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Operation of HPLC system


Solvent delivery system

Put mobile phase reservoirs on the top of the HPLC system or above the level of the pump head.

Purge the lines required for the test with about 5 system volume of mobile phase or other solvents if necessary at 3-5 mL/min (open the
purge valve first, set up the purge flow rate to ensure the mobile phase or solvent directly flow to waste through the purge valve), then connect the tubes with a union, set up the purge flow rate at 2-3 mL/min, close the purge valve and purge the whole system for at least 5 minutes. Purge all 4 line with mobile phase when low UV wavelength is used for detection, i.e. less than 220 nm.

Carefully check any air bubbles trapped in the pump head that is revealed by pressure fluctuation. Remove any air bubbles trapped in the
pump head or any air pockets, which is easily introduced into the system through dropping or swirling the inlet filter as it is transferred between
reservoirs, by purging solvent through the pump head and out the purge valve at high flow rate, e.g. 5mL/min, or priming the system if necessary.

Install the required column. Set flow rate to the required rate (slowly increase the flow rate to the required flow rate to avoid rapid backpressure increase which could damage the column) and allow the column to equilibrate. Approximately ten column volumes of solvent are usually sufficient.

Visually check the column and injector loop for leaks.

Observe the backpressure. The operation pressure for a definite method should be consistent, and this pressure can be used as a reference to
determine if HPLC system parameter setup or mobile phase preparation error exists. The significant changes (>20%) on the system backpressure may result from different system components (column length, diameter or particle size), mobile phase composition, flow rate, or particulate build-up within the system. The maximum operation pressure for regular HPLC system is 400 bar (6000 psi).

Check baseline stable or not by injecting a diluent or running the method without any injection.

Column installation

Protect the column from mechanical shock. Dropping a column can impair its performance.

Check the characteristics of the column, the compatibility of the solvents in the system and in the column and the column itself. Remove
both end plugs from the column and put the end plugs in safe place for re-capping after using, meanwhile visually inspect the column ends to make sure no evidence of previous leaks.

Care should be taken not to pass any mobile phase through the column that might cause a precipitation to occur (i.e. buffered solutions should not be passed through immediately after organics).

Set the pump to a low flow rate (0.1mL/min to 0.2 mL/min), and connect the column head to the tubing while the mobile phase passing
through, The direction of mobile phase flow in the column should match with the arrow marked on the column. Always ensure that connections between injector, column and detector be kept as short as possible, preferably using tubing with an internal diameter of 0.01”
(0.25mm) or less, to reduce the dead volume. For optimum performance, the tubing connecting the column to injection and detector must abut the internal shoulder of the fitting. The use of PEEK fingertight fitting is recommended for such connections.

 Make sure that the seat of the ferrule on the tubing is completely compatible with the column fitting. Position a waste beaker under
the exit of the column and make sure flow commences shortly after the column is attached. Once flow has been established, check the system backpressure and slowly increase the flow rate. Flush the column with 5 to 10 column volumes of mobile phase (estimate as about 1mL per centimetre length for a4.6mm diameter column).

Reduce the flow rate to 0.1 ~ 0.2 mL/min and connect the column to the detector. Once the connection is leak-free, slowly increase the
flow rate to the required flow rate. This procedure minimizes the chances for the introduction of air bubbles, particulates and other contaminants into the detector cell. (Note: if any air bubble is trapped in the pump head, then a big pressure fluctuation may be observed, i.e. pressure fluctuation is more that 5 bar, and the long noise can be observed on the baseline. Purge the pump head at a high flow rate, e.g. 5mL/min, to remove the air bubble in the pump head. If short noise on the baseline is observed, probably some air bubbles are trapped in the detector cell or elsewhere in system, purge the system with mobile phase or with 100% methanol or IPA).

Ensure that the column is properly installed in the column temperature controller if the column is required temperature controlled. Column
temperature controller should be covered well to keep constant temperature. Maximum operating temperature for most columns is60°C.

Ensure that the column is properly equilibrated for an analysis. This is best done by running a few standard injections and observing
any changes in retention times and/or peak area counts. If stable results are obtained, the column is ready for use.

At the conclusion of every test, the column should be rinsed by using 50% MeOH for RP column and 100% IPA for NP column (other washes may be appropriate instead of 50%MeOH such as 50% ACN or 20%MeOH if the buffer conc. is high) and store RP column in ACN, NP column in hexane respectively, unless otherwise specified in the method. Also, the HPLC system should be cleaned after using, and ensure it be in ready to use condition for next analysis.

 Some columns may have particular storage requirements. Always refer to manufacturers’ recommendations. HPLC columns must not be
allowed to dry out during storage. Always cap column ends when not in use and place column in the column storage place which is assigned for a dedicated project.

 Use of the autosampler

Fill sample solution into HPLC vials to the same volume as possible, i.e. about 1.5ml, do not fill too much or too little volume of sample
solution into the HPLC vials. Over filling vials can create a vacuum in the head of the vial and prevent full injection volume.

One vial for multiple injections is acceptable if the sample solution volume in the vial is enough for all injections unless otherwise
specified in the method. A new vial of working standard solution for system drift is recommended for a long sequence run, i.e. over 1 or 2 days sequencerun.

An autosampler with amber cover should be used for light sensitive analytical solutions. If an amber cover autosampler is not available,
the regular autosampler should be covered with aluminium film to protect light sensitive samples. Meanwhile sample solution should be kept in amber HPLC vials. Generally specific requirement for sample preparation or storage conditions should be described in the method.

If the method requires a needle wash, place the HPLC vial for needle wash in correct position in the autosampler tray.

Set up the chiller temperature as method required and allow equilibrate.

Before running samples, run autosampler needle/syringe wash cycles or purge, if applicable.

Use of detectors

The most commonly used detectors are VWD (Variable Wavelength Detector, with High sensitivity, low detection limits and  low baseline noise and drift) and DAD detectors (Diode Array Detector, Full spectral detection for compound identification by spectral libraries or verification of the separation quality with peak purity analysis for conventional and ultrafast LC and simultaneous detection of up to 5 signals).

Turn on the detector lamp and allow the lamp to warm up for at least 30 minutes prior to starting injection. Set the wavelength(s) required
for the test.

Detector lamps have a limited life and should not be left lit unnecessarily. At the end of a test the lamp should be switched off.

System shut down and cleanup

At the conclusion of every test, solvent lines, the pump, detectors and the column must be rinsed using appropriate solvents as following
procedures.

First, wash the column with appropriate solvents and store the column in appropriate solvent as method required, i.e. C18 column in 100%
acetonitrile, silica column in hexane etc. Re-cap the column and put it back to the original package for prevention of any impact.

Set the flow rate to 1.0mL/min, then close the purge valve and pump at least 20mL of 50% methanol through the system with a union
connected the tubes to flush the flow path.

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