determination of flavonoids in red wine, J. AOAC Int., 94 (2011) 786-794 [51] P. Paval, K. Petr, N. Tomas, K. Jiri, Development and validation of rapid UHPLC method for determination of acyclovir, its impurities and preservatives in topical cream, Anal. Meth., 5 (2013) 2610-2614. [52] D. Guillarme, D.T.T. Nguyen, S. Rudaz, J.L. Veuthey, Method transfer for fast liquid chromatography in pharmaceutical analysis: Application to short columns packed with small particle. Part I: Isocratic separation, Eur. J. Pharm. Biopharm. 66 (2007) 475-482. [53] D. Guillarme, D.T.T. Nguyen, S. Rudaz, J.L. Veuthey, Method transfer for fast liquid chromatography in pharmaceutical analysis: Application to short columns packed with small particle. Part II: Gradient experiments, Eur. J. Pharm. Biopharm. 68 (2008) 430-440. [54] B. Debrus, E. Rozet, P. Hubert, J.L. Veuthey, S. Rudaz, D. Guillarme, in: D. Guillarme, J.L. Veuthey (Eds.), UHPLC in Life Sciences, The Royal Society of Chemistry, Cambridge, UK, 2012, p. 67. [55] U.D. Neue, D. McCabe, V. Ramesh, H. Pappa, J. DeMuth, Transfer of HPLC Procedures to Suitable Columns of Reduced Dimensions and Particle Sizes, Pharm. Forum, 35 (2009) 1622-1626. [56] T. Zhang, P. Franco, 3 µm particle-based chiral stationary phases for the fast and efficient resolution of enantiomers, LC-GC Eur. 21 (2008) 430-437. [57] G C nc lli r Ciogli I ’ cqu ric F G sp rrini J Koc rgin Misiti M Pi rini H. Ritchie, P. Simone, C. Villani, Transition from enantioselective high performance to ultra-high performance liquid chromatography: A case study of a brush-type chiral stationary phase based on sub-5-micron to sub-2-micron silica particles, J. Chromatogr. A 1217 (2010) 990-999. [58] C. Hamman, M. Wong, I. Aliagas, D.F. Ortwine, J. Pease, D.E. Schmidt Jr., J. Victorino, The evaluation of 25 chiral stationary phases and the utilization of sub-2.0 μm co t d polysaccharide chiral stationary phases via supercritical fluid chromatography, J. Chromatogr. A 1305 (2013) 310-319. [59] D. Guillarme, G. Bonvin, F. Badoud, J. Schappler, S. Rudaz, J.L. Veuthey, Fast Chiral Separation of Drugs Using Columns Packed with Sub-2 µm Particles and Ultra-High Pressure, Chirality 22 (2010) 320-330. [60] H.G. Barth, G.D. Saunders, The State of the Art and Future Trends of Size-Exclusion Chromatography Packings and Columns, LCGC North Am. 30 (2012) 544-563. [61] S.M. Koza, P. Hong, K.J. Fountain, Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and their Aggregates, J. Liq. Chromatogr. Rel. Tech., 35 (2012) 2923. [62] S. Fekete, K. Ganzler, D. Guillarme, Critical evaluation of fast size exclusion chromatographic separations of protein aggregates, applying sub-2 µm particles, J. Pharm. Biomed. Anal. 78-79 (2013) 141-149. [63] P. H. Roos, Chapter 1 Ion Exchange Chromatography, In: Michael Kastner, Editor(s), Journal of Chromatography Library, Elsevier, 1999, Volume 61, Pages 3-88. [64] http://www.crawfordscientific.com/downloads/Application-Notes/Agilent_BioHPLC_Columns_5990-5195EN.pdf - consulted in February 13, 2014. [65] D.V. McCalley, U.D. Neue, Estimation of the extent of the water-rich layer associated with the silica surface in hydrophilic interaction chromatography, J. Chromatogr. A 1192 (2008) 225-229.

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[66] B. Buszewski, S. Noga, Hydrophilic interaction liquid chromatography (HILIC) - a powerful separation technique, Anal. Bioanal. Chem. 402 (2012) 231-247. [67] A. Periat, I. Kohler, J.L. Veuthey, D. Guillarme, Advances in hydrophilic interaction liquid chromatography for pharmaceutical analysis, LCGC Eur. 26 (2013) 17-23. [68] B. Chauve, D. Guillarme, P. Cléon, J.L. Veuthey, Evaluation of various HILIC materials for the fast separation of polar compounds, J. Sep. Sci. 33 (2010) 752-764. [69] A. Periat, B. Debrus, S. Rudaz, D. Guillarme, Screening of the most relevant parameters for method development in ultra-high performance hydrophilic interaction chromatography, J. Chromatogr.A 1282 (2013) 72-83. [70] L. Nováková, I. Kaufmannová, R. Jánská, Evaluation of hybrid hydrophilic interaction chromatography stationary phases for ultra-HPLC in analysis of polar pteridines, J. Sep. Sci. 33 (2010) 765-772. [71] A. Grand-Guillaume Perrenoud, J.L. Veuthey, D. Guillarme, Comparison of ultra-high performance supercritical fluid chromatography and ultra-high performance liquid chromatography for the analysis of pharmaceutical compounds, J. Chromatogr. A 1266 (2012) 158-167. [72] A. Grand-Guillaume Perrenoud, C. Hamman, M. Goel, J.L. Veuthey, D. Guillarme, S. Fekete, Maximizing kinetic performance in supercritical fluid chromatography using state-of-the-art instruments, J. Chromatogr. A 1314 (2013) 288-297. [73] A. Periat, A. Grand-Guillaume Perrenoud, D. Guillarme, Evaluation of various chromatographic approaches for the retention of hydrophilic compounds and MS compatibility, J. Sep. Sci. 36 (2013) 3141-3151. [74] A. Grand-Guillaume Perrenoud, J.L. Veuthey, D. Guillarme, Analysis of basic compounds by supercritical fluid chromatography: Attempts to improve peak shape and maintain mass spectrometry compatibility, J. Chromatogr. A 1262 (2012) 205-213. [75] E. Lesellier, A. Latos, A. Lopes de Oliveira, Ultra high efficiency/low pressure supercritical fluid chromatography with superficially porous particles for triglyceride separation, J. Chromatogr. A 1327 (2014) 141-148. [76] S. Fekete, A.L. Gassner, S. Rudaz, J. Schappler, D. Guillarme, Analytical strategies for the characterization of therapeutic monoclonal antibodies, Trends Anal. Chem. 42 (2013) 74-83. [77] S. Fekete, J.L. Veuthey, D. Guillarme, New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications, J. Pharm. Biomed. Anal. 69 (2012) 9-27. [78] A. Staub, D. Guillarme, J. Schappler, J.L. Veuthey, S. Rudaz, Intact protein analysis in the biopharmaceutical field, J. Pharm. Biomed. Anal. 55 (2011) 810-822. [79] S. Fekete, D. Guillarme, Reversed-phase liquid chromatography for the analysis of therapeutic proteins and recombinant monoclonal antibodies, LCGC Eur. 25 (2012) 540-546. [80] R.A. Everley, T.R. Croley, Ultra-performance liquid chromatography/mass spectrometry of intact proteins, J. Chromatogr. A 1192 (2008) 239. [81] S. Fekete, M.W. Dong, T. Zhang, D. Guillarme, High resolution reversed phase analysis of recombinant monoclonal antibodiesby ultra-high pressure liquid chromatography column coupling, J. Pharm. Biomed. Anal. 83 (2013) 273-278. [82] S. Fekete, J.L. Veuthey, S. Eeltink, D. Guillarme, Comparative study of recent wide-pore materials of different stationary phase morphology, applied for the reversed-phase analysis of recombinant monoclonal antibodies, Anal. Bioanal. Chem. 405 (2013) 3137-3151. [83] L. Nováková, M. olč p R. Jirásko, M. Lísa, in: D. Guillarme, J.L. Veuthey (Eds.), UHPLC in Life Sciences, The Royal Society of Chemistry, Cambridge, UK, 2012, p. 186. [84] http://www.waters.com/waters/en_US/ACQUITY-QDa-Mass-Detector-for-Chromatographic-Analysis/nav.htm?cid=134761404&icid=i8083&locale=en_US - consulted in February 13, 2014. [85] J. Schappler, S. Rudaz, J.L. Veuthey, D. Guillarme, in: A.X. Quanyun (Ed.), UHPLC and its applications, Wiley & Sons, Inc, Hoboken, New Jersey, US, 2013, 95.

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[86] M. Thevis, A. Thomas, V. Pop, W. Schänzer, Ultrahigh pressure liquid chromatography–(tandem) mass spectrometry in human sports drug testing: Possibilities and limitations, J. Chromatogr. A 1292 (2013) 38-49. [87] J.K. Nicholson, J.C. Lindon, Systems biology: Metabonomics, Nature 455 (2008) 1054-1056. [88] E.J. Want, I.D. Wilson, H. Gika, G. Theodoridis, R.S. Plumb, J. Shockcor, E. Holmes, J.K. Nicholson, Global metabolic profiling procedures for urine using UPLC–MS, Nature Protocols 5 (2010) 1005-1018. [89] L. Novakova, J.L. Veuthey, D. Guillarme, Practical method transfer from HPLC to UHPLC: the importance of frictional heating, J. Chromatogr. A 1218 (2011) 7971-7981.

Captions

Fig. 1. Number of papers published each year in the field of UHPLC and UHPLC-MS, from 2004 to 2013. Blue

bars were obtained with keywords ‘‘UPLC’’ and “UHPLC’’, while red bars were obtained with an additional

filter (keyword ‘‘MS’’). (Source: SciFinder scholar search of the Chemical Abstracts database. Date of

information gathering: January 2014).

Fig. 2. Instrumental constraints for successful operation of UHPLC.

Fig. 3. Fast separation of a cashew-nut extract on 50 x 2.1 mm columns packed with superficially porous

particles (SPPs) of 1.3 µm, 1.7 µm and 2.6 µm. The mobile phase was a mixture of 14% water and 86%

acetonitrile at a flow rate of 0.8 mL/min. {Adapted from [27], with permission}.

Fig. 4. Comparison of several approaches to obtain high-throughput and high-resolution separations, using

kinetic plot representations (t0/N2 = f(N)). This figure illustrates the effect of particle size (5 µm, 3.5 µm or 1.7

µm) and upper pressure limit (400 bar or 1000 bar) on overall performance. For a given plate count, the lowest

curve corresponds to the best conditions in terms of analysis time. The vertical asymptote on the left of each

curve corresponds to the maximal plate count that can be achieved with a given strategy.

Fig. 5. Analysis time versus peak capacity for the separation of peptides. Black circles correspond to 1D-LC data

recently reported in the literature. Green triangles correspond to performance achieved with 2D-LC involving

UHPLC in the second dimension. The dotted line represents the current pareto-front in 1D-LC for recently

published results. The two arrows indicate a possible two-fold gain in peak capacity and 10-fold gain in analysis

time. The numbers beside the round black dots indicate the corresponding article in which the given performance

was demonstrated (see [38] for additional explanations). {Adapted from [38], with permission}.

Fig. 6. (A) Influence of mobile-phase temperature of 30–50°C on the separation of a mixture of 10 model

compounds. (B) Influence of UHPLC column pressure drop from 100 bar to 1000 bar on the separation of the

same mixture of 10 model compounds. The column was an Acquity BEH, C18, 50 x 2.1 mm I.D., 1.7 µm. The

chromatograms in (B) were obtained with increasing flow rates; frictional heating was non-negligible in this

case. {Adapted from [89], with permission}.

Fig. 7. Computer-assisted approach for the generic method development of a UHPLC method.

Fig. 8. Characterization of an intact IgG2 of 150 kDa and its reduced form (fragments of 25 kDa and 50 kDa).

The columns were wide-pore columns of 150 mm and 450 mm at 80°C. {Adapted from [81], with permission}.

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