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 userfriendly
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
2.6
2.4
Reduced Plate Height
2.2
2.0 µm
2.0
2.7 µm
1.8
4.1 µm
1.6
4.6 µm
1.4
1.2
Data fitted to Knox equation
1.0
1
2
3
4
5
6
Linear Mobile Phase Velocity, mm/sec
Reduced Plate Heights (h = H/dp) get smaller as the particle size is increased,
indicating more homogeneity in packed beds for the larger particles.
7
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
Solid Core
1.2 µm
0.4 µm
Shell with 90 Å pores
SEM image of 2 µm HALO particles
Mode – 2.006
Mean – 2.016
Median – 2.004
S.D. – 0.111um
CV – 5.5%
2 µm
Comparing van Deemter Plots (H)
Plate Height vs. Mobile Phase Velocity Plots
Columns: 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; 1.7mm TPP - 48.5/51,5 ACN/water, k=6.3
o
; Temperature: 35 C; Injection volume: 0.2 mL
0.012
SPP Halo 2.0 mm
SPP Halo 2.7 mm
SPP 1.6 mm
SPP 1.7 mm
TPP 1.7 mm
0.011
0.010
Plate Height, mm
0.009
0.008
0.007
0.006
0.005
0.004
0.003
Data fitted using van Deemter equation
0.002
0
1
2
3
4
5
6
7
8
Linear Mobile Phase Velocity, mm/sec
9
10
11
12
Comparing van Deemter Plots (h)
Reduced Plate Height vs. Mobile Phase Velocity Plots
Columns: 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
o
1.7 mm TPP - 48.5/51.5 ACN/water, k=6.3; Temperature: 35 C; Injection volume: 0.2 mL
7.0
SPP Halo 2.0 mm
SPP Halo 2.7 mm
SPP 1.6 mm
SPP, 1.7 mm
TPP, 1.7 mm
6.5
6.0
Reduced Plate Height
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
Data fitted using van Deemter equation
1.0
0
1
2
3
4
5
6
7
8
Linear Mobile Phase Velocity, mm/sec
9
10
11
12
Pressure vs Flow
Mobile Phase/Column Pressure Plots
Columns: 50 x 2.1 mm, C18: Instrument: Shimadzu Nexera; Solute: naphthalene
Mobile 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
900
700
Column Pressure, bar
1.6 µm
Halo 2.0 mm
Halo 2.7 mm
SPP 1.6 mm
SPP 1.7 mm
800
1.7 µm
600
2.0 µm
500
400
300
2.7µm
200
100
0
0.0
0.2
0.4
0.6
0.8
Mobile Phase Flow Rate, mL/min
1.0
1.2
Plates per Bar
Plates per Pressure Required
Columns: 50 x 0.21 mm C18; Instrument: Shimadzu Nexera; Solute: naphthalene
o
Mobile phase: 50/50 - 47/53 ACN/water; Flow rate: 0.5 mL/min; Temperature: 35 C
80
71.8
70
58.8
60
Plates/bar
50
40.9
40
38.1
30
20
10
0
Halo 2.0 µm
Halo 2.7 µm
SPP 1.6 µm
Column Type
SPP 1.7 µm
High Pressure Stability of HALO 2 C18
Columns: 2.1 x 50 mm
Instrument: Shimadzu Nexera
Injection Volume: 0.2 µL
Detection: 254 nm
Temperature: 25 oC
Peak Identities:
1. Uracil
2. Pyrene
3. Decanophenone
4. Dodecanophenone
Mobile Phase A: water
Mobile Phase B: acetonitrile
Ratio A:B: 15/85
Flow rate: 0.5 mL/min
3
2
4
1
N = 14551
N = 15480
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Time (min)
1.4
1.6
1.8
2.0
Column performance is maintained after injections at high pressure (950 bar)
Red trace = before high pressure
Blue trace = after high pressure
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
6 - Halo 2.0 µm
980 ± 22
Before: 181 ± 4
15570 ± 330
After: 186 ± 4
14320 ± 550
Average %
Plate Number
Loss
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 2µm particles for this presentation