advantages of using monodisperse particles in u/hplc columns · 2016. 2. 28. · the kirkland 2.7...

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Advantages of Using Monodisperse Particles in U/HPLC Columns

[email protected]

Richard A. Henry, Technical AdvisorSupelco, Div. of Sigma-AldrichBellefonte, PA 16823 USA

T414098

© 2013 Sigma-Aldrich Co. All rights reserved.© 2014 Sigma-Aldrich Co. All rights reserved.

The Trend to Small Particle Columns, High Separation Speed and High Pressure

2


Small Particle Columns are Not New in HPLC

• 3 µm particles yield lower HETP and more efficiency per unit column length.

• Optimum flow rate is higher for 3 µm columns.

• 3 µm columns maintain high efficiency better as flow increases.

• PE was first in offering high speed systems, but Supelco was a close second with 3 x 3 and 5 x 5 HPLC columns (now 2 x 2).

In 1984, 3 µm particle columns were exploited for Fast LC2, which is a precursor to UHPLC; 2 µm particles were also developed by then.

2. M. W. Dong and J. R. Gant, LC, 2(4), 294-303 (1984).

3


HPLC Column Performance Has Steadily Improved

ColumnParticles

Plate HeightH (or HETP)

Efficiency*N/m

Efficiency of a10cm column

Plates/cm estimate*

5 µm 10 µm 80-100,000 8-10,000 1000

3 µm 6 µm 120-160,000 12-16,000 1500

2 µm 4 µm 200-250,000 20-25,000 >2000*

Sub-2 µm 3 µm 300-350,000 30-35,000 >3000*

* Calculations are based upon the assumption of H = 2dp for a uniform, tight particle bed and a small volume of ideal solute introduced to the column under optimum mobile phase conditions. Efficiencies observed in normal practice may be significantly lower due to many variables that contribute to peak broadening and departure from ideal conditions, especially at higher performance levels.

4* Instrument factors are important


New Interest in Monodisperse HPLC Particles

• In 1999 and 2002, Knox (3-4) suggested that contributions from the A term in the van Deemter equation had been underestimated and that more uniform particle beds should have advantages.

• In 2006, Kirkland (5) commercialized a spherical Fused-Core® silica with very narrow size distribution (PSD) that showed surprising performance and became widely accepted.

• In 2010, Desmet, et. al. (6-7) observed a trend between narrow silica particle size distribution (PSD) and better column performance and suggested that porous particles should benefit.

• In 2012, Titan particle launched (9) to explore performance advantages for columns with porous particles having a very narrow PSD equal to Fused-Core.

5


What Can We Learn from Van Deemter Plots?

• Chromatographic band spreading happens in mobile zones (around the particle exterior) and static zones (inside particle pores or other non-convective transport regions). Monodisperse Fused-Core particles showed improved (lower) values for all three terms.

• Band spreading processes outside the particle (A term) have been the subject of recent research (3-6) that has resulted in preparation of more uniform beds with higher column efficiency.

• With monodisperse (highly regular) particles now becoming available in commercial quantities, further advances in HPLC column performance are anticipated as we gain better understanding of A, B and C terms in the van Deemter equation (Knox variation shown).

H = Au1/3 + B/u + Cu

Flow Uniformity Axial Diffusion Radial Diffusion

6


The Kirkland 2.7 µm Fused-Core Particle (2007)

Reproduced by permission from ref (5). 7

2.7 µm Ascentis® Express C8

Kirkland and AMT blew through the previous barrier of h = 2, suggesting that a limit of h = 1 might be attainable.

B term region A term region C term region

Regions shown where different terms in the van Deemter equation dominate.


8

Comparison: Fused-Core Particle vs Totally Porous Particles

Columns: 4.6 x 150 mm; Instrument: Agilent® 1100, autosamplerVerapamil - Mobile phase: 35% acetonitrile/65% 0.1% aqueous trifluoroacetic acid;

Temperature: 40 C; Fused-Core k = 2.8, totally porous k = 6.31-Cl-4-Nitrobenzene - Mobile phase: 50% acetonitrile/50% water;

Temperature: 30 C: Fused-Core k = 2.7, totally porous k = 4.3

Mobile Phase Velocity, mm/sec

0 1 2 3 4 5 6 7 8 9 10

Red

uced

Pla

te H

eigh

t, h

1

2

3

4

5

6verapamil: 5 m Halo fused-coreverapamil: 5 m totally porousData fitted to Knox equation1-Cl-4-nitrobenzene: 5 m Halo fused-core1-Cl-4-nitrobenzene: 5 m totally porous

verapamil: 5 µm Fused-Coreverapamil: 5 µm totally porousData fitted to Knox equation1-Cl-4-nitrobenzene: 5 µm Fused-Core1-Cl-4-nitrobenzene: 5 µm totally porous


-10

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90Time, seconds

1

3

4

5 6

2

Abs

orba

nce

/ %B

Column: 2.1 x 20 mm, Ascentis Express C18, 5 µmFlow Rate: 2 mL/min/Pressure: 172 barMobile Phase: 11/89: A/BA = Water/0.1% TFAB = Acetonitrile/0.1% TFAGradient: 5-50% B in 60 seconds

Temperature: 40 CDetection: UV 254 nm, PDAInjection Volume: 0.5 μLFlow Cell: 1 μL microLC System: Shimadzu Nexera

Peak Identities:1. Atenolol2. Pindolol3. Propranolol4. Indoprofen5. Naproxen6. Coumatetralyl

Fast Gradient with 5 µm Fused-Core Column*

Time (s) % ACN0 5

60 5063 9566 9569 587 5

Gradient Program

* Unpublished data supplied by Advanced Materials Technology. 9


Evolution of HPLC Particles to Smaller Size and Narrower Distribution

2.7 µm Fused Core

5 µm Fused Core

5.0 µm 3.0 µm ≤2 µm 2.5 µm

Higher Backpressure

≤2 µm Fused Core

• Core-type particles generate higher efficiency and speedat lower pressure.

• Porous particles also have high efficiency plus greater retention and sample capacity.

10


Monodisperse (Narrow PSD) C18 Silica Particles

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

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

11


Polydisperse 1.7 and 1.8 µm C18 Silica Particles*

12

* Particles removed from commercial columns.


0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6

number

count

(

%)

Particle Size (m)

Particle Size Distribution Data for Silicas

Titan 1.9

Fused-Core 2.7Fused-Core 5

Comparing Size Distribution for Different C18 Silicas

13

“Narrow PSD columns will prove to be more efficient, reproducible and stable. They should replace broad PSD in analytical columns.”Conventional

silicas have broad PSD

1.9 µm porous (Titan)1.7 µm porous2.7 µm Fused-Core3 µm porous5 µm Fused-Core5 µm porous


Titan Porous Silica Characteristics

Particle Size1µm

Pore DiameterÅ

Surface aream2/g

Pore Volumecc/g

1.9 80 410 0.76

1. EcoporousTM synthesis results in very narrow distribution (D90/10 < 1.15) without additional sizing.

Monodisperse particles can be defined as having a narrow PSD of 5-10% RSD. Modern core-type silicas are monodisperse. Until recent developments at Supelco, porous silica has been polydisperse with much larger PSD of 20-40% RSD.

14


TitanTM Performance Results

15


Evidence for a Monodispersity Advantage

0.000

2.000

4.000

6.000

8.000

10.000

12.000

0.000 2.000 4.000 6.000 8.000 10.000 12.000

h

mm/s

Toluene (reduced plate height)

Titan C18 1.9um

Competitor B C18 1.7um

Ascentis Express C18 2.7um

Porous Polydisperse 1.7

Porous and Core-type Monodisperse 1.9 and 2.7

Lower h values means more plates for a given particle size; Titan and Ascentis Express advantages can be attributed to monodispersity that creates very uniform beds (low A term).

16


1.0 2.0

NN= 13,573N/m = 271,460h= 1.90= 1.03Pressure: 3,100 psi

50 x 2.1 mm0.4 mL/min

= 3.29 mm/s

1.0 2.0


50 x 3.0 mm0.9 mL/min

= 4.07 mm/s

2

1.0 2.0Time (min)


50 x 4.6 mm2.4 mL/min

= 4.50 mm/s

1

3

45

Dionex 3000 UHPLCMobile Phase: 60% AcetonitrileTemp: 35 CFlow Rate: As IndicatedDetection: 254 nmLow D Connecting TubingInlet: 35 cm x 75 mOutlet: 35 cm x 75 m

QC Test1.Uracil2.Diazepam3.Toluene4.Naphthalene5.Biphenyl

Titan C18 Performance in a Modern UHPLC Instrument

Titan C18 1.9 µm: 13-15,000 plates expected for 5 cm columns; system pressure includes instrument blank pressure.

17


Independent Evaluation of Titan Kinetic PerformanceKinetic plot limit for Pmax = 600 bar (Ascentis, Poroshell, Waters XP)and Pmax = 1000 bar (Titan and Waters BEH)

Titan offers best kinetic performance over entire t0 or N range for Pmax = 1000 bar

Acknowledgement to U. Brussels (Desmet Group)

18

0.1

1.0

10.0

100.0

10000 100000 1000000N (/)

t 0(m

in) Ascentis 2.7µm

Waters XP 2.5µmPoroshell - Agilent 2.7µmTitan - 1.9µmWaters BEH 1.7µm


Independent Evaluation of Titan Pressure Drop

Lower P for Titan compared to similar sized particles: Kv0is 25% higher than Waters BEH and only 7 % lower than2.5µm XP column

Lower P of superficially porous particles due to lower T (higher u0)

Acknowledgement to U. Brussels (Desmet Group)

19

0

150

300

450

600

750

0 1 2 3 4 5 6

u0 (mm/s)

H (µ

m)

Ascentis 2.7 µmPoroshell - Agilent 2.7 µmWaters XP 2.5µmTitan - 1.9µmWaters BEH 1.7µm


Dionex 3000 (Low D tubing)Column: 50 x 2.1 mmMobile Phase:60% Methanol40% 0.1% Ammonium Acetate (pH = 7.1)Temp: 35 CFlow Rate: 0.25 mL/minDetection: 220 nm

1.Uracil2.Quinidine3.Diphenhydramine4.Nordiazepam5.Diazepam

Pressure= 5,370 psi

Pressure= 5,220 psi

Pressure= 4,210 psi

1.0 2.0 3.0

1.0 2.0 3.0

1.0 2.0 3.0

1

23

4

5

Titan C18 Performance Comparison in MeOH

20

Efficiency and asymmetry reported on peaks 3 and 5

N= 9,644= 1.15

N= 11,485= 1.00

N= 6,603= 1.02 N= 8,094

= 1.02

N= 7,420= 1.72

N= 9,470= 1.13

Titan C18 1.9 m

Competitor B C18 1.7 m

Competitor A 18 1.8 m


Titan C18 1.9 mDionex 3000 (Low D tubing)Column: 50 x 3.0 mmMobile Phase: 60% AcetonitrileTemp: 35 CFlow Rate: 0.9 mL/min (4 mm/s)Detection: 254 nm

1.Uracil2.Diazepam3.Toluene4.Naphthalene5.Biphenyl

NNap= 9,783Nnap/m= 195,700 h= 2.84= 1.16Pressure= 4,900 psi

NNap= 9,260NNap/m= 185,200h= 3.18= 1.02Pressure= 4,650 psi

Titan C18 Performance Comparison in ACN

21

1.0 2.0

NNap= 14,710N/m = 294,200h= 1.75= 1.05Pressure= 4,100 psi

1.0 2.0

1.0 2.0

1

23

4 5

Data used in subsequent van Deemter plots Competitor B C18 1.7 m

Competitor A 18 1.8 m


Columns: 50 x 3.0 mm60% Acetonitrile

Performance (h) of Porous Titan and Fused-Core Ascentis Express for Different Molecules

22

See poster 0905

0.000

1.000

2.000

3.000

4.000

5.000

6.000

7.000

8.000

9.000

10.000

0.000 2.000 4.000 6.000 8.000 10.000 12.000

Titan C18 1.9 µm Toluene

Ascentis Express C18 2.7 µm Toluene

Titan C18 1.9 µm Diazepam

Ascentis Express C18 2.7 µm Diazepam

Toluene / Diazepam ComparisonReduced Plate Height

h

(mm/s)


Columns: 50 x 3.0 mm60% Acetonitrile

Performance (H) of Porous Titan and Fused-Core Ascentis Express for Different Molecules

23

DiazepamN/m = 295,000plates/m

TolueneN/m = 293,000plates/m

0.000

2.000

4.000

6.000

8.000

10.000

12.000

14.000

16.000

18.000

20.000

0.000 2.000 4.000 6.000 8.000 10.000 12.000


Ascentis Express C18 2.7 µm Toluene


Ascentis Express C18 2.7 µm Diazepam

Toluene / Diazepam ComparisonPlate Height

(mm/s)

H (m)


24

HminHmin

Optimum Linear Velocities are Solute-Dependent

TolueneN/m = 293,000 plates/mat Hmin = 4.873 mm/s

Flow Rate at Hmin = 1.1 mL/minP = 5050 psi

DiazepamN/m = 295,000 plates/m at Hmin = 1.796 mm/s

Flow Rate at Hmin = 0.4 mL/min P = 1810 psi

Columns: 50 x 3.0mm60% Acetonitrile

Every separation has a different velocity tolerance (poster 0905)

0.000

2.000

4.000

6.000

8.000

10.000

12.000

14.000

16.000

18.000

20.000

0.000 2.000 4.000 6.000 8.000 10.000 12.000



Toluene / Diazepam ComparisonPlate Height

H (m)

(mm/s)


Further Evidence for a Monodisperse Advantage*

25*Unpublished data supplied by Supelco Division of Sigma-Aldrich.

-1.000

1.000

3.000

5.000

7.000

9.000

11.000

0.00 2.00 4.00 6.00 8.00 10.00 12.00

h

mm/s

Column G TolueneColumn G NaphthaleneColumn G BiphenylColumn A TolueneColumn A NaphthaleneColumn A BiphenylTitan TolueneTitan NaphthaleneTitan Biphenyl

Columns: porous C18, 50 x 3.0 mmTest conditions 60% acetonitrile

Column G 1.9 µm, D (90/10) ca.1.4Column A 1.9 µm, D (90/10) ca.1.4Titan 1.9 µm, D (90/10) ca. 1.1


TitanTM Selectivity and Applications

26


0 2 4 6 8Time (min)

0.46

3

2.52

3

2.93

1

3.34

93.

473

4.64

14.

847

5.30

6

5.56

5

7.78

8

TolueneD8

H8

Naphthalene

D8

H8

P-Xylene

D10

H10

Dionex 3000 UHPLC Column: Titan C18, 1.9 mDimension: 100 x 2.1 mmMobile Phase: 50% ACNTemperature: 35 CFlow Rate: 0.4 mL/minDetection: UV / 254 nmPressure: 5650 psi/390 bar

Dia

zepa

m

N,N

-Dim

etyl

anili

ne

Bip

heny

l

N = 25,530N/m = 255,000

Titan C18 1.9 µm: High Speed and High Resolution

Separation of deuterated isomers

27


Comparison of Porous and Core-Shell UHPLC Columns

0.2 0.4 0.6 0.8 1.0 1.2 1.4Time (min)

12

3

4

5

6

Titan 1.9 µm C185800 psi (400 bar)

0.2 0.4 0.6 0.8 1.0 1.2 1.4Time (min)

12

3

4

5

6

Core-Type 1.7 µm C187455 psi (514 bar)

LC-MS Analysis of Benzodiazepine Drugs and Metabolites

instrument: Agilent 1290 TOF 6210column(s): 5 cm x 3.0 mm, as indicated

mobile phase: (A) 0.1% formic acid in 95:5, water:acetonitrile;(B) 0.1% formic acid in 5:95, water:acetonitrile;

gradient: 35% B to 60% B in 1 min; 60% B held for 0.5 min

flow rate: 0.6 mL/mincolumn temp.: 35 C

detector: MS-TOF, ESI+, XIC, 100 -1000 m/z injection: 2 µLsample: 300 ng/mL in 97:3, water:methanolelution: 1. Oxazepam glucuronide (463 m/z)

2. Lorazepam glucuronide (497 m/z)3. Temazepam glucuronide (477 m/z)4. Oxazepam (287 m/z)5. Lorazepam (321 m/z)6. Temazepam (301 m/z)

28


column: 5 cm x 3.0 mm, 1.9 µmmobile phase: water:acetonitrile, pH=3.0 (formic acid)

flow rate: 1.0 mL/mindet.: 225 nm

Selectivity Comparisons of Phenols: Titan C18 vs Titan C18-Amide (in the pipeline- see poster 0915)

29

Titan C18-Amide30% Acetonitrile

p-H p-NO2p-Me

p-OMe

Titan C1825% Acetonitrile

Iso-eluotropicp-CN

p-Hp-NO2

p-Me

p-OMe

p-CN

1.0 2.0


p-Me

p-NO2

p-OMe

p-H

p-CN

Selectivity Comparisons para Substituted Benzoic Acids

30





Iso-eluotropic

p-Mep-

NO

2

p-OMe

p-H

p-CN


p-Mep-NO2

p-CN

p-Mep-NO2

p-OMe

p-CN

Selectivity Comparisons for para Substituted Anilines

31

p-OMe

column: 5 cm x 3.0 mm, 1.9 µmmobile phase: water:acetonitrile, pH=6.5 (ammonium formate)




Iso-eluotropic


C

Z

OCH3O

Selectivity Comparison of Methylbenzoates

32

p-Me

p-N

O2

p-OMe

p-H

p-CN

p-CN

p-Me

p-OMe

p-H

p-N

O2



Titan C18-Amide38.5% Acetonitrile


Iso-eluotropic


1.0 2.0

1.0 2.0

column: 5 cm x 3.0 mm, 1.9 µmmobile phase: 65:35 water:acetonitrile, 0.1% formic acid


Titan C18-Amide

Titan C18

OH

O

H3CO

OCH3

O

O2N

Selectivity Comparisons Polar Mix

33

Hydrophobic selectivity (logP or logD)

Dual mode selectivity


1. Piroxicam2. Ketorolac 3. Tolmetin4. Ketoprofen5. Naproxen6. Oxaprozin7. Nabumetone8. Fenoprofen9. Etodolac

10. Flubiprofen11. Indomethacin12. Ibuprofen13. Diclofenac

0 2 4 6 8 10

1

34

2

5

6

7

8

9

10

11

12

13

Imp

0 2 4 6 8 10

2

6

8,10,9

13

12

13,4

5

7

11

column: 5 cm x 3.0 mm, 1.9 µmmobile phase: 55:45, 0.1% H3PO4:acetonitrile


Titan C18-Amide

Titan C18

Applications: NSAIDS (Non Steroidal Anti-Inflammatory Drugs- Carboxylic Acids)

34


35

Application: Tetrahydrocannabinol and Metabolites

1,2

3

12

3

Titan C18

Titan C18-Amide

column: 5 cm x 3.0 mm I.D., 1.9 µm particles mobile phase: (A) 0.1% formic acid in water; (B) 0.1% formic acid in acetonitrile

gradient: 60% B to 100% B in 6 min; held at 100% B 1.5 minflow rate: 400 μL/min

temp.: 35 Cdet.: UV at 220 nm

injection: 2 µLsample: 50 µg/mL in 95:5, 0.1% formic acid in water:0.1% formic acid in acetonitrile

1. 11-HYDROXY-DELTA-9-THC2. 9-CARBOXY-11-NOR-DELTA-9-THC3. (-)-DELTA-9-THC


Instrument Designs and New Challenges

36


Instrument Designs Must Keep Pace with Columns

• It has been known for over forty years, since the beginning of modern HPLC1, that instruments would have to be steadily improved as smaller column particles became available.

• New instrument requirements include higher pressure operation and streamlined small-volume flow paths from injector to detector. As a result, instrument cost has increased dramatically to more that $100,000.

• Modern columns cannot be used effectively with just any instrument. Development of UHPLC methods requires that higher performance instruments be available to all departments and be validated often. Chemists may have to develop methods for both HPLC and UHPLC instruments.

• The best modern instruments still do not perform perfectly (zero dispersion cannot be achieved) with small ID columns!

37


1.0 2.0


50 x 2.1 mm0.4 mL/min

= 3.29 mm/s

1.0 2.0


50 x 3.0 mm0.9 mL/min

= 4.07 mm/s

2

1.0 2.0Time (min)


50 x 4.6 mm2.4 mL/min

= 4.50 mm/s

1

3

45

Dionex 3000 UHPLCMobile Phase: 60% AcetonitrileTemp: 35 CFlow Rate: As IndicatedDetection: 254 nmLow D Connecting TubingInlet: 35 cm x 75 mOutlet: 35 cm x 75 m

QC Test1.Uracil2.Diazepam3.Toluene4.Naphthalene5.Biphenyl

Titan C18 Performance in a Modern UHPLC Instrument

Titan C18 1.9 µm: 13-15,000 plates expected for 5 cm columns; system pressure includes instrument blank pressure (increases with flow rate) .

38


Flow - Pressure Comparison for Same 1.9 µm Titan C18 4.6 mm Column with HPLC vs UHPLC InstrumentSee QC Test on previous slideMobile Phase: 60% ACNTemperature: 35 CFlow Rate: As IndicatedDetection: 254 nm

Agilent 1290 Connecting Tubing:Injector to Column:

35 cm x 75 µmColumn to Detector:

25 cm x 75 µm(blank P about 600 bar at 5 mL/min)

Agilent 1200SL Connecting Tubing:

Injector to Heat Exchange:400 x 0.17 mm

Heat Exchange to Column:150 x 0.127 mm

Column to Detector:280 x 0.17 mm

(blank P about 100 bar at 5 mL/min)

0

2000

4000

6000

8000

10000

12000

14000

0

100

200

300

400

500

600

700

800

900

1000

0.0 1.0 2.0 3.0 4.0 5.0

Pres

sure

(PSI

)

Pres

sure

(bar

)

Flow Rate (mL/min)

Pressure vs Flow on the Agilent 1200SL and Agilent 1290

Linear (1200 Instrument)

Linear (1200 Instrument + Titan C18 50 x4.6mm Column)

Linear (1290 Instrument)

Linear (1290 Instrument + Titan C18 50 x4.6mm Column)

UHPLC

HPLC

39


Efficiency Performance for HPLC vs UHPLC Instrument

Agilent 1200SL Agilent 1290

k’ N k’ N

Diazepam 1.90 8187 2.25 10107

Toluene 3.14 11982 3.75 15407

Naphthalene 4.11 12524 5.01 14603

Biphenyl 6.14 12648 7.56 14227

40

Same Titan C18, 50 x 4.6 mm; flow = 2.4 mL/min

1200SL is rated at 600 bar; 1290 is rated at 1200 bar; see previous slide to estimate system operating pressure.


Performance Plot for HPLC vs UHPLC Instrument

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0 2 4 6 8

N

k'

Agilent 1200SL: N vs k' at 2.4 mL/min

Diazepam

TolueneNaphthalene Biphenyl

Uracil

0

2000

4000

6000

8000

10000

12000

14000

16000

18000

0 2 4 6 8N

k'

Agilent 1290: N vs k' at 2.4 mL/min

Diazepam

TolueneNaphthalene Biphenyl

Uracil

More loss at low k’ due to larger volume flow path.

41

Titan C18, 50 x 4.6 mm

Plates are wasted here


How Particles and Plates Impact Resolution for a Difficult Separation with Selectivity (α) of 1.1

42

More plates in shorter columns allow faster separation of difficult pairs.

Example for α = 1.1 at different k values from 2-7, where α = k2/k1.

The perfect experiment has zero instrument dispersion uniform column performance. Working at low k values is further hampered by wasting plates due to extra-column effects.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0 5000 10000 15000 20000 25000 30000

Rs

N

Rs vs N for selected k and α

α=1.1 k=7

α=1.1 k=6

α=1.1 k=5

α=1.1 k=4

α=1.1 k=3

α=1.1 k=2

Rs = √N/4 + k/(1+k) + (α-1)/α

Target resolution

k = 7


Variables That Limit Modern Column Performance

1. Peak bandwidth increases with sample injection volume; focusing techniques (weak solvent injection and gradients) become important.

2. With particles from sub-2 µm to 3 µm, peaks are so narrow that any excess sample flow path outside of the column creates bandspreading (called extra-column effect); very low internal volume instruments are required; instrument designs still limit performance.

3. With sub-2 µm particles, high pressure creates frictional heat within the column, especially at high mobile phase velocities; uneven temperature and flow across the column increases bandwidth.

4. For certain (non-ideal) solutes, mass transfer can be slow due to adsorption or restricted diffusion within particle pores; slow mass transfer can increase peak bandwidth and discount advantages of small particle columns operating at high flow rates and pressures.

5. Problems 3 and 4 will be difficult to solve, but most problems do not require UHPLC designs. Don’t sell your HPLC instruments yet!

43


Instrument Designs Must Keep Pace with Columns

• It has been known for over forty years, since the beginning of modern HPLC1, that instruments would require steady improvement as smaller column particles became available.

• New instrument requirements include higher pressure operation and streamlined small-volume flow paths from injector to detector. As a result, instrument cost has increased dramatically to more that $100,000.

• Modern columns cannot be used effectively with just any instrument. Development of UHPLC methods requires that higher performance instruments be available to all departments and be validated often. Chemists may have to develop methods for both HPLC and UHPLC instruments.

• The best modern instruments still do not perform well enough with 2.1 mm I.D. columns!

44


Conclusions on Monodisperse Particles in HPLC

• A new process for making porous silica has been developed that matches the narrow size distribution of Fused-Core particles; no extra sizing step is required; no silica is wasted.

• Particles with 80 Å pores and 410 m2/g have been prepared in 1.9 µm with a 6% standard deviation in PSD.

• Performance results for Titan 1.9 µm particles:• Efficiency matches or exceeds porous particles of 1.7 and 1.8 µm size

while pressure drop for the larger Titan particle is lower.• Uniform Titan particles pack easily into rugged column beds that are

stable over a range of UHPLC flow and pressure conditions. • Excellent reproducibility observed for silica bonded phases.• High stability noted for phases within the pH range 2-8.

• HPLC research will continue about equally on both Fused-Core and Titan porous particle platforms.

45


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

Journal of Chromatography A, 831 (1999) 3–15. 4. J. H. Knox, A Universal Expression for Bandspreading in the Mobile Zone,

Journal of Chromatography A, 960 (2002) 7–18.5. J. J. Kirkland, T. J. Langlois and J. J. DeStefano, Fused-Core Particles for

HPLC Columns, American Laboratory, 39 (February 2007), 18-21.6. 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. Dorina Kotoni, HPLC 2013 Meeting, Amsterdam Download files at: www.sigmaaldrich.com/hplc2013.

9. R. A. Henry, et. al., Monodisperse Particles, poster, HPLC 2012 Anaheim. 46


Acknowledgements

Assistance from the following groups for evaluating particles and columns is acknowledged and greatly appreciated:• Ken Broeckhoven and Gert Desmet, Free University of Brussels, Department

of Chemical Engineering, Brussels, Belgium. • Francesco Gasparrini, Sapienza University, Rome, IT.• Luigi Mondello and Paulo Dugo, U. Of Messina. Messina, IT.• Fabrice Gritti and Georges Guiochon, U. of Tennessee, Knoxville, TN.

The assistance of many dedicated analytical chemists at Supelco and Fluka Divisions of Sigma-Aldrich is also appreciated.

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

47


Acknowledgements and Trademarks

The assistance of Ken Broeckhoven and Gert Desmet, Vrije Universiteit Brussel, Department of Chemical Engineering, Belgium in evaluating columns is greatly appreciated.

The assistance of William Campbell and many other Supelco scientists is also greatly appreciated.

Ascentis is a registered trademark of Sigma-Aldrich Co. LLC; Titan, Ecoporous and BIOshell are trademarks of Sigma-Aldrich Co. LLC. Fused-Core is a registered trademark of Advanced Materials Technology, Inc., Wilmington, DE. The assistance of Advanced Materials Technology for supplying data on Fused-Core particles is greatly appreciated.

48

Thank You!

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