HPLC Method Development

[S. G. Joshua]

Agilent 1100 HPLC - Is it necessary to gradually increase pressure and flow rate when switching methods and before the first run after the system has been turned on? Why?

[unmgvar]

for fragile type of columns it is advisable to increase or decrease the flow rate thus pressure in a gentile manner.
the column might be "fragile" for several reasons, the casing or the packing for example.

in the "old days" the procedure of slowly increasing/decreaing flow was extremely common for all types of columns, including all the silica based, because the packing was not as good as today. then the pressure differences produced by increasing or decreasing the flow would cause the packed bed to compress or relax creating dwell volumes and ruinning columns.
these days we mainly watch the maximum pressure allowed.

if you are uncertain, the best aproach i think is to ask the vendor.

[Consumer Products Guy]

Agilent 1100 HPLC - Is it necessary to gradually increase pressure and flow rate when switching methods and before the first run after the system has been turned on? Why?

We don't do that. And that certainly defeats the automated switching between methods in the Sequence Table (and/or columns using the column switching valve accessory). I agree with first response: was more important in the "old days".

[Uwe Neue]

Never done this in my entire life with silica-based columns.

It is advisable though with compressible packings, i.e. polymer-based columns.

[DR]

I suspect you have, Uwe - Alliance pumps have a setting for Pump Acceleration (Empower) or Flow Ramp (2695 panel). It appears as if it is only for flushing and purging, but I think it is also applied when initiating flow with a column in line. I think most modern pumps take at least a couple of turns of their drive motors before they're operating at their programmed rates, if they are starting from a stand-still.

[rhaefe]

It is advisable though with compressible packings, i.e. polymer-based columns.

Only with gel columns (low cross linked stationary phases like the ones used for carbohydrate and amino acid analysis).
Not necessary for highly cross linked phases (>50%XL).

As DR pointed out, most (all?) modern pumps need a few moments to get up to full flow rate.

[HW Mueller]

It´s a myth, then, that pumps damage packings?

[tom jupille]

It´s a myth, then, that pumps damage packings?

Probably not so much a myth as an old tradition that has (mostly) outlived it's usefulness.

[jzt]

it depends on the method. I have a method that I always have to increase the flow rate graduately at the beginning on 1100 instruments to avoid over pressuring. then again I am pushing the 1100 close to its limits with this method (normal operation pressure around 320 -350 bar depending on the actual system configuration). that's the only way to achieve the desired separation with a 30 min gradient. if I go with the conventional 1 mL/min flow rate and 3 um column, it would take 50 min.

when this method was given to the chemists for them to run their reaction mixture samples, they complained it always shut down their instrument when they first started it up. I set up a flow ramp method and told them to use it at the first line of their sequence in ChemStation. the problem went away.

[HW Mueller]

It is interesting to see, though, that all columns take/took the injection pressure shock quite well. This pressure fluctuation can often be detected even at the outlet of the column via the system peak it produces, for instance with a UV detector.

jzt, is the mobile phase at an optimum condition or was the high flow rate a "convenience" solution?

[mbicking]

Remember that most modern silica-based columns are packed at much higher pressures compared to the usual operating limits, so an initial "shock" to the column is probably not an issue. Most users do not have to worry about this problem.

The pressure changes that jzt sees at the beginning are probably due to differences in the viscosity of mixtures of the solvents. In this case, a slower flow would be advisable.

The Agilent 1100 pump has a "soft start" design, so it has a flow ramp at startup. Most modern pumps have this feature.