Question

Gradient conditions and methods development a) why is method development easier with gradient elution chromatography than isocratic separations? b) in some detail explain why one would use elevated temperatures in liquid chromatography? c) what is DryLab and why use it?

Please provide reference

Answer 1

a.

To find answer for this, first understand the difference between these two-

Isocratic Mode of HPLC means the composition of mobile phase remains constant throughout the run.Isocratic mode is most commonly used for assay, dissolution, and routine analysis where you want to separate 2 or 3 compounds in a single run.
On the other hand in gradient mode composition of mobile phase (polarity of mobile phase) in varies and its not constant. Generally gradient modes are useful when you want to analyse mixture of analytes having differences in the polarity so it can not be separated in a single mobile phase and can be separated by varying polarities of solvents i,e, gradient mode.

So while method development there is scope in gradient mode to play with mobile phase, but same is not possible in Isocratic Mode.

b.

The use of elevated temperature has proven effective for improving the overall chromatographic performance on conventional equipment. The main advantages of using temperature in LC are:

1. Speed Increase speed by increasing the temperature and flow rate.

2. Efficiency and resolution Increase efficiency by using longer columns/smaller particles at elevated temperature.

3. Selectivity Change selectivity with temperature.

4. Lower consumption of organic solvents - Green chromatography Higher temperature requires less solvent.

5. Improved detectability Improve peak shape. Use only water.

6. Temperature programming Replace solvent gradients with a temperature program.

Reference- http://www.richrom.com/application/v2/public/upload/0/default/157.pdf

http://www.sepscience.com/Techniques/LC/Articles/489-/High-Temperatures-for-Liquid-Chromatography-Practical-Approaches-for-Better-Separations

c.

DryLab is the revolutionary HPLC method development and optimization software that predicts chromatograms under a much wider range of experimental conditions than would ever be possible in the laboratory. With DryLab you can quickly and easily determine exactly how your separation behaves as you simultaneously vary multiple method parameters, such as pH, temperature, buffer concentration, and many more. Anybody developing HPLC methods who wishes to optimize complex samples and to economize resources spent developing and running methods will benefit from the many advantages offered by DryLab.

Reference http://molnar-institute.com/drylab/