On-column solvent exchange for purified preparative fractions

1. On-Column Solvent Exchange
for Purified Preparative Fractions
Gilles H. Goetz,1* Emily Beck,2 and Peter W. Tidswell1
1Pfizer Global Research and Development, Analytical Chemistry and Sample Logistics,
St Louis Laboratories, Chesterfield, MO
2School of Medicine, Washington University, St Louis, MO
On-column solvent exchange, using many of the
principles of solid-phase extraction, has been im-plemented
to significantly reduce evaporation cycle time
following reverse-phase preparative HPLC. Additional
benefits, such as a reduced potential for salt formation,
thermal decomposition, and residual solvent, are also
described.
Fractions obtained from preparative separations,
typically in a large volume of acetonitrile:water, are
injected into the preparative HPLC and then eluted in
acetonitrile, creating a new fraction in a volatile organic
solvent. Minimal modification to the instrument was
required, and unattended operation is possible.
Acetonitrile evaporation is achieved within 3 h, compared
with 17 h for aqueous-based fractions; lower
temperatures can be used during the evaporation step;
mobile-phase additives, likely to form salts with the target
compound if concentrated in the fraction, are removed
before evaporation; sample recovery and purity are
unaffected. (JALA 2011;16:335–46)
INTRODUCTION
Preparative reversed-phase chromatography has
become the purification method of choice in the
pharmaceutical industry for small-molecule early-discovery
research. Centralized purification groups
relying heavily on automation are often responsible
for bridging the gap between medicinal chemists
and biologists. Along with customized separation
conditions, the use of mass-directed fractionation
enables the isolation of the target compound from
the crude mixture effectively and efficiently. Focus-ing
on cost- and time-reduction, these centralized
automated platforms provide a high-quality and
high-throughput purification platform (Fig. 1).
Within this environment, post-purification
workup of the collected fractions (including evapo-ration,
weighing, reconstitution, and reformatting
procedures) can be time-consuming activities but
must be performed accurately to ensure that the final
product is of high quality. In our laboratories, the fi-nal
product is formulated as a stock solution at
a standard, known concentration with supporting
purity and identity data. It is then sent for immedi-ate
testing by project teams, and any remaining ma-terial
is held for longer-term storage.
Of the various postpurification procedures, evap-oration
to dryness of aqueous/organic matrices is
the most time consuming and represents the bottle-neck
of the whole purification operation (within
our laboratories). During this procedure, the col-lected
target material must be recovered intact from
a solution containing low-volatility solvents (partic-ularly
water). Precautions against sample decompo-sition,
salt formation, and residual solvent must be
taken, because any errors in this step will affect the
purity and/or concentration of the final product.1
Previous reports, mirroring our experiences, have
shown that the chromatography-enhancing additives
Keywords:
fraction
concentration,
solvent exchange,
solid-phase
extraction,
preparative
chromatography
*Correspondence: Gilles H. Goetz, Ph.D., Pfizer Global Research
and Development, Groton laboratories, Eastern Point Road, MS
8118W-305, Groton, CT 06340, USA; Phone: þ1.860.715.6311;
Fax: þ1.860.715.3345; E-mail: gilles.h.goetz@pfizer.com
2211-0682/$36.00
Copyright c 2011 by the Society for Laboratory Automation and
Screening
doi:10.1016/j.jala.2010.02.004
Original Report
JALA October 2011 335

2. Original Report
routinely used during preparative liquid chromatography
(LC) separations (formic acid, trifluoroacetic acid (TFA),
ammonium hydroxide, and others) get concentrated in the
fractions during the evaporation process, potentially leading
to salt forms of the target base2 (or, less commonly, acid), to
hydrolysis of some functional groups (e.g., esters) or deriva-tization
of others (e.g., formylation of amines).
The evaporation procedure used in our laboratories is an
incompressible overnight run using reduced-pressure and
elevated-temperature programs in Genevac HT24 evapora-tors
(Genevac, Gardiner, NY). These programs have been
shown to isolate the target material consistently as a dry solid
from the solvent matrix (water, organic solvent, and volatile
additive). These programs cannot, however, prevent the for-mation
of salts and, in rare cases, the degradation of the final
product.
To reduce the time taken for solvent evaporation, avoid
salt formation, and reduce exposure to heat (while still ensur-ing
that no residual solvent remains), an investigation into
the removal of water (and HPLC additives) from the frac-tions
before the evaporation step was initiated. Various crite-ria
were defined, not least that the quality of the final product
must be maintained or improved, little or no capital invest-ment
would be required, the existing capabilities of instru-ments
would be maintained, and the overall integrity of the
purification platform would not be compromised.
Other authors have recently described solid-phase extrac-tion
(SPE) approaches for handling reverse-phase prepara-tive
HPLC fractions.3e5 In this study, the Waters (Milford,
MA) on-column SPE concept5 is adapted and customized,
and the term on-column solvent exchange is used to describe
the procedure of loading fractions back onto the preparative
HPLC column under aqueous conditions before eluting
them with acetonitrile to yield a water- and additive-free
fraction.
EXPERIMENTAL
Instrumentation
Instrument for Initial Analysis of Crude Compounds. Two
identical systems consisting of four Waters 1525 micro binary
gradient pumps, Waters 2777 sample manager, Waters 2488
dual-wavelength absorbance detector, Waters MUX inter-face
and system controller, Waters ZQ mass spectrometer,
and four Polymer Labs (Shropshire, UK) PL-ELS2100 evap-orative
light scattering detectors were used.
Preparative Purification Instrument. Waters 2525 binary gra-dient
pump, three Waters 515 HPLC pumps (for at-column-dilution,
modifier and MS make-up solvent delivery), Waters
pump control module, Waters 2767 sample manager (injec-tionecollection),
Waters 2757 sample manager (collection),
two Waters 2487 dual-wavelength absorbance detectors,
ZQ mass spectrometer, Waters column selector, Waters
selector valve (for switching injection mode), two Waters sol-vent
selection valves (for additive selection and at-column-dilution
Genevac
Previous Process
Dry
Powder
17 h
25 min
C 18 Column
8 min 17 h
DMSO Water/MeCN
At Column Solvent Exchange Process Dry
C 18 Column C 18 Column
8 min 5 min
Genevac
3 h
DMSO Water/MeCN MeCN
Powder
3 h
13 min
Figure 1. At column solvent exchange process improvements.
push solvent selection), and Waters flow splitter
(1000:1) made up the preparative instrument. The instrument
was controlled using MassLynx, version 4.1 SCN627 (Wa-ters,
Milford, MA).
Software and Controls. Control of the preparative HPLC
components was accomplished through MassLynx, version
4.1 SCN627, operating with the FractionLynx application
manager (Waters, Milford, MA). MassLynx controls the
pumps, detectors, and the injection arm of the sample man-ager,
and FractionLynx controls the fraction collection part
of the sample manager. The MassLynx sample list provided
all the relevant information for the system to perform an in-jection,
such as method, sample location, and injection vol-ume.
Additional columns were added to the sample list to
indicate the specific target mass or masses for the fraction
collection software to monitor. Once the sample was injected
and the appropriate masses entered into the sample list, the
collection software monitored for the target mass. When
the target mass was identified, the collection software di-verted
the solvent flow through the fraction collector diverter
valve to the waiting fraction tube. The collection software
that operates the system does not differentiate between
a standard collection routine and this trap and elute routine.
The instruments in the system were controlled through the
standard MassLynx software through General Purpose In-terface
Bus (GPIB), Institute of Electrical and Electronics
Engineers, New York (IEEE), Ethernet, and serial port com-munication.
The ultraviolet (UV) detectors operated under
IEEE, the primary pump and mass spectrometer were under
Ethernet control, and the sample managers were controlled
through serial ports. The three 515 pumps, the selector valve,
the solvent modifier selection valve, and the column selector
were all controlled by contact closures from the primary
pump, and the watereacetonitrile solvent selection valve
was operated off-line manually.
Evaporation Systems. Two identical HT24 evaporators were
used for the routine removal of solvents from HPLC
fractions.
336 JALA October 2011

3. Reagents and Materials
Mallinckrodt Baker (Philipsburg, NJ) acetonitrile 20 and
4 L, HPLC grade; and EMD Chemicals (Gibbstown, NJ) wa-ter
20 and 4 L, HPLC grade, were used. SigmaeAldrich (St.
Louis, MO) ammonium hydroxide American Chemical Soci-ety
(ACS) grade reagent and trifluoroacetic acid (TFA) and
Mallinckrodt Baker formic acid 88% ACS reagent were used.
Waters X-Bridge C18 2.1 50-mm, 5-mm column; Waters
SunFire C18 2.1 50-mm, 5-mm column; Waters X-Bridge
Prep C18 optimum bed density (OBD) 19 50-mm, 5-mm
column; Waters X-Bridge Prep C18 OBD 19 10-mm, 5-
mm precolumn; Waters SunFire Prep C18 OBD 19 50-
mm, 5-mm column; Waters SunFire Prep C18 OBD
19 10-mm, 5-mm precolumn; Waters X-Bridge Prep C18
OBD 30 100-mm, 5-mm column; Phenomenex (Torrance,
CA) AXIA Gemini C18 110A 21.2 50-mm, 5-mm column;
Phenomenex AXIA Gemini C18 110A 21.2 10-mm, 5-mm
precolumn; and Waters experimental hydrophilic-lipophilic-balanced
(HLB) 19 50-mm, 30-mm column, were used.
Hardware Configuration
Preinjector Fluidic Pathway. Two modifications to the in-strument
were performed; the first was the introduction of
a solvent selection valve for the at-column-dilution pump
to allow the sample to be loaded onto the column with either
acetonitrile (in preparative mode) or water (in the solvent-exchange
mode). In both modes, this pump delivers 20%
of the total flow through the instrument when the at-column-
dilution injection protocol is used.
The second modification inserted a six-port switching valve
ahead of the in-line T used for at-column-dilution injections.
This valve is connected to the column, the sample loop, the
2525 pump, and the at-column-dilution pump; it is controlled
by MassLynx through contact closures configured in the inlet
editor and allows for automated switching between the prepar-ative
and solvent-exchange modes (Fig. 2).
Autosampler Valve and Syringe Mechanism. A second sam-ple
loop (20 mL), to inject the fractions during the solvent-exchange
procedure, was installed alongside the original loop
(2 mL) for preparative separations. Both loops can be filled
with the required accuracy and speed using a 10-mL syringe.
To maintain autosampler accuracy, the syringe speed is set to
aspirate and dispense at 10% maximum motor power
(speed).
Autosampler Transfer Tubing. The transfer line volume be-tween
the syringe and the needle on the autosampler (also
known as the holding loop) was increased from 1.5 to
15 mL to accommodate the larger volumes of sample being
aspirated in the on-column solvent-exchange mode.
Collection Delay Timing
To minimize the volume of water collected during the
solvent-exchange procedure, a suitable delay time (from
Injector
Sample Loop
Original Report
1ml/min
detection of the compound in the MS to fraction head acti-vation)
was found to be 15 s.
Methods
Initial Analysis of Crude Research Compounds. Aliquots
(10 mL; 0.1% of the submitted volume) of the crude
compounds were diluted with DMSO (140 mL) to create
analytical samples with a concentration typically between 1
and 2 mg/mL.
For acidic LC conditions, formic acid (0.1% v/v) was
added to both the aqueous and acetonitrile mobile-phase res-ervoirs,
and the SunFire C18 2.1 50-mm, 5-mm column was
installed on the instrument.
For basic conditions, ammonium hydroxide (100 mM)
was added to both the aqueous and acetonitrile mobile-phase
reservoirs, and the X-Bridge C18 2.1 50-mm, 5-mm
column was installed on the instrument.
Under both conditions, an isocratic hold at aqueous:or-ganic
(95:5) for 1 min was followed by a linear gradient to
aqueous:organic (5:95) during 6.75 min and an isocratic hold
at the final set point for 1 min. The instrument was then re-turned
to the initial conditions and equilibrated for 1 min.
The chromatographic run time was 10 min, and the overall
duty cycle was 10.5 min because of the ‘‘inject ahead’’ feature
of the autosampler. The method’s flow rate was 1 mL/min;
the injection volume was 10 mL; UV detection performed at
214 nm, and the Evaporative Light Scattering Detector
(ELSD) nebulizer temperature at 40 C.
Preparative Separation of Flavone. A known accurate weight
of flavone (approximately 1 g) was dissolved in DMSO and
then diluted to create a stock solution, typically at either
the 30- or the 50-mg/mL level. Aliquots (1.2 mL) were trans-ferred
to the matrix tubes with prepierced septa, and 1 mL
was injected into the instrument.
Main Gradient
Pump
H2O and MeCN
Load
Modifier
Column
Make Up
Splitter
Waste
MS
UV
UV
Primary
Fraction Lynx
Collection
Valve
Valve
MeCN
H2O
26.5 ml/min
2.5ml/min
Secondary
Fraction Lynx
Collection
1ml/min
Figure 2. Schematic of the modified Waters FractionLynx Instru-ment
configured to perform both preparative separations and
on-column solvent exchange using direct-injection and at-column-
dilution (with acetonitrile or water) injection protocols.
JALA October 2011 337

4. Original Report
For acidic LC conditions, the modifier pump delivered
3% aqueous formic acid at 1 mL/min ahead of a SunFire
Prep C18 OBD 19 50-mm, 5-mm column, equipped with
a guard column (30 10 mm, 5 mm). For basic conditions,
the modifier pump delivered 3% aqueous ammonium hy-droxide
at 1 mL/min ahead of an X-Bridge Prep C18 OBD
30 100-mm, 5-mm column, equipped with a guard column
(30 10 mm, 5 mm).
Under both conditions, the at-column-dilution solvent se-lector
valve was used to select the acetonitrile reservoir
(Fig. 3A). The at-column-dilution pump continuously deliv-ered
acetonitrile (2.5 mL/min) through the injection valve;
the modifier pump continuously delivered an aqueous solu-tion
of the additive (1 mL/min), and the 1525 main pump
delivered a binary gradient (26.5 mL/min) for a total flow
of 30 mL/min. An isocratic hold at aqueous:organic (85:15)
for 1 min was followed by a linear gradient to
aqueous:organic (35:65) during 5.75 min, a column wash in
acetonitrile for 1 min, and then a return to the initial condi-tions.
Column equilibration occurred during the subsequent
aspirate/dispense cycle(s). The run time was 8 min, and the
overall duty cycle was 10 min.
Fractions were collected in tared 18 100-mm glass tubes,
evaporated in an evaporator (see later for conditions), the
gross weight was measured, and the net weight was calcu-lated.
All fraction tube weights were recorded on the same
customized Tecan EVO workstation (Tecan Group, Ma¨nne-dorf,
Switzerland).
Preparative Separation of Caffeine. A known accurate weight
of caffeine (1 g) was dissolved in acetonitrile:water (1:1) and
then diluted to create a stock solution, typically at either the
30- or the 50-mg/mL level. Aliquots (1.2 mL) were trans-ferred
to matrix tubes with prepierced septa, and 1 mL was
injected into the instrument.
Conditions and practices identical to those used for the
separation of flavone were used, except for the gradient pro-file.
An isocratic hold at aqueous:organic (100:0) for 1 min
was followed by a linear gradient to aqueous:organic
(80:20) during 5.75 min, a column wash in acetonitrile for
1 min, and then a return to the initial conditions. Column
equilibration occurred during the subsequent aspirate/dis-pense
cycle(s). The run time was 8 min, and the overall duty
cycle was 10 min.
On-Column Solvent Exchange Using Direct Injection. A
known accurate weight of either flavone or caffeine (approx-imately
1 g) was dissolved in acetonitrile:water (1:1) to create
a stock solution, typically at either the 50- or the 70-mg/mL
level. Aliquots (variable volume) were transferred to
18 100-mm fraction tubes and then diluted to the desired
concentration with acetonitrile:water (1:1; unless otherwise
stated) for a final volume of 10e15 mL.
These samples were injected into the instrument. Research
samples, as fractions (from preparative experiments; up to
18 mL of total volume), were separately injected into the in-strument.
For each injection cycle, samples were aspirated in
aliquots (10 mL) and dispensed into the injection port; mul-tiple
aspirate/dispense cycles before injection valve switching
were required for volumes greater than 10 mL.
The 1525 main pump continuously delivered water
(30 mL/min) through the injection valve ahead of either an
X-Bridge Prep C18 OBD 30 100-mm, 5-mm column, equip-ped
with a guard column (30 10 mm, 5 mm) a Gemini C18
110A 21.2 50-mm, 5-mm column, equipped with a guard
column (21.2 10 mm, 5 mm) or a Waters experimental
HLB (19 50 mm, 30 mm) column. The at-column-dilution
and modifier pumps were turned off.
An isocratic hold in water for 2 min was followed by an
isocratic hold in acetonitrile for 3 min and then a return to
the initial conditions. Column equilibration occurred during
the subsequent aspirate/dispense cycle(s). For a typical frac-tion
volume of 15 mL, the overall run time was 5 min, and
A Column
Main
Gradient
Pump
B
Acetonitrile Water
At-Column
Dilution
Pump
ON
Sample
Loop
Main
Gradient
Pump
Column
Acetonitrile Water
At-Column
Dilution
Pump
ON
Sample
Loop
Figure 3. (A) Pumping configuration in preparative mode using
at-column-dilution injection protocol. (B) Configuration in on-column
solvent-exchange mode using at-column-dilution injection
protocol.
338 JALA October 2011

5. the overall duty cycle was 9.5 min. Post-purification practices
identical to those described earlier measured the final frac-tion
weight gravimetrically.
On-Column Solvent Exchange Using At-Column Dilution.
Flavone, caffeine, and research samples were prepared and
dispensed into the sample loop as described earlier. The same
columns were also installed on the instrument.
The at-column-dilution solvent selector valve was manu-ally
switched over to the water reservoir (Fig. 3B). Initially,
the at-column-dilution pump delivered water (6 mL/min)
through the injection valve, the 1525 main pump delivered
water (24 mL/min) directly to the head of the column, and
the modifier pump was turned off. These conditions are
maintained during the injection cycle (6 min). The at-column-
dilution pump was then turned off, and the 1525
main pump delivered water for two more minutes followed
by acetonitrile (30 mL/min) for 3 min to elute the fraction.
For a typical fraction volume of 15 mL, the overall run time
was 11 min, and the overall duty cycle was 13 min. Postpur-ification
practices identical to those described earlier mea-sured
the final fraction weight gravimetrically.
Evaporation
A new short Genevac HT24 program to support the
solvent-exchange process consists of two parts. In the first
part, rapid ramping from ambient conditions to 250 mbar
at 40 C, then slower ramping to 50 mbar over 15 min, before
rapid ramping to 11 mbar for 45 min, occurs; the second part
is a full vacuum stage lasting 2 h at 40 C. The total cycle
lasts for approximately 3 h.
RESULTS AND DISCUSSION
Effects of Changing the Fluidic Pathway
This study was undertaken while attempting to keep an
existing preparative HPLC instrument as close to its opti-mized
configuration as possible. All modifications performed
needed to have a low impact on the existing purification plat-form.
For example, modifications could only minimally
reduce sample recovery (O90%) and cycle time (!2.5 days).
No reduction in final product purity (O80% by UV detec-tion,
O85% by ELSD detection, and a 1H NMR spectrum
consistent with these purity results) or increase in manual in-tervention
could be tolerated. Additionally, the instrument
had to remain compatible with the existing infrastructure
and work streams.
At the beginning of this study, the instrument was config-ured
to deliver an aqueous/organic gradient from the master
pump (2525 pump), with additives being introduced after
mixing by a 515 pump and the sample being injected from
a 2767 sample manager using a 515 pump and an at-column-
dilution protocol (with acetonitrile as the push sol-vent).
Postcolumn UV detection preceded a 1000:1 flow split,
with most of the flow being returned to the 2767 sample man-ager
for collection, and the remainder of the flow being
Original Report
diluted with acetonitrile:water:formic acid (60:40:0.1) and en-tering
the ZQ mass spectrometer. The MS triggered the 2767
fraction collector. When this primary fraction collector was
not active, material entered the waste stream, where the sec-ond
UV detector (2487 dual-wavelength absorbance detec-tor)
triggered the second fraction collector (2757 sample
manager) when a UV response was detected. This secondary
collection method ensures that any failure of the MS trigger-ing
components does not result in sample loss. It has been in
use successfully in our laboratories for more than 2 years and
is similar to the approach described by FitzGibbons et al.6
The six-port switching valve was installed to enable the in-strument
to be automatically switched from the direct-injection
mode (using the 2525 pump to flush the injection
loop) to the at-column-dilution injection mode.
The first position of six-port switching valve installed dur-ing
this study supports the at-column-dilution injection pro-tocol.
7 In this mode, the 515 pump delivers a push solvent
through the injection loop, and the 2525 pump delivers the
initial conditions to the head of the column. In this way,
the sample is flushed from the injection loop with solvent
and diluted with the flow from the 2525 pump immediately
before entering the column. For preparative separations,
the organic modifier (e.g., acetonitrile) is typically used as
the push solvent (Fig. 3A). This valve setting can also be used
in the solvent-exchange mode for polar compounds, because
a fivefold dilution with water is needed to ensure retention on
the column (Fig. 3B; this is described in more detail later).
The second position of six-port switching valve supports di-rect
injection of the sample. In this mode, the 2525 pump de-livers
the initial conditions through the injection loop to the
head of the columnwhen loading the sample (Fig. 4). Thismode
is not normally used for injection of crude samples for prepara-tive
separation in our laboratories, but was implemented for use
with the on-column solvent-exchange procedure.
The installation of the valve allows the instrument to be
switched between the preparative mode and solvent-
Main
Gradient
Pump
Column
Acetonitrile Water
At-Column
Dilution
Pump
OFF
Sample
Loop
Figure 4. Configuration in on-column solvent-exchange mode
using direct-injection protocol.
JALA October 2011 339

6. Original Report
exchange mode without operator intervention, using a con-tact
closure within MassLynx methods. The instrument
can, therefore, run both preparative separations and the sub-sequent
solvent-exchange procedures unattended.
The addition of the solvent selection valve enables the at-column-
dilution pump to readily switch between acetonitrile,
for at-column-dilution injection of the crude compound for
preparative separation, and water, for at-column-dilution
of the purified compound in the on-column solvent-exchange
mode. This valve was installed on the low-pressure side of the
pump head and had no appreciable effect on the performance
of the injection protocols, chromatographic separation, or
overall instrument performance.
Insertion of the 15-mL transfer line on the autosampler
was required to support the larger volumes of sample being
aspirated and dispensed in the on-column solvent-exchange
mode. The effects on both carryover between modes of oper-ation
and injections as well as sample recovery were not mea-sured
specifically; however, they were measured as part of the
overall instrument performance and are described in the fol-lowing
sections.
Sample Recovery Measurements
One of the most important criteria defined during this study
was to ensure that high sample recoveries of the final product
were maintained. Two separate potential losses of material can
occur when the on-column solvent-exchange procedure is in-cluded
in the overall purification workflowdduring the pre-parative
separation and the solvent-exchange procedure.
Sample recovery from both modes of operation was assessed
to derive an overall recovery measurement.
Sample Recovery During Preparative Liquid Chromatography.
Instrument recovery is routinely verified for all preparative
instruments in our laboratories using a gravimetric assay
of flavone. Figure 5 shows the measured sample recoveries
from two instruments; one instrument (triangles) was modi-fied
to perform on-column solvent exchange during June
2008, and the second instrument was not modified. This
study was completed in October 2008, and both instruments
were subsequently used to support the purification platform.
The sample recovery measured from both instruments is
consistently greater than 85% and typically at or above
90%. Although it could be argued that the unmodified in-strument
recovers more material, the differences are small
and have little effect on the success of the purification
platform.
Autosampler Syringe Volume Selection. The 2767 autosam-pler
supports two fluidic pathways with a single syringe
and drive mechanism. The purification platform in our labo-ratories
has been optimized for the injection of mixtures sub-mitted
in 1 mL of solvent (normally DMSO); hence, it is
critical that the entire sample is injected on to the column
to maximize sample recovery. Therefore, the 2-mL injection
Figure 5. Measured recoveries of flavone from an instrument
modified to perform on-column solvent exchange (triangles) and
an unmodified instrument (squares).
loop currently installed on the autosampler was retained in
one of the flow paths. Based on the maximum volume col-lected
in a single fraction tube (18 mL), a 20-mL injection
loop was selected for the second flow path to support on-column
solvent exchange.
With such a large volume difference between the two flow
paths, the control of two different syringes to fill the loops is
preferred. Unfortunately, this configuration is not supported
within the instrument-control software; hence, a single
syringe capable of accurately filling both loops had to be
selected.
Measuring the gravimetric recovery of flavone (30 mg in
DMSO; 1 mL) during the preparative separation, 25- and
10-mL syringes were compared with the original 1-mL
syringe. Figure 6 shows that nearly-identical sample recov-eries
were observed with the 10- and 1-mL syringes, whereas
sample recovery was much lower when the 25-mL syringe
was used, because the speed of the syringe aspirate and dis-pense
steps could not be adequately controlleddsee the fol-lowing.
Because one criterion for the acceptable performance
of on-column solvent exchange was to maintain purification
recovery levels, we sacrificed loading speed (faster using the
25-mL syringe) for sample recovery (higher when using the
10-mL syringe). This compromise requires any fraction con-taining
a volume greater than 10 mL to be loaded using two
aspirate and dispense cycles before the valve switching.
Effects of Altering the Autosampler Syringe Drive Mechanism.
The 2767 sample handlers enable customization of the sy-ringe
plunger speed as a percentage of full motor power.
When aspirating fractions at full speed, during the solvent-exchange
procedure, a partial vacuum was created inside
the syringe, because the liquid could not travel through the
holding loop fast enough. This resulted in a large portion
of the sample remaining in the fraction tube. Similarly, when
dispensing into the loop at full speed, the sample leaked from
the injection port. Combined, these sample losses signifi-cantly
reduced sample recovery.
340 JALA October 2011

7. Visual inspection of the sample still present in the fraction
tube after completion of the injection cycle demonstrated
that a syringe motor speed greater than 30% could not be
used. To further refine this parameter, recovery of flavone
(50 mg in acetonitrile:water [1:1] 15 mL) was measured at
motor speeds of 10% and 30% after the sample been eluted
from the column in the solvent-exchange mode (Fig. 7).
Sample recovery was closely correlated to both the aspira-tion
and dispensing speeds, with slower motor speeds leading
to higher sample recoveries. However, this slower motor
speed increased the length of the overall injection cycle from
6 to 7 min. For our application, this increase in cycle time
was an acceptable compromise to ensure that high sample
recoveries were obtained, and the motor speed selected was
the 10% level.
Overall, installing a 10-mL syringe and using aspirate and
dispense speeds at 10% of the maximum motor power
Original Report
ensured that more than 90% of the sample was recovered af-ter
both the preparative and on-column solvent-exchange
procedures were completed.
Effects of Using Multiple Aspirate and Dispense Cycles. The
utility of multiple aspirate and dispense cycles, required be-cause
the autosampler syringe volume (10 mL) selected was
approximately half the size of the injection loop (20 mL),
was investigated. This injection protocol did not have any
impact on sample recovery when loading the contents of
a single fraction tube (Fig. 7); hence, sample recovery was
measured after loading from separate fraction tubes. Three
separate flavone samples (15 mg in acetonitrile:water [1:1]
15 mL) were loaded into the loop, then injected onto the col-umn
as a single injection using the direct-injection protocol,
and eluted into a single tube using acetonitrile. 43.2 mg of fla-vone
was recovered, representing a 97% sample recovery.
This high level of sample recovery permits the on-column sol-vent-
exchange mode to be used to pool different fractions if
desired.
Retention Behavior of Compounds on Column. Previous
reports3e5 have used a fivefold aqueous dilution of fractions
before or during the injection protocol to ensure that com-pounds
are retained by the C18 column and do not break
through with the solvent front.
The results from this laboratory showed that, for the over-whelming
majority of compounds purified, no aqueous dilu-tion
of the sample was required, and the sample could be
retained on column using a direct-injection protocol. Only
three compounds from a set of 130 samples, representing
11 different research projects and 30 chemotypes, were not
retained and ‘‘broke through’’ to the solvent front partially
or completely when using the direct-injection protocol to
load, and these three samples could be successfully loaded
(with no breakthrough) using the at-column-dilution
protocol.
These three compounds were the most polar compounds
in the test collection and to mimic this breakthrough behav-ior
we selected caffeine, a highly polar and water-soluble
compound, to act as a model compound. By developing
a method that would retain caffeine, the retention of polar
research compounds from our laboratories would be
possible.
Samples of caffeine were prepared in various acetonitrile:
water ratios and then loaded onto the column using either
the direct-injection protocol or the at-column-dilution proto-col
(with water acting as the push solvent). Figure 8 shows
that the C18 column did not retain caffeine using either injec-tion
protocol when the acetonitrile content was greater than
40% and that caffeine was fully retained by both injection
protocols when the acetonitrile content was 10% or less.
When the acetonitrile content was between 10% and 40%,
caffeine was completely retained only using the at-column-dilution
injection protocol; hence, this injection protocol
should be used for the loading of polar compounds.
Figure 6. Recovery of flavone (30 mg in DMSO; 1 mL) from
a preparative separation using different syringe volumes to perform
the injection. Each bar represents an average of three replicate
injections.
Figure 7. Recovery of flavone (50 mg in acetonitrile:water [1:1]
15 mL) in the on-column solvent-exchange mode, using a 10-mL
syringe with different aspirate and dispense speeds. Each bar rep-resents
an average of four replicate injections.
JALA October 2011 341

8. Original Report
During this study, the duty cycle of each injection proto-col
was monitored, and the at-column dilution protocol was
found to be approximately three times longer than the direct-injection
protocol. The reason for this is that, to achieve
a fivefold dilution of the sample with water, the flow through
the loop is approximately 20% the total-system flow rate
when the at-column-dilution injection protocol is used (com-pared
with 100% of the flow when the direct-injection proto-col
is used). In our laboratories, this equates to a 6-mL/min
flow through the loop and a 30-mL/min total system flow
rate. Under these conditions, the time taken to flush the
20-mL injection loop is approximately 3.5 min, and after
allowing for transport time to the column (including any
broadening of the injection band), equilibration and wash-ing,
a total injection cycle time of 6 min was determined as
optimal. When loading using the direct-injection protocol,
the entire 30-mL/min flow washes the contents of the injec-tion
loop onto the column, allowing complete loading of
the sample in less than 2 min. For polar samples, this in-crease
in duty cycle (and hence, the length of the overall
method) is acceptable, because the corresponding higher
sample recovery is critical; however, for less-polar samples,
where high sample recoveries can be obtained with either
protocol, the direct loading protocol is preferred.
To appropriately select the injection protocol needed for
each sample, the retention time observed during initial anal-ysis
of the crude sample was used. In our laboratories, the
preparative LC method typically uses a focused gradient de-signed
to elute the target compound with a retention time
between 5 and 6 min (using an 8-min gradient). Selection
of this method is based on the observed retention behavior
of the intended target material and impurities during an ini-tial
analysis of the crude material. This initial analysis is per-formed
using a generic gradient of acetonitrile in water from
5% to 100%, on two identical instruments, one containing
the additive 0.1% formic acid, the other 0.1% ammonium
hydroxide. From these analyses, the presence of the target
analyte and its retention time are determined, enabling selec-tion
of the appropriate gradient for the preparative run. Be-cause
this retention time is related to the molecule’s
hydrophobicity,8 the retention time can also be used to pre-dict
the molecules that are too polar to retain on column if
a direct-injection protocol is used in the on-column solvent-exchange
mode.
Correlating the retention time of the set of 130 representa-tive
samples with their respective retention time from the ini-tial
analysis identified the polar samples that would require
the use of at-column-dilution injection protocol. Thirteen
samples (10% of the set) eluted within 3 min (of the 8-min
gradient; approximately 40% acetonitrile) during initial anal-ysis;
of these, 10 could be successfully injected onto the
column using direct injection, and three required the at-column-
dilution injection protocol. A conservative retention
time threshold of 3 min was, thus, established, to ensure that
no samples would break through during the injection proce-dure.
Figure 9 shows a representative chromatogram from
one of the three polar compounds requiring the at-column-dilution
injection protocol during the on-column solvent-ex-change
procedure.
Conversely, a research sample too lipophilic to elute off
the column during on-column solvent exchange is yet to be
encountered. Because the compound must be eluted from
the column during the preparative separation before on-column
solvent exchange, this problem is not anticipated to
occur with any frequency.
Collection Delay Timing. Collection parameters were opti-mized
to minimize the volume of water that was collected
with the target compound without compromising sample-recovery
levels. This principally involved setting a delay time
between target detection by the mass spectrometer and col-lection
valve triggering. During initial instrument
Figure 8. Retention behavior of caffeine (10 mg in various aceto-nitrile:
water matrices; 10 mL) using direct-injection and at-column-dilution
protocols. Each bar represents an average of two replicate
injections.
Figure 9. Chromatograms of a polar research sample loaded
using the direct-injection protocol (top; approximately 75% break-through)
and at-column-dilution injection protocol (bottom; no
breakthrough).
342 JALA October 2011

9. qualification in our laboratories, this delay time is established
with a colored performance test mixture, but for ongoing
performance verification (and this study), sample recovery
was determined gravimetrically using flavone.
The volume of water collected in the fraction tube was
measured visually against a graduated fraction tube after
the first stage of the new evaporation program was complete.
The recovery of flavone was measured gravimetrically after
completion of the entire HT24 program. The results are
shown in Figure 10.
More than 90% of the injected flavone was recovered
when the delay time was between 7 and 15 s. No water was
visible for delay times more than 14 s. Therefore, a delay time
of 15 s was selected, to maximize sample recovery and mini-mize
residual water content.
Correlation of Sample Recovery With Mass. To mimic sam-ples
typically received in our laboratories, accurately weighed
known amounts of flavone ranging from 5 to 300 mg were
dissolved in acetonitrile:water (1:1) in volumes ranging from
10 to 15 mL and prosecuted through the on-column solvent-exchange
procedure using the direct-injection protocol. The
lower-weight samples, from 5 to 100 mg, were loaded onto
a 19 50-mm, 5-mm column using a 30-mL/min flow rate;
the higher-weight samples, from 50 to 300 mg, were loaded
onto a 30 100-mm, 5-mm column using either a 30-mL/
min flow rate (dark diamonds in Fig. 11) or a 75-mL/min
flow rate (light squares). After collection, all the samples
were dried, weighed, and the sample recovery was calculated.
For samples weighing more than 100 mg, sample recov-eries
at or above 90% were observed; for compounds weigh-ing
less than 100 mg, sample recoveries greater than 85%
were observed for most samples. The variability in sample
recovery increased as sample mass decreased for reasons that
are not clear. A common rationale for decreased sample
recovery is that a consistent amount of material is lost
Original Report
(e.g., at the start and the end of the peak) in every injection,
and this loss represents a higher percentage of material as the
total-sample weight decreases. This could explain the lower
recoveries observed when using smaller samples; however,
many recovery values greater than 95% were observed from
essentially identical samples. The robustness of instrument
performance can also affect sample recovery; this data set
was acquired over a period of 2 months and (as shown in
Fig. 5), recovery varies over time independent of the sample
type. These variations are frequently attributed to column
and precolumn conditions, timing adjustments, minor
repairs, and other preventative maintenance procedures,
although no specific investigation was conducted as a part
of this study. Ultimately, recovery values greater than 85%
were regarded as acceptable within our application. Interro-gation
of this data set of 189 recovery experiments did not
show any significant differences in sample recovery between
samples prepared in 10 and 15 mL of solvent (data not
shown).
Total Sample Recovery. Combining the sample recovery re-sults
from the preparative separation with those from the
on-column solvent-exchange procedure showed that between
80% and 90% of the available sample could be reliably re-covered
from the overall workflow. These results were vali-dated
by directly measuring compound recovery through
the entire workflow (Fig. 12).
Injection Carryover
Carryover was assessed by bracketing a solvent blank
(acetonitrile:water [1:1] 10 mL) between flavone fractions
(acetonitrile:water [1:1] 10 mL). The mass of flavone was
Figure 10. Residual water and sample recovery of flavone
(25 mg in DMSO; 0.5 mL) using various delay times to trigger
the fraction collector. Each bar represents an average of three rep-licate
injections.
Figure 11. Sample recovery of flavone (in acetonitrile:water
[1:1] 10 or 15 mL) in the on-column solvent-exchange mode.
Samples were loaded using a flow rate of either 75 mL/min (gray
diamonds) or 30 mL/min (black diamonds) onto either a 19 50-
mm column (!100 mg) or a 30 100-mm column (O100 mg).
JALA October 2011 343

10. Original Report
increased from 20 to 50 mg in 10-mg increments. No flavone
was detected by UV or MS in any of the blank injections, as
shown in the example in Figure 13.
Evaporation
The standard reduced pressure with elevated-temperature
evaporation program used in our laboratories for aqueous
HPLC fractions consists of three stages. The first one, ramp-ing
rapidly from ambient conditions to 300 mbar and 50 C,
then more gradually to 40 mbar over 90 min, and remaining
at the set point for 90 min, distills off volatile organic compo-nents
(such as acetonitrile) without bumping; the second
stage, at 8 mbar and 50 C for 12 h, evaporates less-volatile
components (e.g., water and volatile additives, such as formic
acid and any residual acetonitrile); the last stage, at 2 mbar
and 45 C for 2 h, evaporates any residual solvents and addi-tives.
The whole evaporation procedure lasts more than 17 h,
is on the critical path and so is the bottleneck of our
purification operation.
The revised evaporation program, described earlier, runs
for 3 h at a lower temperature. This removes the restriction
that evaporation cycles have to run overnight, allowing
downstream operations to begin on the same day as the pre-parative
separation; increases the effective capacity of the
evaporators, because multiple evaporation cycles can be
run every day if required; reduces the likelihood of thermal
degradation, because the sample is exposed to lower temper-atures
for a shorter time; and chromatography additives are
not concentrated in the early stages of the evaporation cycle.
Removal of Chromatographic Modifiers
Chromatographic modifiers, such as TFA, formic acid,
and ammonium hydroxide, are widely used in many labora-tories
(including ours) to improve peak shape and enhance
resolution during chromatographic separations. Although
typically used in low concentrations, these additives are col-lected
with the target compound during fractionation and
concentrated during the initial stages of evaporation. The
presence of these additives can, occasionally, lead to the de-composition
of the final product; however, more commonly,
they react with the product to form salts. These salt forms
impact downstream processes and affect final product qual-ity.
In our laboratories, the immediate effect is an error dur-ing
the reconstitution procedure because the final product is
formulated as a solution at a standard concentration
(30 mM), and the incorrect assignment of a salt form can re-duce
the actual concentration of the target material by up to
20%, affecting the interpretation of biochemical assay
results. Later processes, such as biological screening, can also
be impacted by the presence of salts. A dramatic example is
the negative impact of TFA on cultures of osteoblasts and
chondrocytes even at concentrations less than 100 nM.9
Using 19F NMR monitoring, Boughtflower et al.3 showed
the effectiveness of SPE for TFA removal from fractions
eluting from HPLC columns. The advantage of TFA
removal from fractions before evaporation has become a rou-tine
application of on-column solvent exchange in our labo-ratories.
In one example, a project team prepared a series of
compounds using a motif similar to that reported by Buckley
et al.10 (Fig. 14). These compounds could not be purified in
formic acid (because of a risk of formylating an exposed pri-mary
amine), ammonium hydroxide (the azetidine ring was
base labile), or TFA (incompatible with the biological assay).
A purification method using TFA was selected, immediately
followed by on-column solvent exchange using direct injec-tion
and evaporation under the revised evaporation condi-tions.
The free-base form of the final product was
successfully isolated in high recovery (typically 95%; weights
ranging from 1 to 20 mg) and tested without any interference
from TFA.
In a second example, a project team separated diastereo-mers
using supercritical fluid chromatography (SFC) with
an ammonium acetate additive. Because the biological assay
Figure 12. Recovery of flavone (30e50 mg in DMSO; 1 mL)
after preparative separation, evaporation, weighing, reconstitution
in acetonitrile:water (1:1; 15 mL) on-column solvent exchange,
evaporation, and weighing. Each bar represents an average of
three replicate injections.
Figure 13. Chromatograms of flavone (20 mg in acetonitrile:
water [1:1] 10 mL; top and bottom) bracketing a blank sample
(middle).
344 JALA October 2011

11. could not tolerate any residual additive or acetate salts, the
compounds were desalted using on-column solvent exchange
(with the direct-injection protocol). The alcohol matrix from
the SFC separation was evaporated using a rotovap (because
compound stability was not an issue) and then the material
was reconstituted in acetonitrile:water (1:1) (9 mL) for the
on-column solvent-exchange procedure. Subsequent evapo-ration
of the acetonitrile using the revised Genevac condi-tions
yielded the free base of the final product
(approximately 40 mg of each isomer; structure not shown).
Comparison of Solid-Phase Material
Comparison of the X-Bridge or Sunfire columns with the
Gemini columns demonstrated no significant differences in
sample recovery or method run times (data not shown).
Because the Waters columns are routinely used within our
existing purification platform, these were selected for detailed
investigation.
Evaluation of an experimental Waters hydrophobicelipo-philic
balanced copolymer (HLB 30 mm) column demon-strated
performance similar to the X-Bridge column when
measuring loadability and retention capacity for flavone
and cortisone. However, significant breakthrough was
observed for many other test compounds11 (e.g., metronida-zole,
amitriptyline hydrochloride, and labetalol; 25 mg each
in acetonitrile:water [1:1] 11 mL). Additionally, a significant
volume of water was collected with the target compound dur-ing
elution, extending the evaporation time required. Conse-quently,
further studies using the HLB column were not
performed.
Use of Reversed Flow for Elution
To reduce elution time, solvent volume, and the risk of
residual water in the final fraction, the feasibility of eluting
the fraction using a reverse acetonitrile flow through the col-umn
was investigated. Flavone (25 mg in acetonitrile:water
[1:1] 12 mL) was injected onto the column using an unmod-ified
direct-injection protocol, the column was then reversed,
and the sample was eluted with acetonitrile. High recoveries
(O90%) were achieved; however, residual water volumes in
the fraction were also observed. Because there was also no
straightforward solution to automate switching flow direc-tion,
further development of this approach was not pursued.
Future Work
Original Report
Preliminary data show that on-column solvent exchange
could be used to remove DMSO from samples. In our labo-ratories,
excess final product is stored as a dry material, re-quiring
the evaporation of DMSO from the stock solution.
Historically, this has been achieved using high-temperature
and reduced-pressure Genevac programs combined with ly-ophilization
procedures, which are time consuming and,
sometimes, do not completely remove the DMSO.
Using the on-column solvent-exchange procedure, the
stock solution can be loaded onto a C18 column and eluted
in acetonitrile before solvent evaporation. Initial results dem-onstrate
that samples in DMSO must be loaded using an
at-column-dilution protocol to avoid sample breakthrough.
Significant back pressure is observed during the sample dis-pense
step, and this needs to be mitigated before this ap-proach
is robust enough to be used within our purification
platform.
The worldwide shortage of acetonitrile has encouraged
many laboratories to adopt methanol as their organic modi-fier.
In our laboratories, methanol has already replaced
acetonitrile for many preparative separations but not for
on-column solvent exchange. The weaker elutropic strength
(and the corresponding compound solubility) and higher vis-cosity
(leading to back-pressure concerns) of methanol need
to be investigated before making this transition.
CONCLUSION
At the outset of this study, a procedure to reduce the poten-tial
for salt formation, residual solvent, and sample exposure
to elevated temperatures for extended times during evapora-tion
was envisaged. It was critical that this on-column sol-vent-
exchange procedure did not decrease the quality of the
final product or the amount recovered from our automated
purification platform. It was also important that the cycle
time from sample submission to availability for biological
testing did not increase. A final requirement was that minimal
capital expenditure would be required to achieve this goal.
The results clearly demonstrate that all of these objectives
were achieved. Sample recovery is typically at or greater than
85% of the submitted mass. Sample purity, as measured by
UV, ELSD, and 1H NMR, is not affected. Overall sample
quality is frequently increased, because the formation of salt
forms can be avoided, enabling more accurate formulation of
the compounds and eliminating potential interferences in
biochemical assays. Samples can be evaporated to complete
dryness within 3 h of the completion of the preparative sep-aration
(compared with overnight using previous practices),
allowing subsequent procedures to start earlier.
ACKNOWLEDGMENTS
The authors acknowledge Paul Lefebvre (Averica Discovery Services) for
describing the initial concept, Ronan Cleary (Waters) for discussions and
editing comments, and Jason Ramsay (Pfizer) for his contributions to the
hardware modifications.
Figure 14. Generic structure of a compound requiring TFA pu-rification
and on-column solvent exchange.
JALA October 2011 345

12. Original Report
Competing Interests Statement: The authors certify that they have no relevant
financial interests in this manuscript.
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