Building up pressure on HPLC?

We’ve all experienced a pressure error on HPLC at some time or another. Here are my best suggestions on handling them.

pig in pot

Establish ground rules-This will help to define the problem and distinguish if there really is one.

1.     Check your programmed max pressure limit. Be aware of what the operating limit is for your system and if there is also a programmed limit. Please consult your instrument manufacturer for this information or consult the operating manual they provide. When programming a setting, please do not try to override the limit suggested by the manufacturer.  Operating pressure limits for regular HPLC systems (not UHPLC) are often in the range of 5800 – 7000 psi (400 – 500 bar), which is usually also appropriate for HPLC column hardware and (fully porous particle) packing material. Physical pressure limits are usually established based on ratings for valves, pumps and other components and have a lot to do with how much it can handle before leakage occurs. Keep in mind also that solvents like methanol and water inherently generate higher backpressure. Generally, I don’t recommend going above 6000 psi for 3 um or 5 um fully porous particle size columns. (This excludes our Raptor columns (SPP) and 1.9 um particle UHPLC columns.)

2.     Establish a flow rate that will give you best resolution. Chromatographic resolution, on the other hand, is more of a practical limit, determined by what will give you acceptable results for sound scientific data. The following table can be used to determine what the optimal flow rate is to get best resolution for the packing material in your column. It also gives an estimate of expected column pressures that would be generated with acetonitrile or methanol. Please keep in mind that actual pressures for individual columns will vary somewhat and will often increase slightly with usage. Also remember that pressure readings are a reflection of overall pressure, which includes any inherent background pressures from the system itself.

3.     Monitor system pressure without an HPLC column in line. Before you install a column, replace the old one with a union connector and let the system go through a run cycle. Record the system pressure in this configuration and keep it on file for reference. Check this occasionally, as an increase would indicate a blockage or restriction has formed in the flowpath. You may be able to prevent an overpressure error and potential crisis by detecting such a change.

4.      Record pressure for new columns. As soon as you have your column installed to run your method and things have stabilized, record the pressure along with mobile phase and flow rate conditions. This will be used as a baseline for comparison as your column ages. Keep in mind that the following applies only to fully porous particle columns, not the Raptor columns (SPP).

Column Inner Diameter (mm) Column Length (mm) Particle Diameter (µm) Optimal Flow rate (mL/min) Predicted Pressure for ACN (psi) @30C Predicted Pressure for MeOH (psi)  @30C
2.1 30 3 0.31 527 838
2.1 30 5 0.2 122 195
2.1 50 3 0.31 879 1396
2.1 50 5 0.2 204 324
2.1 100 3 0.31 1757 2792
2.1 100 5 0.2 408 649
3.0 50 3 0.65 903 1434
3.0 50 5 0.4 200 318
3.0 100 3 0.65 1806 2869
3.0 100 5 0.4 400 636
3.0 150 3 0.65 2708 4303
3.0 150 5 0.4 600 953
4.6 50 3 1.5 886 1408
4.6 50 5 1 213 338
4.6 100 3 1.5 1772 2816
4.6 100 5 1 425 676
4.6 150 3 1.5 2658 4224
4.6 150 5 1 638 1014
4.6 200 3 1.5 4431 7040
4.6 200 5 1 1063 1690

 

Isolating the source-If you’ve been using a column for a while and notice a pressure increase, compared to when it was new (#4 above), you’ll need to see where it is coming from. It may or may not be the column.

Kent's beep

A.      Disconnect the column outlet and place outlet tubing directly into waste container or beaker. Turn the pump on, then observe and record the pressure. Also take note of any particulates or colored materials that appear to be exiting from the column. Continue to pump for 20 minutes or until visible material cease to exit the column. Observe pressure again and see if it has changed and then record the value. If visible material continues to exit the column after at least 20 minutes, remove and replace the column. Contact Technical Services for help in determining why this might have happened.

B.       If after disconnecting column in step A, the pressure immediately returned to normal range for this column (#4 above) with no visible residue coming out, then there is a restriction in the flowpath after the column, either somewhere in the tubing or valves leading to the detector or possibly in the detector flow cell. Replace tubing and/or clean flow cell as needed, according to instrument manufacturer’s recommendations. Check all fittings and valves for proper assembly and ensure that no tubing is crimped. Look for possible accumulation of buffer salts and clean or replace parts as needed.

C.        If after performing step A, there was no residue observed and no significant reduction in pressure, remove the column and replace it with a union connector. After pumping to reach stability, compare the pressure to those recorded as a baseline in #3 above. If system pressure is close to what it was previously in #3, then you have confirmed the buildup of pressure is from the column and likely a result of particulates clogging the inlet frit. For 3 um and 5 um particle size, fully porous particle columns only, try backflushing the column with your mobile phase for 30 minutes, still pumping into a waste container. (Please note that pumping in a reverse direction is not recommended for 1.9 um UHPLC columns or for the Raptor columns). If pressure is still elevated, repeat backflushing using a series of solvents with varying polarities as described in our Technical Note: Restek Liquid Chromatography (LC) Columns — Cleaning Recommendations. If the mobile phase you have been using contains buffer solvents, flush initially with a solution containing a high amount of water (between 60- 90% for a typical C18 phase) and no buffer salts, acids or modifiers. Please use caution in working with chlorinated solvents, as PEEK material may swell slightly. Contact Technical Services if you have any questions about this procedure. If backflushing does not reduce backpressure, then particulates or active compounds most likely have bound irreversibly to the column and it needs to be replaced. Likewise, if following these steps and flushing in a forward direction for your 1.9 um UHPLC column or Raptor column does not help eliminate the problem, then your column has probably exceeded its lifetime an needs to be replaced.

D.       If you have checked the system pressure without the column in line (see #3 above) and the pressure is higher than it was previously without the column in line, there are blockages or restrictions in the system. Please consult your HPLC Operation manual or contact the HPLC manufacturer.

 

 

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2 Responses to “Building up pressure on HPLC?”

  1. Snigdha Tiwari says:

    hell sir/ mam,
    i am constantly facing a problem of unstable and high pressure during hplc run. i had reverse the column also wash the pump with methanol, wash the column with methanol( hplc). but there is no stability in pressure is seen. plz do me suggest the solution for this. waiting fr ur reply asap

  2. Hello Snigdha,
    I am sorry to hear of the troubles. Have you checked the system pressure without the column in line? This is described in #3 of the blog post. If so, what was the pressure when you did that?

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