The presentation discuses the benefits of micro and nanocolumn LC and makes comparisons to gradient CEC.

1. Recent Advances in
Microcolumn LC and
Capillary Electrochromatography
James N. Alexander, IV
Liquid Separations Group
Rohm and Haas Company
Spring House, PA
CFDV
October 14, 1998

2. Presentation Topics
Micro LC
u
Driving Forces
u
u Instrument Considerations
u High Resolution Separations
CEC
u
Gradient Instrumentation
u
u Comparison of Nano LC and CEC
Conclusions
u

3. What is Microcolumn LC?
Column i.d. Flow Rate Classification
(µm) (µL/min)
4600 1000 Analytical
2100 208 Narrowbore
1000 47 Microbore
500 12 Microbore
320 5 Microcolumn
250 3 Microcolumn
50 0.12 Nanocolumn/
Packed Capillary

4. Driving Forces
Higher efficiency / resolving power
Flexibility
LC-MS
Novel detection schemes
Increased mass sensitivity
Temperature control
Reduced solvent consumption
Two-dimensional chromatography

5. Challenges
Hardware
u
u Detectors
u Automation
u Column availability
u Long analysis times
u Dead volume free
connections
u Detection of micro leaks

6. Flexibility
Lengths up to a 1 meter, 25 - 250 µm i.d.
Any packing material can be used (0.1 g / micro-
column)
Microcolumns can be prepared for 1/16 the cost
of an analytical column (about $25)
Observe packing process
Use for scouting purposes

7. Microcolumn Preparation
Dense bed (‘slam pack’)
Slurry Method Loose bed (‘gradual pack’)
1) Prepare fused silica capillary
2) Slurry stationary phase (1-10%)
3) Sonicate / shake slurry (1-2 hrs)
4) Transfer to slurry reservoir Speaker/
5) Connect fused silica capillary Ultrasonic bath
6) Pack (2,000-10,000 psi, 2-24 hr)
7) Bleed pressure
8) Secure outlet frit
Solvent Pump
Fused silica capillary
Reservoir
Stir plate

8. Microcolumn Assembly
Glass Wool Frit
Window
Column Packing
Teflon Sleeve
250 µm i.d. x 365 µm o.d. 50 µm i.d. x 365 µm o.d.
Fused silica capillary Fused silica capillary

9. Split and Moving Injections
Mobile Phase In
Sample In Splitter Vent
Fused Silica
Column
Waste
Splitter Tee
Injector, internal loop
Moving Injection
Split Injection
M. C. Harvey and S. D. Sterns
T. Tsuda and G. Nakagawa
Anal. Chem., 1984, 56, 837.
J. Chromatogr., 1988, 199, 249.

10. Pre-Injection Split
Mobile Phase In
Splitter Vent
Splitter Tee
Sample In
Fused Silica
Waste
Column
Injector, internal loop

11. Split Ratios
(B i.d.)2
(A i.d.)2
Column Injection Flow Split
i.d. Volume Rate Ratio
(µm) (µL) (µL/min)
4600 50 1000 333 : 1
2100 10 200 67 : 1
1000 2.4 47 16 : 1
500 0.59 12 4:1
320 0.24 5 2:1
250 0.15 3
50 0.01 0.1

12. Detection Schemes for Microcolumn LC
Home Made
Commercially Available
Condensation Nucleation
UV/Vis
Electrospray Ionization
Electrochemical
Gas Phase Detection
Fluorescence
Refractive Index
Mass Spectrometry
Conductivity
Evaporative Light Scattering
Radioactivity Monitor

13. On-Column Detection Cells for
Microcolumn LC
F. J. Yang,
J. Chromatogr.,
1982, 236, 265.
J.P. Chervet, et al.,
LC.GC Int.,
Polyimide Coating
1989, 2, 40.
Column Packing
Mono- Photo-
chrometer multiplier
Light Source
Mono- Photomultiplier
chrometer
‘Z-Shaped’ cell
Light Source

14. Micro-LC Systems
Microflow
HPLC Pump
processor
Autosampler
Detector
Injector
Autosampler/
Injector
Microcolumn
Column Oven
Micro-pump
Detector
System A System B

15. Retention Time Reproducibility
Alltech Adsorbosphere HS C18 5µm
250 µm x 100 cm, 2.9 µL/min, 254 nm, 30°C
100 nL injection, split 1:10 prior to injector
Analyte = Toluene
Isocratic Gradient
ACN/H2O
ACN/H2O
(58:42) (30:70 - 90:10) 90 min.
Injection Retention Time (min.) Retention Time(min.)
1 63.72 77.64
2 63.80 77.65
3 63.83 77.61
4 63.75 77.57
5 63.73 77.63
6 63.84 77.66
7 63.81
8 63.74
9 63.74
10 63.69
Average 63.76 77.63
std. dev. 0.051 0.033
% RSD 0.08% 0.04%

16. Higher Efficiency / Resolution
N = L / h dp N1/2 (α−1) k2
Rs =
α k2+1
4
N = theoretical plates
Rs = resolution
L = column length
α = selectivity factor
h = reduced height k2 = capacity factor
equivalent to a theoretical
plate
μηL
dp = mean particle diameter P=
ko dp2
P = pressure drop
μ = linear velocity
η = mobile phase viscosity
ko= constant

17. Octylphenol Ethoxylate
Reversed-Phase
(OCH2CH2)x OH
C8H17
4.6 mm x 25 cm, PLRP-S
1 mL/min
R
250 µm x 31 cm, PLRP-S
e
s 3 µL/min
p
o
n
s 250 µm x 100 cm, PLRP-S
e 3 µL/min
Time (minutes)

18. Linear Alkylbenzene Sulfonate
Ion-Pair
CH3(CH2)xCH(CH2)yCH3
250 µm x 31 cm, PLRP-S
um -
SO3
3 µL/min
uL/min
R
e
s
p
o
250 µm x 100 cm, PLRP-S
um
n
3 µL/min
uL/min
s
e
Time (minutes)

19. Sample A
Reversed-Phase
Alltech Adsorbosphere HS C18 5 µm
210 nm, 30ºC, 20 µL injection
ACN/H2O (gradient)
R 4.6 mm x 25 cm, 1 mL/min
e
s
p
o
n
210 nm, 30ºC, 100 nL injection
s
ACN/H2O (gradient)
e
250 µm x 100 cm, 2.9 µL/min
Time (minutes)

20. Sample A
Characterization 1
11 2
Sample A 33
3
22
3
R
e
s Isomer 1
p
o
n
s
Isomer 2
e
Isomer 3
Time (minutes)

21. Water Soluble Polymer
Aqueous GPC
Macrosphere GPC 60
4.6 cm x 25 cm
0.1 mL/min
R
e
s
p
o
250 µm x 73 cm
n
0.3 µL/min
s
e
Time (minutes)

22. ELSD - Operation
LC Effluent
Nebulization Gas Nebulizer
(Nitrogen)
Drift Tube
(ambient - 140°C)
Laser Diode
(670 nm, 7
mW) Silicon Photo
Diode
Waste

23. Micro - ELSD Fused silica (effluent )
Control A Capillary
of capillary
Tip Position
Bellows
Nebulization Effluent capillary secured
gas to base of bellows
Extended Withdrawn
Flush

24. Micro - ELSD
Control of Nebulization Height
3
1 5
Insert
Nebulization tube tube 3
1 5
Micro-nebulizer
Effluent capillary
assembly
25 cm from light
source
Drift tube
Aerosol
6 cm from light
source
Light
source
Photodiode

25. Complex Sample A
Micro-ELS Detection
Alltech Adsorbosphere HS C18 5
UV @ 210 nm
µm
250 µm x 100 cm, 2.9 µL/min
ACN/H2O (gradient), 30ºC
R
100 nL injection (0.8 µg)
e
s
p
o 170 180 190 200 210 220 230 240 250
n
s Micro-ELSD @ 60ºC
e
= Peaks not detected at 210 nm
170 180 190 200 210 220 230 240 250
Time (minutes)

26. 200 mw Poly(Ethylene Glycol)
Micro-ELS Detection at Various Drift Tube Temperatures
6
7
5 EO
70ºC 8
9
R 0 10 20 30 40 50 60 70 80
e 5 6
7
s
p 50ºC 8
o 4 EO
9
n
s 0 10 20 30 40 50 60 70 80
e 5 6
4 7
30ºC
8
3 EO
9
0 10 20 30 40 50 60 70 80
Time (minutes)

27. What is CEC?
Liquid Chromatography Capillary Electrophoresis
high efficiency
high separation
(electroosmotic flow)
capacity
(packed column)
Capillary Electrochromatography
packed capillary column
electrically driven

28. Advantages of CEC Over
Pressure Driven Systems
+
+- -
-- +
+-
+
-- --
Flat flow profile
u ++++
-
+
++++
----
increase in efficiency
u
EOF
No pressure drop
u
smaller particles (<3 µm) can be used
u
long columns (>50 cm) can be used
u

29. Challenges in CEC
Column life time
u
Frits
u
retain packing
u
minimize bubble formation
u
Bubble formation
u
degas, pressure, temperature
u
Isocratic separations
u
Analytes
u
neutrals
u

30. Research Objectives
u Build a system that:
u allows for nano LC or CEC use under
isocratic and gradient conditions
u does not require separate columns for
nano LC or CEC
u make it simple and reproducible
u Investigate technique and compare to
nano LC

31. Nano Column Assembly
Polyimide Window
Column Packing Coating
Sintered Frits
Common dimensions
75 µm I.d. x 350 µm o.d. x 39 cm (25 cm eff.)

32. Schematic of Gradient Elution
Nano LC / CEC 11
12
A
6 B
9
5
- 13
10
2 8
3 7 14
1 4
1. Power supply 5. Nanocolumn 9. Ground cable 13. Dynamic mixer
2. Platinum electrode 6. Switching valve 10. Injection valve 14. Micro LC pumps
3. Outlet reservoir vial 7. Stand 11. Plexiglas compartment
4. UV Detector 8. Split device 12. Autosampler

33. Close-up of Split Device and Injector
No Restrictor Restrictor
CEC Nano LC or PEC
6
1
3
2
1. Nanocolumn
2. Knurled nut and ferrule
4 5
3. Split device 5. Rotor
4. Injection valve 6. Outlet port

34. Relationship Between Applied Voltage
and Linear Velocity
1.2
1
Linear Velocity (mm/s)
0.8
0.6
0.4
0.2
5 10 15 20 25
Voltage (-kV)

35. Isocratic Separations at Different
Applied Voltages 4
3
N = 19,713
2
5
6
-7.5 kV
1
0 10 20
N = 27,573
-15 kV
1. Uracil
2. Phenol
3. Benzaldehyde
0 10 20
4. N,N-diethyl-m-toluamide
N = 18,541 5. Toluene
-25 kV 6. Ethylbenzene
0 2 4 6 8 10 12 14 16
Time (min)

36. Isocratic Separation of Test Mixture
by Nano LC and CEC
1. Uracil
3 4
16400
Nano LC 2. Phenol
16200
118 nL/min
3. Benzaldehyde
5
16000 v = 1.2 mm/sec 2
4. N,N-diethyl-m-toluamide
6
15800
1
uV 5. Toluene
15600
6. Ethylbenzene
15400
15200
0 2 4 6 8 10 12
Time (min)
20000
CEC
-17.5 kV
v = 1.1 mm/sec
18000
uV
16000
0 2 4 6 8 10 12
Time (min)

37. Comparison of Efficiencies Between
Nano LC and CEC
Nano LCa CECa CECb
Efficiency Efficiency Efficiency
Analyte (N /meter) (N /meter) Increase (N /meter)
Uracil 57900 95048 64% 167870
Phenol 69072 92936 35% 174700
Benzaldehyde 76288 106724 40% 146183
N,N-diethyl-m-toluamide 58316 108232 86% 199420
Toluene 78284 123492 58% 127937
Ethylbenzene 78468 126568 61% 135750
Average of all analytes 69721 108833 57% 158643
Reduced plate height (h ) 4.9 3.1 2.2
Nanocolumn A, 75 µm i.d. x 39 cm (25 cm eff. length), C18, 3 µm
a
Nanocolumn B, 75 µm i.d. x 39 cm (30 cm eff. length), C18, 3 µm
b

38. Comparison of Retention Times Using
Isocratic Elution
Nano LC CEC
Retention Retention
Time Time
Analyte (min.) RSD (min.) RSD
Uracil 3.6 0.13% 4.0 0.61%
Phenol 4.9 0.11% 5.1 0.54%
Benzaldehyde 6.0 0.19% 6.1 0.47%
N,N-diethyl-m-toluamide 8.0 0.15% 8.0 0.56%
Toluene 8.6 0.13% 8.5 0.64%
Ethylbenzene 10.3 0.17% 10.0 0.42%

39. Comparison of Peak Areas Using
Isocratic Elution
Nano LC CEC
Peak Areaa Peak Areaa
Analyte RSD RSD
Uracil 0.24 2.2% 0.23 4.9%
Phenol 0.48 2.5% 0.52 2.8%
Benzaldehyde
N,N-diethyl-m-toluamide 1.32 1.4% 1.60 2.1%
Toluene 0.84 1.2% 0.77 3.3%
Ethylbenzene 0.74 1.3% 0.74 4.6%
a
Normalized on peak area for benzaldehyde

40. Profiles of Linear Gradients
100%
%B
20 µL/min 10%
30 µL/min
35 µL/min
40 µL/min
50 µL/min
0 20 40 60
Time (min)

41. Pump Noise - Linear Gradients
15800
uV 20 µL/min
15600 30 µL/min
35 µL/min
40 µL/min
50 µL/min
50 60 70
Time (min)

42. Gradient Separation of Test Mixture
by Nano LC and CEC 4
1. Uracil
16000
2. Phenol
Nano LC
3
2 3. Benzaldehyde
118 nL/min
5
6
1 4. N,N-diethyl-m-toluamide
15000
uV 5. Toluene
6. Ethylbenzene
0 10 20
4
Time (min)
CEC
-17.5 kV
3
1
6
2
5
18000
uV
0 10 20
Time (min)

43. Gradient Separations of Test Mixture
CEC Nano LC
10 min
gradient
0 10 20 0 10 20
Time (min) Time (min)
40 min
gradient
0 10 20 0 10 20
Time (min) Time (min)

44. Comparison of Retention Times Using
Gradient Elution
Retention Retention
Analyte Time (min.) %RSD Time (min.) %RSD
Uracil 4.2 0.36% 5.7 0.66%
Phenol 8.4 0.42% 12.4 0.61%
Benzaldehyde 11.6 0.40% 16.5 0.28%
N,N-diethyl-m-toluamide 16.8 0.25% 21.6 0.07%
Toluene 18.6 0.19% 22.7 0.06%
Ethylbenzene 20.8 0.16% 24.6 0.08%
Nanocolumn C, 75 µm i.d. x 37 cm (23 cm eff. length), C18, 3 µm
a
Nanocolumn B, 75 µm i.d. x 39 cm (30 cm eff. length), C18, 3 µm
b

45. Comparison of Peak Areas Using
Gradient Elution
Nano LCa CECb
c c
Analyte Peak Area %RSD Peak Area %RSD
Uracil 0.22 4.0% 0.40 8.2%
Phenol 0.59 6.3% 0.50 2.7%
Benzaldehyde
N,N-diethyl-m-toluamide 1.24 2.9% 1.18 2.0%
Toluene 0.87 2.1% 0.48 1.9%
Ethylbenzene 0.74 1.3% 0.38 6.1%
a
Nanocolumn C, 75 µm i.d. x 37 cm (23 cm eff. length), C18, 3 µm
b
Nanocolumn B, 75 µm i.d. x 39 cm (30 cm eff. length), C18, 3 µm
c
normalized on benzaldehyde

46. Comparison of Nano LC and CEC
Na no LC CEC
RSD RSD
Retention Time, isocratic 0.11 - 0.19% 0.42 - 0.61%
Peak Areaa, isocratic 1.2 - 2.5% 2.1 - 4.9%
Retention Time, gradient 0.16 - 0.42% 0.06 - 0.66%
Peak Areaa, gradient 1.3 - 6.3% 1.9 - 8.2%
N/m e te r (h ) N/m e te r (h )
Efficiency, col 1 69,721 (4.8) 108,833 (3.1) increase of 57%
Efficiency, col 2 158,643 (2.1)
Normalized on peak area for benzaldehyde
a

47. Conclusions
scale separations are useful when high
uMicro
separation efficiency is required
uAutomated separation systems can be
assembled from modular components for
isocratic and gradient elution
uHigher efficiencies are obtained with CEC
versus nano LC using the same column
uRetention time and peak area repeatability's are
acceptable for quantitative analysis using nano
LC or CEC

48. External
Acknowledgments
Prof. Jim Jorgenson Univ. of North Carolina
Prof. Sandy Dasgupta Texas Tech Univ.
Prof. Karin Markides Uppsala Univ.
Prof. Vicki McGuffin Michigan State Univ.
Dr. Doug Gjerde Sarasep, Inc.
Dr. John Stillian Dionex, Corp.
Dr. Frank Yang Micro-Tech Scientific, Inc.