Optimizing the Performance of Solid Core for Today and Tomorrow

Mike OliverProduct Manager, Sample Preparation and Accucore LC Products

Tony EdgeR&D Principal

June 2014

Introduction

• The History of Solid Core• The concept

Th i i l lid ti l d th h ll th f d• The original solid core particles and the challenges they faced

• Solid Core Chromatographyg p y• Understanding the benefits of the technology• Bar for bar greater efficiency• How to optimize the separation• How to optimize the separation

• Understanding chemistry• Optimization of the morphology

• The Future• Where will solid core take us?• Where will solid core take us?• New particle morphologies – their synthesis explained

2

Solid Core Material – The Concept

• Features• Less bed consolidation• Less bed consolidation• Better packing of particles• Less void volume in column• Reduced pore depth

• BenefitsBenefits• More efficient chromatography• Allows the use of low pressure systems

• Competitive Edge• Bar for bar gives better separations than porous materialsg p p

3

Liquid Chromatography Particle Design

2.6 µm80 Å

Solid Core Particles

80 Å

2.6 µm150 Å

Conventional Fully Porous Non-Porous Solid Core

4 µm80 Å

1.x µm80 Å

Reduce Size to improve kinetics at expense of

operating pressure

Low sample capacity

Very high pressure

Small particle kinetics

Reasonable pressureVery High Sample Capacity

Lower Efficiency

Low Sample Capacity

Very High Efficiency

High Sample Capacity

High Efficiency

4

operating pressurey y g y g y

History of Solid Core Technology

• 1960s• Horvath and Lipsky introduce the concept of pellicular/shell particles

• 1970s• Core-shell particles developed:-

• Zipax (DuPont later Rockford laboratories and finally acquired by HP/Zipax (DuPont later Rockford laboratories and finally acquired by HP/ Agilent), Corasil (Waters), Perisorb (Merck)

• Improvement in the manufacturing of high-quality fully porous spherical particles• inhibits success of the shell particles• inhibits success of the shell particles• 10 μm fully porous spherical particles

• 1980s • 5 μm porous particles

• 1990s3 μm porous particles• 3 μm porous particles

• 2000−Present• sub-2 μm porous particles and core shell

5

The Theory … the Knox Equation

A term

B term

H

C term

H=Au1/3+B/u+CuCuBAuH 31

H H=Au1/3+B/u+CuCuu

AuH

Linear velocity / mm/s

6

Linear velocity / mm/s

A–Term Eddy Diffusion or Multiple Paths

Packing Efficiency D90/10~1.5

Porous Silica

Packing Efficiency D90/10~1.1Accucore

7

A–Term Eddy Diffusion or Multiple Paths

Enhanced roughness of their surface compared to porous particles results in less bed consolidation as particles willparticles, results in less bed consolidation as particles will tend to stick together

8

B–Term Longitudinal Diffusion

• The B−term depends on;• Void volume of the column

9

• Void volume of the column

C–Term Resistance to Mass Transfer

• The C term depends on;Diff i diff i th i th ili• Differences in diffusion path in the silica pores

• Differences in the radial diffusion path in the liquid• Size of molecule

10

• Significant for large molecules but not for small molecules

Experimental Van Deemter Plots20.0

tical

10 0

15.0

Theo

ret

5.0

10.0

uiva

lent

Pl

ates

0.00 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 10 0ei

ght E

q

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0He

Linear velocity of mobile phase (mm/s)Accucore RP-MS 2.6µm 5µm 3µm <2µm

Columns: 100 x 2.1 mmMobile phase: H2O / ACN (1:1)Temperature: 30 °CDetection: UV at 254 nm Flow rate range: 0.1 to 1.0 mL/min

Highest efficiency and lowest rate of efficiency loss with flow

rate for solid core

11

Flow rate range: 0.1 to 1.0 mL/minAnalyte: o-xylene rate for solid core

Pressure Comparison

900

1000 BAP 200

2 11

600

700

800

900

600 bar limit

pp ddL 30

230

400

500

600

ress

ure

(bar

)

HPLC pressure limit

100

200

300Pr

u = 8 7mm/s0

0 200 400 600 800 1000

Flow rate (µL/min)

Accucore RP MS 2 6µm <2µm 3µm 5µm

u = 8.7mm/s

Accucore RP-MS 2.6µm <2µm 3µm 5µm

Columns: 100 x 2.1 mmMobile phase: H2O / ACN (1:1)Temperature: 30 °C

Wide flow rate range with P < 600 bar

12

Temperature: 30 °C

Van Deemter – Limitations

• Classical interpretation of how a column is performing

• 3 parameters• A – Eddy diffusiony• B – Longitudinal diffusion• C – Resistance to mass transfer

• Optimization of these parameter will give the best peak shape/efficiency

• However it does not take into account;• Analysis time

P t i ti t• Pressure restrictions on a system

13

Kinetic Plots

• Allows for fairer comparisons of analytical systems• Van Deemter just compares pure separation ability• Van Deemter just compares pure separation ability

• Incorporates time of analysisp y• Analysts want FASTER chromatography• Van Deemter plots do not specify the time of analysis

• Incorporates pressure limitations of systems• Van Deemter does not account for a pressure limitation p

on system

• Based on three very simple classical equations• Based on three very simple classical equations

14

Kinetic Plots – Retention Time1000010000

1000(s)

of p

eak(

100

ion

time

Accucore allows optimisation of retention times

Ret

enti

Solid core produces sharper peaks in less time

101,000.00 10,000.00 100,000.00

Efficiency

p

15

Accucore RP-MS 2.6µm 5µm 3µm <2µm Efficiency

Impedance

• Devised by Knox and Bristow in 1977Defines the resistance a compound has to moving• Defines the resistance a compound has to moving down a column relative to the performance of that column

• Allows for pressure to be incorporated

Often plotted with a reverse axis• Often plotted with a reverse axis• Mimics van Deemter plot• Minimum value optimum conditions 2

PtE p

• Often plotted as a dimensional form2N

E• t/N2

• t0 or tr both used

16

100,000

Kinetic Plots – Impedance100,000

20

NPtE

ce

2N

10,000

mpe

dan

Im

S lid i lSolid core requires less pressure to obtain sub 2 µm

efficiencies

1,000100.001,000.0010,000.00100,000.001,000,000.00

Accucore RP-MS 2.6µm 5µm 3µm <2µm Efficiency

17

µ µ µ µ Efficiency

The Impact of Selectivity on Resolution

Efficiency SelectivityRetentionEfficiency SelectivityRetention2 5

3.0

2 5

3.0

NR=k’

k’+1-14

NR=k’

k’+1k’

k’+1-1-14

2.0

2.5

(R) 2.0

2.5

(R)

k +1 4

k2

k +1k +1 4

k’221.5 N

solu

tion

(

1.5 N

solu

tion

(

=k2k’1

=k2

1 = 2

10.5

1.0kR

es

0.5

1.0kR

esSelectivity () has the greatest impact on 1.00 1.05 1.10 1.15 1.20 1.25

0.0 N

1.00 1.05 1.10 1.15 1.20 1.250.0

Nimproving resolution. 0 5000 10000 15000 20000 25000

0 5 10 15 20 25

Nk

0 5000 10000 15000 20000 25000

0 5 10 15 20 25

Nk

S18

Stationary phase, mobile phase, temperature

The Growth of Solid Core Technology

• Particle sizes available• 1.3, 1.6, 1.7,2.6, 2.7, 4, 5

P i il bl• Pore sizes available• 80, 120, 150, 300

• Stationary Phases Availabley• Reversed phases

• C4, EC-C8, C8, EC-C18, SB-C18, XB-C18, C18, C18+, C30, RP-MS, AdvanceBio C18 HT C28 Peptide ES C18AdvanceBio, C18-HT, C28, Peptide ES-C18

• Polar encapped / embedded phases• Polar aQ, Polar Premium, SB-Aq, Bonus-RP, RP-Aqua

• HILIC / Normal phases• Silica, Amide, Urea-HILIC, EC-CN, Penta-HILIC

• π ‐π phasesp ases• Phenyl, Biphenyl, Phenyl-Hexyl, PFP, Phenyl-X, Diphenyl

• Currently >50 different stationary phases available

19

• >7 manufacturers

Stationary Phase Characterization

• Hydrophobic retention (HR)

Hydrophobic Interactions

y p ( )• k’ of neutral compound

• Hydrophobic selectivity (HS)• α two neutral compounds that have different log P

• Steric Selectivity (SS)• α sterically different moleculesα sterically different molecules

• Hydrogen bonding capacity (HBC)y g g y ( )• α molecule that hydrogen bonds and a reference• Good measure of degree of endcapping

20

• Gives indication of available surface area

Stationary Phase Characterization

• Activity towards bases (BA)

Interactions with Bases and Chelators

• Activity towards bases (BA)• k’, tailing factor (tf) of strong base• Indicator of free silanols

• Activity towards chelators (C)• k’, tailing factor (tf) of chelator• Indicator of silica metal content

21

Stationary Phase Characterization

Interactions with Acids and Ion Exchanges

• Activity towards acids (AI)• k’, tf acid• Indicator of interactions with acidic compounds• Indicator of interactions with acidic compounds

• Ion Exchange Capacity (IEX pH 7.6)g p y ( p )• α base / reference compound• Indicator of total silanol activity

• All silanols above pKa

I E h C it (IEX H 2 7)• Ion Exchange Capacity (IEX pH 2.7)• α base / reference compound• Indicator of acidic silanol (SiO-) activity

22

• Indicator of acidic silanol (SiO ) activity

Column Characterization (Visualization)

HR /10

HSAI

Accucore C18HR /10

HSAI

Accucore RP-MS

SSIEX (2.7) SSIEX (2.7)

HBC

IEX (7.6)BA

C HBC

IEX (7.6)BA

C

HR /10

HSAI

Accucore PFPHR /10

HSAI

Accucore Phenyl-Hexyl

HS

SSIEX (2.7)

AI HS

SSIEX (2.7)

AI

HBC

IEX (7.6)BA

C HBC

IEX (7.6)BA

C

23

Widest Range of Solid Core Selectivity Options

500mAU 1,2,3 curcuminoids

2 00

2.50HR /10

HSAIAccucore RP-MS

Solid Core C18

0.50

1.00

1.50

2.00 HS

SSIEX (2.7)

AI Accucore C18

Accucore 150-C18

Accucore C8

Accucore 150-C4

Accucore Polar Premium1

0.00

HBCC

Accucore aQ

Accucore Polar Premium

Accucore Phenyl-Hexyl

Accucore PFP

23

Polar Premium shows different selectivity and separates the peaks

IEX (7.6)BA

Accucore Phenyl-X

Accucore C30

0.0 1.0 2.0 3.0

0

Minutes

24

Database for Column Characterization

http://www.usp.org/app/USPNF/columnsDB.html

• Not all modes of interactions are covered, specifically;i i HILIC N l h

25

• π‐π interactions, HILIC, Normal phase

System Considerations• Column: Accucore RP-MS 2.6 μm, 100 x 2.1 mm

• Gradient: 65–95 % B in 2.1 min

Dwell volume: 100 µL

95 % B for 0.4 min

• Flow rate: 400 µL/minAccela 1250

Dwell volume: 800 L

Surveyor Accela Surveyor Agilent

800 µL

Minutes0.00 1.00 2.00 3.00 4.00

Accela1250

Surveyor Agilent 1100

Run time (min)

2.5 3.0 3.5Dwell volume: 1000 µL

min0 0 5 1 1 5 2 2 5 3 3 5

Agilent 1100 AveragePW (1/2 Height)

0.02 0.02 0.04

min0 0.5 1 1.5 2 2.5 3 3.5

Solid core can deliver performance on a b f diff t t

26

number of different systems

System Considerations

• Minimize volume dispersion

Always Optimize System Configuration

• Tubing–short L, narrow ID• Low injection volume• Low volume flow cell• Low volume flow cell

• Optimize detector sampling rate Need enough points to define

peak (minimum of 10, >20 for quantitation)

5 pts

Fast scanning MS

• Low dwell volume pump for fast45 pts

9 pts

Low dwell volume pump for fast gradients

27

Reducing the Particle Size

• Industry is fixated with pressure• This is an unwanted artefact of running with smaller particles

R d l lif ti d i t t lif ti i f i ti l h ti• Reduces column lifetime, reduces instrument lifetime, causing frictional heating

• System dispersion is critical with smaller particlesy p p• Injection volumes, connectors, detector volumes become more important

C t l f t t• Control of temperature• Increase in pressure results in greater column temperature gradients• Need to ensure that column temperature is controlled effectivelyp y

• Isothermal / adiabatic control

Is there a finite limit to particle size reduction?

28

reduction?

Analyzing Biomolecules

• Move to produce bigger molecules• Difficult to copy• Difficult to copy• Greater success rates

• Chromatography requirements• Need less retentive phase• Need wide pores to cope with larger molecules• Need wide pores to cope with larger molecules

29

Peptides – Resolution & Peak ShapeRT: 0 11 15 03RT: 0.11 - 15.03

1.12

8.743.76 7.09 13.468.9111 20

Accucore 150-C18

11.2013.63

1.01 C ti l S lid C C181.018.52

6.983.6013.13

15.0310.84

Conventional Solid Core C18

100000 10.20< 2 µm Wide Pore Fully Porous C18

0

50000

uAU

8.491.5914.4912.035.25 14.31

y

2 4 6 8 10 12 14Ti ( i )

-50000

30

Time (min)

Proteins – Excellent Resolution vs < 2 µm Wide Pore

• Sharper and higher peaks80000

100000 Accucore 150-C4Backpressure: 185 bar

higher peaks than < 2 µm Wide Pore Fully Porous C4

40000

60000

uAU

Porous C4• Better resolution

and sensitivity100000

0

20000

• Significantly lower backpressure

60000

80000

100000 < 2 µm Wide Pore Fully Porous C4Backpressure: 320 bar

p

40000

60000

0 1 2 3 4 5 6 7 8 9 10Ti ( i )

0

20000

31

Time (min)

Effect of Pore Depth for Small and Large Molecules

12.00

10.00

equa

tion

small molecule, large ρ

small molecule, small ρ

large molecule, large ρ

6 00

8.00

n Deemter e

large molecule, small ρ

4.00

6.00

pnen

ts of va

2.00

B+C comp

0.000.00 20.00 40.00 60.00 80.00 100.00 120.00

Reduced Velocity, ν

32

What is the Solution?

• Fractals• Mandelbrot / Julia sets

C l t h t bl l ti i• Colors represent how stable solution is

2 czz

1

i

biac

33

1i

Increasing Surface Area

• Koch curve• A straight line has a dimension of 1• A straight line has a dimension of 1• This line is infinitely long• Curve has a dimension of 1.26

• Fractal shape• Same surface no matter what the scale• Same surface no matter what the scale

being observed• Reduced mass transfer effects?

Eff ti l if f ?• Effectively uniform surface?• Reduced issues with mass transfer

into and out of pores?

34

Approaches to Growing Solid Particles

• Sol-Gel process under acidic conditions

35

Growing Solid Silica

• Base activated (Stöber) Process

• Porous particles a polymeric surfactant (porogen) is typically added• e.g. Hexadecyltrimethylammonium bromide, Pluronic 123, • This allows formation of micelles which propagate pore structure on• This allows formation of micelles which propagate pore structure on

growth of particle• Surfactant washed out of end material to leave final structure which is

then calcined

36

then calcined

Growing a Particle

Solid particle grows by stages

Nanotechnology 22 (2011) 275718

37

gy ( )

The Ingredients for a New Particle

• MPTMS - (3-Mercaptopropyl)trimethoxysilane

• MPTES - (3-mercaptopropyl)triethoxysilane

• MPMDS - (3-mercaptopropyl)methyldimethoxysilane

• TEOS - tetraethyl orthosilicate• TEOS - tetraethyl orthosilicate

• TMOS - tetramethyl orthosilicate

• CTAB - (3-glycidyloxy propyl) trimethoxy silane, cetyltrimethylammonium bromidecetyltrimethylammonium bromide

• PVA - poly(vinyl alcohol) (PVA, MW 10 K)

38

p y( y ) ( , W )

The Recipe - Typically

• 0.25g PVP + 0.1g CTAB was dissolved in 5 ml DI water8 ml methanol was added with stirring and mixture left to cool to room• 8 ml methanol was added with stirring and mixture left to cool to room temperature

• 2 ml ammonium hydroxide (5.6%) was added to the reaction and stirred vigorously for 15 minutes

• 0.5 ml MPTMS was added drop wise over 30 seconds and the reaction stirred for 24 hours at room temperaturestirred for 24 hours at room temperature

• Resultant particles were collected and washed by centrifugation with DI water then ethanol (both 3×10 ml, 4500 rpm, 3 minutes)

A. Ahmed, W. Abdelmagid, H. Ritchie, P. Myers, H. ZhangJ. Chrom. A, Volume 1270 (2012) 194–203

39

40

41

Recipe Variables Already Investigated

• Increasing Stirring speed• Bigger nanospheres

200rpmBigger nanospheres

• Reduces particle size distribution 500rpm

1500rpm

• Temperature• Raising the temperature reduces

roughness of surface but provides more uniform particle sizeuniform particle size

• Microwave and conventional heating provide similar particles

42

• microwave is much quicker

Varying the Recipe

Standard Method0.25 g PVA (Mw 10k)0 10 g CTAB0.10 g CTAB5 ml DI Water8 ml Methanol2 ml NH4OH (5.6%)2 ml NH4OH (5.6%)0.5 ml MPTMS

Increasing alkalinity

Smaller, less densely packed nanospheres on surface

43

Varying the Recipe

Altering pH

Larger more densely packed nanospheresLarger, more densely packed nanosphereson surface

Altering polymer concentration

Smooth spheres, no nanospheres, poor dispersity

44

Varying the Recipe

Altering polymer concentration

Smooth spheres no nanospheres poorSmooth spheres, no nanospheres, poor dispersity

Altering additive concentrationg

Small, monodisperse, fuzzy particles

45

Varying the Recipe

Altering polymer type and concentration

Fused smooth spheresFused smooth spheres

Altering polymer type and concentrationAltering polymer type and concentration

Very small nanospheres on surface

46

Varying the Recipe

Addition of extra polymer

Very small nanospheres on surfaceVery small nanospheres on surface

Changing pH

Fused smooth spheres

47

Normal Phase Separations

48

Reverse Phase Separations

49

Separation on an SoS Particle

50

Separation on an SoS Particle

Monodisperse SOS, C4 bonded, endcapped 1. GY40 °C, 300 μL/min 2. VYVGradient: 10−40% B in 4 minutes 3. met-EnkA. 0.02M KH2PO4 buffer, pH 2.7 + 0.1% TFA 4. leu-EnkB. Acetonitrile + 0.1% TFA 5. Angiotensin II

51

Conclusions

• The History of Solid Core• The concept

Th i i l lid ti l d th h ll th f d• The original solid core particles and the challenges they faced• The introduction on a new generation of solid core materials

• Solid Core Chromatography• Understanding the benefits of the technology• Bar for bar greater efficiency• Bar for bar greater efficiency• How to optimize the separation

• Understanding chemistry• Optimization of the morphology

• The Future• The Future• Where will solid core take us?• New particle morphologies – their synthesis explained

52

Acknowledgements

Haifei Zhang Adham Ahmed Kevin SkinleyHaifei Zhang Adham Ahmed Kevin Skinley, (Laura), Peter Myers Richard HayesUniversity of LiverpoolUniversity of Liverpool

Harry RitchieTrajan Scientific

53

Trajan Scientific