optimization of hplc.pptx d

8/3/2019 Optimization of Hplc.pptx d

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By

Tejaswi Reddy 1



HPLC SEPARATION MODE

Normal phase chromatography (NP)

Reversed phase chromatography (RP)

Size exclusion chromatography (SEC)

Ion exchange chromatography (IEX)

Affinity chromatography

2



FEATURES

Mode Stationary phase Mobile phase Interaction

Normal phase Silica gel organic solvent Adsorption

(n-Hexane)

Reverse phase Silica ODS MeOH/water Hydrophobic

(C18 column)

Size exclusion

Chromatography

Non-aqueous Porous polymer Organic solvent THF Gel permeation

Aqueous Aq. Porous polymer Buffer solution Gel permeation

Ion exchange Ion exchange gel Buffer solution Ion exchange

Chromatography

Affinity Packing with Buffer solution Affinity

Chromatography ligand

3



Normal Phase Chromatography

Stationary Phase : Polar ex : Silica gelSilica-CNSilica-OH

Mobile Phase : Non-polar ex : iso-octane/AcOEtn-Hexane/CH2Cl2

4



Reverse Phase Chromatography

Stationary Phase : Non Polar ex : Silica gel

Silica-CN

Silica-OH

Mobile Phase : Polar ex : iso-octane/AcOEt

n-Hexane/CH2Cl2

5



OPTIMIZATION FLOW

SHEETS

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4.Replace ACN with equal strength

MBTE

Inadequate separation

5.Run 1:1 blend of correct solvent

strength hexane : DCM (from step 2)

and Hexane : ACN (from step 3)


6.Run 1:1 blend of correct solvent

strength Hexane: ACN ( from step 3)And

Hexane : MBTE (from step 4)


Inadequate separation ½ > 0.4

Run equal strength mixture of

Hexane and methanol


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7.Run 1:1 blend of correct solvent strength

Hexane : DCM (from step 2) and Hexane :

MBTE (from step 4)


8.Run 1:1 blend of correct solvent strengthHexane : DCM (from step 2),

Hexane : ACN (from step 3) and Hexane :

MBTE (from step 4)


MBTE : Methyl tertiary butyl ether

DCM : DichloromethaneACN : Acetonitrile

Compare runs from 2to 8 blend two or more solvents from these runs

9



1.C18 column,400C , aqueous component of mobile phase at PH- 3.5 with 50 mM H3PO4

2.Run with 1:1 Methanol/ ion pair

reagent(solvent C)

K too small

3.Decrease %Methanol;

If K is too small,

Decrease conc of ion pair agent

4.Seperation OK- stop;

change column

Conditions to improve separation


K too large

Increase % Methanol

Tailing peaks

Add TEA at 1/5 conc of ion pair agent


ION ² PAIR HPLC

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5.Use mobile phase of 1:1 blend of

methanol:solventB


6.Use mobile phase of 1:1 blend of

Methanol:solventA


7.Use mobile phase of 1:1:1 blend of

Methanol:solventA+B



Methanol:solventB+C

Inadequate separation11



Study seperations from step 4 to 10 and blend solvents to provide separation


Methanol:solventB+C



Methanol:solventA+C


Solvent A: Buffer PH 3.5 : 50mM H3PO4 adjusted to PH 3.5 with NaOH

Solvent B: Buffer PH 7.0 : 60mM H3PO4

adjusted to PH 7.0 with NaOH

Solvent C: Ion pair agent 0.02 M heptane sulphonate in water adjusted to PH 5.0

12



Decrease % MeCN until 3<k<20


REVERSE PHASE

90% ACN is the mobile phase K large

Alternative technique

Tailing peaks

Add 0.05-0.1% TEA to mobile phase

K Small

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optimization of hplc.pptx d

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