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Variation in Kinetic Performance of Modern HPLC Columns with Solute StructureRichard A. Henry, Stacy L. Squillario, William H. Campbell and David S. BellSupelco, Div. of Sigma-AldrichBellefonte, PA 16823 USA

T413083


Poster Dedicated to Uwe D. Neue

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Introduction• Since Neue published his important book on HPLC column fundamentals in 1987 (1),

major progress has been made in HPLC speed and performance. While faster LC was predicted (2) and even practiced (3) much earlier, primary advances have occurred since 2004, when sub-2 µm silica particles were introduced.

• Porous silica particles still dominate established HPLC methods and feature the widest variety of selective phases and pore-sizes, but modern core-type particles have grown rapidly in popularity since their appearance in 2007 because similar gains in speed and efficiency can be achieved with larger particles at lower operating pressures.

• Porous silica continues to improve in performance with the development of narrow PSD particles that are similar in specification to core-type particles. There is evidence that a narrow PSD can maximize column efficiency and reduce operating pressure.

• While strides have been made to improve HPLC column efficiency, efforts have focused mainly on the A term of the van Deemter equation and improving packed bed uniformity (4-7). More work is required to better understand the B and C terms of the van Deemter equation and the forces that create mass transfer limitations within particle pores.

• In RP mode with silica particles, highest column performance over a wide velocity range is possible for small, nonpolar molecules that are retained by simple partition mechanisms. Speed restrictions still exist for methods involving larger, polar solutes that interact by more complex mechanisms. This poster will demonstrate mass transfer limitations for silica particles and identify steps that might be taken to overcome them. 3


Results and Discussion

• Neue and others described various forms of the van Deemter equation, including origins of slow mass transfer within pores (C term).

– H increases at higher velocity due to 1) diffusion through stagnant mobile phase in pores, 2) interaction with stationary phase and substrate (multiple processes), and 3) diffusion back into the moving phase outside of the particle.

• In 1988, Horvath (8) described advantages of pellicular particles and use of temperature to improve mass transfer at high flow velocity. – Horvath expanded the van Deemter equation to address the origin of mass transfer

(C term) limitations within silica particles.– Horvath recommended operating short pellicular columns at elevated temperatures and

high velocities to separate larger, polar molecules; theoretical plots were provided to illustrate potential improvement for pellicular silica over porous silica using 3 µm particles.

• In 2004, the launch of ultra-HPLC using sub-2 µm porous particles and special instruments shifted HPLC practice toward much higher speed.

– A serious trend began toward operating at higher velocities, but slow mass transfer for polar solutes still creates significant loss of column performance with small particles. Slow diffusion and kinetics within silica pores improves the B term but increases the C term, driving optimum performance to lower rather than higher velocities. 4


• In 2007, Kirkland (9) reported new Fused-Core® silica particles and showed different van Deemter curve shapes for polar and nonpolar solutes under reversed-phase conditions. Bell observed (10) similar curve shapes on Fused-Core particles for small, polar solutes under HILIC operating conditions. See Figure 1.– The neutral molecule naphthalene showed reduced plate heights as low as 1.5 on

2.7 µm C8 core-type particles. Operating pressure for a 5 cm column remained below 400 bar even at 8 mm/sec velocity.

– The larger, polar molecule Lorazepam showed the same low minimum plate height at lower velocity with greater resistance to mass transfer that reduced column efficiency at higher velocity. A global minimum in plate height may not exist at a given velocity for mixtures having different chemical structures.

– This behavior is not unique to Fused-Core particles and occurs with all silica particles studied over several years. In fact, core-type particles typically show a smaller effect than same-size porous particles because of shorter diffusion paths.

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Results and Discussion (contd.)


Results (contd.)

• Titan™ sub-2 µm porous silica particles with a narrow PSD similar to the core-type particles were first described in 2012 (11); Comparisons are underway to determine whether porous particle performance can be made to rival core-type particles by creating a narrow particle distribution. Figure 2 shows SEM data for two narrow PSD silica particles.

• Formation of highly uniform Titan sub-2 µm beds have generated reduced plate heights below 2 for small, neutral molecules for the first time with silica porous particles. See Figures 3A and 3B.

• Like other porous and core-type silicas, Titan shows mass transfer limitations in transferring larger, polar solutes into and out of pores and stationary phases at high velocities. See Figure 3C.

• Slow mass transfer has been observed with all silica and bonded phases tested and appears to be a general property of current silica HPLC and UHPLC column products.

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Figure 1. van Deemter Plot for Fused-Core C8under RP Conditions (9)

Reproduced by permission from ref (9)

column: Ascentis® Express C8, 5 cm x 4.6 mm I.D., 2.7 µmmobile phase: (A) 60:40 acetonitrile:water; (B) 30:70 acetonitrile:20 mM phosphate buffer, pH 3.5column temp.: 24 ºC

Naphthalene (MW 128)Lorazepan (MW) 321)

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Figure 2. Two Narrow Distribution Silica Particles

1.9 µm Titan Porous, ca. 5% standard deviation (D90/10 < 1.15).

2.7 µm Fused-Core, ca. 5% standard deviation (D90/10 < 1.15).


0.5 mL/min

1.5 mL/min

2.5 mL/min

column: Titan C18, 5 cm x 3.0 mm I.D., 1.9 µmmobile phase: 60% acetonitrilecolumn temp.: 35 C

Figure 3A. Multicomponent van Deemter Test Mix

0 2 4

Min 9


Conditions same as Figure 3A.

Figure 3B. van Deemter Test for Nonpolar Solute

1.000

2.000

3.000

4.000

5.000

6.000

7.000

8.000

9.000

10.000

11.000

0.000 2.000 4.000 6.000 8.000 10.000 12.000 14.000

h

(mm/s)

NaphthaleneC18 1.7 µm porousC18 1.8 µm porousC18 1.7 µm core-typeAscentis C18 3.0 µm porousTitan C18 1.9 µm porous

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Figure 3C. van Deemter Test for Polar Drug Solute

1.000

2.000

3.000

4.000

5.000

6.000

7.000

8.000

9.000

10.000

11.000

0.000 2.000 4.000 6.000 8.000 10.000 12.000 14.000

h

(mm/s)

Diazepam

C18 1.7 µm porous

C18 1.8 µm porous

C18 1.7 µm core-type

Ascentis C18 3.0 µm porous

Titan C18 1.9 µm porous

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• While a complete van Deemter study is not required for every sample mixture, it is essential to collect performance data at several flow rates to establish velocity tolerance early during HPLC or UHPLC method development if high speed separation is a goal.

• Every separation system will show different tolerance toward operating at high mobile phase velocities. Figure 4A shows a test mix with polar and nonpolar model compounds. Figure 4B shows van Deemter plots for three adjacent solutes and monitors loss of resolution as peaks broaden at high velocity. Polar solutes have low tolerance because they broaden faster. The (critical) solute pair that reaches a pre-established minimum resolution value will limit mobile phase velocity and establish maximum separation speed. During method development, velocity tolerance should be tested for different column brands and lots during screening and optimization.

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Results (contd.)


0.2 0.4 0.6 0.8 1.0

1.0 2.0

1.0 mL/min

2.8 mL/min

Diazepam

Toluene

Naphthalene

Impurity

Uracil

Biphenyl

Triptycene


Figure 4A. Test Mix to Demonstrate Velocity Tolerance

Triphenylethanediol

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Figure 4B. Velocity Tolerance Plot

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Rs - Diazepam and Toluene

Rs - Triphenylethanedioland Toluene

Rs

0

1

2

3

4

5

6

7

1.000

2.000

3.000

4.000

5.000

6.000

7.000

8.000

9.000

0.00 2.00 4.00 6.00 8.00 10.00 12.00

h

mm/s

Diazepam (MW 284)Triphenylethanediol (MW 290)Toluene (MW 92)R Triphenylethanediol / DIazepamToluene / Triphenylehtanediol


Summary and Conclusions

• Maintaining high plate counts at high linear velocities is very desirable in order to speed up separation and maintain resolution, but it has only been achieved for small, neutral solutes. Mass transfer slows down considerably when large, polar or ionic solutes leave the mobile phase and enter the silica pore structure, even for UHPLC particles.

• Mass transfer processes within pores include 1) diffusion within the stagnant mobile phase, 2) diffusion within the solvated stationary phase (simple liquidpartition mechanism), and 3) kinetically-controlled processes related to strong polar or ionic interaction with attractive sites on the phase or substrate (adsorption or ion exchange mechanisms). Steric or charge repulsion forces may also be evident.

• Solutes in the same mixture may be retained and separated by different mechanisms, so they may exhibit very different curve shapes ranging from flat to steep in the critical C-term region. Separations using the same column may have different tolerance to higher velocities. Each separation should be tested for velocity tolerance during method development if high sample throughput is an objective.

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Summary and Conclusions, (contd.)

• If a separation does not tolerate high velocity well enough, the main variables that can be changed are particle size, particle design, pore size, stationary phase, substrate type and chemistry; and mobile phase conditions such as temperature and pH.

• Use of 1) elevated temperature to increase diffusion rate and 2) porous-layer(core-type) particles to shorten diffusion distance are known to create flatter van Deemter plots at high mobile phases velocity. Both approaches also create the important advantage of lower operating pressure. These and other variables are under further investigation to optimize separation speed for mixtures of polar and higher MW solutes in modern HPLC.

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References1. Uwe D. Neue, HPLC Columns, Wiley-VCH 1997.2. J. H. Knox and M. Saleem , J. Chromatogr. Sci., 7, 614-622 (1969).3. M. W. Dong and J. R. Gant, LCGC, 2(4), 294-303 (1984).4. J. H. Knox, Band Dispersion in Chromatography- A New View of the A Term, Journal

of Chromatography A, 831 (1999) 3–15. 5. J. H. Knox, A Universal Expression for Bandspreading in the Mobile Zone, Journal of

Chromatography A, 960 (2002) 7–18.6. D. Cabooter, A. Fanigliulo, G. Bellazzi, B. Allieri, A. Rottigni, G. Desmet, Relationship

between Particle Size Distribution and Chromatographic Performance, J. of Chromatography A, 1217 (2010) 7074–7081.

7. K. Broeckhoven, D. Cabooter and G. Desmet, Kinetic Performance Comparison of Fully and Superficially Porous Particles, Journal of Pharmaceutical Analysis 2013;3(5):313–323.

8. F. D. Antia and C. Horvath. J. of Chromatography, 435 (1988), 1-15.9. J. J. Kirkland, T. J. Langlois and J. J. DeStefano, Fused-Core Particles for HPLC

Columns, American Laboratory, 39 (February 2007), 18-21.10. D. S. Bell, et. al., Impact of Solute and Stationary Phase on Efficiency in HILIC, Poster

HPLC 2014, New Orleans.11. R. A. Henry, et. al., Development of a New Monodisperse Porous Silica for UHPLC,

Poster HPLC 2012, Anaheim.


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Trademarks

Ascentis is a registered trademark of Sigma-Aldrich Co. LLC.Titan is a trademark of Sigma-Aldrich Co. LLC.Fused-Core is a registered trademark of Advanced Materials Technology, Inc.


Appendix A

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Horvath and Antia van Deemter Equation

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Graph Comparing PSD of Different Silica Particles

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Van Deemter Plot for Fused-Core Silica under HILIC Conditions (10)

column: Ascentis Express HILIC, 10 cm x 3.0 mm I.D., 2.7 µmmobile phase: 10:90, 100 mM ammonium formate:acetonitrilecolumn temp.: 35 C

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