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Recent Advances of Capillary Electrokinetic Separation Technologies
and their Applications in Pharmaceutical and Biochemical Analyses
Qing Liu1, Soumia Cheddah1, Yuanyuan Liu1,2, Weiwei Wang1, Yan Wang1,
Chao Yan1,2*
1Shanghai Jiaotong University, Shanghai, China
2Unimicro Technologies, Inc., Pleasanton, CA, USA
Sept. 29–Oct. 2, 2019, Bethesda MD, USA
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1. Background
Contents 2. eHPLC-MS
3. Capillary column
with sub-2 μm particles
5. Automated qCE
2
4. Capillary column
with sub-μm particles
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Part 1Background
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Life Science
☺ Proteomics
☺ Metabolomics
☺ Systems Biology
Pharmaceutical
☺ CTM
☺ QC/QA
☺ Chiral Separation
Agriculture/
Food
☺ Pesticides
☺ Additives
Environmental
☺ Pollution in air
☺ Water
☺ Soil
Quest on powerful analytical tools
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Resolution 分辨度
Speed 分离速度
Sensitivity 灵敏度
Source: R&D Magazine
Top three chalenges
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As chromatographers, what do we care?
--- Resolution (R)
When R=1, Peak 1 and peak 2 are separated basically.
When R=1.5, peak 1 and peak 2 are baseline separated.
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Resolution (R)
( )
+
−=
'
'
k
k
1N
1
41R
Separation factor
Column efficiency
Retention factor
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Column efficiency (N)
N = L/H (where L is the column length and H is the plate height.)
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van Deemter equation (1956)
)( pdAv
B
A term + B term + C term
vdC p2)(++=H
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A eddy diffusion
B longitudinal diffusion
C resistance to the mass transfer
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2 = u/d pP Lφ is the flow resistance factor,
η is the solvent viscosity;L is the packed length;
u is the mobile-phase linear velocity;
dp is the diameter of the packed particles.
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Column efficiency
1.7 μm
N≈250 000 plates/m
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Trend in liquid chromatography in the past 50 years
Linear velocity (mm/sec)
uHPLC
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Where do we go from uHPLC?
1
2
3
uuHPLC?
sfHPLC (slip flow HPLC)?
eHPLC (pCEC)?
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Capillary ElectroChromatography (CEC) with submicron particles
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A chromatographer’s dream
pCECSubmicron particles
Linear velocity (mm/sec)
uHPLC
15
N ≈ 1,000 000 plates/m
H ≈ L/N ≈ 1 μm
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Comparison of flow profiles in CE and LC
Electroosmotic Flow
Hydrodynamic Flow
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High Efficiency Separation in CEC: 600,000 p/m
Separation of 4 PAHs on 1.5 µm non-porous ODS .
Column: 100 µm i.d. x 28 cm packed length.
Mobile phase: 70% CH3CN/30% 4 mM sodium tetraborate (pH 9.1).
Voltage: 20 kV.
Injection: 5 kV/2s.
Detection: LIF, ex: 257 nm, em: 400 nm.
Sample:
a) Fluoranthene,
b) Benz[a]anthracene,
c) Banzo[k]fluoranthene,
d) Benzo[ghi]perylene.
Anal. Chem., 70(22), 787, 1998, Dadoo. R.; Zare R..; Annex. D ; Yan, C 18
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Column: 100 µm i.d. x 6.5 cm packed with 1.5 µm non-porous ODS.
Mobile phase: 70% CH3CN/30% 2 mM TRIS (pH 9).
Voltage: 28 kV.
Injection: 1 kV/1s.
Detection: LIF, ex: 257 nm, em: 400 nm.
5 second separation of 5 PAHs by CEC
Anal. Chem., 70(22), 787, 1998, Dadoo. R.; Zare R..; Annex. D ; Yan, C
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Double Separation Mechanism in CEC
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Advantages of CEC
☺ Combination of CE & cLC
-- double separation mechanism: suitable for both neutral & charged compounds.
☺ EOF-driven, no back-pressure, small particles
-- high efficiency, high selectivity & high resolution, plus fast speed.
☺ Micro fluidic technique
-- Economically attractive & environmentally friendly.
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What are the bottle-neck for CEC?
1. Dedicated instrument;
2. Dedicated column;
3. Killer applications.
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Part 2高效微流电色谱(eHPLC or pCEC)
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Pressurized Capillary ElectroChromatography (eHPLC)
pCEC
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Advantages of pCEC(eHPLC)
1. High efficiency, resolution, selectivity and fast speed;
2. Miniaturized;
3. pCEC, capillary-HPLC and HPLC, all in one;
4. Gradient capacity;
5. Quantitative injection, fine tuning of selectivity.
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TriSepTM-2000
TriSepTM-2010GV
TriSepTM-2100
Unimicro 十年磨一剑!
TriSep®-3000 eHPLC
最新一代
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More suitable for the separation of complex compounds!28
LIF
MSECD
ELSDUV/Vis
eHPLC
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Achievements
☆ Program on the Development of National Key Scientific
Instruments and Equipment (2011YQ150072).
☆ National Invention and Entrepreneur Award: Outstanding
☆ Shanghai Science and Technology Advancement Award:
First Place
☆ National Key and New Product.
☆ BCEIA Golda Awards : 2.
☆ Patents: 83, including 4 US patents and 3 PCT.
☆ Articles:300
☆ ………… 29
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pCEC-µELSD coupling
1. 试剂瓶;2, 3. 输液泵;4. 混合阀;5. 六通阀;6. 四通;7. 分流阀;8. 毛细管色谱柱;
9. 高压电源;10. 微型三通;11. 微流雾化器;12. 蒸发管;13. 鞘流装置;14. 光散射池;
15. 激光光源;16. 光电倍增管;17. 光阱
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pCEC-µELSD separation of sugar alcohol
Column:EP-200-150-5-Amide 80 (200 μm×150 mm, 5 μm,);
Mobile phase:ACN:H2O(40 mmoL/L TEA)=8 0: 20;
Voltage:+5 kV;
Current:5.6 µA;
Injection:50 nL;
Carrier:N2;
Evaporative Temp:120℃;,
Flow rate:0.8 L/min;
Pressure:4.3 MPa
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LIF with 4-lasers
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Sensitivity test on LIF
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1,2 Reagent bottle;3,4 Pump;5 Mixer Valve;6 Injection Valve;7 loop;8 splitting cross;9 Capillary restrictor;
10 High Voltage;11 Capillary chromatographic column;12 waste;13 Objective lens;14 Filter system;15 Laser;
16 Photomultiplier;17 Data acquisition unit;18 Data processing unit
Schematic diagram of pCEC- LIF
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Sample: 1.G1;2.B1;3.G2;4.B2
pCEC-LIF separation of aflatoxins
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µMS—eHPLC with flowrate of 150 nL/min
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1. Built in vacuum pump and PC ;
2. Analyze on site;
3. Base on chip-technology, balance within 30 min;
4. Save 80% N2;
5. Easy for use and maintenance, completely tool-less;
6. Compatible with eHPLC,qCE,nano-LC, HPLC, etc;
7. On line dilution and injection to monitor a reaction.
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eHPLC- µMS coupling system
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Run Retention
Time
Peak area
15.55 153520
25.59 159197
35.57 151534
45.57 157320
55.59 153974
65.59 154009
RSD% 0.29 1.8
eHPLC-MS Separation of cyclic hepta-peptides (Microsystin)
Reproducibility of eHPLC-MS separation of Mc
0 5 10 15 20 25
0
100
200
300
400
500
Mc-RR
Mc-RR
Mc-RRMc-LR
Mc-LR
-10 kV
+10 kV
mA
U
Time (min)
0 kV
Mc-LR
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Reproducibility of eHPLC-MS
时间 峰面积
1 2.2 1109616
2 2.22 1064812
3 2.2 1070160
4 2.2 1075024
5 2.2 1080975
6 2.22 1103332
RSD% 0.4 1.6
Condition:
Column:100 μm i.d. x 20 cm, 3.0 μm C18,
+50 μm x 5 cm (decoupler)+15 cm (empty);
Moble:80/20:ACN/H2O+0.1% FA;
Flow:166 nL/min;
Sample:0.01 mg/ml Caffeine;
Injection v:1.7 nL;
Temp:15℃。
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Part 3eHPLC with HALO and sub 2-micron particles
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0 10 20 30 40 50 60 70 80
0
1
2
3
μHPLC
mA
U
Time (min)
1
2 3 4
0 10 20 30 40 50 60 70 80
0
1
2
3
eHPLC
mA
U
Time (min)
1
23
4 eHPLC μHPLC
Run Time 25 min 60 min
Solvent Consumed 4.5 mL 10.8 mL
Sample Consumed 1.0 μl 1.0 μl
Column Efficiency
(naphthanlene)
200000 92000
Resolution(3/4) 9.5 9.7
eHPLC vs μHPLC
eHPLC vs nanoHPLC with 1.8 μm particles
eHPLC相比较μHPLC的优势:
Efficiency: 2X,Speed: 2.5X,Sensitivity: 3X.
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Part 4eHPLC with Submicron particles
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Submicron Monolithic
NPS Core-Shell
Capillary columns
packing material
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Preparation
Preparation of Submicrometer silica particles :
Improved StÖber method
Silica particles were
calcined at 600°C
for 6 h for three times
Rehydroxylated in
50/50% (v/v) nitric
acid/water overnight
reacted with 16% n-octade-
-cyltrichlorosilane and 2%
methyl trichlorosilane
respectively in dry toluene
with the protection of nitrogen
modification :
25℃, NH4OH, ethyl
alcohol, H2O
ethyl silicate in ethyl
alcohol, 2h
hydrochloric
acid, PH~7
filtration,
dry
SiO2 Particles
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The SEM photos of 300-800 nm SiO2 particles
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Submicron SiO2 modified with C18
SEM micrograph of 343 nm SiO2 particles.
SEM micrograph of 454 nm SiO2
particles modified with C18.
a)
b)
Infrared spectra for a colloidal crystal
(a) before and (b) after modification.
νCH2
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A: SEM of 320 nm ODS on a plate.
B-D:SEM of cross section of a packed 100 μm i.d. capillary,
B, X900; C, X4000; D, X10000.
SEM of Photonic Crystal column
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Bragg diffraction (Blue color)
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The most amazing photo in history of science
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Applied voltage: 10 kV;
1) thiourea; 2)а- naphthol; 3) benzophenone; 4) naphthalene; 5) biphenyl; 6) butylbenzene.
Column efficiency N (plates/m): 11,780 to 170,000.
Separation of organic compounds with submicron particles
in eHPLC and cLC
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0 2 4 6 8 10
3.5
4.0
4.5
inte
nsity (
mV
)
t (min)
Instrument: TriSepTM-2100 pCEC
Column: 30 cm total length (10 cm effective) 100
μm id packed with 603 nm of nonporous C18
Mobile phase: 45% v/v ACN and 55% v/v H2O
Applied voltage: -10 kV at outlet
Applied pressure: 15.5 MP
Injection: 0.22 nL (a sample loop of 1 μL with
splitting ratio of 4500:1
UV detection: 280 nm
Sample: 5 mg/mL BSA
eHPLC separation of BSA with 603 nm C18
----N: 1,264,910 plates/m
BSA
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0 2 4 6 8 10
0.4
0.8
1.2
1.6
inte
nsity (
mV
)
t (min)
0 2 4 6 8 10
1.0
1.5
2.0
2.5
3.0
3.5
4.0
inte
nsity(m
V)
t (min)
N (plates/m) :1,391,520.
Lysozyme
溶菌酶
0 5 10
0
2
4
inte
nsity (
mV
)
t (min)
N (plates/m) :1,011,560.
ribonuclease A
核糖核酸酶A
Instrument: TriSepTM-2100 pCEC
Column: 30 cm total length (10 cm effective) 100 μm id packed with 603 nm of nonporous C18
Mobile phase : 42% v/v ACN and 58% v/v H2O
Applied voltage : -10 kV at outlet;applied pressure :13.3 MP
Injection : 0.22 nL (a sample loop of 1 μL with splitting ratio of 4500:1
UV detection : 280 nm;Sample : 5 mg/mL
N (plates/m) :989,730.
cytochrome C
细胞色素C
eHPLC separation of lysozyme with 670 nm C18
-----N :1,391,520 plates/m
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0 10 20 30 40
0.0
0.5
1.0
1.5
2.0
Sig
nal (m
V)
Time (min)
Lysozyme
Myoglobin
Carbonic anhydrase
Experimental conditions: Column: 10 mm x 100 μm, 420 nm/C18-NPS
Isocratic: H2O: ACN (v:v, 57:43), +0.1%TFA
Pressure: 16.6 MPa,
Split ratio: 1/2250,
Linear velocity: 0.13 mm/s
Wavelength: 280 nm,
Applied voltage: 4 kV,
Sample: lysozyme, myoglobin and carbonic
anhydrase
Efficiency: carbonic anhydrase: 280,000 p/m.
eHPLC Separation of Protein Mixture on submicron C18
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0 2 4 6 8 10
0.5
1.0
1.5
2.0
2.5
3.0
3.5
3
1, 2
3
2
1
3
2
10 kV
8 kV
inte
nsity (
mV
)
t (min)
6 kV
1
0 2 4 6 8
5.8
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
3
2
1
3
2
LY-6
DE-11
DE-11P
Peptides mixture
Inte
nsity (
mV
)
t (min)
1
pCEC separation of 3 peptides with 420 nm C18-bonded silica particles;
N (plates/m) at 6 kV: LY-6, 1,752,000; DE-11, 460,000; DE-11p, 230,000.
eHPLC separation of peptides with submicron particles
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EOF vs Voltage
0 2 4 6 8 100.0
0.2
0.4
0.6
0.8
1.0
3000 nm
1800 nm
820 nm780 nm
670 nm505 nm
420 nm
EO
F vel
oci
ty /
mm
· s-1
Voltage / kV
315 nmA
Experimental conditions: ACN/H2O (70: 30, v /v); pH 7.8; 10 / 30 cm and 100 μm.
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Part 5Automated qCE® -3010)
56
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What is the bottle-neck for CE?
1. Accuracy and precession?
2. Detection sensitivity?
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More suitable for the separation of complex compounds!
μUV
μLIF
μMS
μELSD
μECD
Automated qCEqCE®-3010 · qCE- μUV/μLIF/μMS/μELSD/μECD
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Schematic of Fully automated qCE(First in the world)
☺ Accurate injection with nL volume
☺ Qualitative reproducibility <1% (time)
☺ Quantitative reproducibility <2% (peak area)
☺ Excellent New Products Award in the
Instrument Industry.
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0 2 4 6 8 10 12 14 16
0.0
7.8
15.6
23.4
31.2
-3.0
-2.5
-2.0
Time (min)
Ad
so
rba
nce
(m
V)
Thi
oure
a
1 2 3 4 5 6 7 8 9 10 RSD
保留时间 4.673 4.673 4.679 4.672 4.676 4.657 4.697 4.683 4.692 4.699 0.28 %
峰面积 41331 41015 41508 41754 41267 41831 41782 41392 41654 41576 0.63 %
qCE reproducibility
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Constant flowrate Constant voltage
Yuan Xu, Bangzan Ling, Wenjun Zhu, Dong Yao, Lin Zhang, Yan Wang, Chao Yan, Biomedical Chromatography, 2015, DOI:10.1002/bmc.3560.
qCE separation of six nucleosides
Capillary: 40 cm x 50 µm i.d. , Sample:(a) 胞嘧啶, (b) 5-氟-2‘-脱氧尿苷,(c) 腺苷,(d) 尿嘧啶,(e) 尿苷,(f) 肌苷
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ACKNOWLEDEMENTS
This research was supported by:
National Natural Science Foundation of China;
21175092,
21105064,
National Important Instrument and Equipment Development
Special Projects by Ministry of Science & Technology of China;
2011YQ150072,
2011YQ15007204,
2011YQ15007207,
2011YQ15007210.62
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上海交通大学药学院药分和代谢组学课题组Yan Lab, School of Pharmacy, Shanghai Jiao Tong University
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上海通微分析技术有限公司团队Unimicro Team
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Thanks for your attention!
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