To answer your question, the readout should show the measured back-pressure of the system (from the outlet head of the Pump to the Column Outlet plus a few insignificant bars for the flow cell and outlet tubing). This is usually referred to as the HPLC "system backpressure". The HPLC column is responsible for most of the pressure generated as it presents the greatest restriction. *There is no "pushing" of sample in an HPLC system.

Please review the basic concepts of HPLC to better understand the component parts involved and how they are used.

Backpressure. You should still have a reading with no sample but with the column installed.

Remember, the smaller the particle size of the packing of the stationary phase the higher the backpressure for the same flow rate and mobile phase composition.

Total Pressure (your meter reading) = Pressure of HPLC (tubing, UV cell, injector…) + Pressure of column

However the major contributor to the pressure reading is the filled column!

To answer your question, the readout should show the measured back-pressure of the system (from the outlet head of the Pump to the Column Outlet plus a few insignificant bars for the flow cell and outlet tubing). This is usually referred to as the HPLC "system backpressure". The HPLC column is responsible for most of the pressure generated as it presents the greatest restriction. *There is no "pushing" of sample in an HPLC system.

Please review the basic concepts of HPLC to better understand the component parts involved and how they are used.

Thanks for the response all. Is generation of back pressure good or bad for the sample binding, elution and for instrument overall?

Good.! The smaller the particle size the higher the retention capacity (see Van Demeter plots). This is the rationale behind UPLC/UHPLC versus ‘old’ HPLC technology. A UPLC column is 50 mm long while HPLC are 500 mm (5 cm) to 25 cm.

However, this means that any HPLC method you see or have must be revalidated for UPLC/UHPLC conditions!

Harleen: Back-pressure has nothing to do with the HPLC separation at all (with one exception for extremely high pressures which result in large amounts of frictional heating). In HPLC, system back-pressure is a result of forcing liquid through a densely packed tube, the "column" (and narrow ID tubing) filled with support only. The support used (media) and the mobile phase are responsible for the separation. At a constant flow rate using the same mobile phase composition, you should observe the same steady back-pressure reading (it is an indication of flow / pressure stability through the support).

Hi William, Thanks for your reply. If I understand correctly, a steady back pressure is good as it is an indicator of flow/pressure stability. However, if the column load is high (or putting too much pressure on the densely packed column), may lead to generation of excess back pressure (or unsteady back pressure) which is not good for the column/sample analysis. Please correct if my understanding is not correct.

At a constant flow rate of static mobile phase, the back pressure should be stable. This is an indication of the column bed stability using that liquid at that flow rate (not the sample "stability"?). Erratic pressure readings (what we refer to as High or Large "% ripple") usually are the result of: lack of /improper mobile phase degassing, poor mixing, flow rate variation, immiscibility, precipitation, turbulence, valve or hardware problems.

"column load" ? What are you referring to? Sample load is the amount of sample injected per volume.

Observed HPLC Backpressure is the result of the flow being forced through the column. HPLC columns are packed using special equipment under extremely high pressures which EXCEED the ratings of most HPLC systems (by a factor of 1.5 or 2.0). This is needed to insure the column bed is stable and will not shift around or move while it is being used. In normal use, you will never exceed a column's pressure maximum. *Some special types of columns contain very delicate supports. They may compress, fracture or be damaged in some other way as to cause voids in the column bed making them unusable. Sometimes we see damage to very tiny HPLC columns (e.g. 2.1 x 30 mm) due to pressure shock. Ramping up pressure in a tiny column, then releasing pressure in a dramatic way will sometimes disrupt the internal support (because the tiny volume inside the columns does not act as a very good shock absorber as it does in larger columns). This is rare. In those cases, the column manufacturer will provide lower than normal suggested max pressure limits for use. These limits are in place to prevent damage to the support during use. Depending on the support used, it may have a suggested pressure maximum of 34, 60, 680, 1000 or 1800 bars. Always review the column supplier's product documentation before use.

Hi William, Thanks for your repky. Could flunctations in the back pressure be because of the improper/ uneven stationary phase in the column which could eventually led to non-equilibrium between stationary and mobile phase And uneven binding of the analyte of interest to the stationary phase? Regards, Harleen

For HPLC: Unless you are flooding the column with high concentrations of material (something which is never done in analytical scale high performance liquid chromatography), sample and sample binding have little to do with pressure. If you have done something to damage the bed of the column (dropped it onto a hard floor or overloaded it with material?), then the pressure may become initially unstable, but should stabilize. Such disruptions to the bed may only lead to peak width broadening and loss of resolution (not pressure fluctuations). These are basic HPLC concepts, critical to understanding when using the technique. Please read my previous responses for more info.

Perhaps you could share with us your real problem or question? It is extremely difficult to help someone who does not yet have a few years of formal HPLC training. When you first start to learn HPLC, it is extremely beneficial to have someone local assist and guide you through the basics. The web is very poor at providing training for this extremely hands-on scientific technique. I see from your profile that you work for a major pharmaceutical company who does employ liquid chromatographers. Surely someone within your company can answer your basic questions and perhaps suggest or provide you with some training options.

Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline) ; https://hplctips.blogspot.com/2014/01/diagnosing-troubleshooting-hplc.html