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Method Development Guide

All Primesep columns are reverse phase (RP) columns. They differ

from other brands' reverse phase columns by having an additional

charge on a stationary phase surface. This charge allows to exploreanother mechanism of interaction - ion exchange in addition tohydrophobic interaction.

The column surface charge is greatly influenced by the pH of the

mobile phase. Many analytes also change their charge state as aresult of changes in solvent pH. If a compound has a positive or

negative charge, then in order to obtain an ion-exchange interaction in

addition to reverse phase interaction using Primesep columns, the

column surface charge and the analyte charge should be opposite. This

guide helps you to decide which Primesep column to choose when youjust begin to do method development with Primesep mixed-modecolumns.

There are three steps in a process of selecting a correct PrimesepHPLC column for your method:

Step 1.Choose the appropriate bar for your compounds based on their

pKa value or structure, then find the pH range where your analytehas a charge.

Step 2.Choose the appropriate buffering system with the pH at apoint where analyte has a charge which satisfies your detection andscale up requirements that you have found in step 1.

Step 3.Choose an appropriate Primesep column, which has a surface

charge opposite to the analyte charge, found in Step 1.

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Step 1.

Choose the appropriate bar for your compounds

based on their pKa value or structure, then find the

pH range where your analyte has a charge.

Note: Some compounds have more than one charge state within a working pH range (examplesinclude: aspartic acid and glutamic acid). Compounds that have no charge state at any HPLCcondition can be analyzed with any Primesep column via the reverse phase mechanism. Besidesthe reverse phase or hydrophobic mechanisms, other mechanisms of interaction can be utilized inthis case (normal phase interaction, HILIC, and pi-pi interaction with Primesep P column).

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Step 2.

Choose the appropriate buffering system with the pHat a point where analyte has a charge, which

satisfies your detection and scale up requirements

that you have found in step 1.

Note:If the analyte and the column surface have opposite charges, elution of analyte from thecolumn requires the presence of other ions in the mobile phase. Water and acetonitrile alone donot produce enough ions in the mobile phase to efficiently participate in an ion-exchangeprocess. As a result, charged analytes in Primesep column systems result in very long retentiontime and poor peak shape. Typical HPLC buffers are a good source of ions capable of facilitatingan ion-exchange process.

Two distinct types of buffers are available depending on applicable detection requirements: Non-volatile buffers for low UV application, and Volatile buffers for MS, ELSD, and preparatory applications

Only TFA-based buffer can be used for both types of detection. Unfortunately it is not alwaysapplicable for MS detection, and does not permit to use lower UV wavelength.

Cation-exchange Primesep columns allow to use non-buffered solutions. The acidic residueembedded in the column provides the acidic component of the buffer. Ammonium acetate orammonium formate salts can be used for MS, ELSD, and preparative chromatographyapplications while potassium sulfate can be used for low UV applications.

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Step 3.

Choose an appropriate Primesep column, which has

a surface charge opposite to the analyte charge,

found in Step 1.

Note: Primesep columns in transitional pH gradually lose or change their surface charge. Thisprocess is reversible and column restores ionic properties with another mobile phase pH. Whenthe column is used without a buffered mobile phase, the surface charge corresponds to the chargeat pH 7 on this chart. In order to work and get consistent results in transitional pH, the ionicmobile phase modifier should have sufficient buffering capacity.

Unlike the other Primesep columns, the Primesep AB column is a zwitterionic reverse phasecolumn with a surface charge that is affected by the type of the analyte: With basic analytes the Primesep AB column behaves as if it has a negatively charged surface With strong acids the Primesep AB column behaves as if it has positively charged surface.

All column except Primesep B are stable in any buffer system within a pH range of 1.5 - 7.Primesep B column is recommended at a pH range of 1.5 - 4.5.

Use the chart below to help properly select a column that will interact with the analyte by boththe reverse phase and ion-exchange mechanism