Column Life-Time :
Q.: My column lasted only for about 100 injections. After that time, the peaks became distorted and the plate-counts were very low. What’s wrong?
A.: 100 injections is indeed a short life-time. Under normal circumstances, one can expect a column to be in service much longer. In order to determine what is wrong, we need to establish first, if short column life is the rule for your application or not.
There are two fundamental cases:
- previously columns used for the same assay lasted much longer.
- all columns used for this application die after about the same amount of use.
In the first case, one would explore if the assay has remained truly constant. Has the sample composition changed? Strongly adsorbed contaminants in your sample can destroy column performance. Are the seals in the fluid path of your instrument in a good condition? Shedding seals can clog column filters and the top layers of the packing and thus effect the distribution of the sample.
If one can be reasonably assured that there are no changes in the chromatographic conditions, one can safely assume that the cause of the problem is a mechanical weakness of the packed bed. This can be induced by rough handling of the column in your lab (did you drop it?) or during shipment, or it could be a manufacturing defect. Such a defect can not be detected by standard column QC and could show up only after some use of the column. In this case, column manufacturers will replace your column free of charge.
Q.: That is nice of these manufacturers, but this is not my problem. My columns always last only a short time. Sometimes it’s 100 injections, sometimes 200. I could live with 200 injections, but only 100 is not good enough. This really is getting expensive. What can I do?
A.: I agree with you 100%. What we need to do together is to find the cause of your problem and then see, what we can do about it.
The most likely cause of your problem is adsorption of sample constituents on the top of the column. They may either precipitate because of a low solubility in the mobile phase or they may be strongly adsorbed. As you inject more and more samples, these contaminants build up on the top of the column and prevent the sample to properly adsorb and distribute. This results in a distortion of the peak profile. Often this problem is accompanied by an increase in back pressure.
Q.: OK, that could be it. How do I get around this problem?
A.: There are several ways to prevent this from happening. One is to clean up the sample with a suitable sample preparation technique. Solid phase extraction using a SPE
cartridge with a similar chemistry as the separation column works well for this problem.
Another and more powerful approach is to use a guard column. The precolumn serves as a sacrificial column top that is replaced when the problem occurs. For best performance, you should use a guard column that contains exactly the same packing as the analytical column and is packed with the same high performance packing technique as the analytical column. If you use precolumns made with a different brand of packing, you will not get the optimal performance both in separation capability and in protection of your analytical column. Also, do not use a larger particle size. Larger particles or badly packed precolumns can result in a deterioration of the separation due to band- broadening in the precolumn.
Q.: To use a guard column sounds ok. Do you have any other solutions?
A.: Well, not really. There are a few other possibilities, but they all have their drawbacks.
I am not an advocate of column “washing” with solvents that are supposed to dissolve the contaminants on the top of the column. In many cases, this process simply does not work. For example, if the contaminants are proteins that have precipitated on the column top, by the time you try to wash them off they have aged a lot by denaturation and maybe even cross-linking that it may be impossible to solubilize them again. Furthermore, every washing will also remove hydrolyzed bonded phase, which otherwise remains in a local equilibrium at the site where the hydrolysis occurred. Consequently, a repetitive washing can actually result in an accelerated aging of the column. Also, after this washing you have to re-equilibrate your column with the mobile phase, which in some cases like in ion-pair chromatography may be quite time consuming.
Another approach that is often tried is column backflushing. If you do it with a different solvent than your mobile phase, the same objections hold as for column washing. If you do it just with mobile phase, it will take a lot of time until the contaminants are removed or it may not work at all. Also, any backflushing weakens the column. Although today’s columns are packed well enough that they can withstand backflushing, I would still recommend to not make this a standard practice.
Q.: So your best recommendation is to use a precolumn?
A.: Absolutely. They don’t cost that much – depending on the brand and type between $ 10 and $ 50 – and they protect a column that usually costs about ten times as much. Furthermore, they protect your column also from other sources of contaminants that may be more difficult to trace. The source of your column problem could for example be dust in the mobile phase or debris from shedding pump seals. Or it could be the adsorption of contaminants from the mobile phase.
troubleshoot, but the guard column will simply take care of them.
Q.: Are there any other causes of short column life?
A.: Yes, but they are less likely if you follow the manufacturer’s recommendations.
One possibility is that the column is collapsing due to a mobile phase pH outside the recommended range. This can also be caused by a sample dissolved in a strongly acidic or alkaline solution.
Furthermore, there are a few items that are specific to certain columns.
Amino columns for example react with aldehydes and ketones. Amino columns in an unbuffered aqueous solution generate a strongly alkaline pH that leads to a partial dissolution of the silica.
Columns can also collapse when exposed to the wrong solvents. The reason for this is that columns are fundamentally very loose structures. They are partially held together by the adhesion of the particles to each other. When you put them into mobile phases that break this adhesion, there is an increased chance of a collapse of the bed. This happens occasionally to CN columns in solvents of intermediate polarity or to Phenyl columns in THF.
Column Conditioning
Q.: What is column conditioning?
A.: In the last troubleshooting column we talked about column equilibration. Column equilibration comprises all
phenomena that are reversible, while column conditioning changes the column irreversibly. By column conditioning you are changing the product that the manufacturer has delivered to you, and the reproducibility of this step is your responsibility. I generally advice against conditioning, but there are some circumstances where column conditioning is unavoidable.
Q.: Please give some examples of column conditioning!
A.: My first example is a commonly used procedure. However, I want to point out that I do not recommend to use this procedure. If you are using a strongly acidic mobile phase, pH 2 or less, with a fully endcapped C18 column, you will fairly rapidly hydrolyze the endcapping groups. Consequently, the column will have a much larger silanol activity than what has been delivered from the manufacturer. This may influence the selectivity of a separation. It is possible that somebody has developed a method using a strongly acidic mobile phase, and by the time that methods development was complete the column had already changed. When a brand-new column is later used for the same assay, the selectivity of the separation may be different. But the separation comes back, when the column is “conditioned” for a day or two with the acidic mobile phase. As you can see, this is a permanent change of the column outside the manufacturer’s specifications. I recommend against such a procedure, but it is practiced in some labs, and detailed column conditioning protocols have been set up for new columns. Instead of changing the properties of a column with such a conditioning process, I recommend to explore the possibility to redevelop the method on a non-endcapped packing. This avoids such a conditioning step in the future.
Another example of column conditioning occurs when aminopropyl bonded phases are used in aqueous solvents. The most common application is the use of this column for the separation of carbohydrates by hydrophilic interaction chromatography. Typical mobile phases are water/acetonitrile mixtures with 60 to 90% acetonitrile. When an aminopropyl column is exposed for the first time to an aqueous eluent, the high concentration of the amino groups in the pores of the packing creates a basic pH, which results in a slow hydrolysis of the silica and the bonded phase. The amount of bonded phase that is washed off decreases exponentially with time, and soon nearly stable retention times are achieved. However, the column has changed significantly from its original properties and should not be used for normal phase separations any more without specifying the exposure to the aqueous eluents as part of the column history.
If you want to use aminopropyl bonded phases for the separation of carbohydrates, there is just no way around this conditioning problem. Some manufacturers offer columns that are dedicated for this application. In this case, the manufacturer has performed the conditioning step for you. Since this is performed with a fixed protocol to fixed specifications, you are better off to purchase a preconditioned column instead of doing the conditioning yourself.
Another unpleasant, but apparently unavoidable conditioning phenomenon happens with columns used in the separation of proteins, very specifically with diol bonded phases used for the aqueous size-exclusion chromatography of proteins. It has been observed that for some proteins the initial injections give smaller peaks than later injections. This has been attributed to non-specific binding of protein to adsorptive sites on the packing. To avoid this, it has been proposed to inject first a large amount of a protein, for example bovine serum albumin, to condition the column and saturate the active sites. In general, I do not recommend this conditioning procedure and suggest that you should observe for yourself, whether or not your sample exhibits such a phenomenon. When you observe an increase in peak height with subsequent injections, you should make sure that this is not caused by carry-over in the injector.
The last example that I want to discuss does not fit my definition of conditioning, but is considered a conditioning step by many people. The example involves dry reversed phase columns. It is possible that a column has dried out during storage, because the fittings were not tightened well. Also, radial compression cartridges are shipped dry. In these cases, the column needs to be wetted first with an organic solvent, such as methanol or acetonitrile. This drives the air out of the pores and wets the surface. Now you can exchange the methanol or acetonitrile for your mobile phase and obtain reproducible retention times. In the case of a column that has dried out accidentally, a problem may arise if the column has been stored in a mobile phase containing buffer or salt. The precipitated buffer may give rise to high backpressure during the reconditioning step, and you may need to re-equilibrate the column at low flow rates.
A related phenomenon is “hydrophobic collapse”. Very hydrophobic, well endcapped reversed-phase packings may lose retention in highly aqueous mobile phases. It has been observed that this can happen suddenly, for example when the flow through a column is stopped. In other cases a gradual decrease in retention has been observed over the course of a few days. In both cases, retention can be restored by washing the column with an organic solvent for a few column volumes and re-equilibrating it with the mobile phase.
Column Storage
Q.: I asked my colleagues how I should store my HPLC columns. Unfortunately, everybody gave me a different answer. What should I do?
A.: In most cases, there are many different ways in which an HPLC column can be stored that have little effect on column longevity. Therefore, it is quite possible that many of the answers that you got were correct. However, the best way of storing columns depends on the type of the stationary phase. Also, some column storage conditions are more convenient
than others. Let me first discuss this in general terms, then I will discuss specific and special considerations that depend on the nature of the packing.
The most convenient way to store a column is in the mobile phase in which it is commonly used. The biggest advantage of this approach is that reequilibration of the column with the mobile phase is very fast. Therefore, one can get reproducible results within a short time after start-up. This approach is especially recommended for normal-phase chromatography, where a change to a storage solvent different from the mobile phase can result in lengthy reequilibration times. However, one needs to think this approach through very carefully. Bonded-phase columns often change slowly in the commonly used mobile phases. Therefore, the convenience of storing the column in the mobile phase needs to be balanced against the reduction in column life.
In the following, I will discuss storage conditions for various HPLC packings based on the nature of the packing. Let me first talk about silica and alumina. Both of these packings are very stable in the mobile phases in which they are commonly used. These mobile phases commonly comprise organic solvents with small amounts of polar modifiers, including water. Due to the low concentration of the polar modifiers, it often takes a considerable time to equilibrate the columns with mobile phase. Therefore it is best and most convenient to store the columns in mobile phase.
The situation is similar for polar bonded phases used in normal phase chromatography. Equilibration with mobile phase is somewhat more rapid than with silica or alumina; nevertheless, it is still fastest to store the columns in mobile phase. Some mobile phase ingredients are not suitable for certain columns. Therefore, they also should not be considered for column cleaning or column storage. An example is the incompatibility of amino columns with acetone.
Polydivinylbenzene-based packings such as SEC packings and ion-exchangers are chemically very stable, but they swell and shrink in different solvents. The manufacturers usually supply you with information about which solvents are compatible with a particular column. General statements can not be made, since the stability of the columns depends on the packing conditions. However, the best storage solvent for these columns is the solvent in which the columns are used.
For silica-based bonded-phase columns used in reversed phase chromatography, the situation is more complicated. Water attacks the bonded phase, but the process is very slow in the commonly used pH range from pH 2 to pH 8. In addition, it depends on the nature of the bonded phase. The commonly used C18 and C8 columns are sufficiently stable to be used for several months with little change in their hydrophobicity. However, bonded phases based on shorter chains can hydrolyze measurably within a few weeks. Therefore, the best storage conditions for reversed-phase columns depend on the frequency of their use. If they are used every day, or even every few days, it is most convenient to store them in the commonly used mobile phase. On the other hand, if they will not be used for an extended period of time, it is best to store them in a solvent that prevents hydrolysis, commonly acetonitrile or methanol.
However, some reversed-phase columns are not stable in these solvents. Some cyano columns can void when stored in organic solvents. For these columns it is best to use the mobile phase for storage, despite the danger of faster hydrolysis. Read the manufacturer’s recommendations!
Amino columns are often used in acetonitrile/water mobile phases for the analysis of carbohydrates. Unfortunately, the amino group creates a basic pH in the pores of the packing, which leads to a slow loss of the functional group. Therefore, amino columns used for this application are best stored in acetonitrile instead of the mobile phase, at least for long term storage.
Additional considerations need to be made for the long term storage of columns in highly aqueous mobile phases that allow the growth of algae or bacteria. Often, the addition of sodium azide to the storage buffer is recommended. If feasible, organic solvents are better solvents for long-term storage.
As a final remark, it should be emphasized that it is always a good idea to record the storage solvent in a permanent file. This can be done conveniently by recording the column type and the serial number. When you create such a file, it is also worthwhile to record the date and time of the last use of the column, as well as the number of analyses run since the last storage. Such a column history file gives you a permanent record that allows you to go back and check column use and column life time. An alternative and less efficient way is to mark the column with your choice of the storage solvent. Of course, this approach works best if you always use the same storage solvent for this particular column.
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