How long should I prime an HPLC pump?

It depends on the system and how long it stood. On a Waters ACQUITY: 1 minute after 4 or more hours idle with the same solvents, 4 minutes with fresh bottles of the same composition, 5 minutes if the composition changed, and typically 7 to 10 minutes for a dry pump, until the vent line runs without bubbles.

What is the difference between priming and purging an HPLC pump?

None in practice. Waters calls it priming; Agilent, Thermo and Shimadzu call it purging. Both run fresh solvent through the pump to waste, bypassing the column, to fill the pump, change solvent or remove air. When you open a purge or drain valve by hand, close it again afterwards.

What should the HPLC seal wash solvent be?

Waters recommends about 10% organic, for example 1:9 methanol and water. Don't use 100% organic or a nonvolatile buffer, don't recycle it, and don't let it run dry.

How long should I equilibrate an HPLC column?

At least 10 column volumes, until pressure and baseline are steady. A 2.1 x 50 mm column holds about 0.2 mL, so 10 volumes is about 2 mL. Ion-pairing mobile phases can need 100 to 200 column volumes.

Why is my first injection of the day different?

Common causes are air in the lines, strong wash solvent left in the needle, or a column that isn't fully equilibrated. Check the priming, check that the autosampler prime finished, and give the column more time.

HPLC startup in the morning: prime, wash, then equilibrate

By Dr. Nazmul Alam, analytical chemist · October 3, 2026 · Updated October 4, 2026

The shutdown guide covers the end of the day. This is the other end.

Three things can spoil the first injection of the day: air in the lines, the wrong solvent still sitting in the needle, or a column that isn’t equilibrated yet. A good startup deals with all three before the first sample.

flowchart LR
    A["Check<br/>bottles"] --> B["Prime or purge<br/>the pump"]
    B --> C["Seal<br/>wash"]
    C --> D["Prime the<br/>autosampler"]
    D --> E["Flow, then<br/>the lamp"]
    E --> F["Equilibrate<br/>the column"]
    F --> G["Blank, then<br/>standard"]

The order works on any HPLC. The times and volumes below are examples from the Waters ACQUITY operator’s guide, so use your own system’s manual for its numbers.

Check the bottles first

Solvent bottles, seal wash bottle, wash solvents, and the waste container. Priming uses solvent fast. On an ACQUITY the pump primes at 4 mL/min per solvent, so 5 minutes on both A and B uses about 20 mL of each.

Prime (or purge) the pump

How long depends on how long the system stood and whether the solvent changed. The ACQUITY guide gives a good example:

Situation	Prime
Idle 4 hours or more, same solvents	1 minute
Fresh bottles, same composition	4 minutes
New solvent composition	5 minutes
Pump completely dry	Typically 7 to 10 minutes, until the vent line runs without bubbles

Waters also says to prime after you change the sample needle or loop.

Agilent’s pump manual gives its own power-up routine: purge the pump heads at 2.5 to 3 mL/min for 5 minutes, then let the system stabilize for 10 to 20 minutes.

Degassing matters as much as priming. Thermo’s troubleshooting guide names poorly primed lines with badly degassed solvents as the most common cause of fluctuating pressure. Agilent’s LC Handbook says most systems now have a degasser built in, but if it’s bypassed, missing or not working, degas the solvent another way, for example by sparging with helium.

Buffer in the lines? Water in between

If the lines hold a buffer and you’re moving to a high-organic solvent, put water through first. Salt can come out of solution when it meets a lot of organic solvent, and it can land inside the pump.

It’s the same rule as at shutdown, in reverse.

Prime or purge? Same job, different names

Waters says prime. Agilent, Thermo and Shimadzu say purge. It’s the same job: run fresh, bubble-free solvent through the pump and out to waste, without going through the column. You do it to fill the pump, to change solvent, and to get air bubbles out.

Agilent’s own manual uses both words. It says the purge valve is there “for convenient priming” of the pump head.

What changes is the valve and how you open it:

Vendor	Word	How the flow goes to waste	Example from the manual
Waters (ACQUITY)	Prime	The vent valve switches to vent automatically	4 mL/min per solvent; a syringe helps prime a dry pump
Agilent (1260 Infinity II)	Purge	Open the purge valve at the pump outlet by hand	2.5 to 3 mL/min for 5 minutes at power-up
Thermo (Vanquish Core and Flex)	Purge	Automatic, through the autosampler wash port, or open the purge valve knob by hand	5 mL/min for 5 minutes as standard; a syringe for empty lines
Shimadzu (LC-40D)	Purge	Open the drain valve 90°, then press PURGE	Stops after 3 minutes by default, settable from 1 to 20

Thermo’s manual adds two useful points: trapped air causes pressure pulsation, noise and non-reproducible results, and you should purge every channel, including the ones your method doesn’t use.

When the valve is opened by hand, as on Agilent, Shimadzu and Thermo’s manual purge, close it again before you start the method. Agilent’s troubleshooting tables list an open purge valve as the first cause for a failed pressure test and for some pump delivery errors.

Prime the seal wash

The seal wash is the small flow of solvent behind the plunger seals. It keeps the plungers wet and washes away salt that gets pulled past the seal.

Waters says to prime it after a buffered mobile phase, after the pump has stood for a few hours, and if it ran dry. Their rules for the seal wash solvent:

About 10% organic, for example 1:9 methanol and water. The organic stops microbes growing and helps dissolve the mobile phase.
Not 100% organic, and not a nonvolatile buffer.
Don’t recycle it, and don’t let it run dry.

Agilent’s rules are close: don’t recycle the seal wash, and change it every week. Agilent uses 10% isopropanol in water instead of methanol.

Check the seal wash bottle when you check the solvent bottles.

Prime the autosampler

The autosampler has its own syringes and wash solvents, and they need priming too.

When you change wash solvents, Waters recommends 5 to 7 prime cycles. Each takes about 2 to 4 minutes.
Don’t stop the prime halfway. Waters warns that it can leave strong solvent in the needle, and that ends up in your first injection.

Waters calls priming all the syringes together good practice, not only the sample syringe.

Start the flow, then the lamp

The detector cell should be full of degassed, clear solvent before the lamp turns on. Waters says gas in the solvent can form bubbles in the cell and make the detector fail its startup tests. So start the flow first, then the lamp. More on the cell in cleaning a detector flow cell.

Equilibrate the column

Pump the starting mobile phase through the column until the pressure and the baseline are both steady.

Waters asks for at least 10 column volumes, and Agilent’s handbook uses 10 as its rule of thumb for method development. On a 2.1 × 50 mm column that’s about 2 mL.
Ion-pairing mobile phases can need 100 to 200 column volumes, Waters says.
Agilent adds that the equilibration volume includes the system’s dwell volume, which differs from system to system. So a time that works on one system may be too short on another.

The HPLC vs UHPLC guide has the column volumes for common sizes.

Before the first sample

Run a blank, then your standard. Thermo’s guide says to judge the system on a standard, not a sample, and to keep its history. If today’s standard looks like yesterday’s, you’re ready.

If it doesn’t, start with the startup. Was the priming long enough? Is the degasser working? Was the seal wash dry? Did the column get enough time?

Sources

Waters ACQUITY UPLC System Operator’s Guide, 71500082502 rev F: priming times and flow rate, the vent valve, seal wash, autosampler priming, starting the detector.
Agilent InfinityLab LC Series 1260 Infinity II Isocratic and Quaternary Pumps User Manual, G7111-90000: the purge valve and purge function, the power-up routine, seal wash (pp. 19, 53-54), the open purge valve in the troubleshooting table.
Thermo Scientific Vanquish Pumps C, Pumps F Operating Manual, 4820.4405 rev 6.0: purging, automatic and manual (pp. 104-109).
Shimadzu LC-40D X3 CL / XR CL Instruction Manual, 228-97197C: the drain valve and the PURGE key (pp. 19-20, 35).
Waters ACQUITY and ACQUITY Premier Columns Care and Use Manual, 720008980: 10 and 100 to 200 column volumes.
Agilent, The LC Handbook: equilibration and dwell volume (p. 57), degassing (p. 71).
Thermo Scientific HPLC troubleshooting guide: priming and degassing as the most common cause of fluctuating pressure (p. 9); judging the system on a standard (p. 14).

Checked 4 October 2026.