| This section offers quick diagnostic guidelines on how to identify and prevent
problems related to your HPLC column. Many of the subjects presented here are discussed in
more detail in later sections. References to these sections are noted where appropriate.
The HPLC column is the heart of a separation and analysis. But when a problem arises in
HPLC analysis, it can be dfficult to determine if it is caused by the column or another
part of the system. Often the HPLC column is identified as responsible for the problem.
More often than not, these problems are due to other factors, such as changes in the
mobile phase, shifts in flow rate, variations in temperature, differences in the sample,
or problems with the instrumentation.
When a chromatographic problem results in column failure, proper corrective action
should be taken to avoid another failure. Identifying the cause of the problem may seem
like a difficult, time-consuming task. Fortunately, there are only a limited number of
reasons why a column would perform poorly.
The following will summarize several common problems that can result in HPLC column
failure. After the description of each problem there is a list of possible problem causes
along with the recommended corrective action. By referring to these guidelines when a
problem arises, you can be well on your way to isolating and correcting any HPLC column
problem you might encounter.
PROBLEM: An unacceptable increase in system back pressure. |
|
|
Possible Cause: |
|
Corrective Action to Take: |
| 1. |
Plugged inlet frit |
|
Back-flush column to dislodge particulates or replace column inlet frit. |
| 2. |
Plugged in-line filter |
|
Replace filter |
| 3. |
Plugged guard column |
|
Replace guard column |
| 4. |
Using a column with a smaller
internal diameter |
|
Reduce flow rate (see 3-5) |
| 5. |
Using a smaller particle packing |
|
Use shorter columns |
|
| Non-column causes: problems with pressure
transducer, sample injector blockage, blocked tubing, blockage in detector flow cell,
increase in flow rate. |
|
|
PROBLEM: A change in peak retention times. |
|
|
Possible Cause: |
|
Corrective Action to Take: |
| 1. |
Column fouling |
|
Clean or replace column |
| 2. |
Loss in bonded phase |
|
Replace column |
| 3. |
Column not equlibrated |
|
Flush with at least seven column volumes |
| 4. |
Sample overloaded |
|
Reduce sample mass injected |
|
| Non-column causes: change in flow rate, change
in mobile phase conditions (composition, pH, etc.), change in temperature. |
|
|
PROBLEM: A loss in efficiency (theoretical plates). |
|
|
Possible Cause: |
|
Corrective Action to Take: |
| 1. |
Column developed a void |
|
Fill in the void with similar packing material, or replace column |
| 2. |
"Worn-out" guard column |
|
Replace guard column |
|
| Non-column causes: extra column effects
(damaged or improperly made fittings/connections), increase in injected sample volume,
increase in strength of sample solvent, changes in mobile phase composition. |
|
|
PROBLEM: Peak tailing or peak splitting. |
|
|
Possible Cause: |
|
Corrective Action to Take: |
| 1. |
Column fouling |
|
Clean or replace column |
| 2. |
Particulates on column inlet frit |
|
Replace inlet frit |
| 3. |
Loss of bonded phase |
|
Replace column |
| 4. |
"Worn-out" guard column |
|
Replace guard column |
| 5. |
Bad sample distribution |
|
Change to a column with a better inlet design |
|
| Non-column causes: extra column effects
(damaged or improperly made fittings/connections), injected sample overload, changes in
mobile phase composition (additives, pH, buffer concentration). |
|
|
PROBLEM: Mystery peaks. |
|
|
Possible Cause: |
|
Corrective Action to Take: |
| 1. |
Column fouling |
|
Clean or replace column |
| 2. |
"Worn-out" guard column |
|
Replace guard column |
|
| Non-column causes: impurities in the mobile
phase, additional components in the sample, air bubbles in the sytstem, electronic
problems. |
|
For Technical
Assistance Call |
|
| 1-800-441-7508 |
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|
