Develop HPLC method in just 2 steps

PRIMESIL HPLC Column method devlopment KIT

Develop your HPLC method in just two steps

Step 1:
Inject the analysis sample and a proprietary reference mixture (RM) into the filled Primesil C18 or  C18-2 column in order to:

  • Determine the retention time of a reference peak (RP) from the analysis sample and the retention times of the compounds of the reference mixture (RM), and then calibrate the calculation system.

Choose a retention factor for the reference peak (RP): K´RP.
Enter it into the spreadsheet (supplied) along with the retention times of the reference mixture (RM) compounds.
This portion of the step allows you to determine, for each of the columns in the kit you have assembled:

  • The percentages of methanol and acetonitrile to be added to each mobile phase to obtain the retention factor K´RP chosen for the reference peak (PR).

Step 2:
Prepare the mobile phases according to the results of step 1. Inject the analysis sample into each of the kit´s columns (for verification purposes, the sample can also be reinjected in the Primesil C18 or C18-2 column).
Analyse the chromatograms to select the best column for the desired separation.
Proceed to the column optimisation step.

The Primesil Kit™ consists of various components:

a) Primesil C18 or  C18-2 reference column
b) Your selection of one or more selectivities from the list of columns for the Upti-select Kit™.
Other selectivities may be added as dictated by your requirements or budget.
c) A pack of tools containing:
a. A proprietary reference mixture consisting of five probes for calibrating the calculation system
b. A USB flash drive containing the spreadsheet and reference documentation.

The Primesil Kit™ is available in the following configurations:

To order:

  • Indicate code Pc18146 (Pc18-select column) on your order form.
  • Indicate the P/N of the Primesil C18 or  C18-2 reference column that you have chosen.
  • Indicate the P/N(s) of the complementary selectivity columns that you have chosen.(the reference column and the selectivities selected must have the same dimensions and particle size)

The column P/Ns are listed on pages B14 to B37.
Remember to also order the Primesil Kit™ tool pack (P/N Pc182290).

Transposing the chromatographic conditions of a stationary phase to an Interchim stationary phase

If you are dissatisfied with how compounds are separated under your analysis conditions (column, silica, temperature, etc.), you can either:

  • improve the selectivity
  • or change your column (stationary phase)

In general, the selectivity optimisation requires the use of a specific software program or a method of trial and error.
Whatever the case, no fewer than 18 trials will be necessary for a given pH and temperature. And after all that, there is no guarantee that the desired separation will be achieved. The only thing left to do is start all over with a different stationary phase.
Interchim has specially developed C18 stationary phases that make the entire process simpler.
In just two experiments, and using your own results (compounds and column), our method helps you find the right isocratic conditions for analysis for the 5 µm, 3 µm, 2.2 µm and 1.7 µm stationary phases in our range.

LETIAM (S. Héron & A. Tchapla) has determined the thermodynamic parameters that characterise each of our stationary phases for methanol/water or acetonitrile/water eluents. This work is based on fundamental research articles published by L.R. Snyder, P. J. Schoenmakers and P. Jandera.
Two parameters are used as measurements:

  • the hydrophobic character of the silica particles
  • their polarity in a hydro-organic medium

Each of our C18 stationary phases offers a different separation potential that is defined by these parameters, thereby allowing you to choose the silica that best matches your analysis criteria (total separation, elution order, analysis time, etc.).

The process

Step 1:
Determine the peak that to be used as the reference (RP, generally the most retained) by injecting your compounds into your column. Determine the retention times of our reference compounds (RM) by injecting this mixture into your column under your analytical conditions.
Enter this data in the spreadsheet supplied with the Primesil pack or available at www.Primesil.com. You have now defined, for the ten WESLEY TECHNOLOGIES 5 µm stationary phases (C18-2, C18-3, ODS,ODS 2, ODS 3, ODS3V), the organic solvent percentages (methanol or acetonitrile) that will make it possible to obtain a retention factor that is very close to that chosen for the reference peak (use a maximum value of close to 15).

Step 2:
After injecting your sample into the ten columns with the mobile phases from step 1, select the optimal column.

Example of transposition with a sample of solar filters

Chromatograms obtained with acetonitrile/water as the mobile phase

The chromatograms show that the retention factor of the reference peak on the ten Primesil® columns is close to 10. This result confirms the process whereby the theoretical transposition equations are used from one stationary phase to another.
Coelution of some peaks occurred on some stationary phases.
The separation of the four compounds was achieved on the Primesil® ODS3 columns, with reversals of the elution order of the peaks.
Depending on the surface chemistry of our phases, compound D was not always the last peak. The last peak remained in a range of retention close to a retention factor of 10.

Chromatograms obtained with methanol/water as the mobile phase


ADVANCED CHROMATOGRAPHY TECHNOLOGIES PVT LTD. is an instrument representation company in India, distributing the products of various foreign manufactures specializing in CHROMATOGRAPHY Instrumentation.

Read More


    CPHI INDIA 2019

  • From 26 Nov 2019 To 28 Nov 2019

    FROM 26 Nov 2019TO 28 Nov 2019 India Expo Centre, Greater Noida, Delhi NCR, India 26 - 28 November 2019