The last 10 years have seen substantial improvements in both chromatographic columns and instrumentation that have enabled faster and more efficient separations. The once common 4.6 x 150 mm column packed with 5 µm particles has been gradually replaced by smaller columns packed with smaller solid-core particles. To maximize the performance of these highly efficient solid-core particles one must understand the impact that instrument dispersion has on the chromatographic separation.

1. ©2015 Waters Corporation 1
Jonathan E. Turner
Product Marketing Manager
ACQUITY UPLC and CORTECS Columns
Waters Corporation
Understanding the Impact of System
Dispersion on Separation Performance

2. ©2015 Waters Corporation 2
Outline
 Quest for ultra performance
 Benefits and challenges of using smaller particles
 The importance of understanding system dispersion
 Classifying instruments based on system dispersion
 Selecting the correct column based on system dispersion
 Summary

3. ©2015 Waters Corporation 3
The Quest for UltraPerformance
1971 - 30-80 µm
pellicular particles
Efficiency
1970s 1980s 1990s 2000s 2010s
1973 - 10 µm
Irregular
shaped fully
porous silica
particles and
HPLC
Instrumentation
1980s - 5 µm
fully porous
spherical
particles
1990s – 3.5 µm
and 2.5 µm fully
porous spherical
particles
N. Cotta

4. ©2015 Waters Corporation 4
The Quest for UltraPerformance
1971 - 30-80 µm
pellicular particles
Efficiency
1970s 1980s 1990s 2000s 2010s
1973 - 10 µm
Irregular
shaped fully
porous silica
particles and
HPLC
Instrumentation
1980s - 5 µm
fully porous
spherical
particles
1990s – 3.5 µm
and 2.5 µm fully
porous spherical
particles
2004 – 1.7 µm fully
porous spherical
particles
N. Cotta

5. ©2015 Waters Corporation 5
The Quest for UltraPerformance
1971 - 30-80 µm
pellicular particles
Today Sub-2 µm
solid-core particles
Efficiency
1970s 1980s 1990s 2000s 2010s
1973 - 10 µm
Irregular
shaped fully
porous silica
particles and
HPLC
Instrumentation
1980s - 5 µm
fully porous
spherical
particles
1990s – 3.5 µm
and 2.5 µm fully
porous spherical
particles
2004 – 1.7 µm fully
porous spherical
particles
2007 - 2.7 µm
solid-core
particles
N. Cotta

6. ©2015 Waters Corporation 6
1k
k
α
1α
4
N
Rs



Mechanical Contributions
Ultra-low dispersion system
Operate at optimal linear velocity
Particle morphology
Small particles
Well-packed columns
Chemical/Physical
Contributions
Complementary bonded phases
Multiple particle substrates
Ability to utilize high pH
Increase retentivity
Increasing Resolution

7. ©2015 Waters Corporation 7
1k
k
α
1α
4
N
Rs



Mechanical Contributions
Ultra-low dispersion system
Operate at optimal linear velocity
Particle morphology
Small particles
Well-packed columns
Chemical/Physical
Contributions
Complementary bonded phases
Multiple particle substrates
Ability to utilize high pH
Increase retentivity
More Efficiency, More Resolution

8. ©2015 Waters Corporation 8
Improved Productivity
 Well packed columns containing smaller particles have more
intrinsic efficiency than columns packed with larger particles.
The Once Typical 4.6 x 100 mm, 3.5 µm Column
L = 100,000 µm = 28,571
dp 3.5 µm
Replaced with 2.1 x 50 mm, 1.6 µm solid-core Column
L = 50,000 µm = 31,250
dp 1.6 µm
N α L
dp

9. ©2015 Waters Corporation 9
Improved Productivity
2.1 x 50 mm, 1.6 µm
UPLC System
9X Faster
AU
0.00
0.10
0.20
0.30
0.00 0.50 1.00 1.50 2.50 3.00 3.50Minutes
2.10
AU
0.00
0.10
0.20
0.30
Minutes
0.00 4.00 8.00 12.00 16.00
20.00
24.00 28.00
4.6 x 100 mm, 3.5 µm
HPLC System
Tolmetin
Naproxen
Fenoprofen
Indomethacin
Diclofenac
M. Summers
Power of small particles and
Low dispersion Instrument

10. ©2015 Waters Corporation 10
Challenges in Using Smaller Particles
 As the column dispersion get smaller the influence of system
dispersion increases
– More significant for small narrow bore i.d. columns
– More significant on weakly retained analytes compared to strongly
retained analytes
 Instruments must be able to handle the additional back
pressure smaller particles generate when operated at their
optimal linear velocity.

11. ©2015 Waters Corporation 11
Challenges in Using Smaller Particles
 As the column dispersion get smaller the influence of system
dispersion increases
– More significant for small narrow bore i.d. columns
– More significant on weakly retained analytes compared to strongly
retained analytes
 Instruments must be able to handle the additional back
pressure smaller particles generate when operated at their
optimal linear velocity.
Backpressure does not equal performance

12. ©2015 Waters Corporation 12
Performance and Dispersion
 True separation performance is governed by the system
dispersion paired with a flow rate range that yields the
highest possible efficiency for a given analytical column
 Dispersion – n. Broadening of an analyte band due to both on-
column effects (diffusion and mass transfer kinetics which are
both dependent on particle size and linear velocity) and system
effects (tubing internal diameter (I.D.) and length,
connections, detector flow cell volumes, etc.)
2
,
2
,
2
, ColumnvSystemvtotalv
 

13. ©2015 Waters Corporation 13
Where Does Dispersion Occur?
Extra Column
Within Column
Band Spreading:
1) From the Injector (“Sample
Band)
2) Into, through and out of the
column (“Analyte Bands”)
3) Into the Detector
Analytical Column
E. Hodgdon

14. ©2015 Waters Corporation 14
Contributions to Dispersion
22
ectordet
2
ector,detv
2
postcolumn,v
2
column,v
2
precolumn,v
2
injector,v
2
total,v
F 
Injection
volume
+
injector
band-
spreading
Tubing
between
injector
and
column
Column
volume
+
Hardware
Design
+
Packing
Material
Tubing
between
column
and
detector
Band-
spreading
inside the
detector
cell
+
tubing
Time-based
Band-
spreading
in the
Detector
(Sampling
Rate; Time
Constant)
E. Hodgdon

15. ©2015 Waters Corporation 15
Extra Column Dispersion
 Engineering developments have reduced extra column
dispersion
– Injector design
– Reduced tubing volumes
– Reduced flow cell dispersion
22
det
2
det,
2
,
2
,
2
,
2
_, FectorectorvpostcolumnvprecolumnvinjectorvColExtv  
Injection
volume
+
injector
band-
spreading
Tubing
between
injector
and
column
Tubing
between
column
and
detector
Band-
spreading
inside the
detector
cell
+
tubing
Time-based
Band-
spreading
in the
Detector
(Sampling
Rate; Time
Constant)
E. Hodgdon

16. ©2015 Waters Corporation 16
Measuring Extra Column Dispersion
(Bandspread)
 Simple Approach
– Replace column with low volume union
– Run following method conditions:
– 7:3 Water:Acetonitrile at 0.3mL/min
– Sampling rate: 40Hz, λ = 273 nm
– Sample: 0.16 mg/mL Caffeine 9:1 Acetonitrile:water, 1 µL
injection
– Measure peak width at 13.4 % (4σ) or 4.4% (5σ)
 Dispersion can be expressed in two ways
 Extra column dispersion (µL) = peak width (min) * flow rate (µL/min)
 Variance (σ2) = (Extra column dispersion (µL)/σ)2)
A more complex approach of measuring bandspreading:
Gritti_Guiochon_Accurate measurements of true column efficiency_Journal of
Chromatography A, 1327 (2014) 49– 56.

17. ©2015 Waters Corporation 17
Instrument Dispersion Contribution to
Column Efficiency
System contribution: HIGH
Column contribution: Low
wide chromatographic band
System contribution: Low
Column contribution: Low
Narrow chromatographic band
Keeping the instrument dispersion contribution the same:
Larger i.d. columns are less susceptible to instrument dispersion
Column I.D 2.1 mm* 3.0 mm* 4.6mm*
Empty Column Volume 173 µL 353 µL (2X) 831µL (4.8X)
Flow Rate --- 2X Increase 4.8X Increase
Injection Volume --- 2X Increase 4.8X Increase
* For 50 mm length columns
For Small particles packed in narrow bore columns

18. ©2015 Waters Corporation 18
Retention Factor and Peak Volume
 Peak volume = peak width x flow rate
 Peaks with low retention factors have smaller peak widths
 2.1 mm i.d. columns are run at low flow rates
Early eluting peaks have volumes which are low; impact of
system dispersion is high
 4.6 mm i.d. columns are run at higher scaled flow rates (~5x)
with larger scaled injection volumes.
Early eluting Peak volumes are higher than the 2.1 mm i.d.
column; impact of system dispersion is lower

19. ©2015 Waters Corporation 19
Interaction of System Dispersion
and Peak Volume
Peak Volume Decreases
Impact of System Dispersion Increases
Column i.d.

20. ©2015 Waters Corporation 20
% Intrinsic Efficiency vs. Retention
Factor: High Instrument Dispersion
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
0 2 4 6 8 10
Retention Factor (k)
% of Intrinsic Efficiency vs k for 50 mm Columns
with20 uL2
Extra-ColumnVariance and Ni = 11,000
2.1 mm`
3 mm
4.6 mm
Peak Volume Increasing

21. ©2015 Waters Corporation 21
% Intrinsic Efficiency vs. Retention
Factor: Low Instrument Dispersion
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
0 2 4 6 8 10
Retention Factor (k)
% of Intrinsic Efficiency vs k for 50 mm Columns
with5.8 uL2
Extra-ColumnVariance and Ni = 18,500
2.1 mm`
3 mm
4.6 mm
Peak Volume Increasing

22. ©2015 Waters Corporation 22
% Intrinsic Efficiency vs.
Retention Factor
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
0 2 4 6 8 10
Retention Factor (k)
% of Intrinsic Efficiency vs k for 50 mm Columns
2.1 mm`
3 mm
High Bandspread
Low Bandspread
Peak Volume Increasing

23. ©2015 Waters Corporation 23
System Bandspread Influence Across
Different Column i.d.s
AU
0.00
0.02
0.04
0.06
AU
0.00
0.02
0.04
0.06
AU
0.00
0.02
0.04
0.06
Minutes
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50
4.6 x 50 mm
3.0 x 50 mm
2.1 x 50 mm
7200
4800
2900
Efficiency
3.6
2.5
1.4
Alliance HPLC: Bandspread 36 µL
Resolution

24. ©2015 Waters Corporation 24
Matching System Bandspread With
Column I.D.
AU
0.00
0.02
0.04
0.06
AU
0.00
0.02
0.04
0.06
AU
0.00
0.02
0.04
0.06
Minutes
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50
4.6 x 50 mm
3.0 x 50 mm
2.1 x 50 mm
Alliance
ACQUITY Arc
ACQUITY UPLC H-Class
7200
7600
7300
Efficiency
3.6
Resolution
3.8
3.7

25. ©2015 Waters Corporation 25
Dispersion Impact on Performance:
Gradient Separations on UHPLC and UPLC
Column: C18 2.1x 50 mm
USP Assay for Diclazuril
UHPLC
Extra column dispersion 25 µL
UPLC
Extra column dispersion< 10 µL
1
2
3
4 5 6
USP Res= 1.5
USP Res= 2.0 USP Res= 2.7
USP Res= 1.8
No Compound
1 6 carboxylic acid
2 6-carboxamide
3 Diclazuril
4 Ketone
5 4-amino Derivative
6 Des-cyano derivative
AU
0.000
0.012
0.024
0.036
0.048
AU
0.000
0.012
0.024
0.036
0.048
Minutes
1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00

26. ©2015 Waters Corporation 26
Defining the LC Categories by their
System Dispersion

27. ©2015 Waters Corporation 27
Defining the LC Categories by their
System Dispersion
Dispersion > 30 µL
Columns accepted:
• 3.0 – 4.6 mm ID
• 3 - 10 µm particles
Optimal:
• 4.6 mm ID, 5 µm
Typical operating pressure:
• < 6,000 PSI
Dispersion 12 - 30 µL
Columns accepted:
• 2.1 - 4.6 mm ID
• 1.7 - 5 µm particles
Optimal:
• 3.0 mm ID, 2.x µm
Typical operating pressure:
• 6,000 – 15,000 PSI
Dispersion < 12 µL
Columns accepted:
• 1.0 - 4.6 mm ID
• 1.6 - 5 µm particles
Optimal:
• 2.1 mm ID, 1.7 µm
Typical operating pressure:
• 9,000 – 15,000 PSI
Increased flexibility and sample characterization

28. ©2015 Waters Corporation 28
Defining the LC Categories by their
System Dispersion
How are these categories differentiated?
Chromatographic Resolution Increases
Overall Run Time Decreases
Method Sensitivity Increases

29. ©2015 Waters Corporation 29
Maximizing the Separation Performance
based on System Dispersion
Determine your systems
Dispersion
Matching the right LC system
to the right column will yield
the best chromatographic
results

30. ©2015 Waters Corporation 30
Maximizing the Separation Performance
based on System Dispersion
Select the appropriate particle
size to match the HPLC,
UHPLC, or UPLC system

31. ©2015 Waters Corporation 31
Maximizing the Separation Performance
based on System Dispersion
Pair the particle size with the
column i.d. that best matches
the dispersion of your
chromatographic system

32. ©2015 Waters Corporation 32
Maximizing the Separation Performance
based on System Dispersion
Select a flow rate
that gives the
optimal linear
velocity to maximize
efficiency for your
column
characteristics (van
Deemter)

33. ©2015 Waters Corporation 33
Maximizing the Separation Performance
based on System Dispersion
The system must be
able to operate at the
typical back
pressures associated
with the selected
column

34. ©2015 Waters Corporation 34
Maximizing the Separation Performance
based on System Dispersion
The relative cost/per analysis will decrease as you move from HPLC to UHPLC to UPLC
due to shorter run times at lower flow rates

35. ©2015 Waters Corporation 35
Maximizing the Separation Performance
based on System Dispersion
HPLC
UHPLC
UPLC

36. ©2015 Waters Corporation 36
Performance is Impacted when
System and Column are not Matched
UHPLC UPLCHPLC
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
2.1 mm ID
1.6 μm
50mm length
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
3.0 mm ID
2.7 μm
50mm length
4.6 mm ID
2.7 μm
50mm length

37. ©2015 Waters Corporation 37
Performance is Impacted when
System and Column are not Matched
UHPLC UPLCHPLC
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
2.1 mm ID
1.6 μm
50mm length
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00
AU
0.00
0.20
0.40
0.60
0.80
1.00
Minutes
0.50 1.00 1.50 2.00 2.50
3.0 mm ID
2.7 μm
50mm length
4.6 mm ID
2.7 μm
50mm length

38. ©2015 Waters Corporation 38
Maximizing the Separation Performance
based on System Dispersion
Chromatographic System Particle Size Band Spread
Recommended Column ID
Primary Secondary
1.6 µm < 12 µL 2.1 mm 1.0 mm
2.x µm 12 – 30 µL 3.0 mm 2.1 mm
5 µm >30 µL 4.6 mm 3.0 mm
P. McConville

39. ©2015 Waters Corporation 39
Summary
 When developing a chromatographic method system
dispersion is a critical value to know
 Maximum performance can be achieved as long as
you match the sytem dispersion to the proper column
particle size and configuration.
 Increasing column i.d. can mitigate dispersion effects;
however, solvent and sample consumption and
ultimately cost to operate would increase

40. ©2015 Waters Corporation 40
Acknowledgements
 Paula Hong
 Michael D. Jones
 Eric Grumbach
 Patricia McConville
 Dick Andrews
 Peyton Beals
 Brooke Koshel
 Nicole Cotta
 Jacob Fairchild
 Liz Hodgdon

41. ©2015 Waters Corporation 41
Questions