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

Importance of End-capping for Reversed
Phase LC
反相LC色谱柱封端的重要性

Stationary Phase 固定相
C18 (ODS)

Si

C8Si

C4Si

TMS（三甲基氯硅烷）Si

Ph（苯基）

Si

CN（氰基丙基）Si

CN

NH2（氨基丙基）Si

NH2Diol（二醇基）

Si

O

OH
OH

C302

Functionality of reagent(mono, di, tri)
试剂的功能甲硅烷基化试剂的类型
单官能团

双官能团

三官能团

Monofuntional

Difuntional

Trifuntional

ClRR

R

SiSiSi

CH3CH3Cl

Cl与二氧化硅表面上的Si-OH和Si-O-Si硅氧烷键反应ClCH3 ClClCl然而，三官能团试剂在所有三个点位都不能结合硅氧烷
3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

4

End-capping 封端残留硅醇基Si-OH

Si-OH

Si-OH

Si-O-Si(CH3)2C18H37

Si-OH

Si-OH

二氧化硅 Si-OH
Si-OH
Si-OH

Cl-Si(CH3)3
三甲基氯硅烷

Si-OH
Si-O -Si(CH3)2C18H37
Si-OH
Si-OH

Si-OH
Si-OH

Si-O -Si(CH3)2C18H37ODS转换

-OH: 8-9 µmol/m2-ODS: 大约3 µmol/m2

封端-TMS: 1-2 µmol/m25

Ratio of residual silanol group
残留硅醇基的比例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6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

7

Problems with unhydrated silanol group
未水合的硅醇基所引起的问题对于反相如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

流动相：乙腈/20mM醋酸铵pH6.8
峰：阿米替林

C18N

高吸附性硅醇基

CH 3CH 3

低吸附性的硅醇基

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O H

较少受疏水基团影响并且充分水化的硅醇基8How tailing occurs
拖尾如何发生pH3LowSilanol group硅醇基
Basic compound碱性化合物9HighSi-O-Si-OH

R-N+R-(CH3)2NH+

在pH 2.6或更低的pH
條件下No tailing

7R-(CH3)2NH
R-(CH3)2NH+Unhydrated Si-O未水合硅醇基Tailing拖尾

在pH9或更高的pH條件下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Comparison of amitriptyline peak I
阿米替林峰的比较
CH3OH,甲醇,pH7.5, 40 ºC
４Sunniest C181３N(4)=11,000
TF(4)=1.06

2４

Sunniest RP-AQUA
３12N(4)=10,500
TF(4)=1.07

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CHONHOH

1４A company C18
3

2N(4)=8,950
TF(4)=1.76

3 = Nortriptyline
NHCH3

1４B company C18
３

2N(4)=10,000
TF(4)=1.50

4 =阿米替林CH3NCH30246810121416Retention time/min

Pittcon 2010

113CH3Comparison of amitriptyline peak II
阿米替林峰的比较CH3CN,乙腈, pH7.0, 40 ºC
Sunniest C18
1４

３N(4)=14,000TF(4)=1.16

2Sunniest RP-AQUA

４３1

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CH
2 =普萘洛尔ONHCH
3N(4)=13,800TF(4)=1.24

23OH

3 = Nortriptyline
1A company C18
32

1B company C18

24

Pittcon 2010

68NHCH3

4 =阿米替林

N(4)=4,300
４TF(4)=5.24

３20４N(4)=4,000TF(4)=4.33

101214Retention time/min

1618201222CH3NCH324

The difference between acetonitrile and methanol

乙腈与甲醇的差异Methanol 甲醇
４Sunniest C18
1３Acetonitrile 乙腈Sapphiresil C18N(4)=11,000
TF(4)=1.06

1４Sunniest RP-AQUA
３1Sapphiresil C18-AQ

N(4)=10,500
TF(4)=1.07

2４A company C18
32B company C18

2N(4)=8,950
TF(4)=1.76

1４

1

３

204

4

3

12

1N(4)=14,000
TF(4)=1.16

22

14

3

6

8

10Retention time/min

1214A company C18
32N(4)=10,000
TF(4)=1.50

16

2

4N(4)=4,000
TF(4)=4.33

B company C18
320

N(4)=13,800
TF(4)=1.24

46

N(4)=4,300
TF(4)=5.24

48101214

Retention time/min1618202224

Comparison of amitriptyline peak III
阿米替林峰的比较色谱柱拖尾因子

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

Sunniest RP-AQUA

1.24 13,800

Gemini-NX C18

Acclaim PolarAdvantage
II5.19Hiber Purospher STAR RP-18e

Acclaim 120 C18

2.19 14,200

Inertsil ODS-4

ACE C18

3.255,300

Inertsil ODS-SP

柱尺寸：150×4.6mm
2.14 8,700 粒径：5微米
1.29 9,500 流动相: 乙腈/ 20mM磷酸盐缓冲液pH7.0 = 60/40
2.01 11,200
流量：1.0mL / min
1.30 12,000
温度：40℃
2.92 8,000 样品：阿米替林

Ascentis C18

1.748,300

Inertsil ODS-3

2.70

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

X-Bridge C18

1.43 10,000

TSKgel ODS-100V

1.71 10,000

Cadenza 5CD-C18

3.078,500

TSKgel ODS-100Z

2.15 11,500

Unison US-C18

2.52

9,200

Wakopak Navi C18-5

11.12,100

CAPCELLPAK C18 MG II

2.0110,900

YMC-Pack Pro C183.77

7,400

CAPCELLPAK C18 MG III

7,75

3,600

ZORBAX Eclipse Plus C183.28

5,900

3,300

6,100

6,700

CH3NCH314

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 SunShellSunniest和SunShell的封端
hexamethyldichrolotrisiloxane

Final TMS

HOSiOSi
SiSiO

SiO
SiO

SiO

Si
Si O
Si O
O O

Si
SiO
O

SiOOOOSiO

OOO

SiOSiOOO

SiSiOSiOSiOSiOO

SiOHOOOSiSiOOOSiO OOSiSiO OHOSiOOSiSiSiO OO O OOOSi OO Si OSiOOOSiOOOO18

Silanol Activity Control
硅醇基活性控制
Typical no endcapping C18

Sunrise C18-SAC

Silanol Activity ControlTechnology
烷醇活性控制Heat熱19

The amount of residual silanol groups
残留硅醇基的量
Conventional end-capping
C18

C18

C18

C18

C18

End-capping layer

Conventional C18 常規C18
Both end-capping with hexamethyldichrolotrisiloxane and heating
C18

C18

C18

C18

C18

End-capping layer

Sunniest C18 and SunShell C1820

Comparison of Amitriptyline
阿米替林比较1324

TF=2.61

TF=3.12TF=1.89
TF=3.24234567Retention time/min

Cortecs C18Company A C18 (2.7 μm)

PoroShell C18 EC

Company S C18 (2.7 μm)

Ascentis Ex C18

Company A C18 Hybrid

TF=3.59

1Company W C18 (2.7 μm)

SunShell C18 (2.6 μm)

TF=1.55

08

Company P C18 Hybrid
910Mobile phase: Acetonitrile/10mM ammonium acetate pH6.8=(40:60)
Column dimension: 150 x 4.6 mm, Flow rate: 1.0 mL/min, Temp.: 40oC
Sample: 1=Uracil, 2=Propranolol, 3= Nortriptyline, 4=Amitriptyline

Accucore C18

Company T C18 (2.7 μm)

TF=2.73

TF=1.20

Kinetex C18

Company P C18

PoroShell C18 HPH

Kenetex EVO-C18

CH3N
CH3Amitriptyline

Comparison of core shell C18s 核壳C18比较Column:Company P C18, 2.6 μm 150 x 4.6 mm (26.1 Mpa, 30,800 plate )2364Company T C18, 2.6 μm 150 x 4.6 mm (22.7 Mpa, 31,600 plate)
11
Company W C18, 2.7 μm 150 x 4.6 mm (18.5 Mpa, 23,300 plate)
7 Company T C18
5
1 2 k6=7.4
Company A C18, 2.7 μm 150 x 4.6 mm (30.6 Mpa, 30,200 plate)6
3 N6=31,600 422.7 MPa
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)
57 Company W C18
1 2 k6=7.7
46
Company A C18 Hybrid, 2.7 μm 150 x 4.6 mm (24.5 Mpa, 30,200 plate)
3 N6=23,300
18.5 MPa
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 )CompanyAC1875
1 2 k6=9.0
Mobile phase: CH3OH/H2O=75/25
67543 N6=30,20030.6 MPa
Flow rate: 1.0 mL/min
57 Company S C18 Temperature: 40 ºC
2 3 k6=9.7
461Sample: 1 = Uracil, 2 = Caffeine, 3 = Phenol, 4 = Butylbenzene
N6=31,800
22.2 MPa
5 = o-Terphenyl, 6 = Amylbenzene, 7 = Triphenylene
72 3 SunShell C18
k6=10.4
516 N6=31,900
4
k6=5.4N6=30,800

57Company P C18
26.1 MPa21.8 MPa

12312312302

k6=6.84
N6=28,800

氢键性能57 Company A C18 HybridCompany P C18Company T C187Company W C185SunShell C18 5 μm
k6=9.36Company A C18
N6=20,000 48.0 MPa
Company S C186,7SunShell C18
Company P C18 Hybrid 5 μm
k6=4.64 5
N6=15,000 9.3 MPa
Company A C18 Hybrid
SunShell C18 5 μm
4681012141618202224
Company P C18 Hybrid 5 μm
Retention time/min

624.5 MPa

(Caffeine/Phenol)

0.48
0.35
0.38
0.42
0.44
0.39
0.42
0.42
0.40

疏水立体选择性

(Amylbenzene/Butylbenzene) (Triphenylene/o-Terphenyl)

1.54
1.56
1.59
1.57
1.60
1.60
1.58
1.59
1.45

1.20
1.50
1.32
1.251.311.461.19
1.351.02

Stability test under acidic pH condition
在酸性条件下的耐久性 (加速测试）

Relative retention/%

100▲▲

▲

▲

8060
SunShell C18
Company S C18 Ascentis Ex C18
Company W C18
Company T C18
Company P C18 Kinetex C18
Company A C18

402000204060Time/h

80100120

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

Relative plate of butylbenzene/%

Stability test under basic pH condition
在碱性条件下的耐久性
1008060
SunShell C18

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

Company S C18

40Company P C18 Kinetex C18
Company T C18

20

Company A C1800

1,000 2,000 3,000 4,000 5,000 6,000

Elution volume/mL

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24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-cappingskill. 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。