HPLC Tips & Tricks: Proper Storage of HPLC/UHPLC Columns

Dr. Egidijus Machtejevas, , Lead Expert, Chromatography Product & Portfolio Management

Merck

Article from Analytix Reporter - Issue 8

The concept may seem simple. Once the last chromatogram of the day or the project is finished, we disconnect the column and put it into the drawer. However, what exactly should be done with the column before storing it? Does the procedure vary depending on the planned storage time? There is actually quite a lot to: planned storage time, column modification (stationary phase), buffer concentration, pH, etc. In all the column storage scenarios, special care must be taken if buffers, which provide a microbe friendly environment, are used. In such cases, fresh buffers are to be prepared daily and filtered using 0.45 or 0.22 μm membrane filters. Also adding a small amount of organic solvent (~ 10%) or adding sodium azide (~ 0.05%) in the storage solvent - if buffers are used for storing e.g. needed for some HILIC columns - can be sufficient to prevent microbial growth. The easiest and safest way to store the column, however, is by using the same solvent in which it was delivered to you.

This applies in particular to the polymer-based stationary phases. Depending on their material properties, these might not be compatible with some organic solvents.

For silica-based normal phase columns, it is typically recommended that heptane or isopropanol are used. I personally have also had good experience with dioxane, as it nicely removes residual water, but this cannot be generalized. Some stationary phases such as aminopropyl- or diol-modified stationary phases might be effectively stored in 2-propanol, which is in fact, compatible with both Reversed Phase and Normal Phase modes. Size exclusion columns should be stored in a solvent compatible with the swelling properties of the packing.

Column storage may be short, middle, and long term

For short term storage, i.e., overnight, either the mobile phase used in the last analysis can remain in the column, or it is possible that the mobile phase passes at a very low flow rate (especially if the buffer concentration in the mobile phase is high, >50 mM). In these cases, column conditioning can potentially be skipped before continuing the analysis the next day. This option is particularly recommended for normal phase separations, where change in mobile phase composition can result in lengthy re-equilibration. However, if the buffer concentration in the mobile phase is very high (>0.5 M), then the lifetime of the pump parts (e.g. injector & switching valves) could depend on the length of time they are in contact with high concentration buffer. The same is true for the column if the pH is close to the limit of the column (for most silica-based columns - pH 2 to pH 7). Some salts, such as chloride salts used in ion chromatography in particular, are very corrosive to stainless steel and might attack the column wall as well as the inlet-outlet frits. In such cases, column (and all system) should be flushed with a less harsh mobile phase. In this case, I would recommend rinsing the column with a water-rich mobile phase (~90%) with about 10 column volumes (the approximate column volumes for some popular dimensions are listed in Table 1).

Table 1. Approximate column volumes for some popular column dimensions and their multiples

Length (mm)	ID (mm)	Approximate Column Volume (mL)	10 Column Volumes (mL)	15 Column Volumes (mL)
250	4.6	4.15	41.5	62.3
250	2.0	0.79	7.9	11.8
150	4.6	2.49	24.9	37.4
150	2.0	0.47	4.7	7.1
100	4.6	1.66	16.6	24.9
100	2.0	0.31	3.1	4.7
50	4.6	0.83	8.3	12.5
50	2.0	0.16	1.6	2.4
25	4.6	0.42	4.2	6.2
25	2.0	0.08	0.8	1.2
Note: The volume of the recommended mobile phase as indicated in the table must actually pass the column. Be aware that, if you exchange the solvent bottle and remove tubing from one solvent and place it into another container, you have to consider the volume of the tubing (~2-3 mL), degasser (older degassers could be up to 15 mL, newer ~ 4 mL), pump (~1 mL), and injector until the new solvent reaches the column. Depending on the flow rate, additional time has to be added for the rinsing procedure.

If you disconnect a column from the instrument, end plugs should be tightly fitted to prevent solvent evaporation, otherwise a drying of the stationary phase could happen. The worst-case scenario is an improperly washed column previously used with a high salt concentration and allowed to dry over time, resulting in the formation of salt crystals. The column most likely will be irreversibly damaged. However, it might be permissible for some columns to be stored dry, others should not. Please check the manufacturer’s column care guidelines. Standard HPLC columns should only be stored at room temperature and never in a freezer (exceptions are protein modified affinity or active enzyme reactor columns). This paragraph’s recommendations are also valid for mid- and long-term column storage.

Medium interval storage, i.e., 2 days or over the weekend. Columns should be flushed. Flush intensity or volume depends on the buffer concentration used during analysis. It is generally advisable to first flush buffering agents off the column with about 10 column volumes of mobile phase with 10% organic solvent in the water. In this case, washing will be effective, and we would also avoid buffer precipitation and possible column dewetting problems. When the buffer is washed out, pump 100% organic for 15 column volumes. The column could then be left connected to the instrument or disconnected and closed with end plugs. Please consider short term column storage advice too, such as referencing column documentation for recommended storage solvent.

Storing a HILIC column in an acetonitrile–water mixture may take a long time to re-equilibrate if a low ionic strength buffer such as 5 mM ammonium acetate is used for the analytical method. Therefore, for HILIC columns, it is recommended that they are stored in solvents containing 80–90% acetonitrile and buffers containing 5–10 mM ammonium acetate or ammonium formate. But for some HILIC phases this may differ, please check the column product information.

Ion-exchange and mixed-mode phases containing carboxylic acid functional groups (for example, weak cation-exchange phases) cannot be stored in solutions containing alcohols, because of a possible slow esterification and the resulting change in selectivity/capacity.

For long term storage (>2-3 days), silica-based columns, after proper washing with a minimum of 15 column volumes (Table 1) using ~ 10% organic solvent in water, should then be flushed with an organic-rich mobile phase for a minimum of 10 column volumes and should then be stored in an aprotic solvent. If water is also present, it should not be in higher concentrations (less than 50%). The best storing solvent recommended in the literature is acetonitrile or methanol (some exceptions exist, such as columns with amide modification, which should be stored in acetonitrile only). Some studies¹ also indicate that at RP conditions, rates of erosion and corrosion of the stainless-steel components of the HPLC using pure acetonitrile or methanol were higher compared to when they were mixed with water. Therefore, 90% acetonitrile or methanol are perfect long-term storage agents for most reverse-phase columns.

However, my personal favorite storage solution is a mixture of isopropanol and water (80/20), because of isopropanol’s higher vapor pressure and the reduced chance for column dry-out, even if end fittings are not completely sealed. Isopropanol is also a stronger eluent, therefore, after storing in isopropanol, we can be sure that even more impurities will be removed than with acetonitrile or methanol gradients. Last but not least, isopropanol is also less toxic. It is also important to note that all mobile phases used for flushing, washing, or column storing must be of the same quality grade as the ones used for the analysis. Columns should be stored at room temperature (exceptions include affinity columns, as mentioned before) in their original box, with a copy of the certificate of analysis (CoA)/Column Report, and possibly with the column log book to show previous uses and to help the user evaluate the column prior to future use.

How long can columns be stored? This depends on many factors. Some columns do not change even after 5 or 10 years of storage. If you decide to use a column after such a long period of time, assume that the column most likely has dried out, and needs to be rewetted by first flushing with 100% acetonitrile (RP-phases), and then equilibrated in mobile phase for about 1 hour before making any selectivity measurements. Additionally, consider running a column test mix and compare the data to the CoA or previous column tests.

Correct column storage is essential for proper chromatography and prolonged column life. In addition, always follow the manufacturer’s guidelines for column operation details!

For more information on HPLC method development download the A Practical Guide to HPLC - Method Development Guide or watch our webinar HPLC Method Development in 1.5 Days using the Selectivity Concept

LC-MS workflow tools can be found at SigmaAldrich.com/lcms

References

1. Mowery RA. 1985. The Corrosion of 316 Stainless Steel in Process Liquid Chromatography with Acetonitrile or Methanol Carriers. Journal of Chromatographic Science. 23(1):22-29. https://doi.org/10.1093/chromsci/23.1.22