AgilentHplc高效液相色谱仪测定方法开发1

高效液相色谱方法开发使用新的色谱柱加快方法开发速度HPLCMethod

DevelopmentApril,

2007PDF

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version常见的方法开发途径HPLCMethod

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2April,

2007PDF

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version按照“方法开发指南”按步操作–基于选择性改变参数–

比如:

pH值,

色谱柱，流动相等使用方法开发软件–

运行几个预设方法最后选用结果最好的一个方法在多种色谱柱及多种流动相下测试并比较结果，最后选用最好的一个方法＃很多人采用途径1进行方法开发。常见的方法开发从哪里开始?HPLCMethod

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3April,

2007PDF

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version选择C18键合相

–

种类很多，不同的键合特点一根短的色谱柱可以减少方法开发时间较小的色谱柱填料粒径可以在短时间内提高所需的分离度Eclipse

Plus

C18色谱柱可以改进分离结果–

更好的柱效和峰形选择简单的流动相

–

可靠，并适用多种样品水相：磷酸缓冲液

pH3,

三氟乙酸,

或甲酸有机相：乙腈或甲醇调速流动相得到更理想的保留和分离度所有的峰有适当的分离度(Rs

2.0)

–

相应较高的柱效第一个峰的保留值最好至少等于1分离时间在20分钟内

–

更短更新的色谱柱选择，小粒径更短的色谱柱可以在短时间内提高分离度及柱效，加快方法开发速度分离度公式HPLCMethod

DevelopmentPage

4April,

2007PDF

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versionN选择性–受流动相和固定相的影响柱效

–

受柱长和颗粒度的影响k保留因子

(保留)

–

受固定相和流动相，梯度斜率和延迟体积(梯度)的影响常见的方法开发参数–

选择性，柱效，保留值对分离度的影响0.002.001.003.004.005.006.007.00ResolutionIncrease

NIncrease

AlphaIncrease

k'选择性最影响分离度改变键合相改变流动相柱效是最容易预测的因素Rs

=

N½/

4

(-1)/

k’/

(k

’+1)Plates:Alpha:

k’:50001.102.0100001.354.5150001.607.0200001.859.5250002.112.0HPLCMethod

DevelopmentPage

5April,

2007PDF

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version选择性－－选择性

α细微的差异可以使得分离度上有较大的不同Eclipse

Plus

C8Greatest

Resolution

for

this

samplepeaks

1,2peaks

2,3α1.431.29Resolution4.153.55peaks

1,2peaks

2,3α1.331.31Resolution3.553.82peaks

1,2peaks

2,3α1.681.4Resolution6.124.97caffeinebarbitalsulfamethoxazoleCols:4.6x

150,

5

umm.p.:

40%

MeOH,60%

watermin01Eclipse

Plus

C18234min01Eclipse

XDB-C8234min01234HPLCMethod

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6April,

2007PDF

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version在最开始时如何制定方法开发方案?HPLCMethod

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7April,

2007PDF

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version流动相

(改变的第一选择，因为最易行)流动相

–

有机相

(乙腈,

甲醇等)流动相

–

pH–

在一个更宽的pH范围

–

有时会是pH

1-12键合相

(提供多种最优化方法的可能)很多种选择

C18

–

C8,Phenyl,CN等AQ

柱可以用于高水相的流动相体系选择新的色谱柱得到更好的峰形使用两个重要参数改变选择性以提高分离度–

流动相和键合相好的液相色谱柱所应具备的性能HPLCMethod

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8April,

2007PDF

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version好的峰形尤其对于碱性化合物,可以不加额外的流动相添加剂减小拖尾因子以提高分离度和定量准确性高的柱效1.8um,

3.5um和5um，不同的颗粒粒径可以满足任何分离的柱效要求柱效N

–

采用柱效进一步优化分离度好的选择性C18是最受欢迎的键合相，且适用于大部分化合物的分离C8

是在C18之后的受欢迎的键合相在较宽的流动相范围内均可使用简化方法开发适于更多的应用出色的重现性

–

不同批次的色谱柱都有很好的重现性好的使用寿命

–

至少能分析

1000

个样品采用新的色谱柱进行方法开发HPLCMethod

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9April,

2007PDF

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version1.

小颗粒的更短的色谱柱1.8um粒径的高分离度快速液相色谱柱

(RRHT)

–

采用50mm或更短的柱长可以用于快速的方法开发较长的色谱柱可以提高柱效11种键合相可供选择2.

改进的键合相可以提供更好的结果更好的峰形和柱效可以提高分离度对于建立好的方法至关重要新的Eclipse

Plus

色谱柱可以提供更好的峰形一根色谱柱可以满足很多分离要求吗?是!新的

ZORBAX

Eclipse

Plus

色谱柱可以做到!与其它厂商相比，有更好的结果！HPLCMethod

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10April,

2007PDF

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versionZORBAX

Eclipse

Plus

色谱柱ZORBAX

EclipsePlus

色谱柱:使用ZORBAX

硅胶–

采用更好的专利技术处理硅胶采用改进的键合试剂及键合过程更加严格的质控标准，批与批之间更加好的重现性OOOOOCH3SiCH3改进的硅胶基质CH3CH3 改进的双封Si CH3 端技术CH3CH3SiCH3CH3SiCH3CH3SiCH3更好的峰形HPLCMethod

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11April,

2007PDF

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version改进所带来的好处:HPLCMethod

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12April,

2007PDF

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version更好的峰形对于大部分的碱性化合物对于大部分的流动相

–

无需额外的添加剂与其它色谱柱相比对于除了碱性化合物之外的样品同样也有更好的峰形和更好的柱效酸碱混合物中性化合物Eclipse

PlusC18

–分离碱性化合物时没有拖尾

–

标准测试混合样品1Eclipse

Plus

C18Ace

5

C18Discovery

HS

C18Gemini

C18Luna

C18(2)SunFire

C18XTerraMS

C18XBridge

C1800.511.522.533.5

min1.

吡啶

(碱) 2.酚(酸) 流动相:60/40

水/乙腈1HPLCMethod

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13April,

2007PDF

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version111111122222222ZORBAXEclipse

Plus在甲醇和磷酸盐流动相中分离阿米替林峰形最好Eclipse

Plus

C18HPLCMethod

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14April,

2007PDF

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versionPhenomenex

Gemini

C18Phenomenex

Luna

C18

(2)Waters

SunFire

C18Ace

C18(2)Inertsil

ODS

(3)Tf

=

1.00Tf

=

1.25Tf

=

1.26Tf

=

1.20Tf

=

1.92Tf

=

1.11Amitriptyline

~

0.1µg 80%

Methanol

8mM

total

Potassium

PhosphateBuffer

pH

7.0,

Detection:

UV

215

nm, Flow

Rate:

1.0mL/min Columns:

4.6x100mm5µ

columnsPeakShapeisnormallybetterinMethanolmobilephases.

Eclipse

Plus

issuperior

under

these

conditions.Eclipse

PlusFamotidineCimetidinePirenzipine在中等pH范围没有三乙胺添加剂下也可以提供更好峰形Conditions:

Columns:

As

listed Mobile

Phase.:

20%MeOH,

80%

20

mM

phosphate

pH

7.0Flow

Rate

=1

mL/min.

Detection:

UV

254

semi

micro

flow

cellEclipse

Plus

C18

4.6

x

75

mm3.5

µmN=7800

TF:

1.05N=7800

TF:

1.00N=5700

TF:

1.12N=4300

TF:

1.74024 6 8 10 12 14 16Eclipse

XDB-C18

4.6

x

75

mm3.5

µm18 min0 2 4 6 8 10 12 14 16 18 minHighefficiencywitha3.5umparticleiseasilyachievedwithEclipsePlus

No

mobile

phase

modifiers

are

needed

for

perfect

performance!HPLCMethod

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15April,

2007PDF

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version1. 采用RRHT进行快速方法开发：RRHT

SB-C184.6x50mm,

1.8ummin0.511.52 2.5RRHT

SB-CN4.6x50mm,

1.8um1,2

3

4采用短的RRHT

C18色谱柱在短时间内得到更高柱效Column:

4.6

x50mm,

1.8um

Mobile

Phase:

A=

0.1%

Formic

Acid,

B=ACN

+

0.1%Formic

Acid(95:5) Flow

rate:1.5

mL/min

Inj.

Vol:

2ul

Sample:

Xanthines:

1.

1-methylxanthine,

2.

1,3-dimethyluric

acid,

3.

3,7-dimethylxanthine,

4.

1,7-dimethylxanthineHPLCMethod

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16April,

2007PDF

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version12340.5 1 1.5 2 2.5 minThe

C18

gives

the

most

effective

resolution

–

andwith

a

4.6

x

50mm,

1.8um

column

the

initial

resultsare

achieved

in

just

a

couple

of

minutes,with

the

highest

efficiency.The

50mmlength

is

short

and

fast

–

methods

can

be

approximated

or

completed

in

just

minutes!!1. 采用RRHT进行快速方法开发:不同的

ZORBAX

RRHTC18键合相所具有的不同的选择性EclipseXDB-C18Extend-C18Mobile

phase:

(69:31)

ACN:

water

Flow

1.5

mL/min.Temp:

30

°CDetector:SingleQuadESI

positive

mode

scan

Columns:

RRHT4.6x

50

mm

1.8

umSample:anandamide

(AEA)Palmitoylethanolamide

(PEA)2-arachinoylglycerol

(2-AG)Oleoylethanolamide(OEA)1234 Eclipse

Plus

C181234512341234512,34123451234 StableBondSB-C18min12345不同的键合相具有不同的选择性，根据所分析的样品选择适合的键合相。HPLCMethod

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17April,

2007PDF

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version方法开发的起始步骤选择一根性能优越的

C18或

C8键合相作为方法开发的起始，这个键合相应对酸性、碱性及中性化合物都可以提供好的峰形和保留。优化流动相中有机相的组成以改变其选择性。如果必需的话，选择不同的键合相来完成方法的优化。当pH在1-2时，选择

SB-C18如果使用新的RRHT色谱柱，这些步骤可以很快地完成，使得寻找最佳方法变得更为容易。选择

Eclipse

Plus

C18低

pH调节

%ACN

for

0.5

<k

<

20改变

有机相比例改变键合相Eclipse

Plus

C8,

SB-C18,

CN,Phenyl,

其它的反相键合相改变有机相调速有机相比例以使

0.5

<

k

<20STEP

1STEP

2STEP

3STEP

4峰间隔问题峰间隔问题峰间隔问题HPLCMethod

DevelopmentPage

18April,

2007PDF

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version为什么在方法优化中，建议您在改变键合相前应先尝试改变有机流动相？HPLCMethod

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19April,

2007PDF

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version易操作

–

乙腈和甲醇都是实验室常备溶剂。对于任何键合相，都会起一定的作用。以下化合物的方法开发diltiazemdipyridamolepropranololpindololdisopyramideβ-阻滞剂(β-blocker)抗心律不齐药物(Anti-arrhythmic)血管扩张药物(Vasodilator)钙通道阻滞剂(Ca+

channel

blocker)HPLCMethod

DevelopmentPage

20April,

2007PDF

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version从低pH值开始,

调速有机相比例2mAU40030020010002mAU1251007550250min24681012mAU6040200-20采用Eclipse

PlusC18在乙腈流动相中分析几种强心剂Column:

ZORBAXRRHT

Eclipse

Plus

C18,

4.6

x

50

mm,

1.8

mMobile

Phase:

A:

25

mM

NaH2PO4

,

pH

3.0

B:

ACNFlow

Rate:

2.0

mL/min Temperature:

30°C Detection:

UV

240

nmSample:

Cardiac

Drugs 1.Pindolol 2.

Diisopyridamide

3.Propranolol

4.Dipyridamole5.

Diltiazem40%

ACN20%

ACN30%

ACNk=

44!!RRHT

Eclipse

PlusC18

可以在低有机相比例中快速优化方法。好的分离度快速分离节省时间HPLCMethod

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21April,

2007PDF

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version2mAU40030020010002mAU1251007550250min24681012mAU806040200Column:

ZORBAXRRHT

Eclipse

Plus

C18,

4.6

x

50

mm,

1.8mMobile

Phase:

A:

25

mM

NaH2PO4

,

pH

3.0

B:MeOHFlow

Rate:

2.0

mL/min Temperature:

30°C Detection:

UV

240

nmSample:

Cardiac

Drugs 1.Pindolol 2.

Diisopyridamide

3.Propranolol

4.Diltiazem

5.

Dipyridamole70%

MeOH40%

MeOH50%

MeOHk=

49!!HPLCMethod

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22April,

2007PDF

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version采用RRHT色谱柱可以快速地看出不同有机溶剂的影响甲醇改变选择性，但分析时间更长从低pH值开始,

调速有机相比例采用Eclipse

Plus

C18在甲醇流动相中分析几种强心剂方法开发

–

改变有机相种类乙腈和甲醇的比较123mAU140120100806040200-2012mAU140120100806040200-2023450%

MeOH30%

ACN1234Column:

ZORBAXRRHT

Eclipse

Plus

C18,

4.6

x

50

mm,

1.8

mMobile

Phase:

A:

25

mMNaH2PO4

,

pH

3.0

B:

organicFlow

Rate:

2.0

mL/min Temperature:

30°C Detection:

UV

240

nm551HPLCMethod

DevelopmentPage

23April,

2007PDF

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versionSample:

Cardiac

Drugs 1.Pindolol 2.Disopyridamide

3.Propranolol

4.Diltiazem

5.

DipyridamoleResolutionof

pairs(2,3),

(3,4),

(4,5)is

betterin

MeOH.

Peak

5

selectivity

shiftResolutionofcriticalpair

(1,2),

is

better

in

ACN使用不同色谱柱规格的最佳条件比较– 长度，粒径，分离度RRHT

SB-CN4.6x

50

mm,

1.8um1.5

ml/min

1

µlinjection1.5

ml/min

2

µl

injectionEstrilEstradiolEthynyl

EstradiolDienestrolDiethylstilbestrolEthynl

estradiol

methyl

esterSB-CN

4.6x150

mm,

5um1.0

ml/min

3

µl

injectionminmAU1751501251007550250)12 34562.557.510mAU500100200150)234561RRHT

SB-CN

4.6

x100

mm,

1.8um

A:

WaterB:

30

%

MeOH/

70%MeCN40

%

BDetection:

UV

210

nm2.55mAU140120100806040200-20243 5612.5 5 7.5 10 12.5 15 17.5RRHT

Columns

allow

rapid

resolution

optimization

during

initial

methoddevelopment. Column

lengths

comparedquickly.HPLCMethod

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24April,

2007PDF

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version改变有机相–乙腈vs.甲醇采用ZORBAX

EclipseXDB-CN

分离雌激素024681234567min0246810121416181HPLCMethod

DevelopmentPage

25April,

2007PDF

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version234567Column:

ZORBAX

Eclipse

XDB-CN

Column

Dimensions:

4.6

x

150

mm,

5

m

Mobile

Phase:

As

shownFlowRate:

2.0

ml/

min

Injection

Volume:

2.00

l Column

Temperature:

25

°C

Detector:

UV,

210

nm10 12 14 16 18 minMobile

Phase:47.5:52.5MeOH:WaterSample:1.

Estriol

(0.00130

g/

l),2.

-Estradiol

(0.00130

g/

l),3.

Ethinyl

Estradiol

(0.00147

g/

l),4.

Dienestrol

(0.00123

g/

l),Diethylstilbestrol

(0.00128

g/

l)Ethynylestradiol

3-methyl

ether(0.00103

g/

l)Ethynodiol

Diacetate

(0.00139g/

l)Mobile

Phase:

35:65

ACN:WaterSolventcomparisonwithall

samplescanyieldoptimized

methods.RRHT色谱柱可以快速地找出最佳有机相的选择－－更长的色谱柱提供更好的分离度42424Eclipse

Plus-C184.6

x

50

mm

1.8um2Anandamide

(AEA)Palmitoylethanolamide

(PEA)2-Arachinoylglycerol

(2-AG)1(3)-ArachidonylglycerolOleoylethanolamide

(OEA)65%

ACN69%

ACN77%

ACN12,353454213124,5min246869%

ACN1241053Eclipse

Plus-C184.6

x

150

mm

1.8umEndocannabinoids

dissolved

in

methanol

(1mg/mL)

and

diluted

1:100

in

water.Mobile

Phase

is

69%

acetonitrile

at

1.5mL/min.,30°C.

MSisESI,positvemode,

totalionscan.HPLCMethod

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26April,

2007PDF

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version为什么改变键合相是有效的?HPLCMethod

DevelopmentPage

27April,

2007PDF

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version不同键合相对于极性及非极性化合物的作用是不同的。改变键合相有时可以提高选择性/分离度,减小分离时间。采用RRHT进行键合相的选择可以提高工作效率!!前提是，必须有多种RRHT键合相可供选择！不同键合相表现出不同的选择性－－流动相为30%

ACNmAU0

110012mAU010012mAU010012mAU010012mAU0100RRHT

SB-C18

4.6

x

50

mm,

1.8

mRRHT

SB-Phenyl4.6

x

50

mm,

1.8

mRRHT

Eclipse

Plus

C184.6

x

50

mm,

1.8

mRRHT

SB-AQ4.6

x

50

mm,

1.8

mRRHT

SB-CN4.6

x

50

mm,

1.8

m5

种不同的键合相比较每一个运行时间只需2分钟在最佳有机相比例下比较更多的键合相可以优化!HPLCMethod

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28April,

2007PDF

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version极性键合相在分离类固醇时与非极性键合相C18/C8的选择性比较1234

50520605201

2435HPLCMethod

DevelopmentPage

29April,

2007PDF

createdwithpdfFactorytrial

version6Column:

ZORBAX

Eclipse

XDB-CNMobile

Phase:

35:65

ACN:Water6 7Column:

ZORBAX

Eclipse

XDB-Phenyl10 15Column:

ZORBAX

Eclipse

XDB-C18Mobile

Phase:

60:40

ACN:Water7

10 15Column

Dimensions:

4.6

x

150

mm,

5

mFlowRate:

2.000

ml/

minInjection

Volume:

2.00

lColumn

Temperature:

25

°CDetector:

UV,

210

nm01234,55610 15 20

minMobile

Phase:

48:52

ACN:Water7Sample:1.

Estriol

(0.00130

g/

l),2.

-Estradiol

(0.00130

g/

l),3.

Ethinyl

Estradiol

(0.00147g/

l),012

435510 15 20Column:

ZORBAX

Eclipse

XDB-C8Mobile

Phase:

54:46

ACN:Water74.

Dienestrol

(0.00123

g/

l),Diethylstilbestrol

(0.00128

g/

l)Ethynylestradiol

3-methyl

ether

(0.00103

g/

l)Ethynodiol

Diacetate

(0.00139

g/

l)Eclipse XDB-CN

is

the

optimum

bonded

phase

–

the

onlyonethatcanresolveallthecomponentsinunder20

minutes.为什么pH在方法开发中对选择性很重要?HPLCMethod

DevelopmentPage

30April,

2007PDF

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version为什么化合物对流动相的pH值敏感?流动相的pH值如何影响保留和分离度?流动相的pH值如何影响方法开发?流动相的pH值如何影响色谱柱选择?pH

什么时候影响分离度?

化合物类型比较Ionizable

compounds

–

acids

and

bases

can

change

retention

and

selectivity

mostwith

changes

in

pHnon-polarnaphthalenepolarbenzyl

alcoholacenaphthenenitrobenzenepH

3pH

70

1

2

3

4

5

6

7

8

9

100 1 2 3 4 5

6

7

8HPLCMethod

DevelopmentPage

31April,

2007PDF

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version0

1

2

3

4

5

6

7

8

9

10Time

(min)0 1 2 3 4 5

6 7 8Time

(min)ionizablebenzoic

acidbenzanilide0 1 2 3 4 5

6 7 80 1 2 3 4 5

6 7 8Time

(min)分析离子化的化合物时改变pH值对于方法开发至关重要HPLCMethod

DevelopmentPage

32April,

2007PDF

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version不带电的化合物有较好的保留

(比如酸性化合物在低pH值，碱性化合物在高pH)硅胶上的硅羟基在中等pH值时离子化增加对碱性化合物的保留(可以存在离子交换的作用)在方法开发中选择流动相的pH值优化保留及选择性Eclipse

Plus可以用在宽的pH值范围高pH时可以选择Extend

C18碱性化合物抗阻胺剂的保留

vs.pH值

-在低pH值下变化较少DoxylamineTriprolidine

Diphenhydramine345pH6750403020100Retention

Volume

mLColumn:Eclipse

XDB-C84.6

x

150

mm,

5

mMobile

Phase:

75%

25

mM

phosphate

buffer25%

AcetonitrileSlightchanges

in

pH

may

dramatically

change

retention

and

selectivityAlways

evaluate

retention

changes

with

pH

during

method

developmentNo

change

in

retention

in

this

rangeHPLCMethod

DevelopmentPage

33April,

2007PDF

createdwithpdfFactorytrial

versionpKa

4.4,

9.2Chlorpheniramine

pKa

9.1pKa

6.5pKa

9.0方法开发步骤

– 在中等pH值下改变有机相

(MeOH)STEP

6STEP

7峰间隔问题改变有机相比例峰间隔问题从低pH值到中等pH值STEP

5ZORBAXEclipse

Plus

C18pH

7

(6-9)

20

-

50

mM

buffer,调节

%ACN

for

0.5

<k

<

20HPLCMethod

DevelopmentPage

34April,

2007PDF

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version调节有机相比例以使

0.5

<

k

<20从步骤6重新开始峰间隔问题STEP

8尝试采用EclipsePlusPhenyl-Hexyl,Eclipse

XDB-CN,

Phenyl

orBonus-RP从步骤5重新开始在等pH可能可以提供更好的选择性可能与你的样品更兼容在中等pH下方法优化步骤与低pH下相同Eclipse

Plus

在中等pH值下性能优越如果需要改变选择性可以考虑其它键合相Eclipse

Plus

色谱柱是在中低pH值下进行方法开发的理想选择HPLCMethod

DevelopmentPage

35April,

2007PDF

createdwithpdfFactorytrial

version适用于

pH2-9的流动相适合于中低pH值下的方法开发使得对pH较敏感的分离得到优化Eclipse

Plus提供极好的峰形和柱效Eclipse

Plus 可以在宽的pH范围(pH2-9)下优化选择性procainamidebuspironepioglitazoneeletriptandipyridamolediltiazem,furosemidemin0 246812354

67pH

2.7

0.1%

formic

acid/acnmin0246817426534pH

4.8

NH

AC/acn01HPLCMethod

DevelopmentPage

36April,

2007PDF

createdwithpdfFactorytrial

version2342 4 6 8567pH

7

NaPO4/acnSelectivity

andresolutioncan

change

with

pHEclipsePluscan

beusedwithmany

mobile

phases

and

pH’sConditions:

Column:

Eclipse

Plus

C18

4.6

x

100mm,

5um

Gradient:

10

–

90%

in

10

minutes

Detection:

UV

254

nmpH

2和

pH7

对选择性的影响有时非常显著Eclipse

Plus

C8

4.6

x

50mm,

5

umColumn:

4.6

x

50,

5

um

PN

959946-906Gradient:10-60%

B/3min.pH2.7:

A:

0.1%

formic

acid

B:

0.1%

fa

in

ACN

pH7.0:

A

20

mM

Na

phosphate

B:

ACNSample:“

genericExcedrin

tablet”pH

2.7AcetaminophenCaffeineAcetylsalicylic

acidunknown134324212

min.00.511.522.533.5 minpH

7.00 0.5 1 1.5 2 2.5 3 3.5 minSelectivitycanchangedramaticallyfrompH2–7withmanysamples,such

as

this

analgesic

tablet.Eclipse

Plus

can

be

used

under

bothconditionsHPLCMethod

DevelopmentPage

37April,

2007PDF

createdwithpdfFactorytrial

versionFamotidineCimetidinePirenzipineN=7900

TF:

1.05N=8000

TF:

0.99min0Eclipse

Plus

C18

4.6

x

75

mm

3.5

µm20

mM

phosphate

buffer,

pH3.0N=5700

TF:

1.280123 4 5 6Eclipse

Plus

C18

4.6

x

75

mm

3.5

µm20

mM

phosphate

buffer,

pH7.07 minColumns:

as

listedMobilePhase:20%MeOH,80%

20mMphosphate,pHaslisted

Flow

Rate:1

mL/min.Detection:

UV

230

semi

micro

flow

cell在中等pH值条件下选择性的不同是分离的关键Eclipse

Plus

在不同pH值下选择性比较HPLCMethod

DevelopmentPage

38April,

2007PDF

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version1123232 4 6 8 10 12 14 16Forthis

sample

resolution

is

notpossible

at

pH

3,

but

is

at

pH