Guest Author: Dr. Richard McAllister, Phenomenex UK
With a new year, most of us have our new resolutions. Join a gym, get healthier, quit smoking, drink less, basically try to become a better version of our previous year self. Analytical scientists are no different. Most involved with HPLC/UHPLC start new projects & assays, break out the new columns, use fresh batches of solvent, and of course new samples. However, to keep with the theme of “new”, it’s also a good time to answer one of the most commonly asked questions we get at Phenomenex by HPLC/UHPLC analysts—what are the best tips to maintain both our systems and columns to prolong column life?
Before we go further, it’s important to point out that aspects which are positive for a HPLC/UHPLC systems, usually apply to the column as well. Good system maintenance is important, however, not at the expense of your column. Solvents and solutes travel from injection port to detector in a relatively benign fashion. When the column is introduced, things can start to go wrong due to the types of interactions, taking place in the column. For example, in reverse phase (RP) mode, if there are very hydrophobic impurities that are going to accumulate anywhere and cause problems, it will be on the column. Usually on or around the front frit!
Systems can be complicated pieces of machinery and despite best efforts to keep things working smoothly, mechanical parts eventually break down and electrical components can stop communicating with one another. For these reasons alone, it’s imperative to have a maintenance contract to get the system serviced regularly. Like any other piece of complicated machinery—like the car you drove to work in—servicing is paramount to make sure everything works as it should and everything is up to date. Some service contracts can also include the replacement of any parts, so it’s certainly worth investigating with your system manufacture, or a private company who specializes in HPLC/UHPLC system servicing who can look after your instruments for you.
Other than having your system serviced regularly, which can become expensive, there are smaller things you can do to help keep the system engineer away. It sounds silly to even mention, but always use HPLC grade solvents (and always degassed). It might be tempting to use lower grades of the most popular solvents like H2O, MeCN, and MeOH to save costs. However, they can have a detrimental effect on the pumps and cause chromatographic issues as lower grade solvents can vary in density and composition, causing the pumps to pump more or less fluid than they are meant to. Lower grade solvents can also damage detectors, whether UV-Vis, RI, or any other, especially MS.
Another common problem with systems is software. Regular software updates can come under regular servicing as mentioned before, however, it is best to keep your system software and the PC running the HPLC/UHPLC instrument fully up-to-date. The HPLC/UHPLC system failing to communicate with the PC attached to it is a common problem but is easily avoidable.
Tubing can also be an issue. PEEK tubing is the most commonly used due to its flexibility, strength and ease of use with bio-samples. However, PEEK is not compatible with some strong solvents, such as THF, chlorinated solvents, and high concentrations of acids. Ensuring you don’t need to use certain strong solvents like THF before you start your analyses is vital as these solvents will degrade the tubing and will require an entire system flush and professional service. Checking the tube connectors exiting the pumps, into columns, detectors etc., is also a simple but valuable check that can help prevent leaks, solvent wastage, strange backpressure readings, and sensitivity loss.
A common problem we see with systems that are used for multiple assays at any one time is the contamination of tubing. For example, one analyst using a HPLC/UHPLC system for analysing blood samples can easily leave residue of their samples within the tubing or column. When the second analyst comes along to analyse their pharmaceutical compounds in waste water, they find all sorts of peaks and baseline noise and even potential backpressure increases because the previous samples were left over in the system. If systems are shared between assays and their respective analysts, we strongly advise either flushing the system thoroughly before each assay or after every assay.
Here at Phenomenex, we talk with many analysts who have experienced issues with their HPLC/UHPLC columns. This is understandable due to the nature of HPLC/UHPLC. All the chemical interactions (aka partitioning and separating) should take place in the HPLC/UHPLC column only. As mentioned before, solvents and the solutes they carry should travel from injection port to the column in a benign fashion—separated. Then travel uniformly to the detector for identification and quantification in a benign manner. A lot of column issues are avoidable if you can follow some basic rules and tips.
Upon opening up a new column, test its performance immediately to make sure it acts as it should. If you can, always test a new column using standards when its delivered to you, even if you’re planning on putting it in storage for a while. Most HPLC/UHPLC columns are shipped in their typical running solvents, examples for RP columns: 65% MeCN 35% H2O or Normal Phase (NP) columns 100% Isopropanol or Hexane. These shipping solvents are also their storage solvents, unless specified otherwise. Never store columns using solvents containing buffers or ion-pairing reagents. If you do, then your column never stood a chance.
I often get asked “How long can a column stay in storage”?
To be honest there is no strict answer but all I would say is—I wouldn’t want any of my columns in the cupboard for longer than six months. After six months, run the column again, get some fresh solvent through it, and check to see if it still performs as it should. Columns should always be stored in cool, dark conditions (to reduce solvent evaporation) and always on their side. DO NOT stand them up whilst in their boxes. If a column dries out while in storage it will be permanently damaged and unusable.
As with HPLC/UHPLC systems, columns need HPLC grade, filtered, and degassed solvents for maximum longevity. When running any column, especially a new one, set the pump(s) at 0.1mL/min (or lowest setting possible) and increase to normal flow rates (e.g. 1mL/min for 4.6mm ID columns), over five minutes. When shutting down the system you should reverse the above procedure, slowing the flow rate down from normal flow rates to 0.1mL/min. Avoiding any sort of sudden change in pressure while the column is being used will help increase column longevity.
Always run within the columns backpressure specifications helps to increase column longevity. At Phenomenex for example, all our 10µm, 5µm, and 3µm fully porous silica and TWIN technology columns are specified to run at no more than 250 bar (for Phenomenex UHPLC columns, backpressure limits are 1000 bar). To be fair, all our HPLC and UHPLC silica and TWIN technology columns are easily capable of running above and beyond their backpressure specifications, however, there will be a significant impact on column longevity and you may find yourself going through columns very quickly. For most analysts, running below the recommended backpressure limit isn’t a problem. It’s when the backpressure increases over time that problems begin to arise, suggesting the problem is being caused by something elsewhere. Mainly, the introduction of real world samples!
One thing that’s astounds me week-in-week out, is how overlooked sample preparation—especially sample clean-up—is for HPLC/UHPLC analysis. I do understand that sample preparation is often time consuming and can be expensive. However, if you only get 100 injections from a HPLC/UHPLC column, it may be worth investing in some sample clean-up to reduce column expenditure.
Injecting dirty, or minimally prepared samples, is the number one cause of reduced column longevity. Dirty samples contain a lot of particulates and contaminants that can chemically and physically alter HPLC/UHPLC columns.
A common physical problem in HPLC/UHPLC columns is particulates building up on the front frit, creating a blockage and resulting in increased backpressures.
When working with chemical alterations, for example using a RP method with a typical C18 column, an analyst is looking to identify and quantify pharmaceutical compounds in biological samples that are full of phospholipids. Due to their high affinity with the stationary phase (SP) caused by their high hydrophobicity, the phospholipids will quickly bind to the C18 SP. Once they’re bound, it is very difficult to get them off. The main impact to the column will be a loss of selectivity, sensitivity, and retention as the pharmaceutical compounds are blocked from interacting with the SP. Leaving the analyst to get a new column to analyse their compounds of interest.
There is a wide variety of techniques and products that clean up samples. In our phospholipid example, you would be able to use a product such as PhreeTM—a simple phospholipid removal tool that removes >99% of phospholipids. Products like Phree are relatively inexpensive and require a small amount of additional time, but significantly help HPLC/UHPLC columns last longer. For those who already have a dying column, there are column cleaning procedures that can regenerate HPLC/UHPLC columns. However, I would strongly recommend getting specific advice from your column manufactures as the incorrect procedure can irreversibly damage the column. If you can’t contact your column manufactures, don’t worry! At Phenomenex we have an online Column Care Guide. Its free to download from the Phenomenex website and is full of useful hints and tips!
There are, of course, situations where thorough sample preparation, such as Solid Phase Extraction (SPE), cannot remove all the unwanted particulates and contaminants. And there are those times that SPE and other cleaning methods cannot be used as it may remove unwanted components and could also remove the compounds of interest.
As HPLC and UHPLC separations continue to advance and become more complicated, it’s likely there will always be a quantity of unwanted components in the samples that cannot be removed in order to preserve the compounds of interest. For those in this situation that still wish to protect their columns, there are guard columns (also known as pre-columns) that can protect the main HPLC/UHPLC column from particulates and contaminants. Phenomenex’s guard system, SecurityGuardTM, consists of two parts: a holder and a cartridge. The SecurityGuard holder and cartridges (Figure 1) increase the column’s lifetime by preventing both particulates and contaminants from entering your column.
Simply exchanging a cartridge from the SecurityGuard holder can save your column from contaminants and have a significant benefit to your results. And added bonus? It’s exceptionally inexpensive.
Figure 1. A cutaway of a SecurityGuard Holder with the Cartridge inside attached to the front of a HPLC column catching and blocking contaminants
Guard columns are not necessary for everyone, but they are certainly worthwhile if you have dirty samples that cannot go through a cleaning procedure. The best thing I can advise if you experience poor column longevity is to try both sample preparation and the addition of a column guard. This is particularly pertinent if you are using UHPLC columns, which are much more prone to blockage due to the smaller particle size.
For more information on column/system care, including SecurityGuard and sample preparation products, please visit http://www.phenomenex.com, or get in touch with your local Phenomenex office. Till next time, wishing everyone a successful year of HPLC/UHPLC separations!
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