4. Preparation of the Column

This is the stage where the column can be connected to the system.  It is important not to use excessive force to tighten and/or release the column connections.  The column should be hooked with the nuts manually fitted snugly, and then it can be tightened with the appropriate ranch for about ¼ round DO NOT USE EXCESSIVE FORCE!  It is recommended to do it with the flow on, so that the tightening will be just right.  At the adge of every tubing there is a nut and a ferrule, which is a special flexible cone that tightens the connection hermetically, when the cone is pressed against the tubing.  Every over-tightening requires higher force next time it is connected, until the connection is ruined.

 

It should be noted that the column is the heart of the system; it is mostly sensitive part in the system, prone to problems.  The actual separation occurs in the column, and it is very important to know which solvent systems to select and what program to work with to get a good separation.  It needs to be protected from contaminations in the mobile phase solvents or the sample matrix, from particles that block its voids and from air bubbles that will spoil its surface activity.

 

Before using a new chromatographic method the user should check the manufacturer’s instructions for the range of pressure, pH, solvent types and buffers’ types that are allowed to use with the specific column.  It is recommended to prepare a standard test mixture of solutes and a standard method that will enable to examine the column performance between projects, and whether there has been any change in its activity.  This way there is an objective test for the status of the column.

 

When a column is connected for the first time, or after it has been stored for a long time, it needs to be washed first of all with the storage solvent, to get wetted.  At this stage it is recommended that it should not be connected to the detector yet, but to let the solvent elute out freely for 5 minutes to release possible dust particles, silica gel particles that might be released due to problems with packing, or even air.  After passing the storage solvents with no problems (over-pressure under-pressure or fluctuations), only then the solvents can be switched carefully and gradually to the new mobile phase required for work.  At this step it can be washed gradually by intermediate solvents at low flow rate, until using the mobile phase to equilibrate it.  Any switch from salt-buffer to organic solvent should be done through pure or acidic water to prevent precipitations.

 

The use of guard column at the head of the column is recommended to protect it from sample contaminations, unless the samples are simple and clear, such as simple chemical compounds.  Complex samples originate from environmental sources, or industrial formulations or even chemical synthesis mixtures, and can contaminate the separation column.  The guard column serves as sacrificial protector of the separation column, therefore, it should be consisted of a packing identical to the main column.  The connection to the main column should be snug, to prevent extra-column band broadening. 

 

When switching between different solvent compositions, their compatibility should be carefully considered.  They should be gradually and carefully mixed to prevent a chemical shock to the stationary phase.  The transfer from buffer to organic solvent should be done very carefully and a test for their proper mixing should be done.  Micro-precipitation can occur also at column and system connections.

 

Pressures should not exceed the maximum allowed by the manufacturer.  A high pressure is, for example, over 3000 psi in a 4.6x250 mm column packed with 5 uM at flow 1 ml/min of pure Acetonitril at 25 deg.

 

Over pressure is an indication for a problem in the column, and can cause irreversible damage to the packing itself, the most frequent one is creation of voids in it, which makes it unusable.  Other damages are chemical hydrolysis of the bonded phase and irreversible adsorption of contaminations from the solvent and the system itself.

For more information:
http://www.forumsci.co.il/HPLC/topics.html#Trouble
http://www.lcgceurope.com/lcgceurope/data/articlestandard/lcgceurope/262003/61538/article.pdf

Introduction

1-3: Solvent delivery system

4. The column

5 - Detector

6- Injector

7-Gradient and isocratic

8-Data Processing