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7.  Isocratic or Gradient Work

 

It is usually preferred to work in isocratic conditions, whereby the mobile phase composition remains constant.  The system and column are equilibrated all the time and does not suffer from fast chemical changes.  However, the demands from HPLC analysis has increased and the samples are usually complex in nature, the HPLC systems has evolved into very robust reliable machines, and the columns are manufactured to provide thousands of injections, therefore, in recent years the majority of the chromatographic runs has been based on a composition gradient in the mobile phase.

In a gradient work the solvent strength is increased with time during the chromatographic run.  For example, in reversed phase chromatography the mobile phase's composition at the start of the run is highly aqueous and the percentage of the organic modifier (such as methanol or Acetonitril) is increased with time, thereby raising the elution strength.  In normal phase chromatography the initial mobile phase's composition is usually mostly hexane and then a more polar organic solvent is added, such as chloroform, THF, ethanol or isopropanol.  (note that methanol and Acetonitril DO NOT MIX with hexane enough for a proper chromatography).

Creation of a gradient is done by a specified table in the instrumental method where the percentage of each mobile phase component is determined and the time segments are detailed. 

For example:

 

TIME (min.)

 

A%

(buffer+modifier)

B%

Methanol

0

100

0

1

100

0

10

70

30

15

70

30

20

100

0

30

100

0

 

In the above table the composition of the mobile phase is as follows:

 

A graphic display would be:


 

The table might look different a little in various chromatographic data stations or system's panel but it is always various compositions at various time segments.

 

The following requirements should be kept:

 

 

It is recommended to run a blank gradient for the first time before starting a sequence of injections to test the quality of the solvents and the stability of the baseline throughout the run.  Only when the baseline is stable throughout the run, the system is ready for work.

As a rule in chromatography a test run should be performed before starting to work.  The samples' solvent should be injected before the actual standards and samples are injected, to test the system for cleanliness and interferences.  Sometimes extraneous peaks appear, which belong to the system itself, due to injection of pure solvent, even before any other solute is introduced to the system, and they can be mistaken as real sample components.  These peaks are called "system peaks" and they can be part of the chromatographic chemical system and not necessarily contamination.  System peaks originate from injection of a sample whose composition is different than the mobile phase itself, into a multi-component mobile phase whose one of the components is adsorbed on the stationary phase. 

For explanation please see: http://www.forumsci.co.il/HPLC/syspks.html.  For example, when organic buffers made of acetate or ammonium salts are used, there will always be additional peak in the chromatogram if a pure solvent is injected to it and the detection wavelength is below 220 nm.  The additional peak is related to the acetic acid or the ammonium ions.  Once the additional peaks are assigned as system peaks they can be eradicated by adding mobile phase components to the sample. 


 A detailed article on extraneous peaks was published in LC-GC

 

 Introduction

1-3: Solvent delivery system

4. The column

5 - Detector

6- Injector

7-Gradient and isocratic

8-Data Processing


Copyright (C) since 1997:  Dr. Shulamit Levin