Investigation into the design and application of solid core stationary phases has led to a better understanding of how the phases work and has resulted in their design aligned to the structure of the analytes being separated. The current range of columns available is discussed both in terms of selectivities, and also morphologies, allowing informed decisions to be made by the chromatographer. Using real life examples, coupled with advanced modeling, the effects of the particle size and morphology will be given for both small and large molecules, offering an insight into what the future holds for solid core products.

1. Optimizing the Performance of Solid Core
for Today and Tomorrow
Mike Oliver
Product Manager, Sample Preparation and Accucore LC Products
Tony Edge
R&D Principal
June 2014

2. 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

3. 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

4. Liquid Chromatography Particle Design
2.6 µm
80 Å
Solid Core Particles
80 Å
2.6 µm
150 Å
Conventional Fully Porous Non-Porous Solid Core
4 µm
80 Å
1.x µm
80 Å
Reduce Size to improve
kinetics at expense of
operating pressure
Low sample capacity
Very high pressure
Small particle kinetics
Reasonable pressure
Very High Sample Capacity
Lower Efficiency
Low Sample Capacity
Very High Efficiency
High Sample Capacity
High Efficiency
4
operating pressurey y g y g y

5. 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
• 1990s
3 μm porous particles• 3 μm porous particles
• 2000−Present
• sub-2 μm porous particles and core shell
5

6. The Theory … the Knox Equation
A term
B term
H
C term
H=Au1/3+B/u+CuCu
B
AuH  3
1
H H=Au1/3+B/u+CuCu
u
AuH 
Linear velocity / mm/s
6
Linear velocity / mm/s

7. A–Term Eddy Diffusion or Multiple Paths
Packing Efficiency
D90/10~1.5
Porous Silica
Packing Efficiency
D90/10~1.1Accucore
7

8. 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

9. B–Term Longitudinal Diffusion
• The B−term depends on;
• Void volume of the column
9
• Void volume of the column

10. 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

11. Experimental Van Deemter Plots
20.0tical
10 0
15.0
Theoret
5.0
10.0
uivalent
Plates
0.0
0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 10 0
eightEq
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
He
Linear velocity of mobile phase (mm/s)
Accucore RP-MS 2.6µm 5µm 3µm <2µm
Columns: 100 x 2.1 mm
Mobile phase: H2O / ACN (1:1)
Temperature: 30 °C
Detection: 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/min
Analyte: o-xylene rate for solid core

12. Pressure Comparison
900
1000
   BAP
2
00
2
11  




600
700
800
900
600 bar limit
pp ddL 3
0
23
0 

400
500
600
ressure(bar)
HPLC pressure limit
100
200
300
Pr
u = 8 7mm/s
0
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 mm
Mobile phase: H2O / ACN (1:1)
Temperature: 30 °C
Wide flow rate range with P < 600 bar
12
Temperature: 30 °C

13. 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

14. 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 limitationp
on system
• Based on three very simple classical equations• Based on three very simple classical equations
14

15. Kinetic Plots – Retention Time
1000010000
1000
(s)ofpeak(
100
iontime
Accucore allows optimisation
of retention times
Retenti
Solid core produces sharper
peaks in less time
10
1,000.00 10,000.00 100,000.00
Efficiency
p
15
Accucore RP-MS 2.6µm 5µm 3µm <2µm Efficiency

16. Impedance
• Devised by Knox and Bristow in 1977
Defines 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
Pt
E

p
• Often plotted as a dimensional form
2
N
E
• t/N2
• t0 or tr both used
16

17. 100,000
Kinetic Plots – Impedance
100,000
2
0
N
Pt
E 

ce
2
N
10,000
mpedanIm
S lid i lSolid core requires less
pressure to obtain sub 2 µm
efficiencies
1,000
100.001,000.0010,000.00100,000.001,000,000.00
Accucore RP-MS 2.6µm 5µm 3µm <2µm
Efficiency
17
µ µ µ µ
Efficiency

18. The Impact of Selectivity on Resolution
Efficiency SelectivityRetentionEfficiency SelectivityRetention
2 5
3.0
2 5
3.0

N
R=
k’
k’+1
-1
4
N
R=
k’
k’+1
k’
k’+1
-1

-1
4
2.0
2.5
(R)
2.0
2.5
(R)
k +1 4
k2
k +1k +1 4
k’22
1.5 N
solution(
1.5 N
solution(
 =
k2
k’1
 =
k2
1
 =
2
1
0.5
1.0
k
Res
0.5
1.0
k
ResSelectivity () 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 1.25
0.0 
N
improving resolution. 0 5000 10000 15000 20000 25000
0 5 10 15 20 25
N
k
0 5000 10000 15000 20000 25000
0 5 10 15 20 25
N
k
S
18
Stationary phase, mobile phase, temperature

19. 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

20. 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

21. 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

22. 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

23. Column Characterization (Visualization)
HR /10
HSAI
Accucore C18
HR /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 PFP
HR /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

24. Widest Range of Solid Core Selectivity Options
500
mAU 1,2,3
curcuminoids
2 00
2.50
HR /10
HSAI
Accucore 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 Premium
1
0.00
HBCC
Accucore aQ
Accucore Polar Premium
Accucore Phenyl-Hexyl
Accucore PFP
2
3
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

25. 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

26. 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/min
Accela 1250
Dwell volume:
800 L
Surveyor Accela Surveyor Agilent
800 µL
Minutes
0.00 1.00 2.00 3.00 4.00
Accela
1250
Surveyor Agilent
1100
Run time
(min)
2.5 3.0 3.5
Dwell volume:
1000 µL
min0 0 5 1 1 5 2 2 5 3 3 5
Agilent 1100 Average
PW (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

27. 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 fast
45 pts
9 pts
Low dwell volume pump for fast
gradients
27

28. 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?

29. 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

30. Peptides – Resolution & Peak Shape
RT: 0 11 15 03RT: 0.11 - 15.03
1.12
8.74
3.76 7.09 13.468.91
11 20
Accucore 150-C18
11.20
13.63
1.01
C ti l S lid C C18
1.01
8.52
6.983.60
13.13
15.0310.84
Conventional Solid Core C18
100000 10.20< 2 µm Wide Pore Fully Porous C18
0
50000
uAU
8.491.59
14.4912.035.25 14.31
y
2 4 6 8 10 12 14
Ti ( i )
-50000
30
Time (min)

31. Proteins – Excellent Resolution vs < 2 µm Wide Pore
• Sharper and
higher peaks
80000
100000
Accucore 150-C4
Backpressure: 185 bar
higher peaks
than < 2 µm
Wide Pore Fully
Porous C4
40000
60000
uAU
Porous C4
• Better resolution
and sensitivity
100000
0
20000
• Significantly
lower
backpressure60000
80000
100000 < 2 µm Wide Pore Fully Porous C4
Backpressure: 320 bar
p
40000
60000
0 1 2 3 4 5 6 7 8 9 10
Ti ( i )
0
20000
31
Time (min)

32. Effect of Pore Depth for Small and Large Molecules
12.00
10.00
equation
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
pnents of va
2.00
B+C comp
0.00
0.00 20.00 40.00 60.00 80.00 100.00 120.00
Reduced Velocity, ν
32

33. 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
1i

34. 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

35. Approaches to Growing Solid Particles
• Sol-Gel process under acidic conditions
35

36. 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

37. Growing a Particle
Solid particle grows by stages
Nanotechnology 22 (2011) 275718
37
gy ( )

38. 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 )

39. The Recipe - Typically
• 0.25g PVP + 0.1g CTAB was dissolved in 5 ml DI water
8 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. Zhang
J. Chrom. A, Volume 1270 (2012) 194–203
39

40. 40

41. 41

42. Recipe Variables Already Investigated
• Increasing Stirring speed
• Bigger nanospheres
200rpm
Bigger 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

43. Varying the Recipe
Standard Method
0.25 g PVA (Mw 10k)
0 10 g CTAB0.10 g CTAB
5 ml DI Water
8 ml Methanol
2 ml NH4OH (5.6%)2 ml NH4OH (5.6%)
0.5 ml MPTMS
Increasing alkalinity
Smaller, less densely packed
nanospheres on surface
43

44. Varying the Recipe
Altering pH
Larger more densely packed nanospheresLarger, more densely packed nanospheres
on surface
Altering polymer concentration
Smooth spheres, no nanospheres, poor
dispersity
44

45. Varying the Recipe
Altering polymer concentration
Smooth spheres no nanospheres poorSmooth spheres, no nanospheres, poor
dispersity
Altering additive concentrationg
Small, monodisperse, fuzzy particles
45

46. 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

47. Varying the Recipe
Addition of extra polymer
Very small nanospheres on surfaceVery small nanospheres on surface
Changing pH
Fused smooth spheres
47

48. Normal Phase Separations
48

49. Reverse Phase Separations
49

50. Separation on an SoS Particle
50

51. Separation on an SoS Particle
Monodisperse SOS, C4 bonded, endcapped 1. GY
40 °C, 300 μL/min 2. VYV
Gradient: 10−40% B in 4 minutes 3. met-Enk
A. 0.02M KH2PO4 buffer, pH 2.7 + 0.1% TFA 4. leu-Enk
B. Acetonitrile + 0.1% TFA 5. Angiotensin II
51

52. 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

53. Acknowledgements
Haifei Zhang Adham Ahmed Kevin SkinleyHaifei Zhang Adham Ahmed Kevin Skinley,
(Laura), Peter Myers Richard Hayes
University of LiverpoolUniversity of Liverpool
Harry Ritchie
Trajan Scientific
53
Trajan Scientific