In the pharmaceutical arena there is great interest in solid core technology, where there is a broad range of sample types as well as requirements throughout the process of developing new chemical entities. The presentation looks at how solid core technology can be readily adapted to cope with the challenges associated with the pharmaceutical sector, looking at various sample matrices and molecular entities, from small molecules to large biomolecules. The presentation gives an insight into how varying the solid core to porous layer allows the user to optimize separation performance by reducing extra band broadening. Data presented demonstrates how this technology is more robust than fully porous systems when analyzing biological extracts, routinely used in DMPK departments, resulting in longer column lifetimes.

1. Use of Solid Core Chromatography for the
Analysis of Pharmaceutical Compounds
Dafydd Milton
Product Manager, LC & LC/MS columns
Tony Edge
R&D Principal
March 2014

2. Introduction
• The Pharmaceutical Process
• Mapping out different sectors
Wh t t f h ll f d• What types of challenges are faced
• Understanding the drivers in the pharmaceutical industry
• Solid Core Chromatography
• Understanding the benefits of the technology
• Bar for bar greater efficiency• Bar for bar greater efficiency
• How to optimise the separation
• Understanding chemistry
• Optimization of the morphology
• Coupling Solid Core Chromatography to Pharmaceutical Analysis• Coupling Solid Core Chromatography to Pharmaceutical Analysis
• Design of new workflows
• Improving column lifetimes
2
• Improving assay robustness

3. The Pharmaceutical Process – Beginning
3

4. The Pharmaceutical Process – Further Development
4

5. Solid Core Material – Features and Benefits
• Features
• More uniform particle sizing• More uniform particle sizing
• Better packing of particles
• Reduced pore depth
• Reduced mass transfer effects in mobile phase
• BenefitsBenefits
• More Efficient Chromatography
• Allows the use of low pressure systems
• Competitive Edge
• Bar for bar gives better separations than porous materialsg p p
5

6. 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
6
operating pressurey y g y g y

7. 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
7
Temperature: 30 °C

8. The Theory … the van Deemter equation
CC
B
AHETP  uCuC
u
AHETP sm 
8

9. A – Term Eddy Diffusion or Multiple Paths
Packing Efficiency
D90/10~1.5
Porous Silica
Packing Efficiency
D90/10~1.1Accucore
9

10. B – Term Longitudinal Diffusion
Longitudinal diffusion: (HETP = H + H + H )Longitudinal diffusion: (HETP = HA + HB + Hc)
Th l t t ti i l t th d f b d l tThe solute concentration is lower at the edges of a band; solute
diffuses to the edges.
Time
High concentration C t ti t ilib iHigh concentration. Concentration at equilibrium
• The B-term depends on;
10
• Void volume of the column

11. 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
11
• Significant for large molecules but not for small molecules

12. Efficiency Comparison – Van Deemter
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
12
Flow rate range: 0.1 to 1.0 mL/min
Analyte: o-xylene rate for solid core

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. Resolution Equation







 



 '11 50 k




















'1
1
4
1 5.0
k
k
NRs


Efficiency
Particle size / packing
Selectivity
(Chemistry)
Retention
FactorParticle size / packing (Chemistry) Factor
(Surface area)
18

19. 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
19
Stationary phase, mobile phase, temperature

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. Accucore Columns – Selectivity Choices
Columns: Thermo Scientific™ Accucore™ C30 Column
Accucore C18 Column
Ki t C181
Different selectivity for K2 isomers
350 mAU
Accucore C18
Kinetex C18
Dimensions: 2.6 µm, 100 x 3.0 mm
Mobile Phases: Methanol:buffer, 98:2
Buffer = 2 mM ammonium acetate
1
250
300 2+2’
Flow: 650 µL/min
Temperature: 20 ºC
Injection: 5 µL
Detector: UV 250 nm
2+2’
Ki t C18
200
250
Detector: UV 250 nm
Peaks: 1. Vitamin K2, 50 µg/mL
2. Vitamin K1, 50 µg/mL
Other peaks formed by UV irradiation
1
C30 shows better
separation for K1
Kinetex C18
100
150
2
2’
separation for K1
isomers
Accucore C30
50
0
Vitamin K2 Vitamin K10.00 1.25 2.50 3.75 5.00 6.25 8.00
-25
min
25

26. Very Fast Separations with Superb Resolution
• Separation of atorvastatin
i t di t (ATC AT1
Fully porous C18
5 μm, 250 x 4.6 mm
intermediates (ATC-AT1
with ATC-AT1-Difluro)
• All customer requirements 60 min• All customer requirements
were met
• Reduction of run time from 60 min to
60 min
8 min
• Resolution improved using the
Accucore PFP column
Accucore PFP
2.6 μm, 150 x 3.0 mm 8 min
• Mobile phase constituents kept
similar
• HPLC compatible method
(270 bar)
26

27. Available Databases for Column Characterisation
http://www.usp.org/app/USPNF/columnsDB.html
27

28. Some Basic Column Requirements
• Column Ruggedness
• Stable under isocratic conditions
• Stable under gradient conditions
• Stable at low pH
• Stable at high pHg p
• Stable at elevated temperatures
28

29. Solid Core Column Stability – Ruggedness
Accucore RP-MS 2.6 µm 100 x 2.1 mm ID
Mobile Phase: 60/40 ACN/H2O
Flow Rate: 400 µL/min
Accucore RP-MS 2.6 µm 100 x 2.1 mm ID
Mobile Phase A: Water (0.05% TFA)
Mobile Phase B: Acetonitrile (0.05% TFA)
W h H O (0 05% TFA)Injection Volume: 1 µL
Column Temp: 30 °C
Wash: H2O (0.05% TFA)
Injection Volume: 1 µL
Column Temperature: 30 °C
Efficiency (o-Xylene)
Asymmetry (o-Xylene)Asymmetry (o Xylene)
4 000 + isocratic test 6 000 f t di t4,000 + isocratic test
injections with no
decrease in performance
6,000 + fast gradient
injections with no
change in retention
29

30. Solid Core Column Stability – Low pH
40
Column Stability at pH < 2
pH  1.8
(0.1% TFA)
30
35
30,000
l
20
25
tionFactor
Acetaminophen
p-HBA
o-HBA
column
volumes
(5.5 days)
10
15
Retent
Amitriptyline
Nortriptyline
DIPP
DNPP
(5 5 y )
5
0
0 5000 10000 15000 20000 25000 30000 35000
Column Volumes
Solid core columns are stable at low pH
30
Solid core columns are stable at low pH

31. Solid Core Column Stability – High pH
pH  10.5
(0.1% ammonia)
30,000
column
r
column
volumes
(5.5 days)
ntionFactorReten
S lid l t bl t hi h H
31
Solid core columns are stable at high pH

32. Solid Core Column Stability – Elevated Temperature
8
9
Column Stability at 70°C Mobile phase: MeOH/H2O (65:35)
Flow rate: 0.4 mL/min
6
7
8
r
Column temperature: 70 °C
Column: Accucore C18 50 x 2.1 mm
Run time: 5 min
4
5
6
entionFactor
Phenol
Butylbenzene
Run time: 5 min
2
3
Rete
o-Terphenyl
Pentylbenzene
0
1
0 2000 4000 6000 8000 10000
9,000
column volumes
(400 i j ti )
0 2000 4000 6000 8000 10000
Column Volumes
(400 injections)
Solid core columns are stable at high temperature
32
Solid core columns are stable at high temperature

33. Work Flow Solutions – Generic Methods
• Used where sample throughput is critical
• Compound management
• Discovery DMPK
• Ability to run at high flow rates without compromising chromatographyAbility to run at high flow rates without compromising chromatography
• Require robust methods
• Assays cannot afford to fall over
• Many samples means long column lifetime
• For bioanalytical samples need columns that are robust with plasma extractsFor bioanalytical samples need columns that are robust with plasma extracts
• Require orthogonal chemistries
• Reversed phase / HILIC etc.
33

34. Faster than 5 and 3 µm
Fully porous 5 µm,
150 x 4.6 mm
Rs = 2.64
5 µL injection
∆P = 59 bar
 Gradient and flow rate:
• Fully porous 5 μm 150 x 4.6 mm
35–60 %B in 10.0 min
1000 µL/min solvent used 17 mL1µL injection ∆P 23 b 1000 µL/min solvent used 17 mL
•Fully porous 5 μm, 100 x 2.1 mm
35–60 %B in 6.7 min
210 µL/min solvent used 2.4 mL
F ll 3 100 2 1
Fully porous 5 µm,
100 x 2.1 mm
Rs = 1.64
1µL injection ∆P = 23 bar
• Fully porous 3 μm, 100 x 2.1 mm
35–60%B in 4.0 min
350 µL/min solvent used 2.4 mL
• Accucore RP-MS 2.6 μm, 100 x 2.1 mm
Fully porous 3 µm,
100 x 2.1 mm
Rs =1.96
1µL injection
∆P = 97 bar
35–60 %B in 3.5 min
400 µL/min solvent used 2.4 mL
ACCUCORE 2.6 µm,
100 x 2.1 mm
Rs = 2.50 1µL injection ∆P = 218 bar
Reduced analysis time and solvent costs
Minutes
0 1 2 3 4 5 6 7 8 9 10
-100
100 x 2.1 mm
34
Reduced analysis time and solvent costs

35. Shorter Columns – Faster Separations
ACCUCORE 2.6 µm,
100 x 2.1 mm  Gradient and flow rate:
• Accucore RP-MS 2.6 μm, 100 x 2.1 mm
Rs = 2.50
35 –60%B in 3.5 min
400 µL/min
• Accucore RP-MS 2.6μm, 50 x 2.1 mm
35–60%B in 1.8 min
mAU
400 µL/min
Rs = 1.51
ACCUCORE 2.6 µm,
50 x 2.1 mm
Double productivity with 50 mm column
Minutes
0.0 1.0 2.0 3.0 4.0
35
Double productivity with 50 mm column

36. Example of a HILIC Separation
Column: Accucore HILIC 2.6 μm, 150 x 3.0 mm
Flow: 0.5 mL/min
B k 290 b
Separation of decitabine
and α anomer impurity
Backpressure: 290 bar
Temperature: 40 °C
Injection: 5 µL and α-anomer impurityj µ
Detection: UV @ 244 nm
Mobile phase: 5% A (20 mM
ammonium acetate)ammonium acetate)
95% B (acetonitrile)
36

37. Regulatory DMPK – Bioanalysis
• Methods can be optimized
• Possibilities to optimize stationary phase chemistries• Possibilities to optimize stationary phase chemistries
• Analysis times still important for PK studies
• Methods will tend to form final clinical method
• Sensitivity can be an issue due to efficacious nature of drug
• Injections with plasma• Injections with plasma
• Columns must not block
• Can result in peak splitting
• Can result in columns overpressurising
• Can result in retention time shift
• Metabolism studies
• Need columns with high resolution
37
• Ideally limited sample prep, so columns stable with diluted urine

38. Lower Pressure than Sub 2m
• Flow rate: 500 μL/min
• Mobile phase: A Water; B Acetonitrile
Sub 2 µm, 100 x 2.1 mm
• Mobile phase: A–Water; B–Acetonitrile
Accucore RP-MS 2.6 µm,
100 x 2.1 mm
Accucore RP-MS 2.6 µm,
100 x 2.1 mm
Sub 2 µm,
100 x 2.1mm
Maximum
pressure (bar)
171 338
Minutes
0.0 0.5 1.0 1.5 2.0 2.5
Equivalent performance, lower pressure
38
(50% lower)

39. Accucore with TLX – Method Conditions
Sample Preparation
• Acetonitrile with internal standard added to spike plasma sample
• 2 (600 μL) parts acetonitrile : 1 part spiked plasma (300 μL)
Precipitated sol tion mi ed on orte mi er
Autosampler Method LC Method
• Precipitated solution mixed on vortex mixer
• Sample centrifuged and supernatant transferred to 2 mL vial
• 10 μL injected onto system
p
• Wash Solvent A – 20% acetonitrile + 0.1% Formic Acid +
80% water
• Wash Solvent B – 45%IPA + 45% acetonitrile + 10%
Acetone + 0.1% Formic Acid
• Mobile Phase A – 0.1% Formic Acid in water
• Mobile Phase B – 0.1% Formic Acid in acetonitrile
• Columns: Cyclone 50 x 0.5 mm (TFC), Accucore C18 50 x
2.1 mm (Analytical)
• 100 μL sample loop, 100 μL syringe
39

40. Accucore with TLX – Retention Stability
4.4000
Rosuvastatin Retention
4.1000
4.2000
4.3000
3.9000
4.0000
Minutes
3 6000
3.7000
3.8000
3.5000
3.6000
0 500 1000 1500 2000 2500
Injection
~2,400 injections on Accucore column
with TLX system – no change in retention
40
y g

41. Accucore with TLX – Backpressure Stability
140
Backpressure Plots
100
120
60
80
Bar
Linear (Loading Pump Pressure at t=0) Linear (Eluting Pump Pressure at t=0)
20
40
0
0 500 1000 1500 2000 2500
Injection
~ 2,400 injections on Accucore column with
TLX system – no increase in t=0 backpressure
41
y p

42. Accucore with TLX–Pressure Traces
300
Backpressure Traces
250
150
200
Bar
100
Injection 2 Elute
Injection 500 Elute
Injection 2395 Elute
50
0 50 100 150 200 250 300 350
Seconds
~ 2,400 injections on Accucore column
with TLX system – backpressure traces
42
y p

43. Greater Peak Capacity than 5 or 3m
220
240
5 µm,100 x 2.1 mm
 Gradient: 65–95%B in 2.1 min,
95% B for 0.4 min
3 100 2 1
 Flow rate: 400 μL/min
3 m, 100 x 2.1 mm
120
140
160
180
capacity
ACCUCORE 2.6 µm,
100 x 2.1 mm
40
60
80
100
120
malisedpeakc
0 0 0 5 1 0 1 5 2 0 2 5 3 0
0
20
40
Accucore 2.6µm 3µm 5µm
Norm
Minutes
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Higher peak capacity – more peaks can be separated per injection
43
g p p y p p p j

44. More Sensitive than Fully Porous 5 and 3m
 Gradient and flow rate:
• 5 μm, 100 x 2.1 mm
35–60 %B in 6 7 min
S/N = 169 5m, 100 x 2.1 mm
35–60 %B in 6.7 min
210 μL/min
• 3μm, 100 x 2.1 mm
35–60 %B in 4.0 min
350 μL/min
S/N = 368
350 μL/min
•Accucore RP-MS 2.6μm, 100 x 2.1 mm
35–60 %B in 3.5 min
400 μL/min
mAU
S/N = 399
3m, 100 x 2.1 mm
S/N = 399
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
ACCUCORE 2.6m,
100x2.1mm
Higher S/N ratios – detection and quantification
of low level impurities
Minutes
44
of low level impurities

45. Production - Quality Control Workflows
• Robust methods
• Loading capacity can be an issue as looking for impurities• Loading capacity can be an issue as looking for impurities
• Selectivity important
• Resolution also important
• Need to be able to transfer the methods to CRO’s
• Need to be aware of differences caused by different instrumentation• Need to be aware of differences caused by different instrumentation
45

46. Loading Capacity
Columns:
• Accucore RP-MS 2.6 μm,100 x 2.1 mm
• <2 μm,100 x 2.1 mm2,500,000
1 2
Effect of Loading - Accucore
R² = 0.9998
R² = 0.9993
2,000,000
0.8
1
1.2
dValue
A
R² = 0.9721
1,000,000
1,500,000
eakarea
0.2
0.4
0.6
Normalise
As
N
Tr
As
N
Tr500,000
Pe
0
0 5 10 15 20 25
Load on Column (µg)
0
0 0.5 1 1.5 2 2.5
Load on column (µg)
<2µm Accucore 2 6μm Competitor
No loss in performance with 2 μg loaded on a
<2µm Accucore 2.6μm Competitor
46
2.1 mm ID Solid core column

47. Method Transfer and Optimisation
5 μm, 150 x 4.6 mm
Method Transfer Calculator:
www.thermoscientific.com/crcRs = 2.64
5 µL injection
 Gradient and flow rate:
• 5 μm, 150 x 4.6 mm
35 60 %B in 10 0 minRs = 2 50 35–60 %B in 10.0 min
1000 μL/min
• Accucore RP-MS 2.6 μm, 100 x 2.1 mm
35–60 %B in 3.5 min
Rs = 2.50
400 μL/min
• Accucore RP-MS 2.6 μm, 50 x 2.1 mm
35–60 %B in 1.8 minutes
400 μL/min
ACCUCORE 2.6 μm,
100 x 2.1 mm
Rs = 1.51
1µL injection
400 μL/min
ACCUCORE 2.6 μm,
50 2 1
1µL injection
Minutes
0 1 2 3 4 5 6 7 8 9 10
0
50 x 2.1 mm
Scalable from fully porous 5 μm columns
µ j
47
Scalable from fully porous 5 μm columns

48. 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
48
number of different systems

49. System Considerations
• Minimise volume dispersion
Always Optimize System Configuration
• Tubing–short L, narrow ID
• Low injection volume
• Low volume flow cell• Low volume flow cell
• Optimise 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
49

50. 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
50

51. 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
51
Time (min)

52. 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
52
Time (min)

53. Conclusions
• The Pharmaceutical Process
• Mapping out different sectors
Wh t t f h ll f d• What types of challenges are faced
• Understanding the drivers in the pharmaceutical industry
• 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
• Coupling Solid Core Chromatography to Pharmaceutical Analysis• Coupling Solid Core Chromatography to Pharmaceutical Analysis
• Design of new workflows
• Improving column lifetimes
53
• Improving assay robustness