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INTRODUCTION

The formation of column radial thermal gradients in liquid chromatography (LC) employing very small (i.e. <2 µm) particles is detrimental to chromatographic performance.

1 Accordingly, modern LC

instruments employ column ovens to minimize the formation of such radial thermal gradients and provide for excellent chromatographic performance. These column ovens, however, limit the placement of the separation column to the column oven and require lengthy connection tubing to interface the outlet of the column to the inlet of detection such as mass spectrometry (MS). Recently, a novel column insulating technology was developed which facilitates excellent chromatographic performance.

2 This

technology is comprised of a sleeve containing an evacuated chamber which jackets the column in a similar fashion to an insulated beverage container (Figure 1). The evacuated jacket insulates the column and prevents heat flux through the column wall which, in turn, prevents the formation of radial thermal gradients and the associated degradation of chromatographic performance. In addition to minimizing radial thermal gradients on the column, reducing the extra-column system volume is also necessary to maximize the chromatographic performance of the LC system.

3 Since

the vacuum jacketed column (VJC) technology enables placement of the column outside of the column oven without sacrificing chromatographic performance, it can be leveraged to minimize extra-column system volume through novel column placement. For example, in a LC-MS system, the column can be placed as close to the ion source as possible. Therefore, the VJC technology can specifically reduce the post-column system volume between the outlet of the column and the point of ionization within the ion source. Reducing the post-column volume of the system is specifically beneficial to gradient elution LC due to the focusing effect at the head of the column.

4

The VJC technology therefore provides an instrumental

technique to maximize the chromatographic performance and detection

signal-to-noise ratio without modifying the sample preparation

technique, column loading, or LC-MS method conditions. This poster

presents the integration of a chromatographic column to the ion source

of a LC-MS system and explores column thermal equilibration,

chromatographic performance, peak height, and the possibility of

integrating workflow enhancements such as infusion, post-column

addition, and diversion.

NOVEL INTEGRATION OF A SEPARATION COLUMN TO AN ION SOURCE FOR LC-MS

Michael O. Fogwill, Theodore A. Dourdeville, Jacob N. Fairchild, Wade P. Leveille, Joseph D. Micheinzi, Jeffrey Musacchio Waters Corporation, 34 Maple St. Milford, MA 01757 USA

Figure 2. System with the novel VJC-containing ESI probe.

METHODS

Apparatus

A Waters® ACQUITY

® I-Class UPLC system equipped with a

binary solvent manager, a flow-through needle sample manager, and a CH-A column oven was used in these comparisons (Figure 2). A Waters

® ACQUITY

® isocratic solvent manager was employed as an

additional flow source when required. A Waters® Xevo TQ-MS tandem

quadrupole mass spectrometer equipped with an electrospray ionization (ESI) ion source provided detection. Waters

® MassLynx

® software was

employed for data collection and analysis. A novel ESI probe was designed and assembled which contained the column, a mobile phase pre-heater, a vacuum-jacket, a post-column fluidic tee, and an ESI emitter capillary (Figure 3). Conventional and custom-built vacuum-jacketed columns were

employed, each 2.1x100 mm and packed with 1.6 µm Cortecs® C18

media.

Samples

A solution containing 2 pg/µL acetaminophen, 2 pg/µL caffeine, 1

pg/µL sulfadimethoxine, 0.5 pg/µL verapamil, and 50 pg/µL 17-

alphahydroxy progesterone (17-AHP) was prepared in 90:10:0.1 (v/v)

Water:Acetonitrile:Formic Acid.

LC Methods

The mobile phase flow rate was 650 µL/min and, upon injection,

a three minute linear gradient from 1 to 99% of LC-MS grade acetonitrile

in 18.2 MΩ/cm water was executed. Both mobile phase solvents

contained 0.1% (v/v) formic acid. The sample manager wash and purge

solvents were LC-MS grade acetonitrile with 0.1% (v/v) formic acid and

18.2 MΩ/cm water with 0.1% (v/v) formic acid, respectively. Columns

were held at 40 °C, and 2 µL injections were performed.

MS Methods

The ESI source was operated in positive ionization mode with

750 V capillary voltage, 500 °C desolvation gas temperature, 125 °C ion

block temperature, 750 L/h desolvation gas flow rate, and 75 L/h cone

gas flow rate. The MS was operated in selected reaction monitoring

(SRM) mode using the parameters listed in Table 1.

References

1. F. Gritti, G. Guiochon. J. Chromatogr. A 1216 (2009) 1353–1362.

2. F. Gritti, M. Gilar, J.A. Jarrell. J. Chromatogr. A 1444 (2016) 86-98.

3. F. Gritti, G. Guiochon. J. Chromatogr. A 1217 (2010) 7677-7689.

4. K. Vanderlinden, et al. J. Chromatogr. A 1442 (2016) 73-82.

RESULTS AND DISCUSSION

Workflow enhancements such as infusion, diversion, or post-column addition often require fluidic elements to be added between the column outlet and the point of ionization. These fluidic elements (i.e. valves, pumps, tubing, etc.) are of considerable size and contribute considerable post-column volume to the LC-MS system. Accordingly, such elements contribute to band broadening and require physical space to implement. The prototype VJC-containing ESI probe incorporates these fluidic elements remotely through a novel, extremely small and low-volume interface. The performance of the VJC-containing ESI probe (Figure 4C) was evaluated against conventional LC-MS plumbing (Figure 4B) and against a conventional LC-MS system containing infusion/diversion fluidics (Figure 4A). A mixture of small molecules (Figure 5) was employed to evaluate the differences in peak height and peak capacity between the conventional system with (Table 3) and without (Table 2) infusion/diversion fluidics. As represented in these data tables, the reduced post-column volume in the VJC-containing ESI probe significantly improves peak capacities and peak heights over conventional systems.

Of additional interest is the ability to rapidly equilibrate the system after changes in column temperature set point. These equilibration times were explored with conventional column ovens and with the VJC. As shown in Figure 6, each system equilibrates from 30 °C to 60 °C in approximately the same time. This observation suggests that the majority of the heating of an LC column is contributed by the mobile phase pre-heater and not through the column wall. Conversely, when cooling from 60 °C to 30 °C, the VJC reaches target temperature significantly faster. The increase in cooling rate is largely due to the significant thermal mass of the column oven and could be reduced with active cooling.

The diversion functionality of the VJC-containing ESI probe is

shown in Figure 7. A caffeine infusion was employed to demonstrate the

efficacy of diversion at 500, 750, and 1000 μL/min mobile phase flow

rates. The reduction of response to near zero supports the ability of

remote fluidics to affect diversion in a very small space and with very

low system volume contribution.

CONCLUSIONS

VJC technology enables column placement outside of a

conventional column oven

Novel remote fluidics enable post-column addition, diversion, and infusion in a very small space without adding significant

post-column volume

Integrating the VJC into the ESI source improves peak capacity: By 40.1% vs. a conventional system By 61.9% vs. a system with diversion/infusion fluidics

Integrating the VJC into the ESI source improves peak height: By 16.6% vs. a conventional system By 49.2% vs. a system with diversion/infusion fluidics

Reduced VJC thermal mass speeds column equilibration rates

Divert functionality is effective using remote fluidics

Figure 1. Cross sectional representations of the VJC insulating sleeve

(above) and the VJC assembly (below).

Figure 5. Separation of five small molecules employing the novel VJC-

containing ESI probe.

Figure 4. Schematic representation of system fluidic components for a

conventional column with diversion/infusion (A), a conventional system

(B), and the novel VJC-containing ESI probe with diversion/infusion (C).

Figure 6. Comparative column heating (left) and cooling (right) rates be-

tween the VJC technology and conventional column heating technology.

The equilibration time is noted on each figure.

ACKNOWLEDGEMENTS

The authors wish to thank Martin Gilar, Fabrice Gritti, and Andy Jarrell

for the helpful collaborative discussion.

Cone

Voltage (V)

Parent Mass (Da)

Collision Energy

(V)

Fragment Mass (Da)

Dwell Time (s)

Acetaminophen 26 151.94 18 109.98 0.036

Caffeine 34 194.98 20 138.04 0.036

Sulfadimethoxine 33 310.98 24 155.97 0.036

Verapamil 38 455.25 33 96.94 0.036

17-AHP 32 331.12 25 165.02 0.078

Figure 7. Flow diversion in the novel VJC-containing ESI probe at vari-

ous mobile phase flow rates. A caffeine sample was infused to demon-

strate diversion efficacy.

C

B

A

Improvement in Peak Height (%)

Improvement in Peak Capacity (%)

Acetaminophen 32.2 29.4

Caffeine 10.9 41.1

Sulfadimethoxine 43.9 11.4

Verapamil 7.2 84.8

17-AHP -11.3 33.9

Average 16.6 40.1

Improvement in Peak Height (%)

Improvement in Peak Capacity (%)

Acetaminophen 79.8 29.1

Caffeine 31.8 64.8

Sulfadimethoxine 51.0 60.1

Verapamil 53.0 66.1

17-AHP 30.3 66.2

Average 49.2 61.9

Table 2. Comparative peak heights and peak capacities of the VJC-

containing ESI probe (Figure 4C) vs. the conventional system

(Figure 4B).

Table 1. Mass Spectrometer SRM Details.

Table 3. Comparative peak heights and peak capacities of the VJC-

containing ESI probe (Figure 4C) vs. the conventional system with di-

vert/infusion fluidics (Figure 4A).

Figure 3. Schematic representation of the cross-sectional view of the

ESI probe containing a column, a vacuum jacket, a mobile phase pre-

heater, and a post-column tee.