Before you can use a new LC column to perform analyses, there are a few important steps you need to do to prepare it for its new life in your lab.
In this Restek Tip, we show you how to set up your new LC column. We cover certificate of analyses (CofAs), discuss the different column end-fittings you can use, share how to condition your column for your mobile phase, and finally, we’ll look at how to select an appropriate flow rate for your column. Let’s get started!
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Congratulations on your new column! You’ll soon be on your way to performing some high-quality chromatography. But before you can use your column to perform analyses, there are a few steps you need to do to prepare it for its new life in your lab.
In this Restek Tip, we’re going to show you how to set up your new LC column. We’re going to start by showing you where to find your certificate of analysis and review the information it contains. We’ll discuss the different column end-fittings you can use and then connect the column to your instrument. Next, we’ll look at how to condition your column for your mobile phase, and finally, we’ll look at how to select an appropriate flow rate for your column.
Let’s get started.
Certificate of Analysis
Every LC column that Restek makes is individually tested to ensure superior performance. A certificate of analysis is generated for each column and includes the performance report with the conditions that were used. It also lists the storage solvent that’s currently within the column, which we will need to reference later. You can download your certificate of analysis from the Restek website at any time from www.restek.com/documentation which can be helpful to have if troubleshooting is ever required.
Connecting Your Column
Let’s look at your new column. All Restek LC columns are made from 316 stainless-steel, meaning they are corrosion resistant. If your current system is set up with a Restek column, the end-fittings of your new column will be compatible with your current fittings. However, if your system is set up with another manufacturer’s column, you may need new fittings. If your current fittings are permanently swaged on, either cut off the old fittings, or replace the tubing and fittings entirely. While you may already have PEEK or stainless-steel fittings installed on your system, it’s important to check if these are suitable for your analysis.
PEEK fittings are convenient for low pressure—less than 4000 psi or 275 bar—but are likely to leak at higher pressures. If you have moderate to high flow rates, a column with smaller particle sizes, or a narrower column, your backpressure is likely to exceed 4000 psi. Therefore, we do not recommend their use under these conditions.
Stainless-steel fittings and connectors are good choices for higher pressures. However, one major drawback to stainless-steel fittings is that they require wrenches for column installation, and tubing depth can only be set once as the ferrules “bite” into the tubing.
EXP fittings are another option for connecting your new column to your instrument. EXP fittings are a hybrid titanium /PEEK solution that allows for hand-tightened connections that are effective to 8700 psi or 600 bar—and can exceed 20,000 psi or 1380 bar with wrenches.
They also have an adjustable fitting depth, so you can repeatedly make connections with columns of differing seat depth without changing the fitting or tubing. EXP fittings provide most of the high-pressure capabilities inherent with stainless-steel fittings while providing many of the conveniences offered by PEEK fittings, making them an easy, go-to choice.
Connecting your new column is relatively straightforward, but there are some considerations to keep in mind. Make sure your column connectors are properly seated before tightening. Loose connectors can introduce dead volume, resulting in poor efficiency and band broadening. It can also make it harder to remove your column in the future. Finally, double-check that the arrow direction on your column’s label matches the direction of your solvent flow. Failing to do so can shorten your column’s lifetime and result in poor efficiency.
Now that your column is installed, it’s time to check if the mobile phase you’re using is compatible with the storage solvent already in the column. Each column is shipped containing a storage solvent that keeps the column stable for long-term storage. The storage solvent is listed on your certificate of analysis.
If the solvent you wish to use is immiscible with the storage solvent—here’s a chart from restek.com for more information—then you will need to flush the column with an intermediate solvent that is compatible with both the storage solvent and your mobile phase.
If you plan to use a buffer, make sure it won’t precipitate out of your mobile phase or in the storage solvent. Most storage solvents are greater than 50% organic, which can cause buffers to precipitate and plug the column. This can increase backpressure, so make sure you have enough aqueous mobile phase to guarantee your buffer remains in solution.
Your next task is to select an appropriate flow rate for your column. Optimal flow rates are determined by the column’s inner diameter and the particle size of your stationary phase, as shown in this table.
When starting the flow through the column, we recommend you direct the flow into a beaker for 10-15 minutes. Start with a slow flow rate and gradually increase to the optimum flow rate during this time. Abrupt changes in pressure can cause leaks or even damage the column’s particle bed, resulting in poor peak shape or even split peaks.
Once the column has been adequately flushed at the desired flow rate, stop the mobile phase flow. Now you can connect the column to the detector.
And there you go! That’s how to prepare your new LC column. If you have any questions, contact us at www.restek.com/contact-us
Thank you for joining us for this Restek Tip!