Complex LC Method Development Using Method Development System and LC Simulation Software

Koudi Zhu,1 Matthias Pursch,2 Binghe Gu3

1Analytical Technology Center, 1897 Building, The Dow Chemical Company, Midland, MI 48667; 2Dow Stade Produkt. GmbH&Co OHG,

Analytical Technologies, Stade, BN, Germany 21677; 3Analytical Sciences, 1897 Building, The Dow Chemical Company, Midland, MI 48667

The development of a liquid chromatographic (LC) method for complex samples

is a time-consuming, sometimes trial-and error process. Newly developed

analytical tools, such as Agilent LC method development system and LC

simulation software from Advanced Chemistry Development (ACD/Labs), have

the potential to significantly improve the productivity of the “slow” method

development process.

This study explored the capabilities of the method development system and the

simulation software by developing a universal LC method for resolution of a total

of 26 peaks in raw material and in process samples. With the capability of the

multiple solvent selection valve and multiple column compartment of the method

development system, four analytical columns, mobile phases with different acid

additives, three different gradient conditions and two different column

temperatures were screened. The method scouting software in the system has

the capability of flushing the system, equilibrating column automatically when

column and other LC conditions change. It greatly increased the flexibility of LC

column and mobile phase screening.

The data generated were transferred to a computer on which ACD/LC simulator

software was installed. The peaks were identified according to individual

standard retention time match and UV spectra. The simulation software

transferred the chromatography data into a table which contains the retention

times and peak widths of all peaks of interest. The LC simulation software then

built a model to obtain resolution maps. Several method conditions were

generated that have potential to separate all the peaks of interest. In addition,

manual optimization can be performed to attain tailor-made separation by using

the resolution map. The manually optimized LC condition provided by ACD/LC

Simulator software was tested. The experimental run matched the predicted run

very well and separated all 26 peaks of interest in 14 minutes.

Abstract

Conclusions

References

Acknowledgments

1290 Infinity LC/MSD Method Development System

ACD/Labs’ LC Simulation Software

Model Compound Selection

Column Screening

Data Collection

Mobile Phase Selection

Data Transfer

ACD/LC Simulator Mode and Method Suitability Table

LC Simulation for Method Optimization

Dow Chemical Company Analytical Technology Center Leadership

Dow Chemical Company Technology Renewal & Development Program

Grant Von Wald for ACD Software discussion

JD Darland for providing samples

LC method development system from Agilent Technologies is an excellent tool

for automation of column and mobile phase screening.

ACD/LC Simulator software is very helpful in optimizing LC methods for complex

samples that contain multiple critical band pairs.

A combined use of LC method development system and ACD/LC Simulator

software was successfully demonstrated for the development of a universal LC

method for in-process and starting material analysis.

Snyder gradient flow rate scaling principle: Keep tG x F be constant. By changing

flow rate to 1.5 mL/min, baseline resolution of all 26 peaks was achieved in 14 min.

• Challenges: AP new process impurities (AP dimers) out of spec; production

yield sacrificed for quality; data provided on raw material COA insufficient for

process improvement purpose; resolution for critical pair not rugged in the

product release method

• Solution: Develop a universal LC method that can separate all 26 peaks of

interest

Choose to use phosphate buffer to

stabilize mobile phase pH in an

attempt to attain a rugged method

used in QC lab

A: 5% ACN / 95% 20 mM

phosphate buffer, pH at 2.12

B: 70% ACN / 30% 20 mM

phosphate buffer, pH at 2.12

1. M Pursch, R Edam, etc.“Rapid Method Development for Industrial LC

Separations Using Automated Screening of Stationary Phases and Solvents”,

LC-GC 2010, Vol. 28 (4), pg 44–50

2. www.agilent.com

3. www.acdlabs.com/lcsimulator

Multiple solvent selection valve & Multiple column compartment

1. Column Screening

2. Mobile Phase Screening

3. Gradient Screening

4. Temperature Screening

Method Scouting Software

1. System Flushing

2. Column Equilibration

3. Column Storage

4. Auto Sequence Generation

Experimental chromatogram

using optimized method by

ACD/LC Simulator

Predicted chromatogram by

ACD/LC Simulator

Chromatogram overlay above shows excellent retention time match of most

peak of interests in experimental chromatogram using the optimized method

and chromatogram predicted by ACD/LC Simulator.

Accurate Peak identification & integration are essential in generating successful LC

methods

3-D resolution map

Visualized chromatogram

shows separation when

method condition changes

without running samples

Selected