Optimizing Splitless Injections in Gas Chromatography, Part III: Setting Up and Optimizing the Inlet

In the first two parts of this series, we explored the rationales by which splitless injection is most commonly used for trace quantitative analysis, and the band broadening and focusing mechanisms that impact our ability to effectively perform splitless injections. In this installment, we will discuss techniques for setting up the inlet and connections for optimized injections. We will examine the connections, including septum nut, glass liner, and column connections, along with column installation and gas flow setting. While this review is not comprehensive, we see that some general principles can guide inlet setup efforts and ensure successful, rugged, and reproducible splitless injection-based methods.

Most problems in gas chromatography (GC) are caused at or before the sample injection. When the inlet is set up correctly, GC is among the most rugged and reproducible instrumental techniques. When properly installed, set up, and maintained, columns and detectors generally perform properly for hundreds or thousands of injections. The column will separate, and the detector will detect whatever reaches them. The main challenge, as we have seen in parts I and II of this series on splitless injection, is getting the analytes to the column in sharp, reproducible, and quantitative bands (1,2).

Make Sure that Your Inlet is Clean

Cleanliness is paramount when performing capillary gas chromatography. With sensitive detectors that can easily detect nanograms or less material, even small amounts of contaminants can lead to ghost peaks or an elevated baseline. As you perform each of the setups and optimizations discussed below, keep cleanliness in mind and avoid introducing contaminants, including fingerprints, into the inlet.

Make Sure Your Inlet is Properly Set Up and Maintained

There are several considerations for ensuring that the inlet and injection are properly set up. These include the syringe and autosampler, the septum, the glass liner, column installation, setting gas flows, inlet temperature and pressure initial column temperature, and split purge valve timing. Besides setting the various temperature and pressure set points, each of these requires continuous attention and maintenance.

Use a Properly Maintained Autosampler and Syringe

Nearly all gas chromatography today is performed using an autosampler. Prior to each sample batch, visually inspect the syringe for evidence of leaks or damage, such as a slightly bent needle, encrustation, or resistance in the travel of the plunger. If you have any questions about the syringe, replace it. It is a good idea to have spare syringes on hand. Today’s autosamplers generally do not require maintenance, except for periodic maintenance performed by the vendor and syringe replacement.

Choose the Correct Glass Liner

Glass liner selection is possibly the most confusing part of gas chromatographic method development because there is no single glass liner that will serve all analytes and samples. Testing several glass liners should be part of any method development process. When trying di erent glass liners, all other instrumental conditions should remain constant. Several manufacturers of glass liners have provided selection guidelines and they all recommend the following considerations; each makes a recommendation for a specific glass liner to begin method development. Remember that there is no “killer app” with glass liners; they must be tested experimentally with your own samples (3,4).

Sample concentration is usually the first consideration; for splitless injection, we can assume relatively low sample concentration in the low part per million or part per billion range. A single taper liner with (for dirty samples) or without (for cleaner samples) wool is a good place to start. For solvents with higher vapor volume, such as polar solvents like ethanol, a double taper liner can help prevent backflash of excess solvent vapor into gas lines and other tubing. If the analytes are polar or reactive, a deactivated liner should be used. These quick recommendations provide a start; again, glass liners should be tested and tried.

For splitless injections, the glass liner should be inert, deactivated by the vendor. In the past, it was common for laboratories to purchase less expensive non-deactivated liners and deactivate them on their own. I recommend against using such “home brew” methods for liner deactivation; use the deactivated liners provided by the vendors. Vendors o er several levels of deactivation, ranging from none (least expensive) to highly inert materials (most expensive). The glass liner should be checked often for contamination. If obviously contaminated, it should be replaced. Figure 1 shows an image of a contaminated glass liner. Note the discoloration near the area where the syringe has penetrated and deposited sample material.

Properly Cut and Install the Column

An often overlooked, but critical, aspect of inlet performance is properly cutting and installing the column. The inlet-column connection is often blind, unwieldy, and requires practice to perform correctly. One of the most important practical characteristics of working with capillary columns is that the columns themselves are easily trimmed. Column vendors o er videos demonstrating inserting columns into ferrules and connectors and trimming them (5). Trimming columns requires training; a good way to do this is to use an old column for practice, and to inspect your practice column cuts with a magnifying glass or your phone camera, zoomed.

Figure 2 shows the results of some column cuts and handling, both good and bad. The up-close images were obtained using a cell phone camera on maximum zoom, with the phone held steady using a ring stand and clamp. Figure 2a shows a properly cut column end. Note that it is square, and there is no evidence of cracked glass or degradation of the polyimide coating. As seen in the figure, care should be taken with the scoring tool to not cut all the way through the tubing; just make a score and then quickly snap off the end with a flick of a finger.

Figure 2b shows an improperly cut column end, cut by scoring too hard with a column scoring wafer. The uneven cut is easily seen, along with exposed glass surfaces and cracks in the polyimide coating. Unwanted adsorption of polar analytes can happen on these uneven surfaces, causing poor peak shapes and loss of reproducibility. This is an extreme example for illustration purposes. Typical poor column cuts may be more subtle and difficult to see with the naked eye. After every cut, use a magnifying glass or zoomed cell phone camera to closely inspect the cut. If you have any questions about the cut quality, make another cut. A meters-long column will not miss a few extra centimeters cut off.

Figure 2c shows a properly cut column end that was incorrectly threaded through a graphite ferrule following cutting. One subtle point of column trimming and installation is to thread the column through the ferrule prior to trimming. When the column passes through the hole in the soft ferrule, it collects graphite, which then will contaminate the inlet, causing possible sample adsorption or ghost peaks in the chromatogram. This is avoided by threading the column onto the ferrule prior to cutting; the ferrule contamination is removed when the column is trimmed.

Install the Column Correctly into the Inlet

Proper column installation is critical to successful injections and chromatography. Each inlet from each vendor has a specific distance above the ferrule that the end of the column must protrude to be properly inserted into the inlet. If the column is inserted too far or too little into the inlet, analyte transfer into the column may not be complete. Be careful to not mix up the column and detector installation instructions; I have seen situations where the column was inserted all the way into the inlet until it stopped and then slightly pulled back, similar to many detectors, such as a common flame ionization detector. The user then wondered why they had no peaks. Most instrument vendors provide video instructions and devices that assist in properly setting the column length above the ferrule to ensure proper column insertion into the inlet (6).

Figure 3 shows a close-up of a column installed into an inlet the “old-fashioned” way, to illustrate the key point that the installation is blind, yet the length of column above the ferrule protruding into the inlet, is critical. In this figure, an old, used septum is threaded onto the column (prior to trimming!) through the hole made by the syringe, to hold the column nut and ferrule in the proper position; the column nut is then threaded into the inlet and tightened. An old septum can also be used to seal the column ends, if a column is removed from the instrument and stored.

Use Proper Technique When Tightening Column Nuts and Ferrules

Tightening the nut and ferrule, especially for the column connection with the inlet, is an art form. The nut should be tightened enough that the column is firmly held, and the connection is leak-free, but not overtightened so that the soft ferrule material is crushed or cracked. A crushed or cracked ferrule, while it may not leak, can introduce contaminants. Generally, the nut should be tightened finger-tight, plus about one-quarter turn with a wrench. Once tightened, a gentle tug on the column can test whether the column is firmly seated. If the column moves with a gentle tug, re-set and tighten.

At the top of the inlet are two additional nuts that require care in tightening. The glass liner is held in place by a nut with an O-ring that maintains a seal. When changing the glass liner, also change the O-ring and ensure that it is not deformed or discolored. When tightening the nut, again, finger tighten, plus about one-quarter turn until moderate resistance is felt. Remember that the glass liner is rigid; over-tightening of the nut can actually crack or break the glass liner. Septa should be replaced on a regular schedule, typically about every 50 injections. The septum nut should be finger-tightened until moderate resistance is felt. Under-tightening results in leaks; over-tightening can deform the septum, also resulting in leaks.

Be Gentle

In general, when working with connections and tightening nuts, either finger tight or with wrenches, if you feel you are using a lot of force to tighten the connection, it is a good idea to stop and check that the connection is seating properly. As the connections are screw-threaded, the need for too much force can lead to stripping of the threads.

Ensure that the Carrier Gas Flows Properly, and Check for Leaks

Once the column is installed in the inlet, but prior to installing in the detector, establish carrier gas flow. Check for column flow by placing the column outlet into a vial of solvent and looking for bubbles emanating from the column end. The bubbles indicate gas is flowing; then connect the column to the detector. Use an electronic leak detector to check all of the connections: septum nut, glass liner, column nut, and detector nut. If any leak, another one-quarter turn to tighten will usually stop it.

Heat the inlet, column oven, and detector and allow them to equilibrate. Once the inlet is properly set up, the column is installed and not leaking, take your time with this heating step. Although the inlet, column oven, and detector will come to the temperature settings very quickly, they may not be fully equilibrated at the set temperature for an hour or two. Once heated and equilibrated, run the system through 23 temperature cycles of the method (blank runs are fine here) and recheck for leaks. Leaks may appear after a few heating and cooling cycles because of dissimilar materials used in the connections. Again, be gentle and judicious with connections and wrenches. If you feel the need to force the connection to achieve a leak-free seal, it is best to have the connection repaired.

There are many critical steps to follow in properly setting up an inlet. Our current series has focused on the splitless inlet, but the general principles discussed in this installment apply to all inlets. Keep the inlet clean. Use care in making connections. Ensure that glass liners and septa are replaced on a regular basis. Optimize the glass liner and, as discussed in Parts I and II, the temperature and gas flows. The column is often termed the “heart of the separation;” the inlet, then, is the lungs. The gases and samples that flow through the column must first pass through the inlet. If the inlet is not properly optimized, the column and detector will also not perform their best.

References

(1) Snow, N. H. Optimizing Splitless Injections in Gas Chromatography, Part 1: What It Is and What Happens When We Inject? LCGC International 2024, 1 (8), 18–21. DOI: 10.56530/lcgc.int.ho2380j6

(2) Snow, N. H. Optimizing Splitless Injections in Gas Chromatography Part II: Band Broadening and Focusing Mechanisms. LCGC International 2025, 2 (1), 2024. DOI: 10.56530/lcgc.int.wo3487s4

(3) How to Choose a GC Inlet Liner. Restek 2025. https://www.restek.com/articles/how-to-choose-a-gc-inlet-liner (accessed 2025-02-19)

(4) Selecting the Right Inlet Liner for Efficient Sample Transfer. Agilent 2022. https://www.agilent.com/cs/library/posters/public/poster-selection-guide-GC-inlet-liner-5994-1222en-agilent.pdf (accessed 2025-02-19)

(5) Cutting a GC Fused Silica Capillary Column. YouTube 2018. https://www.youtube.com/watch?v=5bzgXiP-FHE (accessed 2025-02-19)

(6) GC Column Installation Parts 1 and 2 – GC Troubleshooting Series. YouTube 2016. https://www.youtube.com/watch?v=Nx1FzgwlCiE and https://www.youtube.com/watch?v=YmkKikaakSI&t=84s (accessed 2025-02-19)

About the Author

Nicholas H. Snow is the Thomas and Sylvia Tencza Professor and Chair in the Department of Chemistry and Biochemistry at Seton Hall University, and an Adjunct Professor of Medical Science. During his 30 years as a chromatographer, he has published more than 70 refereed articles and book chapters and has given more than 200 presentations and short courses. He is interested in the fundamentals and applications of separation science, especially gas chromatography, sampling, and sample preparation for chemical analysis. His research group is very active, with ongoing projects using GC, GC–MS, two-dimensional GC, and extraction methods including headspace, liquid–liquid extraction, and solid-phase microextraction.

Nicholas.Snow@shu.edu