Are Sub-2 µm Superficially Porous Particles Needed for Small Molecules?

J. J. Kirkland, B. E. Boyes, W. L. Miles, and

J. J. DeStefano Advanced Materials Technology, Inc.

3521 Silverside Rd., Quillen Bld., Ste. 1-K

Wilmington, DE 19810 USA

Are Sub-2µm SPP Needed for Small Molecules?

•Controversial question •Theory predicts efficiency advantages of smaller particles •SPP shown to have unusually high efficiency •Sub-2µm SPP already available •General consensus is “Yes” • Previous studies within AMT showed practical limitations

•This presentation •Authors’ opinions on topic; no equations •Large molecule separations not discussed

Upside of Using Sub-2µm Particles

• Smaller particles allow faster separations • High efficiency in short columns

• Improved productivity

• Short run times = less solvent usage

• Sharper peaks for more sensitivity

• High number of theoretical plates possible in longer columns • Improved peak capacity for complex mixtures

• Keeping up with state-of-the-art technology

Downside of Using Sub-2µm Particles

• Specially designed (expensive) instruments required for optimum use • 400 – 600 bar often insufficient for optimum flow

• Low-dispersion design required to minimize extra-column effects for highest efficiency

• Small ID tubing and flow cells significantly add to operational pressure

• Maintenance is expensive and often not user-friendly

Downside of Using Sub-2µm Particles • Column frits with small pores (0.2 – 0.5µm)

required to retain particles in columns • More subject to plugging than 2µm frits • Additional efforts needed to avoid particulate

fouling (filter samples and mobile phases)

• Frictional heating of columns • More pronounced as dp is reduced • Can result in band-broadening and changes in

retention • ≤ 3 mm i.d. columns required to minimize

frictional heating effects

Downside of Using Sub-2µm Particles

• High pressure can cause changes in retention and selectivity vs low pressure separations • Problematic to convert separations made with

small particles to columns of larger particles suited for routine analyses

• Columns may not exhibit expected efficiency or stability • Small particles harder to pack into homogeneous

beds for highest efficiency

Effect of Particle Size on h vs v Plots

Reduced Plate Heights (h = H/dp) get smaller as the particle size is increased, indicating more homogeneity in packed beds for the larger particles.

Linear Mobile Phase Velocity, mm/sec

1 2 3 4 5 6 7

Red

uced

Pla

te H

eigh

t

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.0 µm

2.7 µm 4.1 µm

4.6 µm

Data fitted to Knox equation

Are Sub-2µm SPP Needed for Small Molecules?

• Our conclusion: useful but not necessary • Upsides not sufficient to overcome the

Downsides for most small molecule applications

• Small molecules do not require shorter diffusion paths of small particle size SPP for adequate mass transfer

• A compromise alternative is suggested

An Alternative – 2µm SPP

• Retains most of advantages of sub-2µm – Higher efficiencies than sub-3µm SPP

• Minimizes disadvantages of sub-2µm – Lower pressure requirements

2 µm HALO Particle Design

SEM image of 2 µm HALO particles

Solid Core 1.2 µm 2 µm

0.4 µm

Shell with 90 Å pores

Mode – 2.006 Mean – 2.016 Median – 2.004 S.D. – 0.111um CV – 5.5%

Comparing van Deemter Plots (H) Plate Height vs. Mobile Phase Velocity Plots

Columns: 50 x 2.1 mm; Instrument: Shimadzu Nexera; Solute: naphthaleneMobile phase: Halo - 50/50 ACN/water, k=6.3; 1.6 mm SPP - 48.5/51.5 ACN/water, k=6.3;

1.7 mm SPP - 47/53 ACN/water, k=6.2; 1.7mm TPP - 48.5/51,5 ACN/water, k=6.3; Temperature: 35 oC; Injection volume: 0.2 mL

Linear Mobile Phase Velocity, mm/sec

0 1 2 3 4 5 6 7 8 9 10 11 12

Pla

te H

eigh

t, m

m

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

0.011

0.012

SPP Halo 2.0 mmSPP Halo 2.7 mmSPP 1.6 mm SPP 1.7 mm TPP 1.7 mm

Data fitted using van Deemter equation

Reduced Plate Height vs. Mobile Phase Velocity PlotsColumns: 50 x 2.1 mm; Instrument: Shimadzu Nexera; Solute: naphthalene

Mobile phase: Halo - 50/50 ACN/water, k = 6.3; 1.6 mm SPP - 48.5/51.5 ACN/water, k = 6.3; 1.7 mm SPP - 47/53 ACN/water, k = 6.2

1.7 mm TPP - 48.5/51.5 ACN/water, k=6.3; Temperature: 35 oC; Injection volume: 0.2 mL

Linear Mobile Phase Velocity, mm/sec

0 1 2 3 4 5 6 7 8 9 10 11 12

Red

uced

Pla

te H

eigh

t

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

SPP Halo 2.0 mmSPP Halo 2.7 mmSPP 1.6 mm SPP, 1.7 mmTPP, 1.7 mm

Comparing van Deemter Plots (h)

Data fitted using van Deemter equation

Pressure vs Flow Mobile Phase/Column Pressure Plots

Columns: 50 x 2.1 mm, C18: Instrument: Shimadzu Nexera; Solute: naphthaleneMobile phase: Halo, 50/50 ACN/water, k=6.3; SPP 1.6 mm, 48.5/51.5 ACN/water, k=6.3

SPP 1.7 mm, 47/53 ACN/water, k=6.2; Temperature: 35 oC; Injection volume: 0.2 mL

Mobile Phase Flow Rate, mL/min

0.0 0.2 0.4 0.6 0.8 1.0 1.2

Col

umn

Pres

sure

, bar

0

100

200

300

400

500

600

700

800

900

Halo 2.0 mmHalo 2.7 mmSPP 1.6 mmSPP 1.7 mm

2.7µm

2.0 µm

1.7 µm

1.6 µm

Plates per Pressure RequiredColumns: 50 x 0.21 mm C18; Instrument: Shimadzu Nexera; Solute: naphthalene

Mobile phase: 50/50 - 47/53 ACN/water; Flow rate: 0.5 mL/min; Temperature: 35 oC

Column Type

Plates

/bar

0

10

20

30

40

50

60

70

80

Halo 2.0 µm Halo 2.7 µm SPP 1.6 µm SPP 1.7 µm

71.8

38.1 40.9

58.8

Plates per Bar

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Time (min)

Columns: 2.1 x 50 mm Instrument: Shimadzu Nexera Injection Volume: 0.2 µL Detection: 254 nm Temperature: 25 oC

Mobile Phase A: water Mobile Phase B: acetonitrile Ratio A:B: 15/85 Flow rate: 0.5 mL/min

Peak Identities: 1. Uracil 2. Pyrene 3. Decanophenone 4. Dodecanophenone

N = 15480

N = 14551

Column performance is maintained after injections at high pressure (950 bar) Red trace = before high pressure Blue trace = after high pressure

1

2 3 4

High Pressure Stability of HALO 2 C18

Column Stability Test

Columns: 50 x 2.1 mm, Halo 2.0 µm C18; Flow rate: 2.50 mL/min; Temperature: 25 oC Solute: naphthalene; Mobile phase: 85% ACN/15% water

One sigma results

Columns Average Injection Pressure, bar

Average Test Pressure, bar

Average Plate Number

Average % Plate Number

Loss

6 - Halo 2.0 µm 980 ± 22 Before: 181 ± 4 15570 ± 330

After: 186 ± 4 14320 ± 550 8%

Conclusions

• Sub-2 µm SPP useful for R&D but less practical for most routine small molecule applications

• Larger SPP are less problematic for daily operation

• Columns of 2-µm SPP appear to be a good compromise of speed and efficiency with superior advantages for small molecule applications

Acknowledgements

Thanks go to Stephanie Schuster and Robert Moran who supplied much of the data with 2-µm particles for this presentation