a novel end-capping concept for high performance liquid...
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Importance of End-capping for Reversed
Phase LC
反相LC色谱柱封端的重要性
A Novel End-capping Concept for High
Performance Liquid Chromatography
高效液相色谱的新型封端概念
Norikazu NAGAE, Lingchao LIN, Xiaojing ZHOU
2
Stationary Phase 固定相
C18 (ODS)
C8
C4
TMS(三甲基氯硅烷)
Ph(苯基)
CN(氰基丙基)
NH2(氨基丙基)
Diol(二醇基)
C30
Si
Si
Si
Si
Si
Si CN
Si NH2
Si O OH
OH
3
Functionality of reagent(mono, di, tri)
试剂的功能
R
Si
ClCH
3
CH3
R
Si
ClCl
CH3
R
Si
ClCl
Cl
甲硅烷基化试剂的类型
单官能团
Monofuntional
双官能团
Difuntional
三官能团
Trifuntional
Cl与二氧化硅表面上的Si-OH和Si-O-Si硅氧烷键反应
然而,三官能团试剂在所有三个点位都不能结合硅氧烷
4
ODS(C18)
C18(十八烷基甲硅烷基ODS)与硅胶表面结合
≡Si-OH + Cl-Si(CH3)2C18H37
(二氧化硅表面)
≡Si-O-Si(CH3)2C18H37 + HCl
5
End-capping 封端
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
二氧化硅
Si-OH
Si-OH
Si-OH
Si-OH
Si-OH
残留硅醇基
Cl-Si(CH3)3
三甲基氯硅烷
ODS转换封端
Si-O
Si-O
Si-O
-Si(CH3)2C18H37
-Si(CH3)2C18H37
-Si(CH3)2C18H37
-OH: 8-9 µmol/m2 -ODS: 大约3 µmol/m2-TMS: 1-2 µmol/m2
Ratio of residual silanol group
残留硅醇基的比例
6
二氧化硅表面的硅醇基的量为8〜9μmol/ m2
C18键合后,仍有2/3硅醇基残留
C18键合密度〜3 μmol/m2
作为顶尖的封端技术,C18键合和TMS等封端后
1980年代 1/2
1990年代 2/5
2000年代 1/3 硅烷醇基残留
1/3残留硅醇基的作用几乎被封端剂阻断
7
Interaction of silanol group
硅醇基的作用
1. Adsorption 吸附 Si-OH
2. Ion exchange 离子交换 Si-O- 阳离子(-N+)
3. Ion exclusion 离子排斥 Si-O- 阴离子(-COO-)
4. Hydrogen bond 氢键 Si-OH
Problems with unhydrated silanol group
未水合的硅醇基所引起的问题
8
对于反相如C18,碱性化合物拖尾起因于硅醇基
在Cyano(CN)等的反相中,碱性化合物不因硅醇基而拖尾
高吸附性硅醇基
O H
O H
在由有机溶剂和水组成的流动相中,由于疏水基团(C18)的影响,难以水合的硅醇基
较少受疏水基团影响并且充分水化的硅醇基
低吸附性的硅醇基
流动相:乙腈/20mM醋酸铵pH6.8
峰:阿米替林
NCH3
CH3
C18
How tailing occurs
拖尾如何发生
Low High
pH3 7 9
Silanol group
硅醇基Si-OH Si-O-
Basic compound
碱性化合物R-N+R-(CH3)2NH+ R-(CH3)2NH
R-(CH3)2NH+
Unhydrated Si-O-Tailing
拖尾
未水合硅醇基
在pH 2.6或更低的pH
條件下
在pH9或更高的pH條件下
No tailing No tailing
The difference between acetonitrile
and methanol
乙腈与甲醇的差异
Acetonitrile 乙腈: 氢键相互作用弱,硅醇基的影响较大
Methanol 甲醇: 氢键相互作用强,将甲醇放置在硅烷醇基团周
围.
硅醇基的影响降低
N(4)=11,000
TF(4)=1.06
N(4)=8,950
TF(4)=1.76
N(4)=10,000
TF(4)=1.50
N(4)=10,500
TF(4)=1.07
Sunniest C18
0 2 4 6 8 10 12 14 16
Retention time/min
A company C18
B company C181
1
1
2
1
2
2
2
3
3
3
3
4
4
4
4
Sunniest RP-AQUA
Pittcon 2010 11
Comparison of amitriptyline peak I
阿米替林峰的比较
N(4)=11,000
TF(4)=1.06
N(4)=8,950
TF(4)=1.76
N(4)=10,000
TF(4)=1.50
N(4)=10,500
TF(4)=1.07O
OH
NH CH3
CH3
CH3OH,甲醇,pH7.5, 40 ºC
0 2 4 6 8 10 12 14 16
Retention time/min
A company C18
B company C181
1
1
2
1
2
2
2
3
3
3
3
4
4
4
4
N
CH3
CH3
NHCH3
柱尺寸:150×4.6mm
粒径:5微米
流动相:
CH3OH / 20mM磷酸盐缓冲液pH7.5 = 80/20
流量:1.0mL / min
温度:40℃
样品:1 =尿嘧啶
2 =普萘洛尔
3 = Nortriptyline
4 =阿米替林
O
OH
NH CH3
CH3
NHCH3
Pittcon 2010 12
Comparison of amitriptyline peak II
阿米替林峰的比较
O
OH
NH CH3
CH3
NHCH3
N
CH3
CH3
N(4)=14,000
TF(4)=1.16
N(4)=13,800
TF(4)=1.24
N(4)=4,300
TF(4)=5.24
N(4)=4,000
TF(4)=4.33
0 2 4 6 8 10 12 14 16 18 20 22 24
Retention time/min
Sunniest C18
A company C18
B company C181
1
1
2
1
2
2
2
3
3
3
3
4
4
4
4
Sunniest RP-AQUA
柱尺寸:150×4.6mm
粒径:5微米
流动相:
乙腈/ 20mM磷酸盐缓冲液pH7.0 = 60/40
流量:1.0mL / min
温度:40℃
样品:1 =尿嘧啶
2 =普萘洛尔
3 = Nortriptyline
4 =阿米替林
CH3CN,乙腈, pH7.0, 40 ºC
The difference between acetonitrile and methanol
乙腈与甲醇的差异
N(4)=11,000
TF(4)=1.06
N(4)=8,950
TF(4)=1.76
N(4)=10,000
TF(4)=1.50
N(4)=10,500
TF(4)=1.07
0 2 4 6 8 10 12 14 16
Retention time/min
A company C18
B company C181
1
1
2
1
2
2
2
3
3
3
3
4
4
4
4
N(4)=14,000
TF(4)=1.16
N(4)=13,800
TF(4)=1.24
N(4)=4,300
TF(4)=5.24
N(4)=4,000
TF(4)=4.33
0 2 4 6 8 10 12 14 16 18 20 22 24
Retention time/min
Sapphiresil C18
A company C18
B company C181
1
1
2
1
2
2
2
3
3
3
3
4
4
4
4
Sapphiresil C18-AQ
Sunniest C18
Sunniest RP-AQUA
Methanol 甲醇 Acetonitrile 乙腈
14
Comparison of amitriptyline peak III
阿米替林峰的比较
N
CH3
CH3
柱尺寸:150×4.6mm
粒径:5微米
流动相: 乙腈/ 20mM磷酸盐缓
冲液pH7.0 = 60/40
流量:1.0mL / min
温度:40℃
样品:阿米替林
色谱柱 拖尾因子 理论塔板数 色谱柱 拖尾因子理论塔板
数
Sunniest C18 1.16 14,000 Develosil ODS-UG-5 2.14 8,700
Sunniest RP-AQUA 1.24 13,800 Gemini-NX C18 1.29 9,500
Acclaim PolarAdvantage
II5.19 3,300 Hiber Purospher STAR RP-18e 2.01 11,200
Acclaim 120 C18 2.19 14,200 Inertsil ODS-4 1.30 12,000
ACE C18 3.25 5,300 Inertsil ODS-SP 2.92 8,000
Ascentis C18 1.74 8,300 Inertsil ODS-3 2.70 6,100
Atlantis T3 C18 1.97 10,600 L-column ODS 1.56 10,400
SunFire C18 1.59 10,100 Shim-pack VP-ODS 3.44 6,700
X-Bridge C18 1.43 10,000 TSKgel ODS-100V 1.71 10,000
Cadenza 5CD-C18 3.07 8,500 TSKgel ODS-100Z 2.15 11,500
Unison US-C18 2.52 9,200 Wakopak Navi C18-5 11.1 2,100
CAPCELLPAK C18 MG II 2.01 10,900 YMC-Pack Pro C18 3.77 7,400
CAPCELLPAK C18 MG III 7,75 3,600 ZORBAX Eclipse Plus C18 3.28 5,900
Our end-capping method
我们的封端方法
1)Special end-capping reagent
特殊封端剂
1)High reaction temperature
反应温度高
End-capping of Sunniest and SunShell
Sunniest和SunShell的封端
18
Final TMS
O
Si
OH
Si
Si O
O
OO
SiOO
Si
OSi
O
Si
SiO O
OO
Si
O
Si
O Si
Si
OO
Si
O
Si
Si
OOO
OSi
OSi
O
OO
O Si
Si
Si
O O
OO
Si
OSi
O Si
SiO
O
OOO
O
SiO
Si Si
O
OO
O
O
O
Si
OSi
O
Si
SiOH
O
O
OSi
O
SiO
SiOO O
O
Si
O H
O
hexamethyldichrolotrisiloxane
Silanol Activity Control
硅醇基活性控制
19
Silanol Activity ControlTechnology烷醇活性控制
Heat
熱
Typical no endcapping C18 Sunrise C18-SAC
The amount of residual silanol groups
残留硅醇基的量
20
Conventional end-capping
Both end-capping with hexamethyldichrolotrisiloxane and heating
C18 C18 C18C18 C18
End-capping layer
C18 C18 C18 C18 C18
Sunniest C18 and SunShell C18
Conventional C18 常規C18
End-capping layer
Mobile phase: Acetonitrile/10mM ammonium acetate pH6.8=(40:60)
Column dimension: 150 x 4.6 mm, Flow rate: 1.0 mL/min, Temp.: 40oC
Comparison of Amitriptyline
阿米替林比较
Sample: 1=Uracil, 2=Propranolol, 3= Nortriptyline, 4=Amitriptyline
1
0 1 2 3 4 5 6 7 8 9 10Retention time/min
TF=1.89
TF=2.61
TF=2.73
TF=3.24
Company P C18
Company S C18 (2.7 μm)
Company A C18 (2.7 μm)
Company T C18 (2.7 μm)
TF=3.12 Company W C18 (2.7 μm)
23 4
N
CH3
CH3
Amitriptyline
TF=3.59
Company A C18 HybridTF=1.55
Company P C18 Hybrid
Kinetex C18
Ascentis Ex C18
Accucore C18
PoroShell C18 EC
Cortecs C18
PoroShell C18 HPH
Kenetex EVO-C18
SunShell C18 (2.6 μm)TF=1.20
Column:
Company P C18, 2.6 μm 150 x 4.6 mm (26.1 Mpa, 30,800 plate )
Company T C18, 2.6 μm 150 x 4.6 mm (22.7 Mpa, 31,600 plate)
Company W C18, 2.7 μm 150 x 4.6 mm (18.5 Mpa, 23,300 plate)
Company A C18, 2.7 μm 150 x 4.6 mm (30.6 Mpa, 30,200 plate)
Company S C18, 2.7 μm 150 x 4.6 mm (22.2 Mpa, 31,800 plate)
SunShell C18, 2.6 μm 150 x 4.6 mm (21.8 Mpa, 31,900 plate)
Company A C18 Hybrid, 2.7 μm 150 x 4.6 mm (24.5 Mpa, 30,200 plate)
SunShell C18, 5 μm 150 x 4.6 mm (21.8 Mpa, 31,900 plate)
Company P C18 Hybrid, 5 μm 150 x 4.6 mm (26.1 Mpa, 30,800 plate )
Mobile phase: CH3OH/H2O=75/25
Flow rate: 1.0 mL/min
Temperature: 40 ºC
Sample: 1 = Uracil, 2 = Caffeine, 3 = Phenol, 4 = Butylbenzene
5 = o-Terphenyl, 6 = Amylbenzene, 7 = Triphenylene
Comparison of core shell C18s 核壳C18比较
Company P C18
26.1 MPa
Company S C18
22.2 MPa
SunShell C18
21.8 MPa
1
1
2
2
7
3
3
4
4
5
5
6
67
0 2 4 6 8 10 12 14 16 18 20 22 24
Retention time/min
1 2
3 4
56
7
12 7
3 45
6
1 2
3 4
5
6
71
Company T C18
22.7 MPa
Company A C18
30.6 MPa
k6=10.4
N6=31,900
k6=9.7
N6=31,800
k6=9.0
N6=30,200
k6=7.4
N6=31,600
k6=5.4
N6=30,800
k6=7.7
N6=23,300
Company W C18
18.5 MPa1 2
34
56
7
5 7
1 2
1 2
1 2
3
3
3
45
6
7
45
6
7
4 5
6,7
SunShell C18 5 μm
8.0 MPa
Company A C18 Hybrid
24.5 MPa
Company P C18 Hybrid 5 μm
9.3 MPa
k6=6.8
N6=28,800
k6=9.3
N6=20,000
k6=4.6
N6=15,000
氢键性能(Caffeine/Phenol)
疏水(Amylbenzene/Butylbenzene)
立体选择性(Triphenylene/o-Terphenyl)
Company P C18 0.48 1.54 1.20
Company T C18 0.35 1.56 1.50
Company W C18 0.38 1.59 1.32
Company A C18 0.42 1.57 1.25
Company S C18 0.44 1.60 1.31
SunShell C18 0.39 1.60 1.46
Company A C18 Hybrid 0.42 1.58 1.19
SunShell C18 5 μm 0.42 1.59 1.35
Company P C18 Hybrid 5 μm 0.40 1.45 1.02
Stability test under acidic pH condition
在酸性条件下的耐久性 (加速测试)
耐久性的测试条件柱尺寸:50 x 2.1 mm流动相:乙腈/1.0%TFA, pH1 = 10/90流量:0.4mL/min温度:80℃
测量条件柱尺寸:50 x 2.1 mm流动相:乙腈/水= 60/40流量:0.4mL/min温度:40ºC样品:1 =尿嘧啶
2 =丁基苯
23
0
20
40
60
80
100
0 20 40 60 80 100 120
Re
lati
ve
rete
nti
on
/%
Time/h
SunShell C18
Company S C18
Company W C18
Company T C18
Company P C18
Company A C18
▲▲▲
▲
Kinetex C18
Ascentis Ex C18
Stability test under basic pH condition
在碱性条件下的耐久性
耐久性的测试条件柱尺寸:50 x 2.1 mm流动相:乙腈/ 20mM硼酸钠/ 10mM NaOH = 30/21/49 (pH10)流量:0.4mL/min温度:50ºC
测量条件柱尺寸:50 x 2.1 mm流动相:乙腈/水= 70/30流量:0.4mL/min温度:40ºC样品:1 =丁基苯
0
20
40
60
80
100
0 1,000 2,000 3,000 4,000 5,000 6,000
Elution volume/mL
SunShell C18
Company S C18
Company P C18
Company T C18
Company A C18
Re
lativ
e p
late
of
bu
tylb
en
zen
e/%
24
Kinetex C18
Summary
摘要*Residual silanol groups in C18 stationary phase make basic
compounds be tailing, so that an end-capping is very
important for separation of basic compounds.
*End-capping makes stability increase under acidic and basic
pH conditions.
*HPLC column manufacturers have their own end-capping
skill. ChromaNik has the best end-capping skill.
* C18固定相中的残留硅醇基使碱性化合物拖尾,因此封端对于分离碱性化合物非常重要。*封端将增加在酸性和碱性条件下的稳定性。* HPLC色谱柱制造商各有自己的封端技术,ChromaNik具有最佳的封端技术。
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