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What is the Expiry Date for HPLC Mobile Phase?

We use a wide variety of mobile phases and columns, but nothing out of the ordinary. In this article, we will discuss what an expiry date for prepared mobile phases usually has in the industry?

There are no reliable universal guidelines for the expiry date for HPLC Mobile Phase, as each solution is different and even mobile phases with 20% organic solvent content can still grow bugs in them over time. Only pure solvents which are kept in sealed bottles are generally safe for long-term storage. The moment we add acids, bases, buffers, or any additives to the solution, the stability of the solution is now a variable (e.g. pH, bacterial or alga content, particulate matter). 

Some buffered or aqueous-containing mobile phases will at most be good for one week at refrigerated temperatures (e.g. simple phosphate buffers), but some will still grow bugs in them during this time and be unsuitable for HPLC work. We really need to make up fresh solutions each day. Don't risk contaminating the entire instrument and column using questionable solutions. Also, please Do Not "Top-up" solutions in bottles (a very bad habit, which results in contamination). Dump out the contents to waste, clean the bottle, then refill with fresh solution.

Even "pure" organic solvent solutions (e.g. Methanol) containing just an acid can change over just a few days, so must be made fresh too.

It's good to change the buffered solution every day while running samples for 2 days at the max. There is a risk of microbial growth, as this may affect the chromatogram. As for the mobile phase/organic solvent, try to replace it every week or so as the organic solvent is much more resistant to microbial growth. Making a new buffer/mobile phase is much easier than buying a new column! 

The expiry is usually decided during method development. If we use high-quality water and have very little organic present in our mobile phases, their expiry is likely to be related to wildlife growing, and/or absorption of CO2 and other gases - which may affect unbuffered systems and/or UV characteristics.

If lots of organic are presents (10%+) then most mobile phases will last weeks to months - provided they are sealed and stored OK (eg. to prevent loss of volatiles). Some organics (eg. ethers, acids) can degrade or react, as will some buffers and should be replaced more frequently. 

Regardless of the lab rules, an auditor would want to see how you established the expiry date for all novel mobile phases. The easy solution is to retain some old mobile phases during development/qualification and analyze samples with them and fresh mobile phases after several intervals. We may be surprised how long we can use many mobile phases.

HPLC grades of some ion-pair reagents are fairly expensive, so making up just sufficient for your daily needs is a good idea, as work schedules can change. 

Mobile Phase may not pose a big problem in the case of single peak analysis, but when you are using it for related substances analysis, wherein a number of peaks (probably some of them are pH-sensitive) are more, it is not practically good to use those mobile phases which has been stored for months together.

There is no industrial ‘norm’ for the expiration date assigned to the HPLC mobile phase because the composition of the mobile phase varies so greatly. In general, preparing a fresh mobile phase is best and if possible, it is a good idea to prepare as much as you are likely to need for a particular chromatographic run. Since it is wasteful (in terms of both cost and environment) to dispose of solvents when it is not really necessary, I would make the following recommendations.
  • Buffers are most likely to cause problems in storage due to bacterial and fungal growth but all buffers do not behave in the same way. An example of a buffer in which growth occurs very soon after preparation is a phosphate buffer at pH 7. You should assess how long a buffer should be kept for your particular method. If the buffer (or water) is not mixed with organic solvent (for example when you use the HPLC system to mix the mobile phase for you) then it will probably not last as long. Assessing the buffer for a suitable expiry date consists of checking for visual growth (dispose of the buffer if you can see any), and checking the chromatographic performance.
  • The easiest way to check chromatographic performance is to use the system suitability test for the method, this includes any standard checks. If the system's suitability is well designed, it should be able to confirm that a particular mobile phase is giving the expected results and is suitable for use. You may find that methods, where the pH of the buffer is close to the pKa of an analyte, will be more affected by small pH changes due to buffer degradation.
  • Typically, for blends of aqueous buffers or water with organic solvents, where the aqueous content is less than 20%, an expiry date of 1 month after preparation is suitable and it is possible that you could extend this to 6 months. For blends where the aqueous content is greater than 20%, assess visually and check chromatographic performance.
  • For mobile phases which do not contain buffers and consist of blends of organic solvents, you can easily set an expiry date of 6 months, but you may need to watch out for changes in the relative amounts of solvents due to evaporation. My personal preference is to use the HPLC system to blend solvents in this situation thus a single solvent is in each mobile phase reservoir and changes in the composition cannot occur.

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  1. Adding 0.01% sodium azide towards buffers works aswell. Ensures no growth for 1 week (tested via dynamic light scattering and multi angle light scattering).

  2. Thank you for your great advice. I like your chromatographic knowledge blog