The 15th Multidimensional Chromatography Workshop

Trends in Sample Preparation

Recent Advances and Applications of Passive Sampling Devices

Case Studies Dealing with Cannabis sativa L.

From “Green” to “Sustainable” Sample Preparation in Omics Studies in the Natural Product Field

A Focus on Biopharmaceutical Analysis Column

Characterization of Adeno-Associated Virus (AAV) Gene Therapy Products

The 18th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-18) will be held from 28–31 May 2024 in Leuven, Belgium.

The 18th International Symposium on Hyphenated Techniques in Chromatography and Separation Technology (HTC-18)

 in Leuven, Belgium. The HTC symposium series started in 1990 and is firmly established as a unique meeting place for scientists and practitioners from academia and industry to exchange state-of-the-art knowledge and gather new insights and ideas covering all aspects of chromatography and separation techniques, sample preparation, detection, and data handling. Organized under the auspices of Ghent University (UGhent), the University of Leuven (KU Leuven), the Vrije Universiteit Brussel (VUB), and the Royal Society of Chemistry (RSC), HTC-18 is expected to gather over 250 participants.

Town Hall in center of Leuven at sunset, Belgium | Image Credit: © Mistervlad - stock.adobe.com

The conference will encompass parallel sessions consisting of plenary lectures, keynote lectures, tutorials, and oral presentations. The symposium will also host an attractive technical exhibition where vendors will present their newest instruments and developments, topped with technical seminars.

Techniques to be covered include the fundamental and practical aspects of liquid-phase (LC) and gas chromatography (GC), including multidimensional LC, multidimensional GC, supercritical fluid chromatography (SFC), LC–mass spectrometry (MS), and GC–MS. The programme will also include topics such as automated sample preparation, online analyzers and sensors, electro-driven separations, ion-mobility and native mass spectrometry, emerging detectors and separation modes, method development and artificial intelligence, column technology and stationary-phase developments, system design and optimization, miniaturization, lab-on-a-chip, 3D printing, and data processing, mining, and curation. The symposium will also address bottlenecks and describe trends and new technologies for a wide range of applications, including (bio-) pharmaceuticals, medical diagnostics and clinical applications, forensic analysis and doping control, food analysis and safety, environmental analysis, energy, green approaches, high-throughput analysis, -omics (lipidomics, metabolomics, proteomics), and biomarker discovery, as well as modern industrial applications and natural products.

As always, there will be abundant networking opportunities during the conference, with a welcome reception in the University Hall, an informal Belgian beer and food tasting event at the conference venue, the conference dinner in the Great Beguinage, which dates from the 13th century and is listed as UNESCO world heritage, and lots of dissemination activities.

A number of awards will be presented at HTC-18. The 

 will be offered to a researcher with less than 15 years’ experience after obtaining his or her PhD for “a pioneering contribution to the field of separation sciences by introducing new methodologies, new instrumentation, or new techniques in the field, with a strong focus on applicability”. The most innovative oral contribution presented during the conference will receive the 

 The most innovative poster contributions will receive the 

 will also be organised for young scientists, sponsored by the 

Participants are invited to register and submit abstracts for review using the abstract submission menu available on the website: 

. The deadline for oral abstract submission is 

The poster abstract submission deadline is 

The organisers look very much forward to welcoming you in the historic centre of Leuven!

For more information and registration, visit the HTC-18 webpage at: 

Articles

HTC-18 Preview

The use of split injection minimizes analyte breakdown, reduces solvent on the column, and produces sharp Gaussian peak shapes. The drawback of this technique is the loss of sensitivity, as only a small percentage of the sample reaches the analytical column while the remainder of the analyte is vented. The use of splitless injection allows most of the sample to reach the column. The disadvantage of this technique is solvent interference with target compounds and analyte breakdown in the inlet. This article will detail splitless modes of operation using an optimized pulsed injection.

 has managed a team of chemists in Restek’s innovations laboratory since 2004. Before taking the reins of the laboratory, he spent seven years as an environmental chemist and was critical to the development of Restek’s current line of volatile GC columns. Chris holds a B.S. in environmental science from Saint Michael’s College, USA.

Split and splitless injections introduce the sample into a heated injection port as a liquid where it is rapidly and completely vaporized along with the analytes in the sample. The vaporized sample condenses at the head of the column along with the solvent. In split injection mode, only a small amount of the vaporized sample is transferred onto the head of the column; the remainder of the sample is removed via the split vent line. The use of split injection minimizes analyte breakdown, as compounds spend less time in the hot injection port; however, loss in sensitivity is observed because much of the sample exits the split vent line. In splitless injection, the split valve is closed long enough to allow most of the vaporized sample and solvent in the injection port liner to be transferred to the analytical column; this can take anywhere between 30 and 90 seconds. As the split vent valve is closed, the sample transfer moves at the speed of the column flow—typically in the 1–2 mL/min range (1). Solvent focusing in splitless mode requires an oven temperature 20 °C below the boiling point of the sample solvent, thereby allowing the solvent and the analyte to condense and wet the stationary phase. One exception is analyte focusing, which can be performed if the difference between the analyte boiling point and solvent boiling point is 150 °C (2). There are many analyses that use solvents and volatile organic compounds (VOCs) where solvent focusing and analyte focusing are not possible. This article will evaluate different solvents and compounds that cannot easily be analyzed using classical splitless injection and evaluate pressure pulse injections.

Silhouette of businessman holding target board on the top of mountain with over blue sky and sunlight. It is symbol of leadership successful achievement with goal and objective target. | Image Credit: © Dilok - stock.adobe.com

A 30 m × 0.25 mm, 0.25-μm (Restek) XLB-type column was used with the following oven program: 35 °C starting temperature with a hold time of 4 min followed by a 35 °C/min ramp to 330 °C with no final hold time. Analysis was performed using an Agilent gas chromatograph mass spectrometer (GC–MS) with a scan range of 35 to 550 amu. Seven different methods were performed as follows: 5:1 split analysis, splitless 1 min hold time, splitless 0.5 min hold time, splitless 0.25 min hold time, splitless 0.15 min hold time, pulsed splitless 30 psi pulse 0.64 min hold 0.59 min, and pulsed splitless 30 psi pulse 0.64 min hold 0.64 min. In addition, other pulsed splitless conditions were evaluated and are briefly
mentioned in the results and discussion section.

Pyridine and 1,4-dioxane were made up at a concentration of 10 ppm in both methylene chloride and methanol individually. They were also made up in methylene chloride and separate standard mixes spiked with 2% methanol, 2% benzene, and 2% 2-ethoxyethanol (EGEE) to evaluate the effects of co-solvents on the standards under different injection port conditions.

The first interesting observation is the shift in retention of the analyte related to the amount of solvent entering the column. 1,4-Dioxane eluted at 3.50 min under split conditions where a 5th of a microlitre of methylene chloride was injected onto the column. Because the amount of solvent on-column increased (increase in hold time), the retention also increased, with the most retention occurring for peak number four in Figure 1, which had a retention time of 3.83 min. This phenomenon was documented in a 2021 article in 

, which described increases in retention even when switching from a 200:1 split to a 25:1 split (3). In addition, in splitless mode it requires more time to transfer the analytes from the injection port to the head of the column. The split analysis, not surprisingly, produced good peak shape and lower response compared with the splitless injections. As the boiling point of methylene chloride is 40 °C, we were unable to perform a splitless injection 20 °C below the boiling point of the solvent without cyrogenic cooling. Analyte focusing will be unsuccessful because 1,4-dioxane’s boiling point is 101 °C, which is less than the 150 °C difference in boiling points between the solvent and analyte needed for the technique. Performing a classical splitless injection with a 1-min hold time resulted in split and tailing peak shapes. Only the splitless hold time of 0.15 min produced an acceptable peak shape, with an area 2.5× that of the 5:1 split analysis. Splitless pressure pulses with a 70 psi pressure pulse were evaluated and resulted in no analyte response because the 1,4-dioxane and methylene chloride coeluted. Using previously published optimized splitless pulse conditions produced the best results (4). A slight adjustment to the hold time during the pulse did not change the results, although it is important to remember that the hold time should be the same or less than the pulse time to prevent excessive amounts of solvent from entering the column. Tony Taylor’s 

 Blog details many other factors for the optimization of pressure pulse injections (5). Pyridine and 1,4-dioxane in methanol were analyzed and none of the seven methods produced acceptable peak shapes, with the longer splitless hold time methods producing smeared peak shapes. This was most likely a function of polarity and a higher boiling point for methanol (65 °C) compared with methylene chloride (40 °C).

Figure 2 is the result of using optimized splitless pulse conditions and evaluating cosolvent combinations for method robustness. Ideally the small amount of additional solvents should work for the analysis. Even at low percentage levels (2%), EGEE caused peak distortion because the 1,4-dioxane partitioned in and out of the stationary phase along with the solvent (EGEE), which moved slowly down the column at a similar time. Konrad Grob has documented these cosolvent effects using a variety of solvents and conditions, most notably water (6).

The splitless analysis of solvents and volatile compounds is challenging because classical splitless solvent focusing or analyte focusing is not effective at producing good peak shapes. Split analysis produced excellent peak shape— even at elevated temperatures—but it did reduce sensitivity. Optimized splitless pulsed conditions provided the best peak shapes and response compared with the other five methods evaluated.

(1) Cochran, J. Split Injection GC: The Benefits of "Shoot-and-Dilute” GC. 

(2) Rood, D. Gas Chromatography Problem Solving and Troubleshooting. 

(3) English, C. Selectivity Differences Resulting from Analytes Interacting with the Solvent and Stationary Phase. 

(4) Semivolatile Organics w/Appendix IX on Rxi-5Sil MS by U.S. EPA Method 8270. Restek. Restek Corporation. 

https://www.restek.com/chromatogram-detail/GC_EV1246

(5) Taylor, T. Improve Sensitivity and Reproducibility Using Pulsed Pressure Splitless GC Injection. 

Split and Splitless Injection in Capillary
Gas Chromatography

has managed a team of chemists in Restek’s innovations laboratory since 2004. Before taking the reins of the laboratory, he spent seven years as an environmental chemist and was critical to the development of Restek’s current line of volatile GC columns. Chris holds a B.S. in environmental science from Saint Michael’s College, USA.

Splitless modes of operation using an optimized pulse injection are described.

Splitless Injections: Resolving Target Compounds from the Solvent

To quantify cyanide in different samples, a group of Brazilian scientists created a detection method based around headspace single-drop microextraction (HS-SDME). Their work was published in 

Hydrogen Cyanide dangerous poisonous gas in chemical glassware | Image Credit: © luchschenF - stock.adobe.com

Cyanide anion can be produced by certain plants as a defense mechanism against pests, which can lead to its presence being found in foodstuffs, tobacco smoke, and different types of waters. Its highly toxic nature has led to numerous agencies establishing limits for cyanide levels in water. Alongside this, monitoring cyanide levels in biological samples has been deemed critical for obtaining information on peoples’ health, since cyanide can affect the nervous, respiratory, endocrine, and cardiovascular systems. This has led to a need for creating simple, cost-effective, and reliable analytical methods for quantifying cyanide.

For this study, the scientists created a method for this purpose based on analyte volatilization, single-drop microextraction (SDME) using a selective reagent, and colorimetric quantification using a paper-based analytical device. Single-drop microextraction is a sample preparation procedure that eliminates the memory effect for subsequent procedures, as a fresh drop is used for each extraction. An offshoot of this approach, headspace single-drop microextraction (HS-SDME), also provides advantages in this regard, due to its enabling the preconcentration of volatile or volatilized analytes. This makes it possible to use water-soluble solvents and selective reagents to enhance the efficiency of analyte collection through chemical reactions. However, this approach relies on two key factors to be successful: the extraction solution’s effectiveness in capturing the analyte, and the choice of analytical technique for quantification. Regarding the first factor, colorimetric methods applied to paper-based analytical devices is an approach that, according to the scientists, “[presents] a compelling alternative that fulfills the prerequisites of affordability, portability, disposability, and miniaturization” (1). These factors and more led to the scientists using HS-SDME.

For this procedure, 10 mL of a liquid sample was acidified with phosphoric acid, with the generated hydrogen cyanide (HCN) being collected using 3 µL of a 

 dimethylglyoximate solution (Pd (DMG)22−), which was positioned in the flask headspace using a syringe. Pd(CN)42− was then formed, in addition to the organic ligand being demasked. After 15 minutes of extraction time, the reagent drop was added to a paper-based analytical device containing 3 µL of nickel chloride, creating nickel (II) dimethylglyoximate. After this, digital images were captured.

Under these conditions, the method showed a suitable linear relationship (

2 > 0.99) ranging from 26 to 286 μg/L and a limit of detection of 5 μg/L. The approach offered high sensitivity and selectivity, all while only needing a small volume of reagents. This encouraged the scientists, leading them to believe the method “exhibits a high degree of portability for in-situ applications” (1).

(1) Conrado, J. A. M.; Araújo, D. A. G.; Petruci, J. F. d. S. Combination of Headspace Single-Drop Microextraction (HS-SDME) with a Nickel-Embedded Paper-Based Analytical Device for Cyanide Quantification. 

https://doi.org/10.1016/j.aca.2023.341882

To quantify cyanide in different samples, a group of Brazilian scientists created a detection method based around headspace single-drop microextraction (HS-SDME).

Quantifying Cyanide Using Headspace Single-Drop Microextraction

 is an Assistant Professor of Chemistry at William & Mary. She serves on the ACS SCSC as Secretary. Her research specializes in the application of comprehensive two-dimensional gas chromatography for odor analysis applications, and mentors numerous undergraduate researchers as part of her integrated teaching and research program. Her current interests include odor production from post-mortem microbes, development of GC×GC data processing workflows for dual-channel detection, promoting the adoption of GC×GC in the forensic sciences, and establishing GC×GC curriculum to be taught in undergraduate chemistry classes. Direct correspondence to 

 is the editor of “LC Troubleshooting”. Stoll is a professor and the co-chair of chemistry at Gustavus Adolphus College in St. Peter, Minnesota, USA. His primary research focus is on the development of 2D-LC for both targeted and untargeted analyses. He has authored or coauthored more than 75 peer-reviewed publications and four book chapters in separation science and more than 100 conference presentations. He is also a member of LCGC’s editorial advisory board. Direct correspondence to: 

 is an assistant professor of analytical chemistry in the Department of Chemistry and Biochemistry at the California State University, Los Angeles, USA. He is also the director of the Complex Chemical Composition Analysis Lab (C

AL). His research focuses on characterizing complex chemical mixtures using state-of-the-art techniques, such as two-dimensional gas chromatography (GC×GC) and high‑resolution mass spectrometry (HRMS). His primary focus is on developing methods for analyzing products from plastic waste conversion processes and organic compounds adsorbed on microplastics. He has one patent, six military reports, 25 journal publications, and over 30 research presentations, which include 10 invited lectures and invited presentations. He was invited as a keynote speaker to the 14th Multidimensional Chromatography Workshop in 2023.

The 15th Multidimensional Chromatography Workshop is a free event involving keynote and contributed presentations, a poster session, and discussion groups on all multidimensional techniques, and will be held in-person on the campus of California State University, Los Angeles, from January 10th to the 12th, 2024. The workshop welcomes both experts and new users to the field for training, networking, and sharing the latest trends. Keynote lecturers will present on major trends in both multidimensional gas chromatography (GC) and multidimensional liquid chromatography (LC), offering insights from both academic and industry research perspectives.

Participants will be able to join guided discussions intended to stimulate discussion on key areas of the field. The full program will include popular topics including modulation technologies, data analysis, and applications in different industries and research areas. Additionally, the program features a poster session, and presenters will have the chance to compete for two poster awards:

—Sponsored by the American Chemical Society’s Subdivision on Separations Chemistry and Chromatography—$250 USD Award

This year, the workshop will highlight the work of four keynote speakers. The speakers come to us with experience in either multidimensional GC or multidimensional LC, and they represent academic, industry, and government sectors. In addition, we have asked all the keynote speakers to address the following questions in their presentations to get their perspective on the evolution of multidimensional chromatography:

What sparked your interest in multidimensional chromatography?

What was the moment you were convinced that multidimensional chromatography was a valuable solution to adopt?

What is the biggest challenge facing the field of multidimensional chromatography?

What is the most interesting application of multidimensional chromatography you have seen in the last year?

Why does multidimensional chromatography matter?

 - United States Food and Drug Administration

From Research to Routine Analysis – The Role of GC×GC in the Regulatory Space

 is a Chemist at the U.S. Food and Drug Administration in Maryland as a part of the Office of Regulatory Science at the Center for Food Safety and Applied Nutrition (CFSAN). Sarah worked as a laboratory technician at Procter and Gamble for 2 years after obtaining her bachelor’s in chemistry, before moving to Pennsylvania to obtain her master’s degree in forensic chemistry at Penn State. In 2015, she began working towards her Ph.D. in Chemistry at the University of Washington in Seattle, which she obtained in 2020. After completing a two-year postdoctoral fellowship at the U.S. Food and Drug Administration, she transitioned to her current role as Chemist and is involved in non-targeted method development for foods and dietary supplements using GC×GC. ​

Two-Dimensional Liquid Chromatography for Small Molecule Pharmaceutical Analysis – More Knowledge in Less Time

 is a Scientific Director in Chemical Process Development in Bristol-Myers Squibb Company. He received his B.S. in Chemistry and Ph. D. in Analytical Chemistry from Tsinghua University, Beijing, China. His work has been focused on CMC analytical supports for various projects within BMS portfolio, including small molecules, synthetic peptides and oligonucleotides. He has authored or co-authored 18 peer-reviewed publications and three book chapters in analytical science. His research focuses on application of novel analytical techniques to pharmaceutical analysis. He is the recipient of BMS BMSIARC award for outstanding achievement in analytical science in 2019.

On-Line Multi-Dimensional LC/MS: The Next-Generation Tool for Real-Time Monitoring of Antibody Quality Attributes in Biopharmaceutical Processes

 is currently Senior Scientist in the Department of Analytical Protein Chemistry at Genentech (a member of Roche group) in South San Francisco, California. He received his Ph.D. in analytical chemistry from Sorbonne University, France. After completing a postdoctoral fellowship at the University of Geneva, Switzerland, he joined Genentech. His research involves the development and implementation of novel multi-dimensional liquid chromatography methodologies for the characterization and on-line monitoring of biomacromolecules.

From Wildfire Origins to Courtroom Verdicts: Exploring Arson Investigations with Multidimensional Chromatography

 is an environmental chemist specializing in Environmental Forensics, particularly in wildfire forensics, arson investigation, air monitoring, and risk assessment. She combines analytical chemistry and environmental science to study the origins and behavior of pollutants and their environmental impacts.Over the course of her career, in industry, consultancy, and academia, Gwen has developed technical expertise in the areas of wildfire forensics, environmental chemistry, environmental forensics, air quality, and contaminated land and groundwater. She has worked environmental forensic investigations involving compounds of concerns including ignitable liquids, drilling fluids, petroleum hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, polychlorinated dibenzo-p-dioxins and dibenzofurans, methane, and nitrates. She has also authored numerous scientific articles, edited book series, and successfully competed for research grants both nationally and internationally.​

This year, the workshop will host guided discussions focused on two topics gaining importance in the field of multidimensional chromatography. This will also be an opportunity for individuals to interact more informally to learn more about certain topics, or to contribute their expert opinions. Guided discussions will be led by experts in multidimensional separations.

Advantages and Limitations of GC×GC in Government and Industrial Laboratories

Multidimensional separations are commonly used in government and industrial laboratories, however they are not yet widely accepted due in part to perceptions about complexity of the technique, and lack of standardized and validated methods. This discussion will be focused on strategies to elevate multidimensional separations in government and industrial laboratories.

Development of a System Performance Standard Reference Test Mixture for Comprehensive Two-Dimensional Gas Chromatography

GC×GC system performance is influenced by many parameters, including modulator type, column set, detection method, and data processing software. This discussion will be focused on the development of a standard test mixture that can be used to compare GC×GC system performance across instruments and laboratories.

Registration is completely free of charge and can be completed at 

. We hope that you will join us for this exciting event, whether you are looking to find out what multidimensional chromatography is all about, looking to improve your skills in multidimensional separations, or hoping to improve your network in the field of multidimensional chromatography.

 is at William and Mary University in Williamsburg, Virginia, USA.

 is at University of Liège in Liege, Belgium.

 is at Gustavus Adolphus College in St. Peter, Minnesota, USA.

 is at California State University in Los Angeles in California, USA.

Events

The 15th Multidimensional Chromatography Workshop is a free event involving keynote and contributed presentations, a poster session, and discussion groups on all multidimensional techniques, and will be held in-person on the campus of California State University, Los Angeles, from January 10 to 12, 2024. 

Organic light-emitting diodes (OLEDs) are widely used to create digital displays. To ensure high-performance products, the luminous efficiency and stability of OLED materials are essential and undergo strict quality control (QC). This article introduces a novel analytical method for the analysis of OLED materials using supercritical fluid chromatography (SFC) as well as purification by large-scale prep SFC. Switching from high performance liquid chromatography (HPLC) to SFC for the analysis of these organic compounds enabled a significant reduction of organic solvent consumption, decreasing both the cost of analysis and the environmental impact. SFC technology offers a valuable alternative to HPLC for analyzing OLED materials.

Organic light-emitting diodes (OLEDs) are a hot topic in the lighting industry, with some calling them “the future of lighting." In addition to being thin, flexible, light, cool to the touch, and not containing blinding light sources, they are sustainable as they significantly reduce power consumption, greenhouse emissions, and decrease non-recyclable waste.

OLEDs include a layer of thin organic (carbon-based) material that can emit light in response to an electric current. Unlike LEDs, OLEDs are manufactured in panels, resulting in diffuse light sources for a wide range of applications. Nowadays, they are widely used to create digital displays in smartphones, televisions, and PC monitors. To ensure high-performance products, the efficiency and stability of OLED materials need to undergo strict quality control (QC) (1).

The luminescent materials are classified as polycyclic aromatic compounds, where effective analytical and preparative methods are required to identify their structure and analyze impurities in high-quality OLED materials (2). This article describes the development of a novel analytical method for the analysis of OLED materials using supercritical fluid chromatography (SFC), as well as purification by large-scale prep SFC.

OLED materials (Figure 1) are nonpolar and therefore have low water solubility, which is why conventional high performance liquid chromatography (HPLC) analysis requires a high percentage of organic solvent in the mobile phase. Figure 2 shows an example chromatogram of HPLC analysis of the three OLED materials shown in Figure 1. 

Analytical Conditions for HPLC Analysis of OLED Materials 

System: Nexera XR UHPLC system (Shimadzu); column: 250 mm × 4.6 mm, 5-μm Shim-pack VP-ODS (Shimadzu); flow rate: 3.0 mL/min; mobile phase: A) 9:1 acetonitrile–water, B) tetrahydrofuran; time program: 5% B (0 min) -> 30% B (10 min) -> 30% B (10.01–15.00 min); column temp.: 40 °C; injection volume: 10 μL; detection (PDA): λ = 350 nm.

) as the main mobile phase, which is significantly cheaper to acquire and free to dispose of compared with organic solvents, it could be a more cost-effective technique than HPLC for the analysis of these components. In addition, because carbon dioxide is inert, the risk of degradation of the target compounds is lower than in conventional HPLC. To develop a suitable SFC method, a dedicated software tool (the Shimadzu Method Scouting solution) was used to set up a method screening experiment. Six different columns and four organic modifiers were evaluated creating 24 different analytical conditions to be tested. The different stationary and mobile phases under investigation are listed below.

Analytical Conditions for SFC Method Scouting for Analysis of OLED Materials

System: Nexera UC analytical SFC system; columns: 250 mm × 4.6 mm, 5-μm Shim-pack UC-PyE, Diol II, Phenyl, RP, Sil, GIS II (Shimadzu); flow rate: 3.0 mL/min; mobile phase: A) CO

, B) methanol or acetonitrile or acetone or tetrahydrofuran; time program: 2% B (0–1 min) -> 40% B (6–8 min) -> 2% B (8.01–10.00 min); column temp.: 40 °C; injection volume: 5 μL in tetrahydrofuran (250 mg/L); back pressure (BPR): 10 MPa; detection (PDA): λ = 350 nm.

The Shim-pack UC Diol II column and acetonitrile as modifier were identified as suitable separation conditions. The resulting SFC method was used for quantitative analysis of OLED materials (Figure 3).

Preparative SFC Conditions for Purification of OLED Materials

System: Nexera UC prep, preparative SFC system (Shimadzu); columns: 250 mm × 20 mm, 5-μm Shim-pack UC Diol II (Shimadzu); flow rate: 60 mL/min; mobile phase: A) CO

, B) methanol or acetonitrile or acetone or tetrahydrofuran; time program: 2% B (0–1 min) -> 40% B (6–8 min) -> 2% B (8.01– 10.00 min); column temp.: 40 °C; injection volume: 200 μL in tetrahydrofuran (5 g/L); back pressure (BPR): 10 MPa; detection (PDA): λ = 350 nm; make-up flow: 5 mL/ min (tetrahydrofuran).

Calibration curves for the three analytes of interest were prepared ranging from 1 to 250 mg/L. With 

 > 0.999 and %RSD < 0.7, the method showed excellent linearity and repeatability for all three compounds. Limits of detection (LOD) were determined as 0.13, 0.16, and 0.28 mg/L for TPD, methoxy-TPD, and NPD respectively, while consumption of organic solvent could be reduced by more than sevenfold.

 This reduction in organic solvent consumption was even more significant when looking at preparative purification. Upscaling from analytical SFC to a larger scale preparative method was easily achieved utilizing the same column packing material and column length (250 mm), with a wider inner diameter (20 mm) so that only the flow rate had to be increased.

High sample loading is important to achieve a large amount of purified compound. However, peak separation and peak shape can deteriorate when the injection volume is too large. The so-called on-column dilution function to dilute sample solvent during injection can help to reduce the effect of sample solvent and maintain good peak shape even when using a large injection volume. Experiments were carried out by changing the injection volume from 200 to 1800 μL. As can be seen in Figure
4, good peak separation and shape were obtained even with a sample injection of 1800 μL.

Simple and robust analytical and preparative SFC methods have been introduced as an alternative to the conventional HPLC analysis of OLED materials, namely TPD, methoxy TPD, and NPD. Using the same column material in both the analytical- and preparative-scale resulted in baseline separation and seamless method transfer. Switching from HPLC to SFC for the analysis of these organic compounds resulted in a significant reduction of organic solvent consumption, decreasing both the cost of analysis and the environmental impact. SFC technology offers a valuable alternative to HPLC in the analysis of OLED materials, as well as in QC.

Nakajima, K., Shimadzu Corporation Application Note 01-00135-EN, 2021.

Nakajima, K., Shimadzu Corporation Application Note 01-00136-EN, 2021.

 graduated with a diploma in chemical engineering from the Technical University NTA in Isny, Germany in 2004, and as a master of science in pharmaceutical analysis from the University of Strathclyde in Glasgow, UK, in 2005. She worked until 2006 as a consultant in HPLC method development and validation in an analytical laboratory of the FAO/IAEA in Vienna, Austria. She gained her doctorate for research in pharmaceutical sciences at the University of Strathclyde in 2010 and was employed as an HPLC specialist in the R&D department at Hichrom Ltd. in Reading, UK, from 2009. Since 2013, she has worked as an HPLC product specialist and since 2015 as an HPLC product manager in the analytical business unit of Shimadzu Europa in Duisburg, Germany.

: After earning his bachelor’s degree from Kyoto Institute of Technology in 2014 and subsequently completing his master’s degree in 2016, he began his career journey with Shimadzu in 2016. Starting as an Application Specialist, he specialized in LC, Prep LC, SFC, and MS technologies. His career path eventually took him to Shimadzu Asia Pacific in Singapore, where he assumed the role of Innovation Center Manager, leading pioneering research in state-of-the-art technologies. In 2023, he transitioned to the Shimadzu Headquarters development team, playing a role in enhancing the company’s products and technological advancements.

A novel analytical method for the analysis of OLED materials using SFC is presented.

Analyzing the Light of the Future

Scientists from Essen, Germany recently created a method for simultaneously analyzing fatty acids (FAs) and fatty acid methyl esters (FAMEs). Their work was published in 

Albumin molecules transporting fatty acids, hormones and drugs in the blood | Image Credit: © Juan Gärtner - stock.adobe.com

Fatty acids (FAs) and fatty acid methyl esters (FAMEs) are substance classes that are used in many fields, including medicine, microbiology, industrial production, and more. Simultaneously analyzing these substances can be advantageous in various applications, since they are related, can be converted into each other, and often appear in the same environments together.

Usually, gas chromatography (GC) is used to analyze FAs and FAMEs. However, according to the scientists, there have been no methods capable of analyzing FAs and FAMEs in one run. Part of this can be attributed to classically utilized derivatization techniques for FAs in GC analysis, namely methyl esterification, which makes it impossible to distinguish between FAs and FAMEs. However, this study introduced a new method for simultaneous determination of FAs and FAMEs; specifically, this method includes fully automated solvent-free solid-phase microextraction (SPME) arrow headspace extraction combined with isotope-labeling in situ FA derivatization with deuterated methanol (CD3OD). According to the scientists, using the chromatographic isotope effect(ΔRt = 0.03 min), which refers to changes in chromatographic retention order that stems from differences in the isotopic content of molecules, and the + 3 m

 mass shift, FAs can be selectively differentiated from the FAMEs during gas chromatography tandem-mass spectrometry (GC–MS/MS) operated in the multiple reaction monitoring (MRM) acquisition mode.

GC–MS/MS in MRM mode involves isolating specific precursor ions in the first mass analyzer, fragmenting them, and then selectively monitoring predefined fragment ions in the second mass analyzer. This targeted approach enhances the sensitivity, selectivity, and quantitative precision of the analysis. MRM allows for the detection and quantification of specific compounds in complex samples.

To predict the retention times of deuterated compounds, a different approach was presented using the internal standards. Using design of experiments, the optimized derivatization conditions were found to be 20 min, 50 °C, 4 v/v% CD3OD, and pH 2.1. Calibrating FAs and FAMEs was done in the sample matrix by spiking the required amount of respective mixes to the matrix solution. This was done in different concentration ranges by standard addition in five real matrices and ultrapure water, leading to good linearities and method detection limits for FAs ranging from 1–30 µg/L and for FAMEs from 0.003–0.72 µg/L. Altogether, these compounds were detected in samples from surface water, wastewater treatment plant effluent, and bioreactor samples, held in concentrations ranging from 2–1056 µg/L for FAs and 0.01–14 µg/L for FAMEs.

(1) Tintrop, L. K.; Lieske-Overgrand, J. R.; Wickneswaran, K.; Abis, R.; Brunstermann, R.; Jochmann, M. A.; Schmidt, T. C. Isotope-Labeling In Situ Derivatization and HS-SPME Arrow GC–MS/MS for Simultaneous Determination of Fatty Acids and Fatty Acid Methyl Esters in Aqueous Matrices. 

https://doi.org/10.1007/s00216-023-04930-1

(2) Aslani, S.; Armstrong, D. W. Effect of Position of Deuterium Atoms on Gas Chromatographic Isotope Effects. 

https://doi.org/10.1016/j.talanta.2023.124857

Scientists from Essen, Germany recently created a method for simultaneously analyzing fatty acids (FAs) and fatty acid methyl esters (FAMEs).

Measuring Fatty Acids and Fatty Acid Methyl Esters Using GC–MS/MS

This article provides a guide for laboratories on optimizing chemical procurement to achieve precise and reliable results in chromatography analysis. It explores the critical relationship between the quality of procured chemicals and the accuracy of chromatographic data, emphasizing the importance of purity, compatibility with analytical instruments, batch-to-batch consistency, and cost-effectiveness. The article also outlines best practices in vendor selection, quality assurance, and storage and handling of chemicals.

Chemicals serve as the lifeblood in chromatographic processes, facilitating the separation, identification, and quantification of the components in a mixture. The role of these chemicals is not merely passive—they actively interact with the sample and the chromatographic column.

A study involving pesticide residue analysis revealed that the use of subpar solvents led to inconsistent peak resolutions, ultimately affecting the quantification limits (1). The quality of procured chemicals is intrinsically linked to the precision of chromatography analysis. Even minor impurities can introduce variability, affecting peak shapes and retention times, and compromise the accuracy of results.

Using poor-quality chemicals can result in baseline noise, peak tailing, and even column degradation—issues that are not only disruptive but also costly to rectify.

Key Considerations for Chemical Procurement

The procurement of chemicals is a critical factor that directly influences the quality and reliability of your results. The chemicals you choose—be it solvents, reagents, or standards—must meet stringent criteria.

Failing to optimize these key considerations can lead to compromised data, increased operational costs, and even jeopardize the integrity of the analytical process. It is imperative to review each of the following aspects to ensure that your chemical procurement strategy is aligned with achieving precise and accurate chromatographic analysis.

 The purity of a chemical is not just a specification; it’s a necessity for obtaining reliable chromatographic data. Impurities, even at trace levels, can interfere with the analyte of interest, causing deviations in quantification. For example, the presence of water in organic solvents can drastically affect the polarity and, consequently, the separation efficiency.

Compatibility With Analytical Instruments:

 Chromatography employs
a variety of analytical instruments, such as gas chromatographs (GC), high performance liquid chromatographs (HPLC), and mass spectrometers (MS). Ensuring chemical compatibility with these instruments is paramount, as incompatible chemicals can corrode sensitive parts or contaminate the system. Always consult instrument manuals and Material Safety Data Sheets (MSDS) to verify compatibility.

 Consistency across different batches of chemicals is crucial for maintaining the reliability of chromatographic analyses. Variability in chemical composition can introduce errors that are hard to trace back. Implementing rigorous quality control checks and opting for suppliers who provide Certificates of Analysis (CoA) for each batch can mitigate this issue.

 While quality should never be compromised, laboratories also need to consider the cost implications of their chemical procurement strategies. Bulk purchasing and contract negotiations can offer economies of scale. However, always weigh these cost-saving measures against the potential risk of compromised quality.

These key considerations go well beyond checkboxes to tick off—they are vital parameters that dictate the quality and reliability of your analytical results. By giving due attention, you contribute to the overall efficiency and credibility of your laboratory operations.

Chemical procurement requires more than just a keen eye for quality; it demands a strategic approach. These practices serve as the pillars that uphold the integrity of your chromatographic analyses.

Think of them as essential protocols that ensure you’re not just buying chemicals but investing in the quality and reliability of your analytical results. In a field where precision is paramount, they can be the difference between an average outcome and an exceptional one. Here are some considerations for procuring chemicals. 

 Opt for vendors who are ISO-certified and have a reputation for delivering high-quality products.

Quality Assurance (QA) and Quality Control (QC):

 Implement in-house QA/ QC protocols for incoming chemicals and consider third-party testing for critical reagents.

 Store chemicals in conditions specified by the manufacturer and employ proper handling techniques to maintain their integrity.

The best practices in chemical procurement are a framework that
ensures the quality and reliability of your chromatographic analyses from start to finish and creates a procurement ecosystem that is both efficient and effective, devoid of common chemical sourcing problems. This framework safeguards the integrity of your analyses and fosters a culture of excellence within your laboratory. With chromatography, the quality of your results is often a direct reflection of the quality of your chemical procurement strategies.

Optimizing chemical procurement is not a trivial matter. It’s a critical component that directly impacts the precision and reliability of chromatography analysis. By focusing on purity, compatibility, consistency, and cost-effectiveness, laboratories can significantly improve the quality of their chromatographic data.

1. Wahab, S.; Muzammil, K.; Nasir, N.; et al. Advancement and New Trends in Analysis of Pesticide Residues in Food: A Comprehensive Review. 

 has been a leader for nearly 20 years in consumer products and pharmaceuticals and now runs ChemDirect, a B2B marketplace for chemicals. He has been awarded as a top performer in marketing, sales, and finance. He has an MBA from Stanford University and loves building early-stage businesses.

A concise guide to optimizing chemical procurement.

Optimizing Chemical Procurement for Precise Chromatography Analysis

Molecular spectroscopy techniques, most commonly ultraviolet–visible (UV–vis), are usually thought of as analyzing liquid-phase samples, with techniques such as high performance liquid chromatography (HPLC). Although not as common as in HPLC, molecular spectroscopy can be used with gas chromatography (GC) as well. In this instalment, we examine molecular spectroscopy in combination with GC. The two most common techniques used with GC today are Fourier transform infrared spectrometry (FT-IR) and vacuum ultraviolet spectroscopy (VUV). Both provide structural and quantitative information and can be used in complementary fashion with the more common GC–mass spectrometry (GC–MS) and classical detectors. We will discuss the basics of GC–FT-IR and GC–VUV, when and when not to use them, and how they compare to and complement classical detectors.

 is the Founding Endowed Professor in the Department of Chemistry and Biochemistry at Seton Hall University, and an Adjuncy 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. Direct correspondence to: 

In a recent instalment, we discussed basic care and feeding common to all detectors for gas chromatography (GC) (1). We saw that there are myriad detectors, and that detection in capillary gas chromatography faces many challenges not seen in traditional spectroscopy. Analytes are in the vapour phase, generally at low (ppm–ppb) concentration, and are moving rapidly, often 50–100 cm/s, as they are eluted from the column. A detector must have a rapid response, high sensitivity, and ruggedness. The vapour-phase analytes are often semivolatile; they must be maintained in the vapour phase as they exit the column, so the detector is heated to prevent condensation and deposition of the analyte. Chapters in books by Poole and McNair, Miller and Snow, and CHROMacademy, 

’s online training platform, provide overviews and details about detectors (2–4).

Table 1 provides a summary of many common detectors, grouped into four categories: physical property, ionization, elemental spectroscopic, and molecular spectroscopic. The classical thermal conductivity detector (TCD), a physical property detector, is universal, detecting based on the difference in thermal conductivity between analytes and the background carrier gas. Ionization detectors, including electron ionization mass spectrometry (EI-MS) and flame ionization detection (FID), involve ionization of the analyte by various mechanisms and range from highly selective (electron-capture detection [ECD]) to nearly universal (FID). Spectroscopic detectors can be elemental, such as atomic emission (AED) or molecular. Elemental detectors often use high energy techniques to excite analytes. Like the classical spectroscopic detectors used in high performance liquid chromatography (HPLC), molecular spectroscopic detectors for GC employ lower energy photons that do not ionize or decompose the analytes, and they behave in a very similar manner to their liquid-phase cousins, with a few exceptions. A detailed discussion and analysis of molecular spectroscopic detectors for GC is provided in a 2018 review by Zavahir, Nolvachai, and Marriott (5).

As in HPLC and classical spectroscopy, spectroscopic detectors obey the well-known Beer-Lambert law, a form of which is shown in equation 1:

is the molar absorptivity of the analyte, a physical property dependent on the molecular structure, the wavelength of the incident radiation, and the sample matrix; 

is the pathlength of the cell or flow cell containing the analyte; and 

is the concentration of the analyte. This presents challenges and opportunities particular to gas chromatography that are not seen in liquid-phase analytical techniques. First, the concentration of analyte in GC is necessarily low, as analytes are in the vapour phase and diluted with non-absorbing carrier gas. Low analyte concentration requires a longer pathlength, which provides the main challenge in using optical spectroscopic techniques with GC. The longer pathlength then results in extracolumn band broadening and some resulting loss of resolution and sensitivity.

The vapour-phase analysis required by GC also provides opportunities not easily seen in liquid- or solid-phase analysis. Detection in the vapour phase ensures clean spectra, with no interference from sample matrix or solvent, assuming that the chromatographic separation is complete. It also provides spectral detail and spectral range not generally available when a solvent is present, as we will see particularly with vacuum ultraviolet (VUV) detection.

Spectroscopic detectors can provide a combination of qualitative and quantitative information. Infrared (IR) spectroscopy is a classical method for qualitative analysis; we all remember interpreting infrared spectra in our undergraduate training. IR generates signals through interaction of low-energy electromagnetic radiation that excite rotation and vibration of the chemical bonds in molecules. Ultraviolet (UV) spectroscopy has been implemented relatively recently with GC as VUV, using higher energy wavelengths, which excite high-energy electronic transitions. Traditional UV–visible (UV–vis) spectroscopy has been used with GC, but not widely (6). Interestingly, the two most common spectroscopic techniques used with GC straddle the traditional UV and visible range, with IR at lower energy and VUV at higher energy.

VUV operates in the same manner as traditional UV–vis, except at higher energy wavelengths, typically 120–240 nm, while classical UV–vis detectors used in HPLC typically operate from 200–800 nm. VUV gets its name from a classical need for a vacuum to reduce interferences from air, water, and background; however, today’s benchtop and instrument top detectors use a makeup gas to purge air and water, avoiding the need for a vacuum. A GC–VUV instrument operates like a classical UV–vis spectrometer, coupled to the gas chromatograph. A diagram is shown in Figure 1 (7).

In Figure 1, we see that effluent from the column exit is mixed with makeup gas and flows through a flow cell that serves the same role as the cuvette in a classical UV spectrophotometer. The other components are very similar to the classical instruments and detectors for HPLC. Incident radiation is provided by a modified deuterium lamp, which provides a broad spectrum of electromagnetic radiation to wavelengths as short as 120 nm. After traversing the flow cell, the light is focused onto a diffraction grating, which separates it by wavelength, then to a charge-coupled device (CCD) detector. This configuration allows rapid generation of high-quality UV spectra, enabling VUV as a rapid and highly selective detector.

Figure 2 shows GC–VUV chromatograms of xylene and naphthol isomers. Note the spectral differences between the isomers, which make them easy to identify using a spectral library search. The left side shows the different spectra for the three xylene isomers and for two naphthol isomers. The right side shows a chromatogram of coeluted xylene isomers. Their distinct spectra make them easy to tell apart. In addition to providing qualitative spectral information, VUV also has promising figures of merit with limits of detection in picograms and 3–4 orders of magnitude linear range. It is also fast, with a response time of about 10 ms, making VUV amenable to fast and multidimensional GC.

Nearly all chemists have used infrared spectroscopy, starting as undergraduates and finding it to be a powerful technique, particularly in combination with other instrumental and chemical tests for structure determination. There is a long history of 50 or more years of work with GC coupled to FT-IR, yet, because of sensitivity concerns, GC–FT-IR remains a niche technique. Figure 3 shows a block diagram of a typical GC–FT-IR instrument. Like the VUV example, it is essentially a traditional spectrometer coupled to the outlet of the gas chromatographic column, with the same caveats discussed above for all detectors applied. The most common interface between GC and FT-IR is a light pipe, which is essentially a heated flow cell coupled to the end of the capillary column. Other interfaces include matrix isolation, in which the column effluent is cryogenically trapped under vacuum onto a gold-plated collection disc, and the related direct deposition, which places an IR microscope close to the column outlet. Matrix isolation and direct deposition provide greater sensitivity, while light pipe is much simpler.

Complementary to mass spectrometry, FT-IR provides both universal and selective detection for organic analytes. Nearly all organic compounds absorb in the typical scanning range of IR spectrometers used with GC (4000–600 cm-1) providing universal detection, while scanning a spectrum provides unequivocal structural information. The combination of unique spectra, particularly for structural isomers, and structural library searching, along with retention time data, allows highly selective qualitative analysis with GC–FT-IR.

While not as commonly used as ionization, elemental spectroscopic, or physical property detectors, molecular spectroscopic detectors, most commonly FT-IR and VUV, provide useful and complementary capabilities. They are non-destructive, so they can be used in tandem with other detectors. Both FT-IR and VUV are stars for qualitative analysis, especially for difficult problems in isomer analysis not easily addressed by MS. The longstanding problems with using FT-IR with GC, mostly having less sensitivity than MS, still exist and likely relegate FT-IR to a niche detector; however, there is sufficient sensitivity with relatively simple light pipe interfacing for many applications and FT-IR does not require some of the overhead of MS, such as high vacuum. VUV is a relatively new detector that offers sensitivity between FT-IR and MS, combined with a relatively simple instrument to operate and maintain, and excellent selectivity, especially for structural isomers not easily separated by MS. For problems involving structure determination with gas chromatography, mass spectrometry is not the only option; molecular spectroscopy offers benefits that make it worth considering.

(1) Snow, N. H. Basic Care and Feeding of Your Detector. 

(2) O’Brien, A. M.; Schug, K. A. Molecular Spectroscopic Detectors for Gas Chromatography. In 

, 2nd ed.; Poole, C. F., Ed.; Elsevier, 2021; pp 371–397.

(3) McNair, H. M.; Miller, J. M.; Snow, N. H. 

, 3rd ed.; John Wiley & Sons, 2019; pp 157–175.

(5) Zavahir, J. S.; Nolvachai, Y.; Marriott, P. J. Molecular Spectroscopy – Information Rich Detection for Gas Chromatography. 

(6) Gras, R.; Luong, J.; Shellie, R. A. Direct Measurement of Trace Elemental Mercury in Hydrocarbon Matrices by Gas Chromatography with Ultraviolet Photometric Detection. 

(7) Schug, K. A.; Sawicki, I.; Carlton, D. D. Jr.; et al. Vacuum Ultraviolet Detector for Gas Chromatography. 

is the founding endowed professor 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. Direct correspondence to: amatheson@mjhlifesciences.com

Although not as common as in HPLC, molecular spectroscopy can be used with GC as well.

Molecular Spectroscopy – Underutilized Detection for Gas Chromatography

As chromatography technologies change, our approaches to using them in ways that value reliability, as well as the ways we should approach troubleshooting and fixing problems, should also change. In this instalment, I continue the discussion started last month about how some specific troubleshooting topics have evolved over time, this time focusing on mobile phase degassing. Understanding how approaches to degassing have evolved is helpful for troubleshooting problems with pumps and detection, designing reliability into new methods, and when considering updates to legacy methods that have been in use for decades.

In recent months, I’ve been hearing from several readers of this column that they’ve appreciated the more educational, “back-to-basics” flavour of some of the “LC Troubleshooting” instalments in the last year. So, last month I decided to dip a little further into that theme and provide some perspective on the evolution of various troubleshooting topics over the last few decades. My view is that this kind of perspective is particularly valuable to those who are relatively new to the field. There are some aspects of “the way we do things” that may seem peculiar on the surface, but are in fact very important to the reliability of high-performing liquid chromatography (LC) methods. On the other hand, some aspects of certain methods and ways of doing things are simply unnecessary in 2023 because LC technology has evolved in such ways that the old tricks aren’t needed anymore. Sometimes implementing the old tricks with new technology—though they provide no benefit—doesn’t do any harm, but they do add cost to analyses because they take time and resources to implement. Thus, we really ought to let them go if they are not adding any value to the method. In other words, let’s be smart about the methods we deploy. If we can’t come up with a better explanation for why something about the method is the way it is, then it’s time to let it go. In this instalment, I will discuss the evolution of degassing in liquid chromatography. In general, degassing has become much more convenient, to the extent that most users probably rarely think about it anymore. However, some of the older techniques are still useful, and even with modern techniques it is useful to have a broad sense for how things have changed, as this can impact proper instrument operation when working with different generations of equipment. Readers interested in learning more details about the degassing topic are referred to previous instalments of “LC Troubleshooting” (1,2), Dolan’s book on LC troubleshooting (3), and a very old, but very rich, paper by Bakalyar and co-workers (4).

In most applications, the biggest problem with gas bubbles inside of an LC system is that pumps generally don’t deal with the bubbles very well (see the last section below for other problems that can be significant in some applications). In the worst cases, a gas bubble can cause one or both of the check valves in a high-pressure pump to fail, causing a highly erratic flow or no flow at all. Where, then, do these bubbles come from? Although there are multiple mechanisms that can lead to bubble formation, the most practically relevant one in LC is the situation where two solvents are brought together to make a mixture (here, the mobile phase) that has a lower gas solubility than either of the individual solvents alone. This is most problematic with pumps that use the “low-pressure mixing” design. For a refresher on the differences between low-pressure and high-pressure mixing designs used in LC pumps, readers are referred to previous articles in this magazine (5). In the case of low-pressure mixing, the individual solvent components are brought together under nominally atmospheric pressure conditions before a high-pressure pump provides the force needed to push the mixture through the rest of the LC system.

An example is valuable for illustrating the point here. The solubilities of oxygen gas at room temperature and atmospheric pressure in water or ethanol are about 0.3 × 10

, respectively (4) (albeit on a mole fraction basis, meaning moles of oxygen relative to the total moles of oxygen and solvent). In other words, when saturated with oxygen, ethanol carries about 20 times more oxygen dissolved in the solvent compared with water. If we mix the two solvents in equal parts, then the amount of oxygen present in the mixture initially will be about 3.0 × 10

(mole fraction). However, the solubility of oxygen in a 0.5 (mole fraction) mixture of water and ethanol is only about 1.8 × 10

 (mole fraction). This difference of 1.2 × 10

 is the amount of oxygen in excess of the carrying capacity of the mixed solvent at saturation, and this excess will manifest in the formation of significant bubbles. In a low-pressure mixing pump, these bubbles will be drawn into the pump and may cause failure of one or both check valves. So, in this example, avoiding the problem requires that the concentration of gas dissolved in each of the individual solvents is decreased prior to mixing such that the gas concentration in the mixed solvent (mobile phase) is lower than the solubility limit of the gas in that solvent. If this is achieved, then bubble formation will not be nearly as serious. A different way of thinking about the same problem is to consider the volume of gas that will be evolved as bubbles upon mixing of two solvents. Figure 1 shows data along these lines from the work of Bakalyar and co-workers (who physically measured the volumes of bubbles formed using an apparatus for trapping the bubbles) for mixtures of methanol or acetonitrile with water (4). We see that the volumes can be quite remarkable—60 μL of gas per millilitre of mixed solvent!—and that these volumes roughly maximize around 50:50 mixtures of the organic solvent and water.

The situation with pumps that use a high-pressure mixing design is quite different. In this case the mixing point for the mobile phase components is downstream from the high-pressure pump heads, and the pressure at which the mixing occurs will be nominally the same as the column inlet pressure (that is, well above atmospheric pressure). Under these conditions the gas solubility in the mobile phase will be much higher and bubbles will not form while the liquid is under pressure. However, as the pressure drops towards atmospheric pressure at detector, bubbles may form when the gas concentration reaches the solubility limit in the mixed solvent. If bubbles form in an optical flow cell (for example, ultraviolet/ visible [UV–vis] or fluorescence), this can lead to unstable baselines, including spiking patterns.

Survey of Degassing Techniques, Old and New

Including older discussions of degassing techniques used in LC, the list includes: heating, sparging, refluxing, sonication, offline vacuum degassing, and inline vacuum degassing. I personally am not aware of anyone currently routinely using heating or refluxing for degassing, and I won’t discuss those approaches further here.

I like to think of sparging as a technique that “scrubs” dissolved gases from a liquid. The principal fact in play here is that helium is much less soluble in LC solvents than other gases, including oxygen and nitrogen. In methanol, which is particularly problematic as discussed above, the solubilities of these three gases at room temperature and atmospheric pressure are about 0.7 × 10

 (mole fraction), respectively (4). To use the sparging approach in practice, helium is deliberately bubbled into an LC solvent. Other gases dissolved in the liquid diffuse into the helium bubbles and are carried out of the liquid as the helium bubbles rise to the surface. After an initial scrubbing period (15 min of sparging will remove about 80% of the oxygen dissolved in methanol [6]), the “gas-free” solvent can be maintained on the instrument with a very low flow of helium into the solvent bottle. Helium sparging has fallen out of favour because of other approaches (mostly inline degassing, discussed below), practical inconvenience (nobody wants to deal with compressed gas cylinders if they don’t have to), and the cost of helium, meaning it is not commonly used today. That said, I do still have LC instruments in my laboratory that have the necessary plumbing and sparging stones to support this approach.

A very simple approach to degassing is to simply place a bottle of solvent to be used on the LC instrument in an ultrasonic bath for several minutes before use. Unfortunately, this approach is not very effective; 15 minutes of sonication will only remove about 30% of the dissolved oxygen dissolve in methanol (6).

In the offline vacuum degassing approach, a vacuum is applied to a bottle containing the solvent that will ultimately be used on the LC instrument. The most convenient and safe source of vacuum would be a house vacuum system, as found in many laboratories. The fundamental idea here is that reducing the pressure in the bottle decreases the solubility of gases dissolved in liquid, and any gas present above the solubility limit will spontaneously bubble out of the liquid. The vacuum can be applied using a sidearm type of flask with a hosebarb connection, or a plastic bottletop adapter with a hosebarb fitting that can be used to connect the bottle to the vacuum source. When using this approach, great care should be taken to ensure that the container placed under vacuum is not physically compromised with cracks or other defects, as this could lead to a dangerous implosion of the container. Using a safety shield when the bottle is under vacuum is a good idea. Users should also be careful to use an explosion-proof vacuum pump if using a pump as the vacuum source, particularly when working with solvents that produce explosive vapours.

In my laboratory, we have found it most effective to combine offline degassing and sonication. The additive effects of the two approaches are both visibly obvious and unforgettable. As a demonstration, I suggest preparing a 50:50 mixture of acetonitrile and water, and placing the mixture under vacuum until no bubbles are observed. Then, while the bottle is still under vacuum, place the bottle in an ultrasonic bath. A vigorous rush of bubbles will be observed, and then completed within seconds. Although we only use inline degassing (see below) for routine degassing of solvents on LC instruments in my lab, the combination of offline vacuum degassing and sonication is still very useful in situations where a complementary degassing approach is needed. For example, if one suspects that the inline degasser in a pump is not working and leading to bubble formation, degassing the solvent offline can enable a very informative test of the rest of the system to rule out the possibility that the inline degasser is the source of the problem.

Understanding how approaches to degassing have evolved is helpful for troubleshooting problems with pumps and detection.

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