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?

The determination of an expiry date for prepared mobile phases in the HPLC industry is not governed by universal guidelines due to the diverse nature of each solution. Even mobile phases containing as little as 20% organic solvent content can be susceptible to microbial contamination over time. Only sealed bottles of pure solvents are generally considered safe for long-term storage. However, once acids, bases, buffers, or additives are introduced into the solution, the stability of the mobile phase becomes variable, including factors such as pH, microbial growth, and particulate matter.

Certain buffered or aqueous mobile phases may remain viable for up to one week when refrigerated, such as simple phosphate buffers. However, even within this timeframe, some solutions may still be prone to microbial growth, rendering them unsuitable for HPLC analysis. 

Therefore, it is advisable to prepare fresh solutions on a daily basis to avoid the risk of contaminating the instrument and column. It is crucial to refrain from "topping up" solutions in bottles, as this practice can lead to contamination. Instead, it is recommended to discard the contents, clean the bottle thoroughly, and refill it with fresh solution.

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Even so-called "pure" organic solvent solutions, such as methanol containing only an acid, can undergo changes within a few days, necessitating their frequent preparation. Buffered solutions should ideally be changed daily when running samples, with a maximum duration of two days. This precaution is taken due to the potential for microbial growth, which can adversely affect the chromatogram. In the case of mobile phases with organic solvents, it is generally advisable to replace them every week to mitigate the risk of microbial growth. The ease of preparing a new buffer or mobile phase outweighs the cost of purchasing a new column if contamination occurs.

The determination of an expiry date for mobile phases is typically established during method development. Mobile phases with high-quality water and minimal organic content are likely to be affected by factors such as microbial growth and gas absorption, which can impact unbuffered systems and UV characteristics.

For mobile phases containing significant amounts of organic solvents (10%+), most solutions can last for weeks to months if properly sealed and stored to prevent volatile losses. However, certain organics (e.g., ethers, acids) and buffers may degrade or react, necessitating more frequent replacement. Regardless of laboratory rules, auditors would expect to see documented evidence of how the expiry date for novel mobile phases was determined. One practical approach is to retain old mobile phases during development and qualification, periodically analyzing samples with both the old and fresh mobile phases to assess their performance over time.

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It is recommended to prepare just enough HPLC grade ion-pair reagents for daily needs, considering that work schedules can change unexpectedly.

While mobile phase expiration may not pose significant problems for single peak analysis, it becomes impractical for related substances analysis, particularly when multiple peaks (some pH-sensitive) are involved. Mobile phases stored for prolonged periods should be avoided in such cases.

Due to the vast variations in mobile phase composition, there is no standard industry norm for assigning expiry dates. In general, it is best to prepare fresh mobile phases whenever possible, making only the required amount for a specific chromatographic run to minimize wastage of solvents. Assessing the expiry date of buffers depends on factors like bacterial and fungal growth, which can vary depending on the specific buffer and its pH. Visual inspection for growth and evaluating chromatographic performance are essential in determining a suitable expiry date.

An effective method to evaluate chromatographic performance is by conducting system suitability tests, including standard checks. If the system suitability test is well-designed, it should confirm whether a particular mobile phase yields the expected results and is suitable for use. Methods where the buffer's pH is close to the analyte's pKa may be more sensitive to slight pH changes resulting from buffer degradation.

For blends of aqueous buffers or water with organic solvents containing less than 20% aqueous content, an expiry date of one month after preparation is generally acceptable, with the possibility of extending it to six months depending on visual inspection and chromatographic performance. 

Mobile phases without buffers, consisting solely of organic solvent blends, can typically be assigned an expiry date of six months. However, it is crucial to monitor the relative amounts of solvents for any changes caused by evaporation. In such cases, employing an HPLC system to blend solvents, with each reservoir containing a single solvent, helps prevent unintended changes in composition.

In summary, the determination of expiry dates for prepared mobile phases in HPLC relies on factors such as solution composition, potential microbial growth, and chromatographic performance. While there are no universal guidelines, it is generally recommended to prepare fresh mobile phases, assess buffers for visual growth and chromatographic performance, and employ system suitability tests to ensure the expected results are obtained. Regular monitoring and proper storage practices are essential to maintain the integrity and reliability of mobile phases in HPLC analysis.

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