Guidelines for using Aqueous Buffer Solutions/Mobile Phases in Triple Detector SEC systems

This note gives guidelines for preparation for aqueous samples, system cleaning and maintenance and system storage


For any aqueous solution, it is good practice to filter with a 0.2 micron Nylon filter prior to use in the SEC system. This removes any undissolved material in the mobile phase and it also helps to remove dissolved gas. In order to keep pumps working efficiently, it is strongly recommend to use an in-line degasser. Since most aqueous buffer solutions are in the pH range of 5 to 7, these solutions will be prone to microbiological growth. Therefore, buffer solutions should NOT be kept for more than one week and the mobile phase should not be recycled. Buffer containers should also be cleaned to prevent contamination. To prevent microbes growing in your SEC system, and if it is compatible with the chromatography, it is advisable to poison the mobile phase using for example some sodium azide (0.02% w/v) or alcohol (such as methanol) or acetone/acetonitrile in the buffer solutions.

One additional problem that can occur is the use of water from a contaminated source. In general, purification systems are good as long as proper maintenance is performed on a regular basis. Before using water from these systems, find out if the maintenance has been done. Look for labels or other indications that filters have been changed. Ask how old the main water supply and pipes are. Old age can indicate the potential for general contamination. If you suspect the water, use HPLC grade bottled water. Do not use an open bottle. Start with a new, unopened bottle. Do not rely on an HPLC analysis to indicate the purity of the water.

Buffer Salts

Although the detector is built to be resistant towards most salts and solvents some care is needed when running buffers that contain halides. There is no general reason for not using a buffer such as PBS (phosphate buffered saline, pH around 7). However, due to the presence of chloride ions the buffer is still able to corrode stainless steel if the instrument is not treated well. The reason for halides being aggressive are the high local concentrations that develop when, for some reason, parts of the instrument start to dry out. High concentration halide solutions can extract iron ions from the steel as they form strong complexes together. If proper care is taken of the instrument then you will not experience any difficulties.

If it is possible, a halide-free buffer, e.g. 50 mmol/L phosphate at pH 7 and 0.1 mol/L sodium nitrate or sodium acetate is preferred.

It is recommended that the flow is always left on at a minimum of 0.2 ml/min to waste (no recycling as bacteria will develop and concentrate over time in the reservoir bottle). Do not forget to purge the RI and DP occasionally. When not in use, flush the system with pure water until all salt is removed, then flush the system with a mix of water: methanol or water: acetone 90:10. If non-aqueous solvents cannot be used, water with 0.02% w/v sodium azide is the best solution for preventing contamination and bacterial build-up. Remember to purge the DP and RI to remove the salt solution from the detectors also.

Washing/Cleaning the Instrument's Flow Path

Generally, as long as a flow is maintained in the system, cleaning of the detectors should not be necessary. However, depending on the number and type of samples run and the condition of the column, it is possible that the detectors will become dirty. An indication of this is an increase in noise and background in the light scattering detectors e.g. a doubling of background from 50 to 100 mV. You may also observe a change in the DP level of the viscometer e.g. either an increase or decrease in the overall background for example from 90 to -300 mV. In this case it is usually possible to wash out the detectors by using a solution such as water/methanol 90/10. Most aqueous buffers are miscible with this mixture without precipitation of buffer salts. However it is very important to check the concentration and type of buffer beforehand. An alternative mobile phase for cleaning the system is water/acetone 90/10. Before running this cleaning procedure, remove the analytical SEC column and wash it separately according to the manufacturer's instructions. It is good practice never to wash the column while it is connected to a detector.

Do not forget that any filters in the chromatography system will also need to be replaced regularly to avoid problems. They should certainly be replaced immediately if any contamination occurs and before and after cleaning the system. This includes the dip filter in the buffer, post-pump filter and the post-column filter.

Note that water/methanol is different to the traditional water/ethanol mixtures because the viscosity is much higher with ethanol compared to methanol at the same concentration ratio. Thus, solvent exchange with water/ethanol might take a longer time and requires more care. In addition, the methanol/water mixture is better in keeping the cell windows for the light scattering detector clean.

Instrument Storage

If the system is not going to be used for more than a week or if the system is to be shut off and stored, then use the following procedure: First, remove the column and store according to the manufacturers instructions. Second, flush the system out with plain water at a flow rate of 1.0 mL/min for 30 minutes making sure the RI and DP are purged at least once during the process. Third, use one of the cleaning phases mentioned above, but importantly note that you may need to reduce the flow rate to ensure the viscometer stays on scale. Flush the detector with at least 50 mL.

Once complete, the system can be shut down. To re-start the system, follow the procedure in reverse order. Note that if sodium azide solution is used as a storage medium, it will only be effective for a short time.

Additional Cleaning Procedures

More rigorous cleaning is only advised when the contaminated system is not cleaned effectively by the traditional 'wait for infinite dilution' or water/methanol solvents. Other washing procedures can include cleaning with acids and organic solvents, but they need special instructions on their use we would strongly advise you to contact Malvern-Viscotek service to discuss this before attempting. We will then advise and remind you of the risks! So if there is a problem, call for advice and if cleaning is the correct action, we will explain how it should be done

Sample preparation

Sample solutions should be clear (not cloudy or turbid). We recommend filtering the sample solutions to 0.45 µm or 0.2 µm prior to injection. If you notice that the baselines change or if the detectors become noisy, this may be due to eluting sample.

Let the detector signal return to the baseline with the flow on (infinite dilution principle) before injecting further samples; if the baseline does not return to its expected value, it may be necessary to clean the system (see previous sections). It is then necessary to understand why this sample has caused a problem and correct for it. If analyzing this type of sample is a regular requirement, you may need to clean the system on a regular basis.

Additional Information

This cannot be a fully comprehensive guide describing every possibility you might experience during use. For further help, please do not hesitate contacting the Malvern helpdesk for support.


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