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
