In the process of a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay development for two antimicrobial drugs using their stable isotope-labeled (SIL) analogues as internal standards to correct for matrix–ion suppression effects, an unusually large scatter of data was observed. A systematic observation revealed that the analytes and their SIL analogues (internal standards) were not completely coeluted; therefore, the analytes were not experiencing matrix effects similarly. When a column with lower resolution ability was used to achieve complete overlapping of the analyte and internal standard peaks, the scatter of LC–MS/MS data was minimized, indicating that the maximum correction of matrix effects by internal standards occurs when they are completely coeluted with the analytes. This study highlights the importance of ensuring complete overlapping of analyte and internal standard peaks in eliminating matrix effects when using SIL analogues as internal standards in LC–MS.

In liquid chromatography coupled to mass spectrometry (LC–MS), the ion suppression–enhancement effects because of the sample matrix can significantly reduce or enhance the analyte response (1–6). Ion suppression is more common than ion enhancement. When the analyte is coeluted with other compounds in the sample, the coeluted compounds compete with the analyte for either the total available charge or the available surface area of the droplet in the interface of the MS detector (6), leading to a diminished MS detector signal of the analyte ion. The ion suppression effect has been shown to reduce the accuracy of an assay by as much as 26% (5). Approaches such as removing impurities by sample cleanup have not always been successful in removing the ion suppression effects (7,8). Sample clean-up procedures, such as solid-phase extraction, removes compounds that are dissimilar in physicochemical properties, such as polarity and lipid solubility, from the sample to the analyte of interest. The compounds that are similar to the analyte of interest and thereby likely to be coeluted with the analyte are therefore not removed by the sample clean-up methods (9). As a result, sample cleanup is not necessarily a solution for matrix effects. In addition, it has been shown that even trace levels of compounds in mobile phase solvents can cause ion suppression (10). Therefore, preventing matrix effects in LC–MS is often unattainable. The only practical option to obtain LC–MS data that are not affected by matrix effects is to perform a correction for those matrix effects.

Correction for matrix effects is commonly achieved by using a specialized internal standard calibration procedure; the internal standard used must have almost exactly the same physicochemical properties as the analyte so that it will behave the same as the analyte both in the column and in the MS detector. This condition is commonly achieved in LC–MS by using a stable isotope-labelled (SIL) analogue of the analyte as the internal standard. This internal standard is often coeluted with the analyte but can be resolved from the analyte in LC–MS because of the slight difference in its mass from that of the analyte. As the SIL internal standard is expected to be eluted exactly at the retention time of the analyte, the presumption is that it experiences the same matrix/ion-suppression effects as the analyte in its passage through the MS detector. Therefore, by using the internal standard calibration with a coeluting SIL internal standard, the analyte peak response is usually corrected for the ion suppression matrix effects.

Because each analyte (eluted at a different retention time) is affected by different coeluted impurities, the concentration of each analyte needs to be corrected by using its own coeluting internal standard. Therefore, a coeluting SIL internal standard is required for the quantification of each analyte.

In the process of developing an LC–MS/MS method for the determination of 

-(3-oxododecanoyl)-L-homoserine lactone (H) and fluconazole (F), using their stable isotope labelled analogues as internal standards, we observed an unusually large scatter in LC–MS data. This article describes the systematic investigation of the reason for this scatter and how the problem was resolved.

Compounds were separated using an Agilent 1100 LC binary pump and an Agilent 1100 autosampler. The columns used were as follows: A Zorbax Extend-C18, 3.5 μm 80 Å (2.1 x 50 mm) high performance liquid chromatography (HPLC) column from Agilent for method 1, and a Synergy 2 μm Fusion RP 100 Å (2.0 x 20 mm) HPLC column from Phenomenex for method 2. An API 3000 tandem mass spectrometer with a turbo ion spray interface and the software program Analyst 1.5 from Applied Biosystems were used for detection and quantification.

-(3-oxododecanoyl)-L-homoserine lactone (H) were purchased from Sigma-Aldrich. The stable isotope analogue of 

-(3-oxododecanoyl)-L-homoserine lactone, 

-3-oxododecanoyl)-L-homoserine lactone (Hd), was synthesized by Dr. S.R. Chhabra at the Center for Biomolecular Sciences at the University of Nottingham in Nottingham, United Kingdom. The stable isotope analogue of fluconazole, fluconazole-d

 (Fd), was purchased from BOC Sciences. All solvents used were of HPLC grade.

Preparation of Samples and Standard Solutions

Stock solutions of analytes and deuterated analytes were prepared in acetonitrile and stored at -20 

C. Standard solutions for a six-point calibration curve were prepared to match analyte concentrations in 1000 times dilution of samples (as described below), using 100 μL of combined internal standard solution (300 μM 

-3-oxododecanoyl)-L-homoserine lactone and 40 μM fluconazole-d

 in 10% acetonitrile aqueous solution), appropriate volumes of 100 μM fluconazole, appropriate volumes of 100 μM and 3.36 mM 

-(3-oxododecanoyl)-L-homoserine lactone, and 10% acetonitrile to make the final volume up to 1000 μL. The concentrations of calibration standards were 10, 20, 30, 40, 50, and 60 μM 

-(3-oxododecanoyl)-L-homoserine lactone; 1, 2, 4, 6, 8, and 10 μM fluconazole; 30 μM 

-3-oxododecanoyl)-L-homoserine lactone (in all six standards) and 4 μM fluconazole-d

 (in all six standards) in 10% acetonitrile.

Mobile phase A consisted of deionized water containing 0.1% (v/v) formic acid, and mobile phase B consisted of acetonitrile with 0.1% (v/v) formic acid. Both solutions were filtered through a 0.45 μm polytetrafluoroethene (PTFE) filter (Millipore) before they were used.

Separation was carried out at an ambient temperature of approximately 25 

C. The flow rate was 200 μL/min with an injection volume of 10 μL. Following injection, analytes were separated using gradient elution: For method 1, mobile phase composition was changed from 10% B to 100% B during the first 15 min, then held at 100% B for 2 min before returning to 10% B from 17 to 20 min. The original composition of 10% B was maintained for the final 8 min prior to the next injection. For method 2, mobile phase composition was kept at 12% B for the first 6 min before increasing it from 12% B to 55% B during the next 3 min. Then, it was held at 55% B for the next 6 min before changing it to 100% over the next 2 min. Then, the mobile phase composition was held at 100% for 1 min before returning to 12% B over 2 min; the original composition of 12% B was maintained for the final 6 min to equilibrate the column prior to the next injection.

Multiple reaction monitoring (MRM) was used in positive ion mode. The transitions of 298 

 ion (for Fd) were monitored for each chromatographic run. The MS parameters were optimized for each analyte to obtain the highest sensitivity. The optimized values for H were: orifice-declustering potentials (DP) of 101 V, ring-focusing potentials (FP) of 370 V, collision energy (CE) of 19 V, and collision exit potential (CXP) of 8 V. The optimized values for F were: DP of 56 V, FP of 330 V, CE of 23 V, and CXP of 16 V.

An ion spray voltage (IS) of 5000 V and entrance potential (EP) of 10 V were used. Curtain gas (CUR), nebulizer gas (NEB), and the collision gas (CAD) flows were maintained at 12, 8, and 8 L/min, respectively. The temperature of the ion spray was maintained at 400 

C. A dwell time of 1000 ms was used for all transitions. Resolution of both Q1 and Q3 were 1 amu.

An unusually large scatter was observed with both H and F data obtained from the developed method (method 1). A systematic study was therefore carried out to identify the reasons behind this phenomenon. A combination standard containing H, Hd, F, and Fd was injected and LC–MS/MS was run six consecutive times using method 1, which was the method in use at the time. The upper chromatogram of Figure 1 shows peaks for H and F along with their coeluted internal standards Hd and Fd. The uppermost row of graphs in Figure 2 shows the individual peak areas of H and F, Hd and Fd, and the peak area ratio of the analyte and internal standard for H and F (all presented as percentage deviation from the third of the six runs). It is clear from Figure 2 that the internal standards did not correct for the differences in elution and detection conditions. The chromatograms, shown in the top of Figure 1, further reveal that there were slight differences in the retention times between analytes and internal standards. Differential retention times between analytes and their deuterated analogues have been previously observed (11,12) and attributed to the small differences in lipophilicity of the internal standard and the analyte because of deuteration. The difference is more pronounced with the F/Fd pair as Fd has four deuterium atoms whereas Hd has only three. Because of the differences in the retention times, the elution and detection conditions experienced by the analyte and the internal standard differ, and the resultant ion-suppression effects experienced by the two compounds are not similar. This explains the scatter in the data after correction with the internal standard (the ratio plot on the top right graph in Figure 2). The percent standard deviations for data in ratio plot 1 are 6.67% (H/Hd), and 26.2% (F/Fd), and those for ratio plot 2 are 1.35% (H/Hd), and 1.37% (F/Fd).

To force the two compounds to be coeluted, various gradients were investigated. However, the differences in lipophilicities and the resolution capabilities of the column were too high to achieve coelution simply by changing the elution conditions. Therefore, a column with lower resolution capacity was used to promote the overlap but still to achieve similar retention of both analytes. By using an appropriate gradient with the new column (method 2), we achieved better overlap of peaks (bottom chromatogram in Figure 1). The bottom row of graphs in Figure 2 shows the effect of peak overlapping: Although there is observable scatter in individual peak areas in the first two graphs in the second row, there is insignificant scatter in the third graph (the ratio of analyte to internal standard), indicating the intended function of the internal standard. The extent of overlap in Figure 1 (top vs. bottom) is suggested as the direct cause of the extent of scatter in the last column of Figure 2 (top vs. bottom). Thus, it is clear that despite the seemingly very small difference in peak overlap, its impact on the accuracy and precision of the data is significant.

Therefore, it is advisable to ensure precise coelution by periodic surveillance of chromatograms obtained in routine LC–MS methods as well as in method development because slight changes in the column and in the eluents and solvents may affect the extent of coelution. It is also recommended to examine and ensure the linearity of analyte and the internal standard responses, within the concentration ranges expected, when using the analyte–internal standard response ratio for calibrations (13).

To obtain a significant mass resolution and to prevent cross-talk, the masses between the analyte and the internal standard must differ at least by 3 amu (12). As observed in our study, replacing the three H atoms by deuterium can change the physicochemical properties, likely lipophilicity, of the molecule significantly.

The problem of differential lipophilicity, and its effect on the change in retention, can be minimized by using alternative (to deuterated) stable isotope-labelled analogues such as C-13, N-15, or O-17 as internal standards (11,12). As analysts who develop LC– MS methods are well aware, sourcing a SIL internal standard is a challenge that is often expensive. The most common types of SIL internal standards available are deuterated analogues. The only other effective alternative to using SIL internal standards, to correct for matrix effects in LC–MS, is to use the standard addition with internal standardization (14).

The effects of incomplete coelution of the analyte and SIL internal standard on the scatter and the accuracy of the LC–MS data was studied. Compound deuteration affects the retention of analytes on reversed phase chromatography, causing the analyte and its deuterated analogue to separate slightly. This separation lead to incomplete coelution of the analyte and SIL internal standard, and consequently differential matrix effects on the analyte and the internal standard, giving rise to scattered and inaccurate results with internal standard calibration. Using a column with reduced resolution to achieve coelution of the analyte and the deuterated internal standard proved to be an effective method in overcoming the problem.

This study was funded by an International Association for Dental Research (IADR)-GlaxoSmithKline Innovation in Oral Care Award 2016 awarded to Dr. H.M.H.N. Bandara.

(2) B.K. Matuszewski, M.L. Constanzer, and C.M. Chavez-Eng, 

(3) R. King, R. Bonfiglio, C. Fernandez- Metzler, C. Miller-Stein, and T.J. Olah, 

(5) D.L. Buhrman, P.I. Price, and P.J. Rudewicz, 

(6) R.K. Boyd, C. Basic, and R.A. Bethem, 

Trace Quantitative Analysis by Mass Spectrometry

 (John Wiley, West Sussex, England, 2008).

(7) M. Ivano, V. Viette, F. Badoud, M. Fathi, M. Saugy, S. Rudaz, and J.-L. Veuthey, 

(8) F. Gosetti, E. Mazzucco, D. Zampieri, and M.C. Gennaro, 

(10) H.M.D.R. Herath, P.N. Shaw, P. Cabot, and A.K. Hewavitharana, 

(11) S. Wang, M. Cyronak, and E.J. Yang, 

(12) E. Stokvis, H. Rosing, and J.H. Beijnen, 

(14) A.K. Hewavitharana, N.S. Abu Kassim, and P.N. Shaw, 

 are with the School of Pharmacy at the University of Queensland in Queensland, Australia. 

 is with the College of Pharmacy at the University of Texas at Austin in Austin, Texas. 

 is with the College of Dental Medicine at the University of Sharjah in Sharjah, United Arab Emirates. 

 is with the Bristol Dental School at the University of Bristol in Bristol, United Kingdom. Direct correspondence to: 

Articles

Peer-Reviewed Article

Ensuring complete overlapping (coelution) of analyte and internal standard peaks can be critical for eliminating matrix effects in LC–MS/MS analysis.

The Importance of Complete Overlapping of Analyte and Internal Standard Peaks in Eliminating Matrix Effects with Liquid Chromatography–Mass Spectrometry (LC–MS)

The contribution of hops to the bitter flavors in beer is complicated by a number of factors, including the relative concentrations of humulone homologs in hops, the extraction of bittering compounds from hops, and the conversion of humulones to isohumulones during the brewing process. In this study, we describe a rapid liquid chromatography–mass spectroscopy (LC–MS) method using electrospray ionization (ESI) coupled with selective ion monitoring mass spectroscopy (SIM-MS) for analysis of humulone and isohumulone content in beer. Individual beer samples are monitored directly following filtration and decarbonization. Using multiple injections in a single experimental run (MISER), data were collected in as little as 7 min per beer sample. Results were displayed as a single “misergram,” which allowed for the convenient analysis of relative concentrations of humulones and isohumulones or hops utilization in sets of in-house brewed beers and commercial samples.

Beer is one of the most popular beverages in the world, and consistently ranks at the top of the list for consumers of alcoholic drinks. Growth of craft breweries and consumer interest in beer flavor has led to a wealth of readily available beer styles and flavors. In 1984, there were fewer than 100 breweries in the United States (1). The most predominant beer and perhaps the only style available at many locations in the United States was North American lager. By 2019, there were over 8000 breweries offering hundreds and hundreds of beer styles, as defined by the Brewer’s Association “Beer Style Guidelines” (2). This surprising variety of beers can be obtained from four basic ingredients: water, malted grain, spices (hops, fruits, and other consumables), and yeast. Control of the ingredients, brewing process, fermentation, packaging, and storage has a significant impact on the consistency of the final product, and also presents a challenge for brewing scientists monitoring the components contributing to the flavors in beer (3).

Beer bitterness derives from using hops during brewing, providing an important flavor balance to the sweet flavors of malts and grains (4). The dried hop cones or strobile used in brewing contain significant amounts of polyketides, particularly the humulones, or α-acids (AA), which can represent 2–15% of the total mass of the hops. The major homologs of the AAs are the co-, n-, and ad-humulones, which collectively account for greater than 98% of the α-acid or AA content. (5). During the brewing process, the AAs are isomerized to the more water-soluble isohumulones, or iso-α-acids (IAA), via an acyloin rearrangement with ring contraction (Figure 1). Beer bitterness is often described in terms of international bittering units (IBU), with a scale ranging from values of 5 to 100 (6). With the advent of craft brewing and very hoppy beers, some extremely high values of IBU have been claimed. The IBU numeric value has been suggested as approximately equivalent to the concentration of isohumulones in beer in parts per million (ppm). However, actual sensory beer bitterness is more complex and depends on the concentrations of a number of potential bittering agents (7–9).

The combination of extraction of humulones AA from hops and conversion to IAA during brewing is described as 

. Predicted values of IBU can be determined by calculating the amount of hops, percent AA content, volume of the wort, sugar concentration, and time of hop addition (10). This is particularly important for design and implementation of new beer recipes. The traditional method of analysis of beer bitterness relies on the spectrophotometric ultraviolet (UV) determination of IAA content of an organic extract of beer at 275 nm (11,12). A disadvantage of the spectrophotometric method is the inability to separate absorption of IAA from interfering components that absorb in the same region of 275 nm, but do not exhibit the same contribution to beer bitterness. The growth of the craft beer industry has led to a marked increase in strongly hopped and flavored beers, resulting in significant changes in the absorption profile of organic extracts of beer. Late- or dry-hopped beers, in which hops are added at the end of the boiling process or after primary fermentation, result in higher concentrations of humulone AA and related oxidized products. Their contribution to UV absorption can result in unexpectedly high values of IBU by the spectroscopic method. Complete analytical separation of all the homologs and isomers of IAA has been achieved by high performance liquid chromatography (HPLC) with UV detection (13–16) and by liquid chromatography–mass spectroscopy (LC–MS) (17,18).

We recently introduced a rapid LC–MS method for determining the AA and IAA content in commercial beer samples using selective ion monitoring (SIM) (19). By employing multiple injections in a single experimental run (MISER), we were able to analyze 70 different beer samples in a total of 74 min. The single misergram allows facile comparison of the relative concentrations of AA and IAA in the beer samples in a single chromatographic display (20,21). MISER analysis has been used in our introductory course at the University of Missouri–St. Louis, “Beer Brewing: Chemical and Biochemical Principles” (22). In this report, we take advantage of a set of beers prepared in our beer brewing course to analyze hops utilization determinants of IBU and measure beer bitterness components. Knowledge of the individual brewing recipes allowed direct comparison of predicted IBU from hops additions, measurement of IBU by spectroscopic analysis, and final determination of the concentration of AA and IAA by SIM LC–MS in the finished beer samples. A sample set of commercial beers from a local brewery are also compared for content of AA and IAA.

Methanol and acetonitrile (HPLC grade) were purchased from Fisher Scientific. Ammonium formate (NH

) and formic acid (HCOOH) were purchased from Sigma–Aldrich. Ultrapure water was obtained from a Milli-Q Integral 10 from Millipore. Standards ICS-3 (dicyclohexylamine salts of trans-iso-α-acids) and ICE-3 (hops extract standard) were obtained from the American Society of Brewing Chemists (ASBC Check Sample Service; asbc@scisoc.org).

Unhopped beer, used for preparation of standard solutions, was prepared by addition of 225 g of dry malt extract (DME) to 1 L of boiling water. The mixture held at a boil for 15 min, the hot wort cooled in an ice water bath and subsequently transferred to a 4 L glass fermentation bottle equipped with an air lock. To this mixture, 5 mg of dry yeast (Safale-05) was added. After one week, the beer was transferred to a clean, sanitized 4 L glass fermentation bottle equipped with an air lock. After an additional two weeks, the unhopped beer was transferred to standard beer bottles, capped, and stored in a refrigerator at 4 

C. Humulone and isohumulone standards were prepared by dissolving a suitable amount of AA or IAA in a minimum amount of ethanol and diluted with unhopped beer to provide standard solutions of 5, 10, 25, and 50 ppm.

Beer samples were collected immediately upon opening the specific bottle of carbonated beer. Several milliliters were transferred via gravity filtration through Whatman qualitative P8 filter paper into a 15 mL plastic centrifuge tube. Samples were capped, frozen, and stored for several days. Aliquots of the thawed samples were transferred into HPLC analytical vials for placement in the LC–MS autosampler. Samples were analyzed directly without dilution. Standard solutions of known concentration of humulone and isohumulone were prepared in unhopped beer.

Multiple injections in a single experimental run (MISER) LC–MS experiments were performed on a Shimadzu LC–MS-2010A equipped with two LC-20AD mobile phase pumps and a SIL-10AD autosampler. The mass spectrometer was operated in electrospray (ESI) negative mode with capillary voltage of 1250 V. SIM was conducted to continuously monitor ions at 347 and 361 

, corresponding to the deprotonated molecular ions of the AA and IAA homologs. The source and desolvation temperatures were 275 and 250 

C, respectively. Nitrogen nebulizing and drying gas flow rates were optimized at 1.5 L/min and 0.1 MPa, respectively.

Chromatographic separation of AA and IAA was achieved using isocratic conditions with an Agilent Poroshell 120 EC-C18 HPLC column having dimensions of 30 mm x 2.1 mm i.d. and 2.7 μm particle stationary phase. Mobile phase consisted of an isocratic mixture of 2 mM ammonium formate (HCOONH

) in acetonitrile:water (1:1) with a flow rate of 0.20 mL/min. MISER experiments were performed using sequential 5 uL injection volumes for each beer sample at intervals of 5–7 min.

Students in our introductory science course on beer brewing (22) have been preparing small batches of beer using recipes developed in class (Table I). Over two semesters with 40 students, we brewed 13 different recipes. Students worked in pairs and some teams chose identical recipes. We obtained a wide range of beer styles from very light ales (2A, 5A, 7A, and 12A) to dark stouts and porters (4A, 8A, and 9A).

The hop addition sequence is very dependent on the specific recipe. Timing of hops addition during brewing and the amount added has a significant impact on the sensory taste profile of the final beer. Hops added at the beginning of the boil contribute to beer bitterness and undergo comparatively significant conversion from AA to IAA. Additions of hops near the end of the boil contribute to flavor and aroma, but have less impact on beer bitterness because of low levels of conversion of AA to IAA. The sugar concentration of the wort (boil gravity) also has an impact on extraction of AA with higher sugar concentration resulting in less incorporation of bittering components. Hops utilization can be modeled as a function of boil time and wort boil gravity and used to calculate IBU for specific recipes. The calculated IBU value was determined using the Tinseth method from published utilization tables and free online homebrew calculators (10,23,24).

Values of IBU were determined using the industry standard spectroscopic method (11,12). Absorption of an iso-octane extract of acidified, decarbonated beer at 275 nm can be related directly to IBU. For a number of the beer samples, the spectroscopic IBU showed significant deviation from the calculated value. This is not unexpected, because variations can occur because of age and storage of the hops as well as deviations from the brewing process. Aged or improperly stored hops can result in higher concentrations of oxidized products, such as humulinones, that can contribute both to higher UV absorbance of the isooctane extract and higher perceived bitterness in the beer (25). Four of the beer samples, English brown ale, weissbier, imperial IPA, and porter (1A, 2A, 3A, and 9A) exhibited significantly higher spectroscopic IBU measurements compared to the calculated values. The imperial India pale ale (IPA) showed the greatest UV absorption, resulting in an IBU of 120 compared to the calculated value of 48. The solubility of IAA in beer is dependent on the concentrations of sugars and pH but limited to approximately 100 ppm (26). In this case of beer 3A, the unusually high absorbance and IBU value indicates the presence of additional UV absorbent compounds.

Analysis of the beer samples by LC–MS was achieved using MISER. This provides visualization of all of the beer samples in one chromatographic output or "misergram" (20,21). The beer samples were filtered, degassed, and added to sample vials for direct injection and analysis by LC–MS (Figure 2). A single 120 min misergram of beer samples 1A–13A along with IAA and AA standards was obtained using SIM in negative ion mode at 361 

. It is worth noting that the chromatographic conditions employing a short column and strongly eluting mobile phase result in the coelution of the homologs co-humulone (MW 348), ad-, and 

-humulones (MW 362). However, the isomeric AA and IAA are well resolved because of their inherently different physical properties. Therefore, we observe a single chromatographic peak for the homologs of IAA and a second more retained peak for the homologs of AA. The misergrams obtained from either set of negative parent ions (SIM at 347 or 361 

) are very similar in profile and useful for qualitative assessment of relative amounts of IAA and AA. The misergram is divided into 7-min intervals with vertical hash marks differentiating the results for each beer sample.

The IPA (11A) contains the greatest amount of IAA, while light ales 5A and 12A contain the least IAA. These chromatograms are also useful for assessing the utilization of hops or conversion of AA to IAA. Imperial IPA (3A) and English porter (9A) show low levels of utilization as reflected by greater residual amounts of AA. Both of these recipes (3A and 9A) included late hops additions during the boil, which would be expected to result in higher concentrations of un-isomerized AA. Quantitation of AA and IAA in each beer sample was determined by combined content of the individual homologs of co-, ad- and 

-humulones and isohumlones, respectively. Comparison of each beer sample was made to the standard curves obtained from commercial samples of IAA and AA in unhopped beer, as shown in Figure 3. For instance, the total concentration of AA was obtained by the sum of the ppm concentrations of cohumulone (MW 348) and n-ad-humulone (MW 362) obtained from two separate LC–MS chromatograms. The content of all homologs of AA and IAA was obtained for each beer style using the summation the concentrations in ppm obtained from two misergram plots using SIM of 347 and 361 

A set of beers was also collected from a local brewery for evaluation using MISER LC–MS (Figure 4). The sample set included a range of products from a lemon shandy prepared from a light lager and lemon soda (2B) to the more bitter pilsners (6B and 7B), double IPA (8B) and American IPA (9B). From this small set of nine beers, the American IPA (9B) shows the greatest amount of AA, as evidenced by the second peak (blue).

A comparison of peak heights of AA and IAA at 361 m/z in the MISER chromatograms (Figure 2 and 4) can also be displayed as a single scatter plot (Figure 5). This analysis is qualitative in nature because we are comparing peak heights rather than integration values and only the co- and n-IAA and AA at 361 

 are displayed. However, the scatter plot provides useful visualization of two important aspects: 1) relative amounts of AA and IAA in different samples, and 2) ratios of AA/IAA in a single sample. Similar beer styles also appear as clusters that can help identify features of particular recipes. For example, commercial weissbeer (1B) and lemon shandy (2B), known to have low bitterness and very little hops aroma, appear in the lower left hand corner of the plot. A number of student beers (weissbeer 2A, lampo bianco 5A, cream ale 7A, and cherry ale 12A) were also characterized by its low bitterness and hops aroma, appearing in the same general area of the scatter plot. Using this scatter plot from both the student and commercial samples provides a comparison between our classroom recipes with standard beer styles.

The results of this investigation illustrate the utility of using multiple injections in a single chromatogram MISER coupled with single ion monitoring LC–MS to obtain graphical, qualitative analysis of humulones/isohumulones ratios (AA/IAA) in sample sets of beers. By comparison to standard curves obtained at both 347 and 361 

, it is possible to determine concentrations of the combined homologs of AA and IAA. The technique is rapid, allowing analysis of large sample sets with a minimum of sample preparation. A simple decarbonation and filtration is all that is required prior to transfer to HPLC sample vials. The method is also useful for monitoring the brewing process and hops utilization during the boiling of the wort. Components that interfere with the traditional UV spectroscopic determination of IBU are largely eliminated by SIM and chromatographic separation. The ability to view both AA and IAA concentrations allows direct comparison of hops utilization and the sequence of hops additions in beer brewing recipes.

We are grateful to the students who participated and prepared beer samples in the University of Missouri–St. Louis course, “Beer Brewing: Chemical & Biochemical Principles” (CHEM 1021). This three credit hour course is specifically designed to fulfill the Life/Natural Sciences requirement of undergraduate degrees in the College of Arts and Sciences.

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Bruce C. Hamper, Nicholas Viriyasiri, Aaron Boland, Lorna Espinosa, Hunter J. Campbell

are with the Department of Chemistry and Biochemistry at the University of Missouri–St. Louis in St. Louis, Missouri. 

 is with the Urban Chestnut Brewing Company in St. Louis, Missouri. Direct correspondence to: 

Columns

This 7-minute method enables easy analysis of bitter flavors in beer.


Liquid Chromatography–Mass Spectrometry (LC–MS) Analysis of Hop-Derived Humulone and Isohumulone Constituents in Beer: The Bitter Truth of Hops Utilization During Brewing

For context, I work in Bristol Myers Squibb’s Chemical Process Development department, where (to grossly oversimplify) our remit is to transform discovery medicinal chemistry syntheses into scalable, safe, economical, and sustainable commercial pharmaceutical processes. For my analytical colleagues and me, our day-to-day work can span the gamut of identifying new chemical impurities, inventing and optimizing analytical methods, troubleshooting synthetic hiccups, shaping process control strategies, and researching new ways to speed development timelines. Our dominant tools are separations-based, with liquid chromatography (specifically, reversed-phase) being by far the most widely utilized. Hence, one would assume we would look for the best chromatographers, either in the fields of fundamentals or applications.

My personal observation is that, at least within North America, candidates lacking industrial experience (which is the vast majority of recent graduates) predominantly have not pursued in-depth chromatographic research. Indeed, there are a shrinking number of university groups studying the theory of separations (but if anyone reading this is writing an update to Giddings’ 

 [1], I know many who would preorder it). Perhaps the field is too mature, everything has been studied already, or a fancy software package can develop all of my separations methods for me. I hope that we can agree none of these are true.

To not limit our potential talent pool, we must expand beyond attempting to find plug-and-play scientists already possessing keen chromatographic skills. This is not to say job seekers should be totally naïve, because separations are a cornerstone of any quality chemistry education. Indeed, one component to the “What are we looking for?” question is a comprehensive analytical chemistry foundation, either learned formally or acquired experientially. As my American Chemical Society Subdivision on Chromatography and Separations Chemistry (ACS SCSC) colleague James Grinias noted (2), the topics that could be covered under the analytical umbrella far outpace the time afforded to it in an undergraduate or graduate education. Do we skip burets in favor of capillary electrophoresis, drop graphite furnaces for CCD optics, study specific types of instrument or statistical moments, and on and on?

We hope that students get broad exposure to all facets of chemical measurements, as well as opportunities to delve deeper into a select few (typically a natural result of hands-on research). The most bang for the buck will be topics that transcend subfields. Sampling theory, data evaluation, error analysis, and basic laboratory skills (meaning the ability to weigh, pipette, and use glassware, and do all these things correctly) cannot be underemphasized. Electronics, programming, or in silico modeling experience are not only valuable for more advanced courses and research, but can help differentiate otherwise similar candidates.

Nothing can replace hands-on research experience. Even at an undergraduate level, time in an academic laboratory or interning in industry will yield enormous dividends. This exposure will not only afford an opportunity to hone one’s investigative and technical skills, but it will also provide an environment that will require working within a team and being able to communicate effectively. Hopefully, it’s more than a resume builder, and gives broader insight into the world of research, either igniting a passion for scientific exploration or a realization that another field may be more fulfilling.

If I had to distill it all down to address the question “What am I looking for in a candidate?,” my answer would be “A passionate, innovative, analytical chemist.” If you do not believe me, some of my more recently hired colleagues’ PhD research areas were as varied as:

fundamentals and applications of nanopipette electrospray emitters;

single-molecule fluorescence microscopy studies of speedy DNA robots; and

development and studies of novel bio- analytical stationary phases for liquid chromatography (LC) and supercritical fluid chromatography (SFC).

If our department had limited its hiring scope to “trained separations scientists,” we would have missed out on two out of three of these successful scientists. It is not too difficult to learn how to prime a pump or light a flame ionization detector (FID). Passion, innovation, and problem-solving skills backed by a solid chemical education is where the real gold lies.

 (John Wiley and Sons, New York, New York, 1991).

 is an Associate Scientific Director at Bristol Myers Squibb. Direct correspondence to: 

When selecting new candidates for multifaceted analytical separation science jobs, we rarely find an exact match. So what should we be looking for in job candidates?

What I Am Looking for When Hiring a Candidate: An Analytical Chemist’s Perspective

We were recently flattered to participate in ChromTalks, a virtual symposium put on by 

 and CHROMacademy. Unlike conventional scientific symposia, this was touted as highly experienced speakers from industry and academia imparting key learnings from their long experience with chromatography, from heroic failures that have taught them valuable lessons, to moments of inspirational serendipity, and everything in between. This month, we present additional perspectives from our presentation. Particularly, we discuss health and safety considerations along with the role of training programs, and other often overlooked factors that may improve laboratory efficiency in the long run.

I have to admit I was a little bit flattered when asked, as an experienced chromatographer, to share the wisdom of our group’s years with a broad audience at ChromTalks. However, when I was asked to focus on our group’s failures, my emotions were crushed for a moment, until I realized they must know me too well.

For the balance of this column, I capitalize on the numerous mistakes made during the course of our career (and trust me, there have been plenty), so that you can either learn from my failures in the sample preparation realm, or laugh at my expense. Let’s discuss seven different case studies, some rather simple and somewhat humorous, and others much more serious.

The first of these failures happened in my first job after I earned my Master’s degree, where I primarily used liquid chromatography (LC). Because it took place in a regulated laboratory, I had very little experience in maintaining a chromatographic system, and, especially back in the day, I was not a very mechanical person. Later, at my first professional meetings, I heard Colin Poole claim that in the parking lot of any chemistry facility, the old, beat-up cars likely belonged to the chromatographers because they’re the ones that know how to fix equipment.

When I began my first job, I was replacing a long-time technician, and she was more interested in the social aspects of saying goodbye to all of her friends. As a result, my initial training involved reading the instrument manuals for a Varian 3700 gas chromatograph (GC) and a Hewlett-Packard 5880 GC, the two workhorses of the laboratory. Those of you that remember a Hewlett-Packard 5880 are not only getting old, like me, but remember that you had to be a bit of a master plumber to really understand its operation.

As you can imagine, those instrument manuals were not the most exciting reading in the world to a young twenty-something embarking on a new career. I had a good half day, maybe a full day, of actual training, during which I was shown some of laboratory’s practices and developed methods. The following Monday, I was on my own. During my undergraduate and MS programs, I had heard about the safety concerns associated with gas cylinders, so I was proud of myself that Monday morning when I was able to replace the hydrogen cylinder without any problem, until I injected the my first sample and saw no peaks. There would be several feet of paper on the floor (this was in the days of the strip-chart recorder) with nothing but a flat line. I checked everything; there were no leaks, all the valves were correctly open or closed, and so forth. 

Finally, over lunch, I realized that yes, the flame ionization detector requires a flame and, as observed in Figure 1, the flame requires air and some kind of fuel gas. That fuel gas is hydrogen. When the hydrogen is disconnected to replace the cylinder, the flame goes out. Once the flame was reignited, everything worked great, and I was well on my way toward what has been a somewhat successful career.

The lesson from this case study is to know your system and its operation. When I teach chromatography troubleshooting, one of the first lessons taught is that whenever you encounter a unique situation, you need to ask questions—what did you do differently, what changes were made, and so forth. Perhaps ask the questions repeatedly, in different ways, to uncover the causes of the problem you are troubleshooting. A second lesson is to evaluate your training programs for new laboratory employees.

The next case study occurred when I was in graduate school in Milton Lee’s group at Brigham Young University in the late 1980s, as he was developing Lee Scientific to bring capillary supercritical fluid chromatography (SFC) to the market. The company was evaluating different pumps and ovens from different vendors. Throughout the laboratory, we had a variety of SFC systems that Professor Lee and his graduate students had developed over the years. Each graduate student had a dedicated system, with unique detectors, cosolvent pumps, and injectors. Students often swapped system components depending on the needs of a project.

Because of all of the changes, including software upgrades, the system I was using had a software bug. First, you must know that these systems had a rotary valve, like an injector in an LC system, connecting the carbon dioxide supply tank, the CO

 pump, the cosolvent pump, and the chromatography system. What happened in my particular system was that after the syringe pump was refilled, the valve entered a continuous loop, cycling through all positions. Since I was aware of this, I simply exited the program each time I refilled the pump, and things would return to normal.

That is, until one evening when I went to the other end of the laboratory to talk to some of my fellow students. Before long, there was a loud explosion, not unlike a shotgun had been set off next to my ear. One of the plastic-coated glass solvent bottles had shattered. What happened was every time it connected the CO

 cylinder to that bottle, it released a little pressure into that solvent bottle, eventually causing it to explode. Luckily, no one was hurt.

The Lee Scientific folks came down the next morning and did a very thorough investigation. I found out a lot about how companies perform failure analysis of their equipment. I think the likelihood of this happening again is much more remote than back in the mid-1980s, given that there’s much more awareness of safety and training, as well as the development of the safety culture.

Once again our lesson is to know your system, but the corollary is that if something isn’t working properly, don’t just try to work around the system as I did. Don’t be afraid to ask questions, find the appropriate cause (the software, in my case), and actually fix the problem.

This one occurred after I started at the university and I was teaching instrumental analysis, around the time of the height of popularity of the television show “CSI.” I would end the semester with a forensics project. We would have a mock crime scene, and each student would receive one piece of evidence for analysis, with half of the students working for the prosecution and half for the defense. At the closing trial, they would provide testimony on their evidence with cross-examination by their classmates.

I would develop an elaborate crime scene; one year, that was based on a fire at a meth laboratory. Much of the evidence involved the household products (cold and cough pills, common solvents, and so forth) used in the illicit manufacture of methamphetamine. For example, at the time, you would see the kind of posters shown in Figure 2 in the back of certain retail stores. One of the items was matches used for the phosphorus. To obtain a piece of evidence and somewhat conceal it, I took the matches, cut off the head of the match, then ground it with a mortar and pestle.

All of a sudden, there was a giant flame! It should have been common sense, but if you think about what happens when you grind a “strike anywhere” match with a mortar and pestle, yes, the matches are going to ignite.

The lesson to be learned from this case study is to know your sample. The corollary is to not allow our familiarity with the sample lull us into a false sense of safety as we process our samples.

Our fourth case study is another simple, and humorous, example. While I worked at Procter & Gamble, we were looking at the fat content of Pringles brand potato crisps. I believe I was comparing different extraction procedures like pressurized liquid extraction (PLE), supercritical fluid extraction (SFE), and microwave assisted extraction (MAE).

To improve the extraction, I needed to grind the sample and mix it with diatomaceous earth. For almost all of our sample extraction methods for solids, we generally want our sample to be in the form of a free-flowing powder. In this case, I used an inexpensive coffee mill rather than a standard analytical mill. Keep in mind that this was in the mid-1990s, so coffee mills weren’t as sophisticated as the one you might have in your kitchen today. Nowadays, you can disassemble the mill to clean the blades and chamber. Our mill was one solid plastic casing, so it couldn’t be taken it apart for cleaning. I chose to clean it between every sample by wiping the chamber with a tissue moistened with acetone.

I routinely keep my hand on the lid of these inexpensive mills during operation (a lesson some contestants on “Chopped” could learn). After about a half dozen or so samples, I could feel the pressure starting to build, and then the lid popped off the mill in a kind of a mini-explosion. Evidently, this was loud enough that the personnel in the laboratories on either side of mine came running into my laboratory. Of course, I was wearing my lab coat and safety glasses. Apparently, a white powdery mix of Pringles and diatomaceous earth covered my hair and face. After ascertaining that I was fine, laughter ensued.

While to this day, we’re not entirely sure what happened; the likely cause was either acetone vapors or very fine particulates from the grinding that got down into the motor, causing the small explosion.

The lesson from this case study is to know your process—specifically in this case, the process being not only how the mill works, but how you’re going to clean it. Similar to the “strike anywhere matches” example, don’t overlook things that seemingly are simple.

On another occasion, our laboratory needed to wash or extract a piece of fabric about the size of the bath towel in boiling or, at least, hot ethanol. Because I had a meeting at one of the other technical centers, I asked our newer technician if she could perform this task. Being fairly new to the laboratory, she looked around for something that would be big enough to hold the fabric and the appropriate amount of solvent. The only sizeable container she could find was a developing chamber for thin-layer chromatography (TLC). She proceeded to place the TLC chamber on a hot plate in the fume hood, put the fabric in the chamber, cover it with probably one to two liters of ethanol, and turn on the hot plate. As things were heating, she needed to walk down to the stockroom to get some other materials.

Meanwhile, the second technician was in the laboratory working at his desk, and noticed flames emanating from the fume hood. He dutifully activated the fire alarm, and then noticed that the TLC tank had a crack in it, causing the liquid ethanol to dribble down the tank onto the hot plate, where it caught fire. He grabbed the fire extinguisher, and put out the fire before the building was completely evacuated. Again, I was off-site and this was before cell phones were prevalent, so I knew nothing about this until the next day, when I returned to the laboratory and was called into a meeting with my boss, the associate director.

Most of our common laboratory glassware is Kimax, Pyrex, or some other type of borosilicate glass. This material has a low thermal expansion coefficient and is designed for heating, because it is resistant to thermal shock. Common glass, like TLC chambers, is soda lime or other “soft” glass that is not meant for heating. When I was an undergraduate, I seem to remember having lessons in bending glass tubing and a little bit of elementary glassblowing and glassware safety training, so I learned about the difference between types of glass. These days, this is not taught, so the appropriate experience is necessary to gain this knowledge.

My associate director asked if we had completed a job safety assessment for this task. My reply was that this was not a new procedure; chemists have been heating organic solvents on hot plates for decades. His claim was that the scale was new or, at least, new to our laboratory.

The lesson from this case study is to perform a job safety assessment, even if it is for a process with which you are familiar, if it is performed in a different manner. It is also important to consider our onboarding and training programs we offer new employees and students in our laboratories: What knowledge do we assume that they have? When preparing a job safety assessment, break down the process to the component tasks, enumerate the risks associated with each task, along with the severity and likelihood of each risk. Details on job safety assessments are found on the internet if your organization does not offer specific training.

For the past 15 years or so, part of our research program has dealt with bioprocessing—that is, converting biomass from plants to materials and energy. Key to this is to separate the major components (cellulose, hemicellulose, and lignin) from each other. This is not an easy process; plants aren’t readily soluble in conventional solvents. This led us to our foray with ionic liquids. For those less familiar with ionic liquids, one example that was most relevant to our research is 1-butyl-3-methylimidazolium chloride (BMIM Cl), shown schematically in Figure 3.

When our first order of BMIM Cl arrived, a graduate student and two of her fellow students came to my office. To this day, in my entire career it’s the only time I’ve ever had someone come into my office with the Safety Data Sheet (SDS) (formerly Material Safety Data Sheet [MSDS]) and say, “Are you sure you want us to work with this material?” The former MSDS sheets were not as harmonized as the current SDS forms. The MSDS that came with the BMIM Cl looked like is was prepared by a team of lawyers working with a team of toxicologists. The MSDS had copious descriptions of acute and chronic toxicity, aquatic toxicity, warnings, and danger statements.

We have to keep in mind that with toxicity, it really depends on the route of exposure, and we only have three primary routes of exposure—oral, dermal, or inhalation. One of the advantages of ionic liquids is that they’re nonvolatile, so we don’t have to worry about the inhalation hazard, especially if we work in the fume hood. We don’t eat in the laboratory, so we don’t have to worry about oral exposure. We wear lab coats, gloves, safety goggles, and other personal protective equipment (PPE), so dermal exposure is minimized.

Our lessons with this case study are to know your reagents, and read the SDS when you order new materials. Follow the appropriate safety practice. We’re the ones as professional chemists that must deal with the consequences of the compounds we work with, yet most of us have never had a course in toxicology. Perhaps we should be lobbying our local universities to include courses in chemical toxicology in their degree programs.

This was also in the mid-1990s. For much of my career, I’ve been involved with SFE, generally with carbon dioxide. CO

 is nonpolar, or polarizable. We were interested in extracting triglycerides, or lipids, from foods. Generally, we work at temperatures of at least 80 °C and pressures of 680 atm (or 10,000 psi), or else we add a small amount of an organic cosolvent, such as methanol, for this application. Researchers have been searching for alternative solvents, and literature reports showed some use of nitrous oxide for SFE. Its critical parameters are very convenient to use, similar to that for CO

, which means we can we work at lower temperatures and pressures.

Again, looking to the chemical literature, there were a few reports on supercritical nitric oxide. Each of those reports noted to use caution with nitrous oxide, because it is a strong oxidizing agent. Yet only one of those papers took any special safety precautions to deal with this concern, and another report used nitrous oxide mixed with small quantities of methanol that resulted in a small explosion. I found one paper where a group was using nitrous oxide at 60 °C and reasonable pressures, milder conditions than I used previously for the SFE of lipids from ground coffee beans. I used an unmodified commercial SFE system with vessels that were rated for use at 10,000 psi. Everything worked fine. Being a methodical scientist, I next performed the extractions of the same condition as we did previously with CO

 (80 °C and 680 atm) and, within minutes of starting that extraction, the vessel exploded. You can see the results in the photos in Figure 4.

If you look at the bulge in the photo, the vessel split right along the wrench flats and then peeled back along the end caps. The only damage, other than ruining this vessel, was a dent on the inside of the SFE oven. If we think about it, for a fire or explosion to occur, you need an oxidizing agent (nitrous oxide), an organic fuel (the coffee grounds), and some kind of activation energy (provided by free radicals in the coffee grounds). Again, the instrument had minimal damage, and no one was hurt. We did some rough calculations, and determined that the instantaneous pressure inside that vessel was between 80,000 and 100,000 psi. The original tubing to make this vessel, prior to any machining, was rated by the manufacturer to 100,000 psi. There has only been two or three reported instances of catastrophic failure like this on any SFE equipment, at any scale.

Based on this experience, none of the safety features in any commercial SFE, as well as the one paper that they took extra precautions, would not work because the pressure buildup is so instantaneous within the vessel. How do you report this incident, so that no one else would repeat our experience? Keep in mind that, again, this was back in the time before online journals and internet search engines. Typically, laboratory safety incidents were reported in letters to the editor of 

, which are not abstracted. So, again, how would people find out about these safety incidences?

 (1). I’d like to reiterate what we said there: nitrous oxide should not be used as extracting solvent in SFE, especially with organic samples, and it should not be mixed with organic cosolvents.

This combines all of the previous lessons in this column: Know your system, know your sample, know your process, and know your reagents and solvents. Always think of all the safety considerations, don’t blindly trust what somebody has done in the literature, and think about what are the consequences if something catastrophic happens. I entered this set of experiments with full knowledge of the safety concerns, yet a potentially serious accident occurred.

A few years after this event, I attended a workshop on supercritical fluid processing taught by the Phasex Corporation. Those of you that may be acquainted with Val Krukonis, the owner of Phasex, know that he’s perhaps a bit eccentric. On the first day of the workshop, he arranged a limousine to take the participants to his laboratory. It’s the first day, so we didn’t know each other yet. In the back of the car, there are two groups of conversation and I’m in between those groups, talking to the group in front of me. I then overheard the conversation behind me on high-pressure safety. The person to my immediate right was mentioning work by Doug Raynie at Procter and Gamble with nitrous oxide and a resulting explosion. I simply turned around, offered him a handshake, and said “Hello, I’m Doug Raynie from Procter and Gamble” and then they got the full version of the story.

It was fun to relive the past and tell some stories a little bit different from our typical scientific presentations. I’ve related some of my failures, some of them humorous, some of them with quite serious potential consequences. Keep in mind, we’re all going to make mistakes, regardless of how much attention to detail we have; we just need to make sure to learn from them. As we’re undergoing the planning stages of any of our experiments, we need to make sure to pay attention to every aspect of the analytical process. I hope you have learned from my mistakes.

These seven case studies capture and expand on lessons presented at ChromTalks.

Heroic Failures in Sample Preparation

The May 2021 ChromTalks, presented by LCGC and CHROMacademy, brought together 12 world-renowned chromatography experts discussing some of the most important lessons and experiences that shaped their careers. This month, we look back at ChromTalks as I share the lessons that I learned from these great speakers, with topics including sample preparation, gas chromatography (GC), and GC–mass spectrometry (MS). We share both the successes and failures (and how these experts overcame them), and enjoy some of the hidden stories behind the work of today’s chromatography leaders. The titles of some of these presentations alone—“(MisTAQuES) in QuEChERS,” “SPME Mysteries (Causing Miseries?),” “Everything I Learned Not to Do,” “Weird Observations Straight Out of the Twilight Zone,” and “Go With the Flow”—tell us that even the greatest scientists observe strange occurrences when working through problems and developing methods. Fortunately, strange observations often breed new developments. We see key lessons from the GC speakers as we review ChromTalks 2021.

ChromTalks provided a new approach to a scientific symposium: Get the speakers to talk about mistakes and hard lessons learned that ultimately led to some of their successes. There were four days of presentations, each including industrial and academic speakers and vendor presentations. The four days encompassed sample preparation, liquid chromatography, mass spectrometry, and gas chromatography. The full presentations are available online at 

https://view.ceros.com/mjh-life-sciences/lcgc-ca-symposium/p/1

 or point your browser to chromatographyonline.com and click the link for ChromTalks or search “ChromTalks.”

The sample preparation sessions focused largely on techniques that are commonly used with gas chromatography (GC), especially QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe), presented by Dr. Steven Lehotay, of the United States Department of Agriculture, one of the inventors of the QuEChERS approach, and solid-phase microextraction (SPME), presented by its inventor, Professor Janusz Pawliszyn of the University of Waterloo (Ontario, Canada).

Mass spectrometry was discussed by Koen Sandra of the Research Institute for Chromatography in Belgium, Kevin Schug of the University of Texas-Arlington, and Susan Richardson of the University of South Carolina. All three speakers spoke of successes, failures, and strange stories as they worked to figure out two instrumental techniques at once: chromatography and mass spectrometry.

The gas chromatography session was led off by Dr. John Hinshaw, the former editor of this column for nearly 25 years. John’s chromatography career spans over 40 years with stints at Varian, PerkinElmer, and currently with Serveron. John spoke of experiences and incidents over his career that many scientists can relate to. In a broad sense, John shared the following key points for experimenting, optimizing, and troubleshooting: focus on safety first, not results; be observant and ask the right questions (What? When? Who?); get to root causes (How? Why? Test!); and make appropriate changes (desired, possible, and practical). 

Lesson #1: In practical troubleshooting or optimizing, the result may be cost effective, time bound, or high performance—you get two out of three, and, perhaps most importantly, only change one thing at a time.

John discussed the early computers and electronics that provide the foundations for today’s modern instruments and data systems, discussed in some recent “GC Connections” installments (1). These included starting on a plug-board manual computer (have you ever heard of one of these?), time shared BASIC (Beginners All-Purpose Symbolic Instruction Code) programming in the late 1960s, and working with punch card computing. The original version of the famous DryLab chromatography optimization software was written in BASIC. If a punch card had one error (or even a typo), the program would fail. Additionally, other adventures were discussed, including building a pirate AM radio station that was subsequently shut down. 

Lesson #2: Understand how your electronics and data systems work, and beware of federal and governmental regulations.

John started working for Varian as fused silica capillary columns were becoming available. They used static coating in a fish tank (complete with fish!) to apply the stationary phase to the inside of the capillary tubing. The big challenge with making early columns was drawing out the fused silica with traditional glass coating machines that did not work well, so they changed to purchasing the fused silica. Early gas chromatographs, especially when used with capillary GC, had many interesting engineering challenges. John related a troubleshooting problem that the baseline on a flame ionization detector (FID) would elevate at seemingly random intervals. They eventually solved the problem: Opening the laboratory door caused pressure changes in the laboratory that caused flow changes in the detector, so they re-engineered the detector to eliminate the flow problem. I encountered a similar problem some years later, from accidentally using a contaminated air supply. 

Lesson #3: Troubleshooting is a perfect example of John’s first admonitions: Ask the right questions, get to the root causes, and make practical changes, one at a time.

Most of John’s career was spent at PerkinElmer as an applications manager, a principal scientist in instrument and applications development, and later as a GC engineering manager. He described development of a quadrupole GC–mass spectrometry (MS) system with software development in California, product management in Connecticut, development of the quadrupoles in Scotland, and the vacuum system in Germany. Multidisciplinary teams at multiple sites for the development of new products were somewhat unusual then, but are the norm today. 

Lesson #4: Get as much multidisciplinary training and experience as you can.

Dr. Jaap DeZeeuw of Restek has been making and working with capillary columns for over 40 years, and he shared perspectives and stories from his work as a long-time column maker. He spoke about practical experiences with capillary columns, how they have evolved, and how to extend their application and lifetime. He provided true inside stories about how the chemistry behind stationary-phase coating and bonding has evolved significantly in the four decades since invention of the fused-silica capillary columns commonly in use today.

Jaap discussed how we came to use fused silica tubing, how fused silica columns are drawn and made, and why they have become so popular. Prior to the introduction of fused silica in 1979, stainless steel and glass were used, and, later, aluminum. Glass is not user-friendly and is fragile, although it is very stable. Aluminum seems promising, but coating is difficult, special connectors are needed, and aluminum is not robust for aggressive temperature programming. Deactivated stainless steel can be used, but is difficult to work with, and the thermal mass is higher. Fused silica solves most of these problems and can be run up to about 360 

C because of the polyimide coating on the outside of the column. Fused-silica columns can break if scratched by accident. 

Lesson #5: Long-term operation of fused silica columns at high temperatures can cause the polyimide coating to degrade and reduce column lifetime. Handle columns with care.

In the past, most stationary phases were made from industrial polymers and were very “dirty.” Today, major column manufacturers have in-house polymer laboratories that produce very “clean” materials. This is one reason that the temperature stability of capillary columns has improved greatly over the years. Today, there are at least a dozen polysiloxane-based stationary phases in use. Over time, however, even the newest stationary phases will degrade with temperature, so stabilizing groups and cross-linking or cross-bonding are used to make the polymer chains more rigid. 

At ChromTalks, experienced speakers shared mistakes made during their careers.

July 2021 | LCGC North America

The Importance of Complete Overlapping of Analyte and Internal Standard Peaks in Eliminating Matrix Effects with Liquid Chromatography–Mass Spectrometry (LC–MS)

Liquid Chromatography–Mass Spectrometry (LC–MS) Analysis of Hop-Derived Humulone and Isohumulone Constituents in Beer: The Bitter Truth of Hops Utilization During Brewing

What I Am Looking for When Hiring a Candidate: An Analytical Chemist’s Perspective

Heroic Failures in Sample Preparation

Hard-Won Lessons in Gas Chromatography

Recent Developments in Open-Tubular Liquid Chromatography Columns

Where Has My Efficiency Gone? Impacts of Extracolumn Peak Broadening on Performance, Part IV: Gradient Elution, Flow Splitting, and a Holistic View

Solving Challenges in the Agricultural Industry Using Separation Science: An Interview with Mary Ellen McNally