Overview of preparative HPLC - AIS-Indiaais-india.com/UploadedApps/Preparative HPLC...

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Overview of preparative HPLC Analytical Technologies Limited

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Overview of preparative HPLC

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1.Review of liquid phase chromatography

origin

develop

prospect

Separationresult

Mobile phase

Stationary phase

origin

Tewett 1903

Kuhn

Martin & Synge

Stanford Moore & William Howard Stein

Liquid-stationarychromatographic theory

Paper chromatography andplate theory

AA analyzer

Develop

Chromatography developschronology

1903’s propose chromatographic method1931’s liquid- stationary absorption chromatography1940’s paper chromatography1950’s thin-layer chromatography50-60’s fast develop period of GC70’s HPLC1975’s ion-exchange chromatography80’s rip period of HPLC80’s development of capillary electrophoresis2003’s UPLC

develop

chromatography

Liquid Chromatography Gas Chromatography

PC TLC CCC LPLCMPLC

HPLC

analyzeprepare

analyzeprepare

analyzeprepare prepare

analyzeprepare

develop

develop

HPLC

Analyzequalitativelyquantitativelyanalyze theconsist of sample

prepareextractseparatepurifythe valuablecompound of rowmaterial

analyze

HPLC

prepare

High /Ultra PerformanceLiquid Chromatography

(H/UPLC)

Dynamic Axial compression(DAC)

Simulated Moving Bed(SMB)

prospect

2.ATL preparative HPLC system

From analysis topreparation

Correlation technique ofpreparation

ATL instrumentsystem

Preparative high pressure chromatographyhas no essential difference with traditionally classical columnchromatography. It has higher efficiency and achievesautomatic operation. About the instrument, a high pressurepump is adopted to deliver mobile phase. At the same time, adetector is connected to the outlet of the column which candetect the fraction on-line constantly.

FromAToP

The preparative high pressurechromatography has several features asfollowing:1.shortter column length;2. larger column ID;3. higher flow rate.Sometimes it is also called Highperformance preparative liquidchromatography

FromAToP

The difference and relation of analysis and preparativechromatography

analysis preparative

Applied area qualitatively analyze: sensitivityquantitatively analyze: resolution,repeatability

Extract, separate, purify compounds frommixture of row materialObtain specific fraction with high purificationfrom the sample; pilot preparation and low-costpreparation

Particle size ≤5um ≥10um

Flow rate 0.001-9.999ml/min 100~80000ml/min

Sample size 0.01-2mg 1-500000mg

Capillary ID 1/16” 1/8”~1/4”

Injectionmethod

Inject valve Inject valve and inject pump

Mobile phase No recovery recovery

Separate way linear Non-linear

FromAToP

Typical analysis HPLC system

Mobilephase

pump

injector

column

Work station

detector

waste

FromAToP

Largertank

Larger flowrate pump≥50ml Sample loop ≥1ml

Injection pump≥50ml

≥10mm flange columnDAC

Biggervolume flow

cell

Solventrecovery

Preparative system

Mobilephase

pump

injector

column

Work station

detector

waste

CorrelationTechnique ofpreparation

Samplefeature

cost

Time

Purity ofproduct Output

The five elements for preparative process

0.1 10 1000

Cost of solvent

Cost of manpower

Cost ofequipments

10000

Lab scale Process scale

cost

Output/g

CorrelationTechnique ofpreparation

Cost of Packing material

LinearchromatographyAnalysis

preparation

CorrelationTechnique ofpreparation

Non-Linearchromatography

Concentration in mobile phase

Concentration instationaryphase

Concentration in mobile phase

Langmuirian type

Concentration instationaryphase

Concentration in mobile phaseAnti-Langmuirian type

Concentration instationaryphase

The peak shape of preparative chromatography

signal

signal

Retention timeLangmuirian type

Retention timeAnti-Langmuirian

type

signal

signal

Retention timeLangmuirian type

Retention timeAnti-Langmuirian

type

CorrelationTechnique ofpreparation

Asymmetric peak shape

The retention time of peak ismodified by increasingsample concentration

The peak height (or peak area)is not proportional tosample concentration

FeatureOf

Non-linearchromatography

CorrelationTechnique ofpreparation

The most important thing in preparativechromatography:

Sample overload

CorrelationTechnique ofpreparation

Concentrationoverload

VolumeoverloadPK

Langmuir Type: the capacity factordecreases; the peak shape changes from Gaussian curve to triangle

Increasing sample concentration and keeping theinject volume invariantly; the sample size depends onthe solubility of sample in mobile phase; the outputdepends on selectivity and the particle size of packingmaterial has little influence on it

Invariant peak height;the peak width increase and presentrectangle

The sample size dependson inject volume; theoutput depends on columnID; it needs small particlesize packing material

CorrelationTechnique ofpreparation

Analysis

Preparation

amplification

Optimizing theanalytical method

Overloading onanalytical column

Amplifying onpreparative column

CorrelationTechnique ofpreparation

Adjust sample size:mp/ma=lp/la * rp /ra

L: column length;m:sample size;

r:column ID p: preparative column;a:analytical column

Keypoint 1

2 2

CorrelationTechnique ofpreparation

Adjust flow rate:

Fp/Fa = Vp/Va = Lp/La * rp /ra

F :flow rate ;V:column volume;L: column length;r:column ID;

p: preparative column;a:analytical column

Keypoint 2

2 2

CorrelationTechnique ofpreparation

Adjust gradient:

tp/ta = Vp/Va * Fp/Fa

F :flow rate ;V:column volume;t:gradient elution time;

p: preparative column;a:analytical column

Keypoint 3

CorrelationTechnique ofpreparation

10ml 3000 Seriesanalytical HPLC

system

Analytical Technologies 3000 SeriesHPLC System

50ml binary 3000Series

Semi-preparativesystem

3000 Series Semi PREPsystem

100~1000ml 6000Series

Preparativesystem

6000 Series PREPsystem

6000 Series100~1000mlPreparative

system 50~200mlDAC

PREP with DAC system

3.Industry preparative system

Composition of 2800 DAC system

pump85L/min

Injectpump

Control unitMCU2800

2800DAC

detector

8 fractioncollector

450LSlurry tank

Industry preparativesystem

Columnbody

Sieveplate

Flange ofcolumnbottom

Industry preparativesystem

2800DACPiston head

80Lpump

Industry preparativesystem

Pneumaticcollector

valve

explosion-proof

equipment

Industry preparativesystem

AutomaticSlurrytank

Industry preparativesystem

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example

Sample :The row material contains 68% target compound. It iswhite solid powder and stability for hot and light (cantolerate 100℃).

Custom requirement:After the purification, the purity must reach greater than99%; the single impurity must reach less than 0.8%; the

recovery must reach 70%.

Step1:Meet the requirements of custom

Step2:The analysis of original sample

example You must have an analysismethod to detect the purity ofthe fraction and final product.

A case of application

example

Analytical condition:methanol:acetonitrile:water=81:12:7Wavelength :246nmFlow rate:1.0mL/minColumn :C18,5μm,4.6*250mm

resultA case of application

Step3:Simulate preparation on analytical column

Particle size of packing material≥ 10um 4.6*250mm

No nonvolatile acid, alkali and salt in mobile phase

Overloading when target and impurity has good resolution

Divide the target peak to about ten fractions and collect them

Detect the purity of the fraction and calculate the recovery

A case of application

Select a suitable composition of mobile phase

Further study of optimization

example Study of preparativemethod

Mobile phase Packing material

90%methanol、75%methanol、75%acetonitrile、70%acetonitrile、

60%acetonnitile。

Four kinds ofdifferent brand

packing material

60%acetonitile XX brandC8-10um-100A

select select

A case of application

2.645'3.177'

3.715'

3.968'

4.508'

5.589'

6.181'

7.728'

8.381'

9.348'

12.040'

14.171'

15.813'

16.641'

21.966'

35.288'

39.085'

52.157'

53.655'

76.363'

86.672'

100.659'

110.063'

119.893'

0

300

600

900

1200

1500

1800

2100

mV

10 20 30 40 50 60 70 80 90 100 110 min

exampleThe chromatogram of simulation on

analytical column

Result

A case of application

Step4:Confirm the maximum sample size

Enlarge the sample size continuously untilthe recovery decreases obviously.Theory sample size:3‰---8‰ of packingmaterial

A case of application

example

60% acetonitrile,sample size5‰,recovery 36.3%

60% acetonitrile,sample size3‰,recovery 78.9%

The optimization ofsample size is 3 ‰

A case of application

The final preparative method:Mobile phase:60% acetonitrile-waterSample size :3‰ of packing material weightPacking material:XX brand-C8-10um-100ASolvent of sample:mobile phase

A case of application

Step5:Amplification

Linear amplification of the simulative preparative method on analytical column topreparative column

Repeat this process several times, after the technology is mature, the targetpeak can be divided to only 4 to 6 fractions

Divide the target peak to about ten fractions and collect them

Detect the purity of the fractions and calculate the recovery

The formula is in front; the column size is decided by sample size

A case of application

12.298'

14.003'

15.378'

16.932'

-40

0

40

80

120

160

200

240

280

320

360

400mV

3 6 9 12 15 18 21 24 27 min

───────────────────────────────────────NO. Retention time percentage peak area resolution theory plate capacity factor tailing───────────────────────────────────────1 12.298 0.5167 42956 2.32 4218 -0.000 1.36 2 14.003 99.3 8255744 1.71 6614 0.139 1.14 3 15.378 0.1307 10865 1.74 4727 0.250 0.58 4 16.932 0.04702 3909 1.70 6200 0.377 0.95 ───────────────────────────────────────total 100 8313474

Chromatogram of the purification result

A case of application

Optimization ofpreparativemethod isendless!

Thank youFor your attention!