Gradient Method for Peak Shape Improvement 

Keeping all other Method Conditions the same, we demonstrate how you can improve Peak Shape with the use of a Gradient compared to an Isocratic Method.  Changing the original Isocratic Method, (Figure 1.) to the Gradient Method (Figure 2.) we demonstrate good Retention, and improved Peak Shape.

Using standard HPLC Columns, gradient methods often suffer long equilibration times or lack of precision if 100% equilibration is not achieved after each run. Silica Hydride HPLC Columns, such as the one in this AppNote, do not require many column volumes to achieve 100% equilibration. This allows a quick turnaround in total run cycle times without observing Retention Time (RT) shifts, as shown in our low RT % RSD values.  (More information about this quality is located HERE.)


Figure 1. 
Isocratic: 80% B

This Gradient Method shows excellent Peak Shape and RSD values less than 0.2% for analysis for Ambroxol HCL in this 10 injection overlay, below in Figure 2.

 
Figure 2.
Gradient

Peaks:
 Ambroxol

Method Conditions:
Column: Cogent Diamond Hydride™, 4μm, 100Å
Dimensions: 4.6 x 75mm
Mobile Phase:
A: DI Water with 0.1 % Formic Acid
B: Acetonitrile with 0.1 % Formic Acid
Isocratic Mobile Phase:  80% B
Gradient:

Time (minutes) %B
0 95
1 95
3 70
4 70
5 95
6 95

Post time: 2 minutes
Injection vol.:
1μL
Flow rate: 1.0mL / minute
Detection: UV @ 254nm
Sample Preparation: Ambroxol HCL 0.1mg / mL in 50:50 Acetonitrile / DI Water

Note: Ambroxol is a secretolytic agent used in the treatment of respiratory diseases associated with viscid or excessive mucus.