No matter where you live or who you work for, you’re likely to encounter one or more of these liquid chromatography (LC) column problems at some point.

	I have recently returned from a tour of teaching liquid chromatography (LC) classes to users in Minnesota, USA, the United Kingdom, Poland, and Malta. One thing that always impresses me on such trips is that no one group has a corner on the LC problem market. The same problems pop up in most laboratories, no matter where they are located, the role of the laboratory (for example, analytical, forensic, production, research), what industry is involved, or the brands of instrumentation used. As I teach, I tend to jot down questions as they come up, because these give me some of the topics for this “LC Troubleshooting” column. This month I share some of these question topics (and their answers) that are brought up in a majority of the classes I teach.


	We would all like our LC columns to last forever, but that just isn’t realistic. As I discussed in an earlier article (1), approximately three-quarters of all users get at least 500 injections through a column before it fails, and nearly half exceed 1000 injections. I’ve stated many times that I believe the break-even lifetime of a column is about 500 injections - with typical analysis costs, the contribution of a column to the per-sample cost is 2% or less with a 500-sample column lifetime.

	One of the ways to maximize the column’s lifetime is to prevent or correct for buildup of unwanted contaminants on the column. The column cleaning process is often called column regeneration. The simplest way to remove such materials is to increase the eluting strength of the mobile-phase strong solvent. For reversed-phase LC, the strong solvent is usually acetonitrile or methanol. When considering the effect of solvent strength in reversedâphase mode, I like to use the Rule of Three as a guideline. The Rule of Three states that the retention factor, 

, will drop by a factor of about three for each 10% increase in the strong solvent (B-solvent or %B). For wellâretained compounds, which is the case for strongly retained contaminants, we can consider that retention time will also change by threefold for a 10% change in %B. For example, let’s consider a 10-min isocratic method that uses 60% acetonitrile as the mobile phase and has contaminants that are eluted at 100 min. These contaminants would normally start being eluted in the 11th injection, and with a retention that strong would likely be so broad that they wouldn’t show up as peaks, but only as broad humps or a rolling baseline. So at the end of the batch of samples, we’d want to flush all the remaining contamination from the column. If we switched to 70% acetonitrile, we’d expect the retention to drop by threefold to 100/3 ≈ 33 min. A 20% change to 80% would cause a 3 × 3 = ninefold change, and running at 100% acetonitrile should reduce retention by approximately 3

 = 81-fold. This would cause our 100-min peak to be eluted at 100/81 < 2 min. It is good to take a cautious approach here, because the Rule of Three is an approximation based on an “average” compound of ~400 Da, and each additional 10% change in %B will result in a propagation of errors. However, even with allowance for such uncertainties, a 10–15 min flush with 100% acetonitrile would be very likely to elute anything that was going to come off the column with acetonitrile. This example uses an isocratic method, but you can easily understand that you would get similar results with a gradient method that ran to 100% B with a short hold (for example, 5 min) at the end of each run.

	The column flushing process is the same whether we are using traditional LC separation conditions or ultrahighâpressure LC (UHPLC). For universal application, we should consider flushing in terms of column volume and flow rate rather than time, and it will apply for all circumstances. Take for example, the two most popular column sizes: 150 mm × 4.6 mm for LC and 50 mm × 2.1 mm for UHPLC or LC–mass spectrometry (LC–MS) applications. The 150 mm × 4.6 mm column has a volume of ~1.5 mL, whereas the 50 mm × 2.1 mm column has a volume of ~0.1 mL. A flow rate of 1.5 mL/min and a 10-min flush for the isocratic method discussed above would result in a 1.5 mL/(1.5 mL/min) = 1 column volume/min flushing rate, or 10 column volumes of total flush. The 50 mm × 2.1 mm column operating at 0.5 mL/min would take only 0.2 min/column volume or 2 min to flush 10 column volumes of solvent through the column.

	I generally recommend attempting a 10–20 column-volume flush with 100% of the mobile-phase strong solvent to clean the column. If this flush isn’t successful, I don’t think it is worth the trouble going to any more work trying to clean the column unless there are extenuating circumstances. As mentioned above, after a 500-injection threshold is reached, I don’t feel that the column owes me anything, and additional work at cleaning the column may have diminishing returns. There are techniques to use stronger solvents (for example, methyl tert-butyl ether or dichloromethane), but extra care needs to be taken to ensure solvent miscibility, additional time is needed, and the cleanup efforts may have marginal results. The more extreme the cleanup technique, the more risk there is that the “cleaned” column will not return to a comparable chemical state, so even if you are successful at removing the contaminants, you may not get the same separation with the “regenerated” column.

	If your method falls into one of the categories where the column lifetime is <500 injections or the column cost is exceptionally high, such as for chiral columns, you may want to consider one or both of two additional techniques that you can find detailed in reference 1. The first is to use a guard column. Guard columns will extend the life of an analytical column, but they are fairly expensive, often costing 20% or more of the cost of the analytical column. Guard columns will work for most method applications. You can do the math and figure out if it is worth adding a guard column or not. The second technique is to add sample cleanup steps. Sample cleanup can be very effective and sometimes is essential if column lifetimes are otherwise too short. However, sample cleanup can remove things from the sample that you want to analyze. For example, cleanup may not be an option if you are trying to analyze impurities in a product. Sample cleanup can also rapidly add expense to the chromatographic process that eclipses the cost of the column. If a reversed-phase column costs $500 and you get 500 injections before it fails, the cost per sample for the column is $1. Solidâphase extraction (SPE) cartridges or wellâplates can cost $2–3/sample plus solvents, supporting equipment, and technician time. As a result, SPE may not be an economically wise choice in trying to reduce overall analysis costs. On the other hand, if sample cleanup results in improved data quality, the added cost may be easily justified.


	I occasionally get a question about the negative impact of air on an LC column. This question is usually about one of two problems: injections of air, or pumping air through the column as a result of running a reservoir dry. Let’s consider both of these next.

	What happens if you inject air instead of sample or have air bubbles in the sample that is injected? This injection problem could result if the autosampler was not adjusted properly, the amount of sample in the sample vials was insufficient, or for some other reason. Most of the time, an injection of a few microlitres of air will have no observable effect - the increased pressure in the column will compress the air into solution, and it will pass harmlessly through the column. At the end of the column, when the pressure returns to nearâatmospheric pressure, the bubble may come out of solution and can result in a baseline spike when the bubble passes through the detector. However, a slight back pressure on the detector flow cell will usually keep the bubble in solution until it exits the cell. Just be careful that the back pressure does not exceed the limits for the flow cell, or leakage may occur. In one of my previous jobs, I intentionally injected a sample from an airâsegmented sample stream. I modified the injector to compress the bubbles into solution before injection, and the method worked quite well.

	Sometimes, an injected air bubble may be retained and will show up as a positive or negative peak in the chromatogram (2). If you suspect that injected air is the source of a problem, intentionally make an air injection from an empty vial and see what happens. Then make an injection where you are sure no air is injected. Comparison of the two chromatograms should allow you to determine if injected air is the source of the problem. Usually injected air can be avoided by proper adjustment of the autosampler and ensuring that sufficient sample is in the vials. Rarely is it necessary to degas the samples to remove dissolved air.

	A more common question regards what happens when the pump is run dry, such as when you run out of solvent in a reservoir - does filling the column with air ruin it? My first response to this question is to ask what happens when the pump runs dry? Does it really pump the column full of air? The answer, of course, is no. This is because LC pumps are very good at pumping liquid, but do not pump air at all. As soon as the pump loses prime by filling with air, the piston moves back and forth, but no air is expelled from the pump. Yes, you may pump a bit of air into the column, but you won’t “pump the column dry”. However, you may ruin the pump seals because of increased seal-to-piston friction when operating the pump with no solvent. This can be avoided if you use the lower-pressure cutoff for the pump, so that if a reservoir runs dry, the pump will turn off when the pressure drops below a preset limit.

	What happens if the column is somehow filled with air? Does it ruin it? It probably will not harm the column. I remember doing an experiment years ago where we evaluated a column, then intentionally ran compressed nitrogen through the column to remove all the solvent. When we refilled the column with solvent, it performed as it did before drying it out. The primary problem was that pumping degassed solvent through the column to remove all the air took a lot of time.

	A more likely problem of column damage resulting from air in the column has to do with neither injecting air nor pumping air into the column, but from improper storage of the column. If you store the column with a nonvolatile buffer in it, such as phosphate, and leave the end plugs out of the column, it is possible to evaporate the solvent at the ends of the column. When the solvent evaporates, it will leave a buffer deposit in the column. Solid buffer crystals inside the particle pores will dissolve extremely slowly, if ever, and it is likely that the column would be ruined under these conditions. This problem, of course, is easily avoided by never storing the column when it contains buffer and tightly capping the column when it is not in use.

The Result of “Bad” Method Conditions


	There is one question that I can almost guarantee occurs when one of my lectures contains guidelines regarding conditions that should be avoided for a successful method. For example, the standard silica-based reversed-phase columns that most of us use have a recommended pH range of 2–8. Below pH 2, the bonded phase falls off, and above pH 8, the silica dissolves. There are plenty of columns available today that can be used outside this range, but they are specifically designed for extended pH stability. During the lecture I will see someone very busy taking notes or there’s an “oh, no” expression on a face. Usually this is followed by a question regarding the method. Most recently, one of the students described a method that required a mobile-phase pH of 10 and he complained about short column lifetimes. A batch of samples comprised approximately 50 injections, and he had to replace the column every three or four batches, for a column lifetime of 150–200 samples. This is definitely a problem. When I investigated the method a bit more, I found that the method is validated, the mobile phase cannot be modified, and the use of another column is not allowed.

	What can we do in a situation like this? My first reaction is to examine the method a little more closely. Is it true that the column really cannot be changed to another column? For example, a compendial method, such as those published in the 

 (USP), do not usually state a specific brand and packing for the column, but instead describe the column size, particle size, and stationary-phase classification (such as C18). For other methods, often a certain column may be specified, but many times the column specification is followed by an “or equivalent” statement. This is to anticipate that the specified column may not be available and another may work. If either of these cases applies to the method in question, I would suggest substituting the current column for a column with an extended pH range that would cover the pH 10 mobile phase. Of course, a little work will be involved in this substitution. You’ll have to show that the separation is equivalent between the two columns, verify that you get the same results from real or spiked samples, and document the change; in many cases, revalidation will not be required. How can you find an equivalent column? One excellent source is a free database of column equivalency that is available on the USP website (3); I described this database in an earlier “LC Troubleshooting” article (4). In this case, look for columns equivalent to the current column, then click the link to the manufacturer’s website to see if the column is stable over an extended pH range.

	If you have to live with the current high-pH conditions and designated column, you may be able to reduce your costs by using a guard column. The guard column will be attacked by the pH 10 mobile phase and will fail, as well, but the goal is to use the guard column as a sacrificial element of the system to neutralize the mobile phase before it reaches the column, hopefully extending the life of the column. You’ll have to experiment to see how long a guard column will last and how effective it is at extending the life of the analytical column. For example, it may be possible to replace the guard column after each batch of samples (≈50 samples) and extend the useful life of the column beyond the current 150–200 sample limit. The key, of course, is to replace the guard column early so that it provides maximum protection to the analytical column. After you determine the replacement frequency of the guard column, you’ll have to check the economics of using a guard column versus replacing the analytical column more frequently. Don’t forget to also observe the effect of the guard column on the data quality and consider it in your decision.


	We’ve looked at three questions about LC columns that are encountered by many users at one time or another. Column cleaning is a technique that we should use routinely, but we need to remember to balance the expense and effectiveness of column cleaning with the overall cost of analysis and the quality of the data. Next, we saw that the injection of air rarely ruins the column, but it can be irritating because of the work required to remove the air from the system. A better approach is to ensure that the autosampler is working properly, that there is enough sample in each vial, and that there is enough solvent present in the reservoir for the analysis of the desired number of samples. Finally, we considered how to mitigate problems related to methods that use conditions that are not recommended for good column performance. The best way to avoid such problems is to develop methods that use columns that are stable under the desired operating conditions. If that is not possible, see if there is another column that gives the same separation, but is more stable. Another option is to use a guard column to help protect the analytical column.

		http://www.usp.org/app/USPNF/columns.html	

has been writing “LC Troubleshooting” for LCGC for more than 30 years. One of the industry’s most respected professionals, John is currently the Vice President of and a principal instructor for LC Resources in Lafayette, California. He is also a member of LCGC Europe’s editorial advisory board. Direct correspondence about this column should go to: “LC Troubleshooting”, LCGC Europe, Hinderton Point, Lloyd Drive, Ellesmere Port, CH65 9HQ, UK, or e-mail the editorâinâchief, Alasdair Matheson, at 

Articles

LC Troubleshooting

LC Column Problems Everywhere

The 42nd International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2015), chaired by Gérard Hopfgartner was held 21–25 June in Geneva, Switzerland. This instalment covers some of the highlights observed at the symposium including stationary-phase developments, particle technology, and areas of growing application of HPLC. In addition, trends and perspectives on future developments in HPLC culled from the conference are presented.

	The 42nd International Symposium of High Performance Liquid Phase Separations and Related Techniques, or HPLC 2015, convened in Geneva, Switzerland, on 21–25 June 2015. The conference was held at the International Conference Center Geneva (CICG) in the very country that the conference began in 1973 (Interlaken). This conference, which has grown into the premier event bringing together leading scientists in the field of liquid separations, attracted about 1100 delegates from 50 countries. This was a considerable increase compared to HPLC 2014 in New Orleans, Louisiana, USA, but similar in numbers to previous European events. HPLC 2015 was chaired by Professor Gérard Hopfgartner of the University of Geneva and emphasized fundamental aspects of separations sciences, sample preparation, and novel developments and applications as well as hyphenation with mass spectrometry (MS).

	The conference included 169 oral presentations including several plenary lectures and sections dedicated to Professor Georges Guiochon, who sadly passed away on 21 October 2014. There were also 560 poster presentations and several short courses, vendor seminars, and tutorials within the vast programme. In this instalment of “Column Watch”, observed highlights and trends from the conference are reported.
	
	

 Figure 1 provides a breakdown of the categorization of the presented oral lectures at the meeting. Although the HPLC technique continues to dominate, several areas of increased emphasis are noted. Much like HPLC 2014 where the conference took on the personality of its chair, Professor Michael Ramsey, and a consequent increase in microseparations was observed, this year a strong emphasis was placed on liquid chromatography coupled with mass spectrometry (LC–MS). Techniques such as capillary electrophoresis (along with related techniques) and supercritical fluid chromatography (SFC) have continued to grow in popularity over the past several conferences. Microseparations, although not as prevalent as last year, continue to hold a strong position within the field. Multidimensional HPLC continues to see a rise in popularity and is finding application in areas such as food science, proteomics, metabolomics, and many others. In terms of specific application areas, the oral presentations were heavily dedicated towards large-molecule separations including special sections of biopharmaceuticals and phosphoproteomics. Pre- and post-separation requirements were also well represented within the oral sessions. Sample preparation was well represented not only within the specific sample preparation sections, but also throughout the symposium. Data analysis and design of experiments were likewise discussed in a number of sessions.

Breakdown of Separations Technologies Presented:

 Figure 2 presents a rough breakdown of the separations techniques presented during the oral sessions. As noted previously, the conference was heavily stacked with presentations related to MS. HPLC column technology, developments, and fundamentals continue to be a cornerstone of the conference series; however, this area was not as dominant as it has been in the past. SFC, which laid rather dormant for much of the 2000s, has gained in popularity over the past several years. The increase in interest is likely because of the improvement of instrumentation, the recognition of applicability to mainstream separations, and the desire for “greener” technologies. Likewise, multidimensional chromatography continues to increase its presence at the HPLC symposia. An improved understanding of fundamentals, commercially available instrumentation and software, along with the everâincreasing complexity of samples seems to have driven the technique to new heights. Electrophoretically driven techniques continue to show a strong presence with an increase in protein and peptide separations within the technique. Although understandably down from last year, microseparation techniques continue to be an integral part of the HPLC conference. Thinâlayer chromatography (TLC) was mentioned a few times during the symposium, especially when related to TLC–MS. This may be a sign of an increased interest in this classical technique.

	Digging a little deeper into HPLC technologies, Figure 3 lists oral sessions that specifically called out the use of HPLC modes of chromatography. Reversed-phase LC continues to be the dominant technique, however, more specialized techniques such as hydrophilic-interaction chromatography (HILIC) and chiral modes were well represented. HILIC has become increasingly popular over the past decade because it can retain and separate polar molecules that are difficult to deal with in reversed-phase LC. Issues with reproducibility and long equilibration times in HILIC have prompted much research into the fundamental retention mechanisms and dynamics. James Heaton, PhD, for example, presented a lecture noting some of the issues associated with the multitude of interactions that occur in HILIC (1). Heaton demonstrated deleterious effects of chelation using a set of catecholamines. He also noted issues with certain sample solvents and showed the impact of buffer concentration in the mobile phase on retention and peak shape of certain compounds. The area of chiral separations has also seen some resurgence. Areas of chiral research not only include pharmaceutical concerns, but also areas of metabolomics, agriculture, and food science, to name a few. Size-exclusion chromatography (SEC), ion-exchange chromatography, and affinity chromatography all appear to be increasing in popularity within the oral sessions at HPLC. This trend likely parallels the increase in largeâmolecule separations interest as noted in sections to follow.

	One of the major areas of interest in HPLC over the past decade has been in the development of improved particle technologies. Figure 4 shows a rough breakdown of the particle technologies highlighted during the oral sessions at HPLC this year. Subâ2-µm or ultrahigh-pressure liquid chromatography (UHPLC) technology continues to be a dominant area of interest. Superficially porous particles (SPPs) also made a strong showing. Both technologies appear to have matured for the most part and, as mentioned by Tividar Farkas in his presentation (2), most current efforts in these technologies appear to be focused on alternative chemical modifications and expansion into niche areas (3). An excellent presentation by William Barber demonstrated that further innovative efforts in preparing SPP particles are continuing. Barber described a patented preparation of highly ordered pores through a process called 

 (PMT) and compared this process to standard preparations of superficially porous materials (4). Figure 5 shows the resulting highly ordered pore structure that is obtained using this process. Barber went on to describe the high efficiencies that are obtained when these particles are packed in HPLC columns. These high efficiencies are expected to be a result of the diffusion pathways resulting from the ordered pore structure. In another interesting presentation, Alberto Cavazzini covered the development of narrow particle size fully porous sub-2-µm particles (5). These particles, when packed in HPLC columns, were shown to produce high efficiencies of about 300,000 theoretical plates/m as shown in Figure 6.

	Detailed studies investigating the origin of the high efficiency concluded that the A term of the van Deemter equation for the narrow particle size particles was smaller than that for columns packed with particles with a more disperse particle size. Although monolithic HPLC columns continue to be commercially available from a few sources, research in this area continues to grow. In a similar fashion to developments in fully porous and superficially porous technologies, monoliths continue to be modified with new chemistries to improve selectivity space and application in an abundance of arenas.

	Figure 7 provides a breakdown of the primary application areas discussed during the oral sessions. It is clearly apparent that large-molecule separations dominated discussions where separations were applied. There were even a few specialized sessions on specific large (or larger) molecules such as phosphopeptides and biopharmaceuticals. Dwight Stoll presented an interesting lecture on the characterization of monoclonal antibodies (mAbs) using multidimensional chromatographic approaches (6). The complexity of mAbs necessitates the use of a number of techniques to adequately characterize them. Stoll explained some of the complexity, noting that variations in glycosylation, oxidation, and isomerization of mAbs led to an estimated 108 different molecular forms. He went on to compare different forms of multidimensional LC separation techniques including heart-cutting as well as comprehensive and selective two-dimensional (2D) LC. Selective 2D LC as depicted in Figure 8 was shown to be a powerful and flexible approach that enables the acquisition of multiple dimensions of chromatographic information for mAbs in a single analysis. When coupled with MS, molecular information can also be obtained directly in the same analysis.

	Several talks focused on techniques for sample preparation of mAbs and alternative techniques of analysis, such as Chervet’s discussion regarding the use of electrochemical reduction of disulphide bonds coupled with MS (7). Barry Karger discussed glycoprotein structural characterization using topâdown high-resolution capillary zone electrophoresis coupled to mass spectrometry (CZE–MS) (8). From these few examples, it is clear that the focus on the analysis of large molecules will continue to bring alternative sample preparation, separation, and detection techniques to the forefront of future HPLC meetings. On the small-molecule side, biomarkers, bioanalysis (namely the analysis of small molecules in biological fluids), and metabolomics are areas of high interest. The complexity of the separations and the matrices being investigated also necessitate improvements in sample preparation, separation efficiencies, alternative selectivity, and novel detection schemes. Research in each of these areas is expected to continue and play a large role in future HPLC programmes. Other areas of significant interest included pharmaceutical, environmental, and industrial applications.

Poster Presentation Highlights and Trends

Poster presentations are a vital component of the HPLC symposia. This is evidenced by the 560 posters that were presented at HPLC 2015 in 27 session topics. Half of them were presented on Monday and Tuesday and the other half were presented on Wednesday and Thursday. The conferees have three breaks between talks in each day to see the posters and interact with the authors. The number of posters presented at HPLC 2015 increased 30% compared to HPLC 2014 in New Orleans (Table 1). Clearly, HPLC 2013 was an “outlier” since it had almost twice the number of posters than the adjacent years, which was consistent with its higher attendance compared to the other years.

	The number of posters in each session topic is shown in Figure 9. The two busiest session topics were LC–MS and column design or stationary phase with 58 posters each. This highlights the continuous trend in coupling LC and MS and the never-slowing interest in developing new separation media. It is also pleasing to see that the fundamentals topic is well represented with 50 poster; these efforts will continue to help the development of new instruments and columns. The next three top areas are more focused on applications including food sciences, sample preparation for LC, and bioanalysis or biopharmaceuticals. Clearly scientists are motivated to develop better methods to analyze complex biomolecules and need better tools to deal with the complex sample matrices in food analysis.

	The tradition of Best Poster Award sponsored by Agilent Technologies at HPLC conferences continued in Geneva. This year 306 posters entered the competition, and 38 reviewers devoted a great deal of their time to judge the posters. Each poster was evaluated for inspiration (novelty and originality), scope (amount of work, technical quality, and execution of the experiments), and presentation (clarity, readability, and author’s explanation). There were 10 posters nominated in the PSA session (Monday and Tuesday) and another 10 were nominated in the PSB session (Wednesday and Thursday) to enter the second round evaluation, where each nominee gave a 5-min oral presentation on their posters. Then, 11 reviewers (different from the first round reviewers) evaluated these oral presentations to select the final awardees. Five posters from the PSA session were given the Best Poster Award while six posters from the PSB session were awarded because of a tie in the fifth place. These 11 final awardees are listed in Table 2. It is interesting to note that eight out of the 11 winning posters were presented in the top seven poster session topic areas mentioned above.

	
	The 11 Best Poster Awards were presented at the closing session on Thursday afternoon by the symposium chairman Gérard Hopfgartner and Tom van de Goor, PhD, of Agilent Technologies (Figure 10). Each winner received a cash prize of 500 Swiss francs (CHF). The final winners and the nominees of the final round come from 11 countries and represent the best research in the field of HPLC around the world.


	HPLC 2015 was an eventful symposium bringing together researchers interested in separation science from around the globe. To borrow some words from Ron Majors, the overall impression of the conference was “evolutionary” rather than “revolutionary.” The majority of the presentations provided information on steps forward with improvements in existing technologies and coupling multiple techniques to meet the challenges of complex separations. As mentioned, however, there were several presentations that offered insight into promising new technologies that might, in fact, become revolutionary in the near future. The most evident trend was the focus on largeâmolecule separations. The complexity of such separation challenges has necessitated improvements in existing technologies and the coupling of multiple techniques to gain as much information as possible with minimal time and effort. This trend is expected to drive innovation and will continue to be discussed in future HPLC and related symposia.


	The 43rd International Symposium of High performance Liquid Phase Separations and Related Techniques Symposium on High Performance Liquid Chromatography and Related Techniques takes place in Beijing, China, this month (21–25 September 2015), chaired by Professor Guibin Jiang of the Chinese Academy of Sciences. The 44th International Symposium of High performance Liquid Phase Separations and Related Techniques Symposium on High Performance Liquid Chromatography and Related Techniques will be chaired by Professor Robert Kennedy of the University of Michigan and held 19–24 June 2016 in San Francisco, California, USA.


	Coverage of such a large symposium is impossible without a great amount of assistance. The authors would like to acknowledge the invaluable assistance from William Barber, PhD (Agilent Technologies), Professor Dwight Stoll (Gustavus Aldophus College), and Professor Alberto Cavazzini (University of Ferrara) for their kind permission to discuss their contributions. In addition, we’d like to thank Stacy Shollenberger and Gaurang Parmar (SigmaâAldrich/Supelco) as well as Laura Bush (LCGC), James Heaton, PhD, Professor David McCalley (University of the West of England), and Professor Gert Desmet (Vrije Universiteit Brussel) for providing notes and discussions regarding content of various sessions. Finally, we’d like to thank Ron Majors for valuable discussions regarding the content and format of the present article.
	
	
	

		J. Heaton and D. McCalley, “Some Factors that Can Lead to Poor Peak Shape in Hydrophilic Interaction Chromatography, and Possibilities for Their Remediation”, presented at the 43rd International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2015), Geneva, Switzerland, 2015.

		T. Farkas, L. Loo, L. Abadilla, M. Chitty, I. Rustamov, and T. Tran, “Has Core-shell Particle Technology Reached Maturity?” presented at 43rd International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2015), Geneva, Switzerland, 2015.	

		W. Barber, T.-C. Wei, W. Chen, A. Mack, J. Liu, M. Dittmann, and X. Wang, “Next Generation Superficially Porous Particle Technology: Highly Ordered Pore Structures Formed By Pseudomorphic Transformation”, presented at 43rd International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2015), Geneva, Switzerland, 2015.	

		A. Cavazzini, O. Ismail, A. Ciogli, C. Villani, F. Gasparrini, D. Kotoni, D.S. Bell, and N. Marchetti, “Considerations on the Kinetic Performance of Columns Packed with New 1.9 μm Fully Porous Particles of Narrow Particle Size Distribution”, presented at 43rd International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2015), Geneva, Switzerland, 2015,	

		D. Stoll, D. Harmes, E. Wagner-Rousset, S. Fekete, D. Guillarme, and A. Beck, “Online Selective Comprehensive Two-Dimensional Liquid Chromatography–Mass Spectrometry for Efficient, High Resolution Characterization of Monoclonal Antibodies”, presented at 43rd International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2015), Geneva, Switzerland, 2015.	

		J.-P. Chervet, L. Rieux, A. Kraj, H.-J. Brouwer, “Controlled Reduction of Disulfide Bonds in Protein Therapeutics Using an Electrochemical Reactor Cell Online with MS”, presented at the 43rd International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2015), Geneva, Switzerland, 2015.	

		B. Karger, “Advanced Glycoprotein Structural Characterization Using Top-Down High Resolution CZE-MS”, presented at the 43rd International Symposium of High Performance Liquid Phase Separations and Related Techniques (HPLC 2015), Geneva, Switzerland, 2015.	

		D. Stoll, J. Danforth, D. Harmes, E. Wagner, S. Fekete, D. Guillarme, and A. Beck, 

 is a manager in pharmaceutical and bioanalytical research at Sigma-Aldrich/Supelco. With a B.S. degree from SUNY Plattsburgh and a PhD in Analytical Chemistry from The Pennsylvania
	State University, Dave spent the first decade of his career within the pharmaceutical industry performing analytical method development using various forms of chromatography and electrophoresis. During the past 15 years, working directly in the chromatography industry, Dave has focused his efforts on the design, development, and application of stationary phases for use in HPLC and hyphenated techniques. In his current role at Supelco, Dr. Bell’s main focus has been to research, publish, and present on the topic of molecular interactions that contribute to retention and selectivity in an  array of chromatographic processes. Direct correspondence to: 

 is currently an R&D manager in Agilent CrossLab Group at Agilent Technologies in Wilmington, Delaware, USA. He earned a PhD degree in analytical chemistry from the University of Minnesota in 2006 and then worked in the pharmaceutical industry at AstraZeneca for four years. Xiaoli joined Agilent Technologies in 2010 and his main focus has been the development of novel chromatographic columns.

 is the editor of “Column Watch”, an analytical consultant, and a member of LCGC Europe’s editorial advisory board. Direct correspondence about this column should go to: “Column Watch”, LCGC Europe, Hinderton Point, Lloyd Drive, Ellesmere Port, CH65 9HQ, UK, or e-mail the editorâinâchief, Alasdair Matheson, at 

Column Watch

Highlights from the HPLC 2015 Symposium

As flame ionization detection (FID) approaches its 60th anniversary in 2017, this instalment examines the crucial role that it has played and continues to play for all types of gas chromatography (GC). Without FID, the early development of GC would have proceeded more slowly, especially in the petroleum industry and related hydrocarbon application areas.

	Flame ionization detection (FID) is without a doubt the most often used gas chromatography (GC) detection method. While the older thermal conductivity detection (TCD) responds universally, it lacks the sensitivity and linearity that FID can deliver. Indeed, it is fortuitous that FID responds so well to the chemical moieties that constitute a majority of compounds of interest across many disciplines. The development and widespread acceptance of FID strongly boosted the rapid growth of GC, especially with capillary columns, during the early years.

	The invention and development of FID is a story of simultaneous multiple discovery in two locations by researchers who were unaware of each other’s efforts to improve on an existing detector (1,2). The predecessor to the flame ionization detector was the hydrogen flame detector, described by R.P.W. Scott in 1955, that measured the temperature increase of a hydrogen flame as GC column eluant passed through. The hydrogen flame detector was not particularly more sensitive compared to the ubiquitous thermal conductivity detector, and in 1957 two separate research groups began to investigate how to improve upon it: the Central Research Laboratories of Imperial Chemical Industries of Australia and New Zealand (ICIANZ), and the Department of Physical Chemistry of the University of Pretoria in South Africa.

	Starting in early 1957, R.A. Dewar and I.G. McWilliam of ICIANZ endeavoured to measure the ion current in the hydrogen flame instead of its temperature, while at about the same time V. Pretorius at the University of Pretoria measured the changes in the resistance across the hydrogen flame as peaks were eluted. Both groups submitted papers to Nature in late 1957. Pretorius’s paper was published first, but only because of delays in the editorial process around Dewar and McWilliam’s submission. In the end, it was the ICIANZ group that continued to develop FID, characterizing and optimizing several configurations and presenting a detailed report at the Second International GC Symposium held in Amsterdam, the Netherlands, in May 1958.

	This event created large interest in FID and a number of additional investigators began to work with it, in particular with the then newly developed capillary columns. The relatively low flow rates and small sample capacities of capillary columns, compared to the prevalent packed columns, had stymied their wider adoption. The higher sensitivity and tolerance for low column flow rates in FID made it an ideal match for capillaries. FID was soon made available in the Perkin-Elmer Model 154-C GC system introduced at the 10th Pittsburgh Conference in 1959. Arguably the first commercialization of FID, it was soon followed by offerings from other companies. The Wilkens Aerograph Model 600 “HyâFi” GC system, introduced in 1961, was reportedly named for its hydrogen flame ionization detector. Wilkens Instruments was the predecessor to Varian’s chromatography division.

	ICI obtained patent rights for the flame ionization detector in Australia as well as in a number of other countries including the United States, and the company subsequently issued licenses to as many as 37 instrument companies. More than 60,000 detectors were manufactured under license from ICI. Subsequent chromatographyârelated FID patents were obtained by Shell, HewlettâPackard, PerkinâElmer, Beckman, Pye Ltd., Varian, Microsensor Technology, and Phillips Petroleum, to name a few. Innovations around FID continue today as well, with some of the most recent work encompassing supercritical fluid chromatography (SFC) and microcolumn GC.

	Nearly 60 years after its invention, FID continues to be the dominant GC detection method. While benchtop and portable mass spectrometric detection provides more information via spectral analysis, and selective detection methods such as electron-capture detection (ECD) offer unparalleled sensitivity and selectivity, FID brings sensitivity, low cost, simplicity, and reliability to GC detection. 

	A fraction of the carbon-based compounds that pass through the FID flame are ionized, something like one in 10,000 (3). In most modern designs, the electrons formed inside the detector are impelled by an electric potential towards a collector electrode, producing a minute current on the order of picoamperes (10

A). This current is converted to a voltage, filtered, and amplified as required. Even though not all of the carbon molecules are ionized, FID can deliver a limit of detection on the order of a few picograms of carbon per second.

	The internal arrangement of a typical flame ionization detector is shown in Figure 1. Carrier gas from the column enters at the bottom of the detector and is mixed with hydrogen combustion gas plus optional makeup gas in the area below the flame jet. This mixture is then combined with an excess of air and burned just above the jet tip. A negative polarizing potential is applied between the jet tip and a collector electrode; as electrons are formed they are accelerated across the jet tip–collector gap by the electric field. Depending on the detector design, either the collector or the jet tip is kept at ground potential; Figure 1 shows a grounded collector design with the electrometer input at virtual ground and the jet tip charged to -200 V. Air, carbon dioxide, and water exhaust gases are vented from the top of the detector body. In most modern FID systems a glow plug operates momentarily to ignite the flame.

	The electric current from the collector is converted to a voltage by an electrometer and then is amplified, filtered, digitized, and processed as required. Signal zeroing, scaling, and attenuation are performed in the instrument firmware. The detector signal is filtered to remove unwanted high-frequency noise. Noise is produced by instabilities in the flux of ionizable impurities in the carrier gas, by the flame itself, by the electronic circuit, and by induction of stray electromagnetic signals from, for example, nearby cell phones. Excessive noise is usually the result of contamination from a buildup of carbon particles in the detector, poor electrical connections, or incorrect operational settings.

	FID sensitivity depends on the combustion gas flow rates, the carrier gas flow rate, the flame jet exit diameter, the electric field formed by the relative positions and shape of jet and collector, and to a lesser degree the detector temperature.

 Combustion gas flow rates must be set correctly for proper detector operation; follow the manufacturer’s recommendations for air and hydrogen flow settings. In general the air–hydrogen ratio should be approximately 10:1. A hydrogen flow rate of 30–45 mL/min with a corresponding air flow of 300–450 mL/min is common with standard laboratory detectors. FID sensitivity will be reduced if the hydrogen flow deviates above or below optimum. The linear dynamic range (LDR) is affected by hydrogen flow: higher flows tend to reduce the LDR. There is little reason to operate a flame ionization detector far away from the manufacturer’s gas flow settings; they have been carefully optimized for that specific detector. Air flow is less critical than the hydrogen flow, but too much air will destabilize the flame causing noise and possible flame-out. Too little air will reduce sensitivity and shorten the LDR.

 Hydrogen is highly flammable and can cause a serious explosion if allowed to build up in an enclosed space such as the GC oven. Never turn on the hydrogen flow without a column or plug fitting attached to the detector base to prevent hydrogen from leaking into the oven. Available GC oven hydrogen monitors provide an additional safety level by shutting off hydrogen flow if dangerous levels are detected.

 The carrier-gas flow rate is an important consideration for detector sensitivity. FID does not require any special attention to flow considerations with standard 1/8-in. diameter packed columns or 0.53-mm and greater capillary columns operating at or above 8–10 mL/min. When capillary or micropacked columns are operated closer to their optimum flow rates of less than 5 mL/min, FID can benefit from the addition of makeup gas to the carrier stream before it enters the jet area.

	Carrier-gas type, other than hydrogen, does not materially affect detector operation. Hydrogen, while seldom used for packed column carrier gas, is commonly used with capillary columns and has advantages that have been discussed in detail in “GC Connections” (4,5) and many other articles, conference talks, and seminars. Hydrogen carrier gas gives a wider range of optimum linear velocities, is less expensive than helium, and may be generated from water on demand with an appropriate hydrogen generator.

	Since FID performance is affected, the hydrogen flow rate through the detector should be kept at an optimum level by considering the total flow. With a hydrogen carrier gas flow rate of 5 mL/min, for example, detector hydrogen flow should be reduced correspondingly by 5 mL/min. Carrier flow rates may change as the column oven temperature changes, depending on the mode of carrier control. Electronic pressure control systems can maintain constant total hydrogen flow through the detector by adjusting FID hydrogen dynamically as capillary column flows change during temperature or carrier-gas programming.

	Makeup gas has two important effects. First, it establishes an optimum gas flow rate through the jet when carrier flow is low, which keeps the FID system operating at the best sensitivity and LDR. Second, for some detectors makeup flow sweeps out the area under the jet and inside the detector base, alleviating any peak broadening that might be produced as capillary peaks encounter largeâdiameter internal passageways. With FID, use carrier gas as makeup gas. If hydrogen carrier gas is used, consider nitrogen makeup gas if maintaining the total hydrogen flow from three sources - carrier, makeup, and flame hydrogen - is impractical. Typical makeup gas flow rates are in the 10–20 mL/min range, but in all cases follow the manufacturer’s recommendations.

 The sensitivity of a flame ionization detector does not depend strongly on its temperature, provided some conditions are met. The appropriate FID temperature is determined by the greater of a minimum temperature of 150 °C for stable detector operation, and a minimum temperature of approximately 20–50 °C above the highest column temperature, up to the maximum permitted detector temperature. FID produces a large amount of water vapour that may condense in the cooler upper areas around the collector if the detector base temperature is less than about 150 °C: this condensed water vapour can produce noise and baseline drift. On the other hand, the detector base must be hot enough to prevent condensation of peaks as they are eluted from the column, so it should be kept somewhat warmer than the highest operating column temperature. Some GC systems have an insulating cap that surrounds the detector column fitting. To avoid cold spots, always make sure to reinstall the cap after connecting a column to the detector.

	If a capillary column is installed with its end inserted into the detector base up to the jet, and is operated at oven temperatures approaching the column’s maximum rated temperature, it is possible that the end of the column will be overheated if the detector base is significantly warmer. Such overheating can produce excessive detector noise from decomposing stationary phases, cause solute adsorption onto subsequently exposed column surfaces, and reduce column lifetime. A capillary column detector adapter that positions the end of the separation column in the oven and conducts carrier gas flow along a deactivated connector into the detector jet will help alleviate such problems.

	Carrier and combustion gas purities and flow rates as well as detector and column temperatures are all important considerations when setting up a flame ionization detector. Be sure that all gases are of sufficiently high purity. Then, with the instrument turned on but unheated, set the required gas flows. Finally, heat the injector, detector, and column to their operating temperatures and ignite the flame.

	Flame ionization detectors are sensitive to hydrocarbon impurities that may be present in gas cylinders or connecting tubing. Impurities in the combustion gases will cause increased detector noise levels as well as higher baseline signal levels. Hydrocarbon filters are highly recommended for installation at the external GC bulkhead fittings for air, hydrogen, makeup gas, and, of course, the carrier gas. It’s not necessary to remove oxygen from the FID hydrogen stream, but an oxygen filter on the carrier line is always recommended, certainly if hydrogen is used for carrier gas as well.

	Hydrogen for FID alone should be of 99.995% purity or better. If used for carrier gas then 99.999% or better purity is best. There are several excellent commercial hydrogen generators that can produce sufficient carrier-grade hydrogen to supply dual flame ionization detectors plus one or two carrier channels with split injectors. If an electrolytic hydrogen generator is used be sure the water you add is free of hydrocarbon impurities.

	Air for FID should contain less than 100 ppb of total hydrocarbon impurities. Besides standard compressed gas tanks, various suitable purified air generators are available with capacities ranging from a couple of chromatographs up to an entire laboratory’s worth. Older air compressors or so-called “house” air supplies should not be used with GC systems except to supply operating pressure for pneumatic valve actuators.

	Carrier-gas purity is also important for proper FID operation - with or without makeup gas. Makeup gas impurities affect FID in much the same manner as combustion-gas impurities. Even without makeup gas, impurities in the carrier gas can eventually pass through the column and onto the detector. In temperatureâprogrammed operation, such impurities may appear as broad ghost peaks during a run or as a steadily rising baseline similar to column stationaryâphase bleed. In isothermal operation impurities may appear as a slowly rising baseline with increasing noise, often over a period of hours to days.

	Connecting tubing from the gas source to the instrument can also sometimes cause problems with contamination. Be sure to use copper or stainless steel tubing specially cleaned for chromatographic applications. Never use plastic tubing because significant amounts of plasticizer or monomer may be present. In addition, plastic tubing is permeable to atmospheric oxygen. Avoid leakage by ensuring that all fittings and ferrules are in good shape and not over-tightened.

 Two situations arise when setting FID flow rates depending on whether the gases are electronic pressure controlled (EPC) or manually controlled. With EPC systems the flows are set on the instrument keypad. Don’t assume, however, that the flows are correct - regular flow validation is highly recommended. I like to measure the detector flow rates anyway. Be careful to enter the related settings that control the carrier-gas mode of operation, makeup gas flow, and the identity of the gases in use. Also, bear in mind that in some GC systems with manual detector gas controls, flow rates depend on the supply gas pressure - if the pressure changes then the detector flows should be recalibrated.

	For manually controlled detector gases, as well as when directly measuring detector gas flow rates, it’s easiest to operate with the column connection in the oven blocked off with a blank ferrule or plug fitting. If the column is installed then carrier flow should be enabled for capillary column installations where the column end is in the detector; in this situation the operator will need to correct measured combustion gas and makeup flows for the column flow rate.

	To measure detector flows, attach a calibrated flowmeter to the detector exit with the appropriate adapter and turn off the air, hydrogen, makeup, and carrier-gas flows. Hydrogen flow is the first to set. Turn on the hydrogen, wait a minute or so for air to be purged, and set the correct flow rate, following the adjustment instructions in the manual.

	Next, set the makeup flow, if used. Turn off the hydrogen flow and then turn on, measure, and adjust the makeup flow. If the hydrogen cannot be turned off conveniently then subtract the measured hydrogen flow to find the makeup flow rate. Be careful, however, when using an electronic flowmeter. If your meter has settings to select the type of gas being measured, then it will produce inaccurate readings for gas mixtures. This is not a problem for a simple soap-bubble flowmeter, nor for electronic meters that measure the volumetic flow rate.

	Third, set the air flow rate. This may require a larger-volume flowmeter to accurately measure the 10-fold higher flow. Again, it is best to turn off the hydrogen and makeup flows, but you can correct the measured air flow rate if necessary.

	Finally, if it’s not already on then set the carrier-gas flow. If you want to measure the carrier-gas flow rate directly at the detector then turn off the air, makeup, and hydrogen flows. Adjust the carrier-gas flow controller, pressure regulator, or EPC system as required. After column flow is established, and not before, the inlet, column, and detector may be heated to their operating temperatures

	Accurate direct measurement of capillary-column flow under about 5 mL/min requires a suitable low volume flow measuring device. For an EPC system, remember that the type of carrier gas and the column dimensions must be entered by the operator. If the entered dimensions don’t accurately reflect the actual dimensions then column flow and velocity errors will result.

 While the instrument is heating up, turn the combustion gas and makeup flows back on if necessary. You can ignite the flame as soon as the detector temperature has passed 150 °C. Most flame ionization detectors require that the air flow be temporarily reduced during ignition. Like a choke on an automobile, this reduced air flow creates a momentarily rich mixture that is easier to ignite. Some instruments have built-in igniters that are operated by push-button or from the keypad, while others have manual igniters that must be held over the detector as an internal glow wire is heated electrically. Some rely on a piezoelectric igniter. In any case, ignition is most often accompanied by an audible “pop.” EPC-equipped GC systems will automatically reduce the air flow and turn off the makeup flow during flame ignition.

 Do not lean over the detector to see the flame (it is invisible), and always wear appropriate eye protection. Do not allow any clothing or other flammables to come near the detector exit.

	After the flame appears to have been ignited, check for combustion water vapour by holding a cold, shiny object such as a mirror or the polished end of a wrench directly over the detector exit - you should observe “steam” condensing on the cold surface. If you do not, the flame probably hasn’t ignited or has gone out immediately. Many GC systems will report the FID background signal level as an indication of the presence of a flame. The minimum signal level is settable, but the default is usually suitable.

	Flame ignition problems have several causes. Foremost is an incorrect flow setting, or possibly you forgot to turn on one of the flows. With some configurations it may be necessary to use a bakeout or startup GC method that sets the hydrogen flow rate above optimum for an even richer ignition mixture. Be sure the analytical method has the correct hydrogen flow.

	Make sure that all flows are correct and that the gases are connected correctly at the back of the instrument. Flame ionization detectors will produce a very loud “pop” on ignition if the hydrogen and air lines are reversed, but the flame will usually go out immediately. Be very cautious because a large, invisible hydrogen flame that extends several centimetres above the detector may result from reversed connections.

	Continued ignition difficulty may be caused by a defective igniter or other hardware problem. To check a built-in igniter, first turn off the hydrogen flow. Then press the igniter button while indirectly observing the inside of the detector with a small angled inspection mirror. For a manual igniter, observe the internal element; you should see an orange glow, or with a piezoelectric igniter you’ll see the spark. If not, then check the igniter connections and replace the igniter element if needed.

	Other hardware problems that cause difficult ignition include a broken or cracked flame jet, poor detector or column installation causing leaks around the detector body, or a poorly fitting flowâmeasurement adapter plug that gives inaccurate flow measurements. If the detector had been operating well and then suddenly quit, check for a blocked jet tip by measuring the hydrogen flow. If required, replace or remove and clean out the jet carefully with a cleaning wire, following the manufacturer’s maintenance procedures.

	It is possible to insert a capillary column through the flame jet. This will cause a large detector background signal, poor sensitivity, and usually some difficulty igniting the flame. If ignition does occur, the tip of the column can be observed - with a mirror - glowing brightly in the flame. Establish the correct ferrule-to-tip distance when installing a column.

	Sometimes the flame may blow out just after injection; the solvent peak may be large enough to interrupt the flame. If this occurs often, change to a flame jet with a larger internal diameter if possible, and adjust the hydrogen flow to more closely match the carrier flow rate, being mindful of a possible sensitivity compromise. If the problem persists, you should try reducing the amount injected, using a lower carrier-gas flow rate, or both. If using a 0.53- or 0.75-mm i.d. capillary column, the problem may be caused by the proximity of the column exit to the flame jet. It may be helpful to withdraw the column somewhat or install a deactivated detector-column adapter.

	The flame ionization detector is generally reliable after it is properly set up. Operators can check a few key areas immediately when previously good detector performance falls below the minimum required for the application. FID is subject to two broad trouble categories: contamination and electronics. Of these, contamination is by far the more common.

 Everything that passes through a flame ionization detector is burned in the hydrogen flame. For carbonâbased substances within normal levels, carbon dioxide and water are formed. Large amounts of chlorinated compounds or carbon disulphide, however, are not burned as efficiently as hydrocarbons. These materials can produce significant quantities of carbon particles (soot) as well as hydrogen chloride in the case of chloromethanes and carbon tetrachloride. Carbon particles tend to aggregate between the jet and the collector, forming an electrical leakage path, and the result is a high, noisy baseline. Hydrogen chloride from chlorinated solvents can be tolerated in small quantities, but after extended exposure in combination with the water of combustion hydrogen chloride may corrode the detector’s inner surfaces, producing electrical leakage paths and a high, noisy baseline.

	Another common contamination source is stationary-phase bleed from the column. Although this is not generally a problem for most capillary columns, packed columns as well as thick film capillaries can emit significant amounts of stationary phase, especially at elevated temperatures. Siloxane polymers produce silicon dioxide when burned in a hydrogen flame. In a flame ionization detector these silica particles tend to adhere strongly to the jet and collector surfaces. These, in turn, can reduce sensitivity and increase the background signal level.

	To check for detector contamination, shut off the combustion-gas flows and turn off the power to the instrument. After the instrument has cooled sufficiently, remove the detector covers and examine the outside of the detector body near the exit. It should be clean and completely free of deposits. Look down into the detector. Again, the surfaces should be clean and free of deposits. If you observe some material inside the detector, remove the collector electrode for a closer look. A black deposit indicates carbon formation. White or grey deposits are typical of silicon dioxide contamination, and green or blueâgreen deposits or corroded areas are a sign of excessive acid formation.

	Light deposits of silicon dioxide or carbon can usually be removed from the collector by gentle scrubbing with distilled water and surfactants or in an ultrasonic bath. Be sure to first remove the collector electrode from any attached electrical connections. Ceramic insulators can also be cleaned in this manner. In general, follow the manufacturer’s recommended maintenance procedures. Detector parts that have been corroded should be replaced because cleaning is ineffective.

	When the detector is reassembled, be sure that internal connections for the polarizing voltage or the collector electrode are secure. Electrical contacts can be cleaned by gently wiping them with a clean pencil eraser. Do not use any abrasives or emery cloth on detector parts - you will cause more harm than good.

FID produces minute picoampere currents. The electrometer–amplifier circuit is thus very sensitive. Although modern amplifiers and power supplies are very reliable, they do fail occasionally. Often, however, what appears to be an electronic problem is actually because of operator error. Check all instrument settings and external connections before assuming that the problem is electronic. Detector electronic and thermal parts should be tested or replaced only by a trained service technician. If the detector does not heat or the instrument reports that the temperature sensor is defective, you should not attempt to fix the problem yourself. Call in a qualified technician.

	The flame ionization detector is the most familiar and widely used GC detector. It provides high sensitivity to a wide range of compounds as well as reliable routine operation. Common FID problems are few and easily identified. However, it is very important to remember that a gas chromatograph is a system that relies on the proper functioning of all its components. A problem that appears to be detector-related may in fact originate elsewhere. Perform at least a brief check of all related instrument components before concluding that the detector is at fault.

Chapters in the Evolution of Chromatography

 (Imperial College Press, London, 2008), pp. 303–320.	

 is a senior scientist at Serveron Corporation in Beaverton, Oregon, USA, and is a member of the 

 editorial advisory board. Direct correspondence about this column should be addressed to “GC Connections”,

, Honeycomb West, Chester Business Park, Chester, CH4 9QH, UK, or email the editor-inâchief, Alasdair Matheson, at 

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