HPLC Technical Hints | - Operating Presures as a diagnostic
- Gradient HPLC non-reproducibility
- Biological growth problems in aqueous phases
-
Buffer precipitation
- Modifying EPA methods
|
Operating Presures
as a diagnostic | Tracking your column
backpressure can be a valuable tool in detecting and correcting problems with your HPPOC column or system.By recording the back-pressure when the column is first installed and then monitoring it with use, potential problems can be prevented resulting in increased lifetime and more reproducible results
The normal operating backpressure for a Pinnacle 5um 150mm x 4.6mm column using 75/25 methanol/water mobile phase at a flow of 1ml/min is approx 100Bar for a Pinnacle 3um (150x4.6mm) it is
approx 230Bar.
Pressures will vary depending on column length and mobile phase composition |
Gradeint HPLC non-reproducibility | Separations achieved using
gradient HPLC methods are often difficult to reproduce between different labs and instruments.
Variables such as high versus low pressure mixing and flow path configuration ( including tubing volume, mixing chambers and pulse dampeners ) affect the gradient profile, delay time and volume.
There can also be disparity in mobile phase composition due to factors such as proportioning valve differences or the use of premixed solvents.
These variations can usually be overcome with
minor modifications of the gradient program times and mobile phase composition |
Biological growth problems in aqueous phases | A frequently overlooked and
predominant scource of HPLC problems is biological growth in aqueous mobile phases and reservoirs.
This material can then clog inlet filters, check valves, and proportioning valves resulting in varying flow and mobile phase composition. It can also plug in-line filters, guard columns and column inlet frits resulting in high backpressure, distorted peak shape and diminished column lifetime
To prevent these problems mobile phases containing less than 50% organic solvent should be replaced
and their solvent reservoir scrubbed regularly.
In addition, biological growth can be minimised by the addition of 0.01% sodium azide to aqueous mobile phases |
Buffer precipitation | When using buffers it is important to always consider the solubility of the buffer in the mobile phase, storage solvent and rinse solvents.
This is especially important when changing a column to and from it's storage solvent.
For most columns it is best to store them in at least 50% organic mobile phase to prevent biological growth.
At this concentration many buffers can precipitate out of solution and ruin the column. To prevent precipitation when
changing mobile phases the column should be first rinsed with 10 column volumes of 20% organic solvent in water, then with 10 column volumes of the mobile phase or storage solvent |
Modifying EPA methods | All EPA methods specify a recommended column and set of conditions, however, the analyst is permitted to modify either or both to improve separations.
By optimising the column and run conditions dramatic improvements in resolution and and analysis time can be achieved.
This is especially true of application specific columns such as Pinnacle TO-11, PAH and Carbamate. These columns have been specially designed to improve resolution
and decrease analysis times for these particular applications |
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