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。