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Beginner's Guide to Convergence Chromatography


Understanding the Founding Principles of Convergence Chromatography
Expanding Selectivity for the Chromatographic Laboratory
Of the chromatographic tools available today, the two most commonly utilized are gas
chromatography (GC) and liquid chromatography (LC). Due to significant advancements in
the performance of systems designed to manage supercritical fluids, convergence
chromatography (CC) is a viable chromatographic approach for addressing both complex and
routine separation challenges.

Figure 1: Three complementary chromatographic techniques used in the analytical laboratory.

Each of these three chromatographic techniques has gone through numerous iterations and
improvements over time, enhancing the capabilities of the chromatographer. In gas
chromatography, the separation is achieved by varying the temperature of the column over
the course of the analysis. This technique provides high efficiency (separation power), but
with limited selectivity. Selectivity is determined solely by the interaction of the analytes with
the column chemistry (stationary phase); the carrier gas (mobile phase) is inert. The advent
of capillary GC, 30 years ago, affected significant performance improvements, but only small
incremental advancements have occurred since.

Due to a narrow polarity range and volatility requirements, gas chromatography is limited
relative to the types of analytes that can be separated. To address a more diverse range of
compounds than is possible to analyze by GC, liquid chromatography, in reversed-phase
mode, has proven an invaluable analytical technique.

In LC (unlike in GC), the analyte has affinity for both the stationary and mobile phases.
Separation is achieved by creating a competition between these phases. This allows for more
options when changing selectivity. Advancements in LC systems were small and incremental
until the introduction of UltraPerformance LC (UPLC) in 2004.

Convergence chromatography is a new category of separation science that provides an


exceptional increase in selectivity to the chromatographic laboratory. CC is orthogonal to
reversed-phase LC and provides significant potential in streamlining the entire analytical
workflow. In CC, the separation is achieved by manipulating the density and composition of a
supercritical fluid-based mobile phase. Because of the very high diffusivity of the mobile
phase, high separation efficiency can be achieved. Additionally, the diversity of stationary
phase and mobile phase (co-solvent) options give the chromatographer access to the largest
selectivity space available to any separation technique.
Figure 2: The ACQUITY UPC2 System.

The term convergence chromatography originates from a concept Professor Calvin Giddings
discussed in 1964. In one of his publications, Giddings states, One of the most interesting
features of ultra high pressure gas chromatography would be convergence with classical
liquid chromatography.1 Convergence chromatography is the realization of this idea, taking
the benefits of GC and merging them with the benefits of reversed-phase and normal-phase
LC.

In order to harness the theories originally proposed by Giddings and realize the benefits of
converging GC and LC, Waters has developed the ACQUITY UltraPerformance Convergence
Chromatography (UPC2) System (Figure 2) - a holistically designed chromatographic
system that utilizes compressed CO2 as the primary mobile phase. The system leverages the
chromatographic principles and selectivity of normal-phase chromatography while providing
the ease of use of reversed-phase LC. Built upon proven, low-dispersion, high-efficiency
UPLC technology, the ACQUITY UPC2 System offers a level of reliability, robustness,
sensitivity, and throughput never before possible when utilizing supercritical fluids and
convergence chromatography.

Streamlined Workflow
Equally as important as its separation capability is how CC impacts the overall workflow of
the laboratory. Often, the largest bottleneck in a chromatographic laboratory is the
preparation of samples for further analysis. Most common sample preparation methods result
in the analytes of interest being dissolved in a solvent incompatible with reversed-phase LC.
Many analytes are easily dissolved in and therefore best extracted with an organic
solvent. Because of this, additional steps are often required to convert the organic solution or
extract into something compatible with reversed-phase chromatography (Figure 3).

Figure 3: Examples of various sample preparation techniques, which often conclude with the sample
being dissolved in an organic solvent.

CC is compatible with direct injection of samples dissolved in organic solvents. Evaporation


and reconstitution (often the most time-consuming steps) required for reversed-phase
separations are no longer required, resulting in considerable cost savings in the overall
assay. In addition to the reduced time required to prepare the sample, the analysis can be
significantly shorter a significant cumulative impact, especially with multiple systems.

Diverse Applicability and Selectivity


CC utilizes the same eluotropic spectrum as normal-phase LC. The flexibility of this
technique, however, also allows the use of some conventional reversed-phase columns such
as C18, yielding similar retention characteristics to reversed-phase LC when analyzing highly
lipophilic compounds. The ability to span this wide range of selectivity gives CC clear
advantages.
An overlap of approximately 80-85% of compounds can be analyzed by both convergence
and reversed-phase chromatography. In addition, CC can also analyze those compounds
typically analyzed by normal-phase LC. The ability to analyze chiral compounds,
stereoisomers, and diasteroisomers dramatically increases the utility of CC. Essentially, any
compound soluble in an organic solvent is a candidate for analysis via CC. Unlike normal-
phase LC, CC is compatible with gradients, enabling utilization of detection techniques such
as ultraviolet (UV), photodiode array (PDA), and evaporative light scattering (ELS).

The primary mobile phase in CC is dense CO2 in either a supercritical or subcritical state. This
mitigates the use of harmful solvents necessary in normal-phase LC mobile phases.
Additionally, the variability expected from normal-phase LC, due to the adsorption of water
to the stationary phase, is completely eliminated. The nonpolar nature of the mobile phase
also makes injection in organic solvents preferable. These advantages are currently being
leveraged in two application areas: fast chiral screening, and normal-phase replacement
methods.

In a chiral screening example (Figure 4), analysis time is reduced from 20 minutes to only 3
minutes, a seven-fold reduction in time, with an increase in resolution. Along with taking less
time, significantly less solvent is consumed, resulting in substantial reduction in cost.
Figure 4: The utility of CC for fast chiral screening using UPC2.

Similar reductions in analysis time and cost per analysis occur with achiral normal-phase
replacement (Figure 5). Replacing typical normal-phase organic solvents with a mobile phase
composed of primarily compressed CO2reduces the cost per analysis from just under 6 dollars
to only 5 cents per sample. The overall financial impact from shorter analysis time, lower
solvent purchase costs, and reduced solvent disposal costs achieved by using CC as a
normal-phase LC replacement is exceptional.

Figure 5: The utility of CC for normal-phase replacement.


The Underlying Principle: Supercritical Fluid Chromatography
The underlying principle of convergence chromatography is supercritical fluid
chromatography (SFC). A supercritical fluid is any substance at a temperature and pressure
above its critical point, where distinct liquid and gas phases no longer exist (Figure 6).
Essentially, the substance takes on properties of both a gas and a liquid, including high
diffusivity and low viscosity, enabling fast and efficient chromatography.

Figure 6: Phase diagram depicting the physical change of a substance from one state to another.

Carbon dioxide is the most common substance utilized as a supercritical fluid mobile phase.
The physical state of CO2 is easily manipulated at temperatures and pressures in an
acceptable range. Extreme conditions needed to put other substances into their supercritical
states, as well as undesireable properties when in that state, make CO2 the best candidate
(Table 1).

Substance Critical Critical Comments


temp C pressure
(bar)

Carbon 31 74 Physical state


dioxide easily changed

Water 374 221 Extreme


conditions
needed
Methanol 240 80 Extreme
temperature
needed

Ammonia 132 111 Highly corrosive

Freon 96 49 Environmentally
unfriendly

Nitrous 37 73 Oxidizing agent


oxide
Table 1: Conditions required to transition a particular substance into a supercritical fluid.

While in reversed-phase LC, water is typically used as the weak mobile phase, in CC,
supercritical CO2 is used instead. Pure CO2 has limited solvating power, so it is often mixed
with different cosolvents and modifiers.

Pairing supercritical CO2 with different co-solvents opens up a significant range of selectivity
options (Figure 7). Reversed-phase LC can access only a small piece of the eluotropic series,
whereas normal-phase and convergence chromatography can explore a much larger range
(Table 2). One caveat of normal-phase chromatography is that not all organic solvents are
miscible with one another, resulting in incompatibility of certain mixtures. Supercritical CO 2,
however, is miscible with all other solvents across the entire eluotropic series, opening up a
wide range of solvent choices to influence the selectivity of separations. Importantly,
different eluotropic strengths can be obtained by using solvents outside of the normal-phase
tool kit.

Figure 7: Column selectivity can be an exceptionally powerful tool when developing methods in CC. In
this example, an active pharmaceutical ingredient and its related compounds were analyzed on multiple
stationary phases (typical to reversed-phase and normal-phase) under a fixed set of conditions.

With CC, although the full range of normal-phase solvents can be accessed to increase
selectivity, in many cases it is not necessary. By simply combing supercritical CO 2 with an
organic co-solvent at the other extreme of the eluotropic spectrum, in addition to the diverse
range of available stationary phases, an exceptionally large selectivity space can be
explored, making this technique applicable to a wide variety of separation challenges. By
combining CO2 with methanol, for example, the mobile phase can be dialed to eluotropic
strengths ranging from 0 to 0.73Eo. The realization of the analytical power of CC is made
possible because of the advancements in instrumentation found in the ACQUITY UPC2
System.

Table 2: Solvent selectivity options for reversed-phase, normal-phase, and convergence


chromatography. The supercritical CO2 used in CC is miscible with the entire eluotropic series, opening
up a wide range of solvent selectivity choices to develop a separation.

Table 3 shows several of the available stationary phases commonly utilized for different
chromatographic techniques. Reversed-phase and normal-phase LC utilize limited column
options as compared to CC (Table 3). Most reversed-phase separations are performed on
C18 stationary phases. Some columns cannot be used in reversed-phase separations at all
because of the phase polarity. Normal-phase chromatography is also limited because of
incompatible polarity between phases. CC allows for use of all of the column chemistries,
opening up a wider range of selectivity choices.
Table 3: Stationary phase options for reversed-phase, normal-phase, and convergence chromatography.
Convergence chromatography can utilize both traditional normal-phase and reversed-phase column
chemistries, opening up a wide range of selectivity choices to develop a separation.

The Enabling Technology


UPLC technology improved separations by utilizing lower dispersion systems, smaller
particles, and higher pressure tolerances.

Figure 8: Relationship between efficiency, particle size, and required back pressure.
There are limits to increasing efficiency in liquid chromatography through improved system
design and reduced particle size columns. Significant technological hurdles need to be
overcome in order to further evolve in this manner. For example, while a 1.0-m particle
column delivers 70% more efficiency than a 1.7-m column (Figure 8), the demands on the
system increase dramatically. Dispersion must be further minimized and an exceptionally
large increase in available system back pressure capability is needed.
In this chapter we discuss how the ACQUITY UPC2 System harnesses the significant
developments in both instrumentation and column chemistry, putting to use the improved
performance of lower dispersion instrumentation and smaller particles for greater efficiency
separations (Figure 9). Due to the improved diffusivity of supercritical fluids, decreased
runtimes, and the ability to exploit selectivity factors, convergence chromatography is able to
overcome separation challenges that are insurmountable with todays techniques.
The Evolution
A number of innovations in the design of the ACQUITY UPC2 System clearly differentiate how
Waters has advanced the science of supercritical fluid chromatography. To gain perspective
on this, we first must review the challenges of past analytical SFC systems (Figure 9).
Historically, managing supercritical fluids for analytical separations has been plagued with a
lack of robustness. This is primarily due to the use of repurposed HPLC and/or GC
instrumentation for SFC (as a majority of analytical or hybrid SFC systems are today). This
repurposed equipment is unable to control the density of CO 2, which results in a mobile
phase with shifting solvation power, shifting retention times from injection to injection. In
addition, accurately delivering low percentages of co-solvent and shallow gradients has not
been possible. Due to the poor accuracy and precision of partial loop injections, these
instruments are often restricted to full loop injections.

Other factors adversely affecting analytical SFC performance include unreliable pumping
systems for delivery of gas/liquid mixtures, as well as poor back pressure regulation. The
large system dispersion inherent to these systems causes unwanted bandspreading,
preventing the adoption of more efficient columns using smaller particles. These
inadequacies limit the potential throughput.

The lack of specifically designed pumping systems also causes problems with sensitivity;
these systems inherently produce a great deal of baseline noise. The detectors utilized are
also not designed to accommodate the refractive indices of supercritical fluids and augment
problems with the signal-to-noise observed.

The ACQUITY UPC2 System is the first analytical system purposefully designed to manage
supercritical fluids.

Figure 9: van Deemter curves depicting the evolution of HPLC to UPLC and SFC to UPC2. Interestingly
both HPLC and SFC are able to achieve similar optimal efficiencies. The key difference is that the
increased linear velocity allows for shorter analysis times. As you can see both UPLC and UPC2 are able
to achieve this decrease in required analysis times with much better separation efficiences.
Figure 10: Flow path of the ACQUITY UPC2 System.

A full understanding of the technology within the ACQUITY UPC2 System requires a review of
the innovations made to each individual module of the instrument (Figure 10).

Solvent Management Technology (The Pump)


The first challenge in the flow path of the system is the accurate and precise control of
mobile phase density and composition. Using a repurposed HPLC pump requires the incoming
CO2 to pass through a device for precompression and chilling. On some instrumentation, this
device is a separate unit that stands beside the chromatographic system (Figure 11). The
farther away from the pump this device sits, the less mobile-phase density control, as the
density can change between precompression and pumping. In addition, the pumping
algorithms (internal control software) of traditional SFC systems tend to be designed to
deliver compressed liquid not a vastly different mobile phase with the properties of a
supercritical fluid. As a result, compositional accuracy, precision, and retention time
reproducibility often suffer. Another downfall is the inability to reliably deliver low
percentages of co-solvent (less than 5%), making it difficult to control retention profiles by
manipulating the co-solvent, as well as increasing baseline noise in the detector.
Figure 11: Comparison of the ACQUITY UPC2 Solvent Manager to an HPLC pump repurposed for use with
supercritical fluids.

In contrast, the ACQUITY UPC2 Binary Solvent Manager (BSM) is specifically designed to
manage supercritical fluids and is fitted with a fully integrated precompression device, which
provides exceptional density control for reliable, reproducible retention times and negligible
baseline noise. In a supercritical fluid system, solvent density controls mobile phase
solvating power, so precise control is critical to reproducibility. Separate control algorithms
for compressible supercritical fluids and essentially noncompressible liquid components result
in the ability to accurately blend different mobile phase compositions, including low
percentages of co-solvent (Figure 12), and reproducibly deliver gradient profiles (Figure 13).

Figure 12: Accurate and precise blends of compressed CO 2 and desired modifier in increments of 0.5%
from 1% to 2.5% methanol.

Analytical SFC systems never before exhibited such fine levels of control, especially in
gradient separations. The technological advances within the ACQUITY UPC2 System provide
precise control of pump intake, compression, and delivery, allowing for the type of
reproducibility expected from UPLC.
Figure 13: A 0.5% difference in the programmed solvent composition for gradient analysis. Overlay of
10 injections. The top chromatogram is starting at 1% modifier, whereas the bottom chromatogram is
starting at 1.5% modifier. A very controlled shift in retention results from the accurate and precise
delivery of the mobile phase.

The volumetric density control utilized within the ACQUITY UPC2 BSM models mass flow,
giving exceptional precision. This leads to controlled elution times and exceptional solvation
strength control. The pump heads themselves are independently cooled, improving both
compressibility and density control of the CO2. The pump and integrated compression
algorithms are so effective and the control so precise that either liquid or gaseous CO 2 can be
used to compose the mobile phase.

Figure 14 shows the inner workings of the BSM. The co-solvent pump is a UPLC pump,
whereas the CO2 pump is behind the insulated black cover. Because the compression and
chilling device is integral to the pump, this insulated cover enables more precise density
control of the incoming CO2.
Figure 14: The ACQUITY UPC BSM accurately and precisely blends CO and
2
2

the desired modifier, even at compositions less than 5%. This ability is the
result of the separate control algorithms used to compensate for the
separate compression of modifier and CO as well as the ability to
2

compensate for changing pressure and refractive index effects.

Sample Injection
Traditional analytical SFC systems struggle to reproducibly inject low volumes. In most
cases, only full loop injections can be performed; with partial loop injections it is difficult to
maintain the supercritical state of the mobile phase. Accuracy, precision, and linearity
therefore suffer, preventing quantitation from being a viable option when using partial loop
injections. Large amounts of sample are often wasted with every injection. Thus, to change
the viable injection volume, a manual change of the sample loop is necessary, limiting the
flexibility of the system.
The ACQUITY UPC2 Sample Manager has a novel dual-injection valve design (Figure 15). This
vents the primary sample loop to waste, enabling the sample to enter the loop under
atmospheric pressure while maintaining the supercritical state of the mobile phase. In
addition, the auxiliary injection valve was designed to reduce pressure pulsation from the
injection sequence and mitigate carryover, enabling repeatable and reproducible partial loop
injections (Figure 16). Injections of 0.1 to 50 L can be made in 0.1-L increments (Figure
17), and with dual needle wash options, sample carryover is negligible.
Figure 15: Comparison of the ACQUITY UPC Sample Manager to an HPLC
2

injector repurposed for use with supercritical fluids.

Figure 16: Chromatographic example of the repeatability and reproducibility


of partial-loop injections.
Figure 17: Injector linearity with partial loop injections from 1 to 10 L in 1-
L increments.

Optical Detection
Optical detection is troublesome with analytical SFC systems. Large dispersion flow cells,
baseline noise, and refractive index (RI) effects all result from using a detector designed for
liquid chromatography. A detector intended for the refractive index properties of liquid
solvents, not supercritical fluids, results in significant baseline noise and curvature,
amplifying the noise produced by the pumping system. Solvents such as methanol and
water, which are commonly used in reversed-phase chromatography, have very similar RI
values (Figure 18), and so RI effects in reversed-phase methods are typically not that
significant. CO2 has a value that is very different from methanol (the most commonly used
co-solvent) making the range of refractive indices of substances larger than in LC. As a
further challenge the density, and therefore refractive index, of a supercritical CO 2-based
mobile phase changes over the course of a gradient analysis.
Additionally, the high dispersion of HPLC flow cells also prevents the adoption of small
particle-sized columns and excludes the use of column diameters less than 4.6 mm in
internal diameter (I.D>). To use these columns and gain the associated benefits of high
efficiency and sensitivity, a purposefully designed detector is required.

Figure 18: Refractive indices of different substances.

The ACQUITY UPC2 PDA Detector is specifically designed for supercritical fluids. Instead of
using sapphire lenses, which reduce energy throughput at lower UV wavelengths, the
ACQUITY UPC2 PDA Detector uses lenses made of high-strength silica which are able to
withstand the pressure requirements. This helps to maximize sensitivity, reduce baseline
noise, and compensate for differences in RI effects between CO2 and the organic co-solvent.
The optics bench is thermally controlled to further improve baseline stability and mitigate RI
effects. A low-dispersion, stainless steel flow cell accommodates narrow peak widths, while
the 10 mm path length maximizes sensitivity while maintaining optimal spectral
performance. The exceptional level of sensitivity attainable enables the quantitation of trace
level impurities (Figure 19).

Figure 19: Impurity profile of metoclopramide demonstrating the


applicability of the ACQUITY UPC System for trace level impurity analysis.
2

Back Pressure Regulation


One of the most critical parts of any supercritical fluid-based system is the ability to
accurately control back pressure. This greatly affects mobile phase density, and therefore
analyte solvation and retention times. Traditional SFC systems are challenged with accurate
and precise control of the backpressure due to multiple factors such as: poor pressure
monitoring at the back pressure regulator (BPR), slow-to-respond feedback loops, low-
resolution stepper motors, poor control of pressure and flow at the pump, and degradation of
BPR components over time.
As system pressure is increased, the density of CO2 increases, resulting in stronger solvating
strength and decreased retention times. The ACQUITY UPC2 System exhibits improved back
pressure and density control through an innovative dual stage active and static BPR (Figure
20). Through this combination of active and static back pressure control, the static BPR
keeps the system at a minimum pressure while the active BPR grants enhanced control,
which can be used to fine-tune retention times (Figure 21). In efforts to further improve
robustness, the static cartridge BPR is also heated to mitigate freezing.
Figure 20: The ACQUITY UPC System Convergence Manager consistently
2

delivers back pressures less than 5 psi deviation from the set point. This
precise level of control enables exceptional retention time reproducibility
and baseline stability.
Figure 21: Two-stage dynamic and static BPR enables consistent
performance and ability to finely control retention to adjust methods as
necessary.

The dual stage BPR resides within the ACQUITY UPC2 Convergence Manager (CM) (Figure
22). This module also houses the inline particulate filter for the incoming CO2, CO2 leak
detector, vent valve, pressure relief valve, and auxiliary injection valve.

Figure 22: The ACQUITY UPC Convergence Manager.


2

Overall System Performance


Finally, the ACQUITY UPC2 System has inherently low dispersion, enabling the use of smaller
I.D. columns and smaller particle size columns (Figure 23). Narrow I.D. columns increase
sensitivity, conserve solvent, and utilize flow rates more amenable to mass spectrometry.
Smaller particle size columns increase separation efficiency and improve resolution.
Figure 23: Comparison of 5-m and 1.7-m columns at the same flow rate
and column dimensions.
By reducing particle size from 5 to 1.7 m, efficiency was improved
threefold, and sensitivity and resolution nearly doubled.
Getting Started: Utilizing Convergence Chromatography in the Chromatographic
Laboratory
This chapter reviews the terminology used in both supercritical fluid and convergence
chromatography and describes what types of samples and analytes are candidates for
analysis by the ACQUITY UPC2 System. We lay out the role of co-solvents, mobile-phase
additives, and sample diluents, as well as the effects of pressure and temperature on density
and how this affects separations. Finally, we introduce a screening protocol for developing a
method.
Terminology
CC is similar in some ways to reversed-phase LC, but instead of mobile phase A (the weak
solvent) being aqueous, it is supercritical CO2.

Mobile phase A: Mobile phase B:

Convergence chromatography: Convergence chromatography:


CO2 Organic solvent (methanol)

Reversed-phase LC: Aqueous Reversed-phase LC: Organic


solvent (buffer) solvent (acetonitrile)

Conventional SFC terms such as solvent, co-solvent, and modifier all refer to the primary
liquid component(s) of mobile phase B, the strong eluting solvent. Typically, the co-solvent is
methanol, but can also be other organic solvents such ethanol, isopropyl alcohol, acetonitrile,
or combinations of these. An additive is a salt or liquid added to the co-solvent at a low
concentration to improve peak shape and/or analyte solubility. The additive can also
influence chromatographic selectivity. Typical additives include diethyl amine, ammonium
hydroxide, formic acid, trifluoroacetic acid, ammonium formate, ammonium acetate, or small
amounts of water. The appropriate concentration is additive dependent; for example, the
addition of water at above 5% risks the formation of a biphasic mobile phase at high co-
solvent concentrations.

Can My Sample Be Analyzed Using Convergence Chromatography


One of the first questions asked about any new analytical methodology is, Can my sample
be analyzed by this technique (convergence chromatography)? The simplest answer is that
if the sample can be dissolved in an organic solvent, it is a candidate for CC. There is no
universal answer to this question, however, and some experimental work is needed to
confirm. This suitability to organic injection solvents is very useful, as many sample
preparation techniques produce samples dissolved in an organic solvent (for example,
liquid/liquid extraction, solid phase extraction, protein precipitation). These can be directly
injected onto the ACQUITY UPC2 System and do not require the laborious and time
consuming evaporation or reconstitution steps often required in reversed-phase LC. Chapter
4 deals with this topic in more detail.

Figure 24: A basic compound and some of its useful physical and chemical
properties.

For the analytical scientist, it is always useful to learn as much about a sample as possible
the more known, the better the chance to develop a robust method (Figure 24). One useful
piece of information regarding the solubility of compounds in various organic solvents relates
to the partition coefficient, P (usually referred to as log10 P). The partition coefficient (P) is
the ratio of concentrations of a compound in the two phases of a mixture of two immiscible
solvents at equilibrium, usually water and 1-octanol (Figure 25). These coefficients are a
measure of the differential solubility of the compound between these two solvents. The
partition coefficient measures how hydrophilic or hydrophobic a compound is.

In terms of CC, the partition coefficient could help determine whether a target compound is a
candidate for analysis by the ACQUITY UPC2 System. A rule of thumb is that compounds with
log P values between -2 and 9 make suitable candidates for CC.

Figure 25: Formula for measuring partition coefficient, P.

Co-solvents in Convergence Chromatography


The co-solvent plays two roles. First, it increases the solvating power of CO2. Second, the co-
solvent acts as the strong, eluting solvent. Changing co-solvent (for example, methanol to
acetonitrile) affects both retention and selectivity. The role of the co-solvent in CC is
analogous to that of the strong solvent in reversed-phase LC; CO2 alone has approximately
the eluting strength of heptane (a very weak eluting solvent).
Table 2, in Chapter 1, shows the eluotropic (eluting strength) series for a range of organic
solvents and highlights the four most common co-solvents used in CC: acetonitrile, isopropyl
alcohol, ethanol, and methanol. The solvents listed in this table are all miscible with CO2,
resulting in a wide range of available retention and elution strengths.
Co-solvents added to the CO2 mobile phase generally decrease an analytes retention time.
When the co-solvent concentration is increased, the polarity of the mobile phase is changed,
resulting in decreased retention time(s). Figure 26 illustrates the effect of changing co-
solvent concentration on retention in an isocratic separation. As the concentration of the
strong, eluting co-solvent (methanol) is decreased, the retention of the analytes increases.
This is the same phenomenon observed in reversed-phase LC.

Figure 26: Effect of changing co-solvent concentration in an isocratic


separation.

Figure 27 demonstrates the effect of changing the strength of the mobile phase through
using different co-solvents. Methanol is the strongest co-solvent and elutes the analytes the
fastest. Isopropyl alcohol is weaker than methanol, but stronger than acetonitrile, while
acetonitrile is the weakest of the three co-solvents and retains the analytes longest. The
same relative type of chromatographic behavior occurs in other modes of chromatography;
stronger solvents lessen retention and elute analytes faster.

Figure 27: Effect of changing the type of co-solvent in a gradient


separation.
Different co-solvents can be mixed, changing solvent strength and creating differences in
retention. Figure 27 illustrates the effect of adding a weaker co-solvent (acetonitrile) to
methanol, for a gradient separation of metoclopramide and related impurities. As the
acetonitrile concentration is increased, the methanol concentration and therefore solvent
strength decreases, and longer retention times are observed. Slight changes in selectivity,
improved resolution, and sharper peak shapes also occur with the introduction of a different
co-solvent for this separation.

Additives in Convergence Chromatography


As in reversed-phase LC, additives are used in CC to improve peak shape and/or resolution
of the separation. In Figure 28, the additive ammonium formate was added to all mixtures of
co-solvents for all four chromatograms. Additives can modify the stationary phase surface or
act as ion pairs, changing selectivity. Basic additives tend to improve peak shape for basic
compounds and may slightly change the selectivity. Examples of basic additives include
ammonium hydroxide, 2-propylamine, and triethylamine. Acidic additives can improve peak
shape for acidic compounds, and may change the selectivity. Common acidic additives
include trifluoroacetic acid, formic acid, and acetic acid. Figure 29 shows a separation of
acidic analytes and, in this example, when the concentration of the acidic additive was
increased, peak shape improvements were realized.

Figure 28: Effect of mixing co-solvents in a gradient separation.


Figure 29: Effect of changing additive concentration on peak shape in CC.

Changing between different additives can have a drastic effect upon peak shape and
retention (Figure 30). For these basic analytes (beta blockers), a methanol co-solvent with
no additive gives poor peak shape. Unlike separations of acidic analytes (Figure 29), the
addition of formic acid actually makes the peak shape worse. Formic acid also absorbs at the
220 nm detection wavelength, resulting in a sloping baseline. For these strong bases, the
addition of ammonium acetate (20 mM) dramatically improves peak shape, as does
diethylamine, as basic compound peak shape often improves when using basic additives.

Figure 30: Effect of changing the type of additive on peak shape for basic
compounds in CC.

Sample Diluents in Convergence Chromatography


Sample diluent strength can strongly affect peak shape and solubility in CC. As with other
modes of chromatography, it is recommended that the sample diluent be as weak as
possible, balancing analyte solubility and peak shape. With CC, that means that the sample
should be dissolved in an organic solvent near the top of the eluotropic series (Table 2).
Waters recommends heptane/2-propanol (90:10) as a good generic solvent, balancing
solubility (isopropyl alcohol) and peak shape (heptane). Water content in the sample should
be reduced, or eliminated if possible. Figure 31 shows seven overlaid chromatograms of
peaks for the neutral compound butylparaben. As the injection volume increases, the effects
of injection solvent strength on peak shape appear. In the strong co-solvent methanol, peak
fronting occurs as injection volume increases. The weaker isopropyl alcohol shows less
fronting and slightly taller peaks as compared to methanol. The recommended sample
diluent isopropyl alcohol/hexane provides sharp, symmetrical peaks for all injection volumes.

Figure 31: Effect of sample diluent strength on peak shape in CC. Waters
recommends 90:10,
but 70:30 was chosen in this case.

Pressure, Temperature, and Density


The Automatic Back Pressure Regulator (ABPR) setting affects retention time by changing the
density of the supercritical fluid before the release of pressure and expansion of the CO 2. As
the ABPR pressure setting increases, the density increases, decreasing retention time.
Despite the fact that mobile phase composition has the greatest effect upon the separation,
the adjustable pressure and its affect on density can be used to optimize or fine-tune a
method. In Figure 32, an example of changing the ABPR setting (pressure) while leaving all
other parameters unchanged is shown. As can be seen in these separations, increasing the
APBR setting results in shorter retention times.
Figure 32: Effect of pressure (density) on retention. Typical operating ABPR
ranges are 1500 2200 psi (100 150 bar).

As in reversed-phase LC, column temperature affects both selectivity and retention in CC,
and different analytes are affected to differing degrees. Like pressure, column temperature
affects the mobile phase density in the column. As column temperature increases, the mobile
phase density decreases, which increases retention time (Figure 33). The retention time
effects in CC are the opposite of those in reversed-phase LC, where higher column
temperatures result in shorter retention times. Also, note the presence of an additional
(small) peak at 50 oC. This results from slight selectivity differences; temperature affects
different analytes in different ways.

Figure 33: Effect of column temperature (density) on retention and


selectivity in CC.

Peak shape, retention, and selectivity can be manipulated by varying and understanding the
roles of co-solvent, additive, sample diluent, pressure, temperature, and stationary phase.
Combining everything shown in this chapter, Table 4 shows how CC separations can be
optimized using these tools. Note, however, that for a given separation, the importance of
each parameter may be different than in this example. For example, sometimes the
stationary phase plays a larger role than choice of co-solvent in manipulating selectivity. In
other situations, switching from methanol to 50:50 methanol/acetonitrile plays a larger role
than changing column chemistries.

Peak shape Retention Selectivity


Stationary 4 2 1
phase

Co-solvent 3 1 2

Additive 1 4 3

Pressure* 3 4

Flow-rate* 3 4

Temperature* 3 3

Injection 2
solvent

Table 4: Optimizing separations with CC. (*) Affect density. 1 = Greatest effect. 4 = Least effect.

Recommended Screening Protocol


Figure 34 describes a protocol to quickly determine whether the target sample can be
dissolved in an injection solvent compatible with CC. If the log P of the analyte is a value
between -2 and 9, it can. If it is less than -2, the analyte is only soluble in an aqueous
solvent and therefore not compatible with CC.
If the log P value of the analyte is unknown, it must dissolve in a suitable organic solvent
prior to injection.

Figure 34: Protocol for determining whether a target analyte is a candidate


for analysis by CC.

Remember that convergence chromatography is a normal-phase separation, and as such


methanol is a very strong solvent (the opposite of reversed-phase LC, where it is a fairly
weak solvent). Consequently, the sample needs to be dissolved (or diluted) in a weaker
solvent such as heptane/isopropyl alcohol. A balance must be struck between sample
solubility and peak shape; a discussion of the effect of sample diluent on peak shape in CC
appears later in this chapter.

In addition to selecting a sample diluent, there are other considerations in starting to


develop a method. For example, what chromatographic conditions are appropriate for the
analyte in question? Figure 35 shows a recommended generic set of starting conditions
known to successfully retain and separate a wide range of analytes.

Figure 35: A recommended set of screening conditions for CC.

As with any mode of chromatography, these conditions are not appropriate in every case,
and strategies for improving peak shape and changing selectivity and retention can be
employed.

For such cases, figures 36, 37, and 38 show the systematic approaches for improving peak
shape, changing retention, and altering selectivity, and are not that different from those
used in reversed-phase LC. Figure 36 provides a protocol for improving peak shape. The
initial starting point is a recommended set of conditions based upon whether the analytes of
interest are primarily basic or acidic in nature. Acidic compounds tend to have better peak
shape under acidic conditions, and basic compounds tend to exhibit better peak shape under
basic conditions. Strategies for improving peak shape include trying a different additive,
changing the concentration of additive, and changing the column chemistry.
Figure 36: Strategies for improving peak shape in CC.

Figure 37 gives a protocol for increasing retention. As in any form of liquid chromatography,
the first option is the use of a different (weaker) co-solvent. Methanol is the strongest co-
solvent used in CC; using a weaker co-solvent such as acetonitrile or another alcohol
increases retention time. Flattening or reducing the slope of the gradient (lowering the final
percentage of co-solvent or increasing the gradient duration) can be effective, as well.
Different co-solvents can also be mixed; reducing the concentration of methanol will weaken
the overall co-solvent strength, thereby increasing retention. The ability to manipulate the
density of the mobile phase in the column is a property unique to SFC and CC. Doing so
changes the overall retention, as higher density equals reduced retention. This is done by
decreasing the ABPR setting and/or increasing the temperature. Lastly, a different column
chemistry can be used.

Figure 37: Strategies for improving retention in CC.

Figure 38 illustrates a protocol for changing the selectivity (elution order, relative retention)
of the separation. This approach includes trying different co-solvents, such as acetonitrile
instead of methanol. Changing from an alcohol-based, protic co-solvent to a non-alcohol,
aprotic co-solvent such as acetonitrile has a much greater effect on selectivity than moving
from methanol to another alcohol. Co-solvents can also be mixed, reducing the concentration
of methanol and weakening the overall co-solvent strength. This can increase retention and
change selectivity. Manipulating the density of the mobile phase in the column can also
change the overall retention of the analytes, as these density effects can vary for specific
analytes, which may be enough to optimize a given separation. Finally, if necessary, this
protocol recommends a different column chemistry.

Figure 38: Strategies for changing the selectivity in CC.

Example of Problems Solved by Convergence


Chromatography
Novel Applications
Due to the expanded selectivity range of convergence chromatography described previously,
the technique is suited to a wide variety of applications (Table 5).

Fine Chemicals/Materials: Food & Environment:

Organic Light Emitting Diodes Vitamins


(OLEDS)

Agrochemicals (pesticides) Pesticides

Polymer additives Lipids (i.e., glycerides)

Surfactants Edible oils

Tobacco

Pharmaceutical/Life Science: Forensics/Research:

Lipids (profiling) Opiates

Vitamins Drugs of abuse

Natural products Steroids

DMPK/Bioanalysis Fatty acids


Reaction monitoring/Medicinal Anti-depressants
chemistry

Table 5: Selected applications for CC by market area and compound type.

Regardless of the market area or exact nature of the analytes tested, CC helps overcome
analytical challenges in three key ways:

Convergence chromatography simplifies the workflow

Convergence chromatography separates compounds with structural similarity

Convergence chromatography provides a mode of separation orthogonal to reversed-phase


LC
Using various applications to highlight the key benefits of CC, these three attributes can be
highlighted and the benefits explained.

Simplicity
Any simplification in the workflow, from initial sample collection through to the final analysis,
generally has the greatest business impact in any analytical laboratory. CC has the ability to
drastically simplify the workflow for many applications, resulting in cost and time savings,
reduced potential for error, and increased productivity. Some of these simplifications include:

Combining multiple techniques (LC and GC)

Combining multiple methods (normal-phase LC and reversed-phase LC)

Reducing sample preparation time

Combining Multiple Techniques Lipid Analysis


Lipids are analyzed for a variety of reasons in many markets. In the pharmaceutical industry,
lipid profiles are studied to determine the impact of drug efficacy in control and dosed
subjects. In clinical research, lipid levels are studied as biomarkers in different diseases, as
well as for efficacy of treatment. In food applications, certain lipid classes such as
triglycerides are profiled for nutritional purposes or to determine product authenticity. In
chemical materials, fatty acids and triglycerides are analyzed in petroleum products (e.g.,
biodiesel). In all cases, lipids need to be analyzed by different techniques to obtain the
desired outcome.
Free fatty acids are typically analyzed by GC. This requires derivatization of the free fatty
acid to the fatty acid methyl ester (FAME) in order to improve peak shape and detection
limits, especially for longer carbon chains. Derivatization can take several hours, and the
resulting GC analysis can take up to thirty minutes. More polar lipids, such as phospholipids
and sphingolipids, often require HILIC or normal-phase LC to separate the different lipids
classes (based on the nature of the polar head group). Then, reversed-phase LC can be used
to target more hydrophobic lipids within a class based on the carbon chain length and/or
number of double bonds. Complete lipid profiling therefore requires multiple techniques. With
CC, a single technique can be used for all these classes.

Figure 39 shows the comprehensive lipid profile of a mouse heart extract using the ACQUITY
UPC2 System. A BEH column is used with a generic gradient to separate the different lipid
classes, resulting in a separation similar to that given by normal-phase LC or HILIC.
Figure 39: Comprehensive lipid profile of a mouse heart extract using the
ACQUITY UPC System with MS detection.
2

For the neutral lipids (i.e., triacylclycerol (TG), diacylglycerol (DG), cholesterol esters (CE),
and free fatty acids), simply changing the column and gradient conditions retains and
separates the different lipids within each class based on the fatty acid chain length and
number of double bonds (Figure 40).

Not only is this faster than what can be accomplished by GC (up to 10 times faster), but it is
accomplished with no derivatization, thus dramatically simplifying the overall workflow for
lipid analysis. The absence of derivatization minimizes the time taken as well as the potential
error that can occur when adding these extra steps into the workflow. Without CC, this type
of profiling and targeted analysis can require up to three different techniques, resulting in
lower sample throughput, higher solvent usage, longer run times, and overall increased cost
of analysis.

Figure 40: Targeted analysis of free fatty acids (FFA), triacylglycerols (TG),
and cholesterol esters (CE) using the ACQUITY UPC System with MS 2

detection.

Combining Multiple LC Methods Fat-soluble Vitamins


Fat-soluble vitamins are another class of compound that are analyzed in all market areas,
from the pharmaceutical industry to clinical research and diagnostics, to food and fuels.
Analysis of fat-soluble vitamins and carotenoids is typically done by either reversed-phase or
normal-phase LC (Table 6). Due to the difficulty in separating these compounds, they are
analyzed individually using different columns and mobile phases, with analysis times
between 10 and 30 minutes.

Vitamin Analysis Column Mobile phase Analysis time


technique (minutes)

A NPLC PVA- Hexane/ethyl 12


functionalized acetate
silica

D3 NPLC PVA- Hexane/t-butyl 20


functionalized alcohol
silica

E RPLC C18 ACN/MeOH//H2O 30


+ additives

- NPLC Amino Hexane 10


carotene

Lycopene NPLC Amino Hexane + 10


additives

K1 RPLC C18 MeOH/MeCl2 12


+ additives

Lutein RPLC C18 MeOH/ACN 10

Table 6: Typical analysis conditions for fat-soluble vitamins and related


nonpolar compounds.

Using CC, a single method of analysis is possible, providing analysis of all vitamins and
related compounds in less than 10 minutes (Figure 41). Unlike the traditional methods of
analysis listed in Table 6, CC allows for the use of a single column, one mobile phase
condition, and one detector, streamlining methods of analysis where large numbers of
samples are analyzed. CC is also compatible with direct injection of the solvents typically
used to extract or dissolve these compounds (e.g., isooctane and hexane), and does not
require the solvent exchange that is typically required for reversed-phase analysis.
Figure 41: Overlay of 10 vitamin standards, demonstrating a single method
for analysis using CC.

Reducing Sample Preparation Time


In addition to providing faster separations due to the ability to combine multiple techniques
and methods into one, CC allows for a reduction in sample preparation time. The
compatibility of CC with direct injection of organic solvents can result in the following
benefits:

Elimination of hydrolysis and/or derivatization steps

Elimination of evaporation and reconstitution for solvent exchange

Reduction in the number of handling steps, thus reducing experimental error and improving
data quality
Consider, for example, the multiple sample preparation steps and analyses required for fat-
soluble vitamins in food. A typical sample workflow for analysis of vitamins A, D, and E in
food is shown in Figure 42. Notice that vitamins A and E require a different sample
preparation procedure as compared to vitamin D. Also, three separate HPLC analyses are
required (both normal and reversed-phase) for each vitamin. The sample preparation for
vitamin D analysis is particularly complex and can contain many steps, including a
semipreparative HPLC step in some cases.
Figure 42: Typical sample workflow for analysis of vitamins A, D, and E in
infant formula.

The sample preparation workflow for the analysis of the same vitamins using CC is much
simpler (Figure 43) because of the compatibility of the technique with the nonpolar organic
solvents used early in the extraction process. The sample is injected directly from the hexane
extraction to quantify vitamin E. It is then concentrated for analysis of vitamins A and D3,
making the run time twentyfold faster than traditional methods of analysis. In addition, the
use of CC requires only three sample preparation steps, one method, and a single
instrument, whereas the traditional workflow shown in Figure 42 requires 12 sample
preparation steps and three methods on two different instruments.

Figure 43: Sample workflow using CC for analysis of vitamins A, D, and E in


infant formula.

Table 7 summarizes the advantages of CC for the applications discussed, and highlights
other application areas where analytical scientists would benefit from similar simplification.
Example ACQUITY Other applications
UPC2advantage

Combining Single technique for Glyceride analysis in


multiple neutral and polar lipids food and fuels
techniques and
methods, lipids
No derivatization for Lipid profiling in drug
neutral lipids (needed for discovery studies
GC)

Faster baseline
separation than LC

Combining Direcly inject organic Pre-mixed and


mutiple methods, extracts formulated samples
fat soluble
vitamins, and
carotenoids One technique to replace Raw materials testing
RPLC and NPLC

Sample prepared by
LLE

Reducing sample Reduced sample prep Samples prepared by


prep and steps SPE, LLE, or protein
analysis times precipitation that need
evaporation and
Faster run times reconsitution

Higher overall Bioanalysis/DMPK


throughput

Directly injecting Direct injection of SPE


organic solvent extract No evaporation
extracts and reconsitution = less
experimental error

No solvent exchange
needed prior to analysis

Table 7: Benefits of using the ACQUITY UPC System for simplifying the
2

workflow.
Separation of Structurally Similar Compounds
Isomers and structural analogs can be challenging to separate due to small differences in
structure, especially for optical isomers. We now discuss the use of CC for the following
structurally similar compounds:

Chiral separations (enantiomers and diastereomers)

Positional isomers (differing in location of functional groups)

Structural analogs

Biomarkers (conjugated/unconjugated)

Drugs (metabolites, impurities, degradants)

Chiral Separations
Chiral separations are performed in a variety of applications, including pharmaceuticals and
agrochemicals. Often, different enantiomers of a compound can have varying potency and
toxicity profiles, and thus need to be monitored throughout the research, development, and
production phases. Chiral separations are predominantly performed by normal-phase LC
using cellulose or amylose-based stationary phases. In normal-phase LC, there is a limited
ability to perform gradient separations, and therefore separations are developed using
different, isocratic analyses on different columns, with different mobile phase combinations
and often with highly toxic solvents. This method development process can be quite time
consuming. With CC, the ability to run gradients and cover a wide selectivity space, with
nontoxic solvents, gives analytical scientists the ability to develop chiral separations in a
single day.
Figure 44: Separation of warfarin enantiomers by CC from plasma.

Purification chemists have recognized the value of SFC for this type of separation for many
years. Analytical separations have proved challenging, but are highly desirable, especially in
the areas of fast chiral screening, chiral method development, enantiomeric excess
determination, and chiral inversion studies. Unlike normal-phase LC, CC is highly compatible
with mass spectrometry detection, providing the ability to identify and characterize
enantiomers and their formation during reactions, manufacturing processes, and in biological
systems (Figure 44). Figure 45 compares normal-phase and convergence chromatography
for the separation of warfarin enantiomers. The enantiomers are baseline resolved in a
fraction of the time with convergence chromatography versus normal-phase (up to 30 times
faster). Also, the elimination of the toxic solvents, which can be costly both to purchase and
dispose of, can reduce the cost of chiral separations by as much as 100 fold per analysis. All
of these advantages make CC the preferred technique for chiral analyses of all types.
Positional Isomers and Structural Analogs
CC is also useful for the separation of positional isomers and other structural analogs.
Positional isomers are compounds with the same molecular weight (isobaric), but differ in the
location of their functional groups. They are often found in applications involving analysis of
starting materials, reaction monitoring, and asymmetric catalysis. Often, these compounds
are derivatized prior to GC analysis to aid in separation of the isomers. Normal-phase
methods suffer from an inherent lack of robustness and speed. The selectivity of separations
in CC enables positional isomers to be easily separated without derivatization under a
generic set of conditions. The ability to separate these isomers in such a rapid analysis time
on the ACQUITY UPC2 System (Figure 46) can help give near real-time assessment for
optimization of reaction starting materials, intermediates, and final products.

Figure 46: Separation of dimethoxybenzoic acid (DMBA) positional isomers


by CC.

Structural analogs are also difficult to separate due to their similarity to each other, and can
include biomarkers that can be conjugated or unconjugated (that is, glucuronides, sulfates),
as well as metabolites, degradants, and impurities of drug compounds. One of the most
popular classes of structural analogs is steroids (Figure 47). The similarity in structure
between different steroids makes them difficult to separate and analyze, even when using
MS detection, because of small mass differences.
Figure 47: Structures of unconjugated (free) steroids.

These can be easily resolved with CC using a generic screening gradient on multiple columns
in fewer than 2 minutes (Figure 48). This type of separation remains challenging for
reversed-phase LC because of the non-polar nature of the compounds, and GC requires
derivatization in order to improve peak shape and detection limits. Using the ACQUITY
UPC2 System, however, this separation can be easily coupled with MS detection for
identification and quantitation assays.

Figure 48: Separation of nine steroids using convergence chromatography.


Structural analogs are more difficult to separate when they are conjugated. Free steroids
(Figure 47) are water insoluble, so the body converts them to water-soluble derivatives by
converting them to their sulfated form. This process results in a negatively charged
hydrophilic side group, making them water-soluble (Figure 49). These types of compounds
are isolated from natural sources for therapeutic use, and can also be used as biomarkers to
study disease and determine efficacy of treatment. Analysis of these compounds presents
two major challenges. First, sample preparation for 30-minute GC analysis requires
enzymatic hydrolysis of the sulfate group followed by derivatization, which takes 2.5 hours.
Second, some of these estrogens are isobaric (same m/z) and thus cannot be distinguished
by mass spectrometry. This results in the need to separate different forms of the isobaric
compounds via chromatography.

Figure 49: Structures of sulfated estrogens. Molecular weights with the


same color are isobaric.

Using CC, all 10 sulfated estrogens can be separated in 15 minutes (Figure 50). This
separation includes two closely eluting peaks (that is, peaks 6 and 7) that can not easily be
separated by a GC analysis of over 30 minutes (Figure 51). With CC, the sample does not
have to be hydrolyzed and derivatized because the sulfated compounds can be analyzed in
their native form. This significantly reduces the amount of steps needed for analysis of
therapeutic formulations and thus increases throughput and productivity.
Figure 50: The separation of 10 sulfated estrogens using CC.

Figure 51: GC-FID separation of ten estrogens using the USP method for
conjugated estrogens. Sample preparation includes cleavage of the sulfate
group from the conjugated estrogen prior to chemical derivatization of the
sample. Total sample preparation time is greater than 2.5 hours. Two of the
compounds, peaks 6 and 7 (circled in red) are not completely resolved.

Table 8 summarizes the advantages of using CC for the applications discussed above, and
gives other application areas where analytical scientists would benefit from using this
technology for separating enantiomers, positional isomers, and structural analogs.

Application UPC2 Advantages Other Applications


Example

Enantiomers and Faster and cheaper than Chiral screening


diastereomers NPLC
(chiral
separations) Chiral method
development
10X faster, >85% solvent
savings

Chiral inversion
studies
Meets green initiatives

Enantiomeric excess

Positional isomers Faster separation than NPLC Starting materials


analysis

Low solvent usage and waste


production Reaction monitoring

Compatibility with NP Asymmetric catalysis


diluents and extraction (chemical materials)
solvents

Separation of both geometric


and enantiometric isomers

Structural analogs No derivatization required Non-polar biomarkers


steroids/estrogens (takes 2.5 hrs)

Natural product API


Faster and better separation and formulation
than LC or GC analysis

Resolution of conjugated
steroids

Directly compatible with MS

Table 8: Benefits of CC for separating structurally similar compounds.

Orthogonality
Orthogonal modes of separation are defined as having different relative retention of peaks.
The ability to separate peaks using different techniques is extremely important for the
following reasons:

Confidence that an impurity, degradation peak, or similar compound (e.g., isobaric


compounds or co-eluting compounds) can be identified and characterized
Ensuring full characterization of a sample

Ability to obtain more information about a sample

Separating desired compounds from matrix interferences


Examples of orthogonal separation techniques include complementary modes such as
normal- and reversed-phase chromatography. CC can provide a similar type of selectivity to
normal-phase chromatography, but is much more powerful as a separation technique
because of the robustness and reliability of the system (see Chapter 2), and the inherent
irreproducibility of normal-phase methods. The following section shows examples of how CC
can be used as an orthogonal mode of separation to achieve the goals listed above.

The separation of an active pharmaceutical ingredient (metoclopramide) from its related


substances using both ACQUITY UPLC and ACQUITY UPC2 systems demonstrates this
orthogonality (Figure 52). Peaks that are not resolved by one technique are resolved by the
other, and vice versa. CC also has the ability to provide longer retention for polar compounds
that are difficult to retain by reversed-phase (for example, peaks 1 and 2) chromagraphy. In
this example, the ACQUITY UPC2 System resolves the critical pairs (peak 5 and
metoclopramide), thus facilitating larger scale purification and isolation of unknown
compounds for subsequent identification and characterization. All this makes CC an ideal
technique to use in parallel with other, routine techniques to help solve a variety of
separations challenges.

Figure 52: Demonstrating orthogonality of convergence chromatography


with the separation of metoclopramide and related substances using the
ACQUITY UPLC and ACQUITY UPC systems. 2

Orthogonal methods of separation are important because of the potential for separating
analytes of interest away from matrix interferences, for instance in bioanalysis or food
analysis. Figure 53A shows a typical example of an LC-MS/MS chromatogram of clopidogrel
extracted from human plasma using protein precipitation. Due to the hydrophobicity of
clopidogrel, it elutes rather late during the analysis. Interfering phospholipids (with a
choline-containing head group) also elute in this same region (Figure 53B), potentially
causing ion suppression of the clopidogrel peak and variable results in quantitation.
Interestingly, the interfering phospholipids elute in about the same region on the ACQUITY
UPC2 System (Figure 53C). Due to the orthogonality of CC with respect to reversed-phase LC,
the analyte of interest elutes much earlier, and away from these interfering phospholipids
(Figure 53D). This minimizes the chance of matrix effects, ensuring more accurate and
precise quantitation.

Figure 53: Analysis of clopidogrel in human plasma after protein


precipitation using RPLC and ACQUITY UPC with MS detection (MRM mode)
2

systems.

Table 9 summarizes the advantages of CC for the applications discussed above, and gives
other application areas where analytical scientists would benefit from a technology offering
orthogonal separation.

Example UPC2 advantages Other applications

Full Different elution order Impurity/degradant


characterization than RPLC analysis
(Metoclopramide)

Resolves peaks no Agrochemical APIs and


resolved by LC formulations

Compatible with MS for Stability indicating


identification of methods (OLEDs)
unknowns
Non-polar compounds
that elute late in RPLC

Orthogonal to Direct injection of organic Complex mixture analysis


RPLC extracts (e.g., microwave
extraction)

MS compatibility

Ability to see Separation of isobaric Isobaric separations


more (isobaric) species

Separation from Analytes of interest are Bioanalysis, DMPK


matrix eluted away from matrix (hydrophobic compounds)
interferences,
bioanalysis
(Phospholipids) Less matrix interferences Other matrices containing
= less potential lipids (food, fuels, tissues)
suppression

More precise and


accurate quantitation

Table 9: Benefits of CC as an orthogonal mode of separation.

These benefits all illustrate the three key attributes of CC, as mentioned earlier in this
chapter:

1. Convergence chromatography SIMPLIFIES the workflow

a. Combines multiple techniques into one

b. Reduces sample preparation and analysis times

c. Can be used for direct injection of organic solvents/extracts

2. Convergence chromatography separates compounds with STRUCTURAL SIMILARITY

. Enantiomers (chiral)

a. Positional isomers

b. Structural analogs and conjugates

3. Convergence chromatography provides ORTHOGONALITY

. More confidence in identifying impurities/degradants

a. Full sample characterization


b. Separation of analytes from matrix interferences

Conclusion
Convergence chromatography is not designed to replace conventional liquid chromatography
or gas chromatography. Rather, it is a complementary technique to the other separations
methods used today.

The improvements made in the ACQUITY UPC2 System, over and above traditional analytical
SFC, finally make supercritical fluid separations robust and reproducible, so that CC can be
as routinely applicable as any other separation technique.

Convergence chromatography can be applied to many types of separation challenges, and


has proven to be a successful and viable solution to many chromatographers for many
different applications.

http://www.waters.com/webassets/cms/library/docs/local_seminar_presentations/sp_semin
ario_upc2_presentacion.pdf

https://www.quiminet.com/articulos/analisis-de-vitaminas-por-cromatografia-de-
convergencia-de-ultra-desempeno-2866101.htm

http://www.industriaalimenticia.com/articles/86127-anlisis-de-vitaminas-por-cromatografa

http://www.waters.com/waters/en_US/Convergence-
Chromatography/nav.htm?cid=134717180&locale=en_US

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