HPLC A Praactical User''''S Guide Part 5 pot

1–3 cm in length, they can be inverted and backwashed without causing them

to void. Please do not wash the guard column down the main column. Discon￾nect and reverse it, reconnect and use the pump to pump a strong solvent

through it into a beaker. This may seem obvious, but I had to troubleshoot a

persistent detector problem that turned out to be caused by a chromatogra￾pher who washed a guard column into his main column.

Since the guard column is placed in the injector/column path, it does con￾tribute to the separation. Methods development should be completed with the

guard column in place. The increased separating length usually overcomes the

effect of extra tubing as long as the connecting tube between the guard and

analytical columns is kept as short and as fine as possible. The wrong diame￾ter tubing can really mess up a separation. Changing guard columns in the

middle of a series of runs generally has little effect on the separation. However,

it is usually a good idea to follow the change with a standard QA run as a

check.

The other type of protective, in-line column is the saturation column. This

column is used when operating conditions tend to dissolve the main column

bed (i.e., high pH, high temperature, etc.). In theory, the packing in the satu￾ration column dissolves first and protects the main column packing. As long

as the same bonded phase is used in the pre-column, the column running char￾acter does not seem to change. Using this technique, I had a customer who ran

taurine separations at pH 12 for a year on the same C18 column. Care must be

taken that the saturation column does not break through; erosion of the main

column will begin immediately if this happens. A guard column will serve as

a saturation column, but is not recommended, since the pre-column bed is con￾sumed and band spreading will occur. Usually, the saturation column is placed

in the flow from pump to injector. At this point, the column to be used can be

slurry packed with no regard given to packing efficiency. I have even seen

columns dry packed with tamping, wetted with solvent, and placed in line as

a saturation column. I’m not entirely satisfied with the explanations as to why

this technique works. I offer it to you as a tool that other chromatographers

have used to produce separations at pH high enough to separate many amines

in their free amine form. Silica appears as a solid on evaporating fractions and,

occasionally, coats out on detector windows. I would recommend using this

technique as an analytical tool only when other methods have failed.

COLUMN SELECTION 71

6

COLUMN AGING,

DIAGNOSIS, AND HEALING

73

HPLC columns have a reputation of being fragile things that only have a

limited lifetime and, therefore, are expensive to buy and maintain. Much of

this reputation is undeserved and in this section we will explore the aging of

columns, the symptoms of aging, and methods of regenerating columns and

extending their operating life. The typical new chromatographer gets about 3

months life from a column; an experienced operator gets about 9 months. I

hope to help you extend column life to 1–2 years.

I know this is possible from a bonded-phase column because I had a cus￾tomer who averaged this on his columns. He ran a clinical laboratory and

rotated C18 columns through a series of four separations, each less demanding

than the one before it. When the column failed on separation 1, it was washed

and reequilibrated for a less demanding separation 2 and so on.

Over the years, I have collected hints, ideas, and tips that were not then

available, allowing us to get the same performance from each column without

rotation. The key to treating column problems is to know when problems are

occurring, catch them as early as possible, and treat them. The main tool for

early detection of problems is column QA with standards described and illus￾trated in Figure 6.1.

There are five basic types of “killers” of column efficiency: 1) effects that

remove the bonded phase; 2) effects that dissolve the column surface, or the

packing itself; 3) materials that bind to the column; 4) things that cause pres￾sure increases; and 5) column channeling.There are definite symptoms of each

of these and either treatments or preventions for each type of killer (Fig. 6.2).

HPLC: A Practical User’s Guide, Second Edition, by Marvin C. McMaster

Copyright © 2007 by John Wiley & Sons, Inc.