Initially, the introduction of liquid chromatography–mass spectrometry (LC–MS) into the field of doping analysis focused primarily on substances that were difficult to detect by other techniques. The first application of LC–MS in antidoping analysis was the detection of the highly polar and thermally labile diuretics. Further research also revealed that good results for nonpolar doping agents can be achieved with LC–MS. Corticosteroids, for example, exhibit a sensitivity, selectivity, and specificity not reached by any other analytical technique. In the last decade, research on the detection of all groups of doping agents has been investigated by LC–MS and routine LC–MS screening applications are now available for almost all classes of doping agents. Improvements in MS technology with respect to scan speed, sensitivity, and polarity switching allows for the combined detection of different classes of doping agents. Low physiological levels in urine and plasma mean the detection of peptide hormones is still difficult by LC–MS. In general, it is predictable that the application of LC–MS in antidoping analysis will increase.

The drive to compete and win is as old as humankind. Throughout history, athletes have sought foods and potions to transform their bodies into powerful, well-tuned machines. 

The Roman gladiators of Circus Maximus used stimulants mixed with alcohol to overcome fatigue and injuries, and Scandinavian warriors (the Berserkers) ate hallucinogenic mushrooms to gear up for battle (1). The first competitive athletes believed to be charged for doping were swimmers in Amsterdam in the 1860s. Doping controls started in 1967 after Tom Simpson died on the Mont Ventoux from a combination of exhaustion, amphetamines, and alcohol. 

In contrast to forensic sciences, in which blood is the most important matrix, urine is preferred in doping analysis. The advantage of urine is the relatively large volume that can be collected when compared with blood. Analytes also are present in urine in higher concentrations than in blood.  

In addition, urine collection is noninvasive, and its use takes into account the fact that top athletes are controlled several times during major events.  

Before the introduction of liquid chromatography–mass spectrometry (LC–MS) in the field of doping analysis, MS information was acquired with capillary gas chromatography–mass spectrometry (GC–MS). Unfortunately, this technique is only suitable for volatile or thermostable compounds. Because many doping agents have polar functional groups in their structure, their volatility is limited and before GC–MS analysis, a derivatization step is required. Unfortunately, some derivatives are thermally labile and cannot be detected. 

LC–MS analysis of these compounds does not require a derivatization step and in the 1980s, many efforts were made to interface liquid chromatography with mass spectrometry. At the beginning of the 1990s commercial LC–MS instrumentation became available in doping control laboratories and research focused primarily on substances for which GC–MS detection was difficult or impossible. In 1991, a first approach for the analysis of diuretics by LC–MS showed promising results (2). Despite these good results, technical limitations impeded its routine application. In the coming years, the quality of LC–MS instrumentation improved and concurrently, the purchase costs decreased. As a result, routine LC–MS detection methods became available in many doping control laboratories by the end of the 1990s. As a consequence, routine detection methods for several classes of doping substances were developed. These early LC–MS methods were reviewed in 2005 (3). The aim of this work is to describe the current state in the application of LC–MS in the field of human antidoping analysis. In this article. the classes of doping substances will be discussed as they appear on the WADA-prohibited list (4). An overview of common LC–MS detection strategies for doping substances is presented in Table I. 

Table I: Sample preparation and mass spectrometric conditions for the detection of doping agents by LCâMS 

Compounds Prohibited in and out of Competition 

 Anabolic steroids are synthetic derivatives of the male hormone testosterone that are used by athletes to increase their strength. Although steroids have been banned since 1976, they remain the most detected doping substances by accredited laboratories (5). The detection of anabolic steroids in human urine normally is performed by GC–MS after hydrolysis and derivatization.  

LC–MS opened the possibility of directly detecting anabolic steroids without derivatization. Unfortunately, LC–MS analysis of these analytes also presents several difficulties including limited ionization when using conventional atmospheric pressure interfaces electrospray ionization (ESI) and atmospheric chemical ionization (APCI). Due to this problem, the number of steroids included in LC–MS screening methods was originally limited to those substances for which the GC–MS detection was problematic. Three steroids (gestrinone, tetrahydrogestrinone [THG], and 39OH-stanozolol) have been included in a screening method for 28 doping agents (6), achieving limits of detections of 2 ng/mL for 39OH-stanozolol and 5 ng/mL for THG and gestrinone, respectively. Similar limits of detection (LODs) were obtained in our laboratory for the screening of these three anabolic steroids and 17α-trenbolone (7). In addition, the applicability of LC–MS for the identification of different stanozolol metabolites was pointed out. 

A more detailed study on the detection of stanozolol metabolites has been described by Thevis and colleagues (8). In this article, a method for the detection of stanozolol and three metabolites is reported with LODs lower than 0.2 ng/mL after solid phase extraction.  

Recently, ESI of anabolic steroids has been investigated in our laboratory (9), indicating that anabolic steroids containing a conjugated keto function or a nitrogen can be easily ionized as [M+H]

  while other steroids can be better ionized after the formation of adducts with mobile phase components such as ([M+NH

). These ionization experiments were subsequently used to develop a comprehensive screening method for the detection of 34 anabolic steroids (10). 

One of the main challenges of LC–MS in doping analysis is the detection of new and unknown designer steroids (11). Different approaches have been described. Thevis and colleagues (12) developed a method based upon precursor ion scan by selecting several ions typically appearing when the steroids have a common structure. This approach has been shown useful for analytes having a preselected structure. LC–MS also has shown its potential to detect unknown steroids as in the case of the new long-term metabolite of metandienone (13) or endogenous interferences, which can hamper the detection of exogenous anabolic steroids (14). 

 When LC–MS was introduced in the 1990s, doping laboratories focused on the implementation of this technique for the detection of small molecules (nonpeptide substances). Currently, these LC–MS methods are well optimized, and doping laboratories are investigating the LC–MS detection of peptide hormones in urine or blood.  

Sensitivity is the major problem for the detection of peptide hormones. Peptide hormones generally are administered in low doses and consequently, the concentrations in blood and urine are fairly low (picrogam-per-milliliter or low nanogram-per-milliliter level). In general, they are detected by immunological techniques, but because scientific evidence obtained with MS is prefered, doping control laboratories are developing LC–MS methods to confirm the presence of peptides. 

Insulin is used medically to treat some forms of diabetes mellitus. Insulin gained interest among athletes because it helps the cellular uptake of glucose and amino acids. Athletes suffering from diabetes can obtain a therapeutic use exemption (TUE). Endogenous insulin concentrations vary between 0.1 and 3.2 ng/mL. Although insulins have been on the prohibited list since 1999, the first doping-related LC–MS application was described in 2005 by Thevis and colleagues (15). Reversed-phase LC–ESI-MS-MS detection of the synthetic analogues Humalog, Novolog, and Lantus in plasma focused on the fivefold protonated molecules [M+5H]

  after immunoaffinity clean-up (Table I). The detection limit of 0.5 ng/mL should be sufficient for antidoping control purposes. 

Tetracosactide is a synthetic analogue of human adrenocorticotropin (ACTH). ACTH stimulates the release of cortisol. Because corticosteroids are on the prohibited list, this cortisol-releasing hormone also is prohibited. Recently, the detection of synacthen has been described by Thevis and colleagues (16). The plasma samples were purified using immunoaffinity chromatography and solid-phase extraction. The compounds were separated by reversed-phase LC. ESI-MS-MS detection was carried out in selected reaction monitoring. The developed method required 2 mL of plasma and allowed for an LOD of 100 fmol/mL (300 pg/mL). 

Hemoglobin-based oxygen compounds are used as artificial oxygen carriers. They can be used in endurance sports to increase oxygen transport. An important compound of this group is Hemopure. Hemopure is derived from bovine hemoglobin. Bovine and human hemoglobins differ by 15% in amino acid sequence and after tryptic digestion, bovine specific fragments were detected and selected as target compounds for the LC–ESI-MS-MS detection (17). In contrast with other peptide compounds, immunochromatographic purification was not needed because concentrations in blood are fairly high (1 mg/mL).  

 agonists) including clenbuterol, salbutamol (albuterol), fenoterol, formoterol and salmeterol are bronchodilator drugs. The use of beta2-agonists is prohibited out of competition because of their possible anabolic side effects at high concentrations. Beta2-agonists also are prohibited in competition due to their stimulating effect at higher levels than therapeutically recommended. Because beta2-agonists are used frequently for the treatment of exercise-induced asthma, athletes can obtain a therapeutic use exemption for some beta2-agonists used in metered-dose inhalers. 

The extraction and analysis of this group of substances is frequently performed together with other classes of doping substances (Table I). In our laboratory, beta2-agonists are included in the LC–ESI-MS-MS screening method for anabolic steroids and corticosteroids. The sensitivity of LC–MS for this class of substances combined with frequent abuse of inhaled beta2-agonists result in numerous adverse analytical findings (AAF).  

In Figure 1, chromatograms obtained after the extraction of a urine sample in which salmeterol was detected are presented together with the chromatograms of a control urine fortified at 0.5 ng/mL. The concentration in the real sample was estimated below 1 ng/mL. 

This class of substances is divided into aromatase inhibitors (for example, anastrozole, letrozole, exemestane), selective estrogen receptor modulators (SERMs) (for example, raloxifene, tamoxifen, and toremifene), and antiestrogenic substances (for example, clomiphene and  cyclofenil). 

Most of these substances are used in anticancer therapy. Tamoxifen, for example, is the most widely used drug in breast cancer treatment. In sports, agents with antiestrogenic activity can be used for the treatment of adverse side effects of anabolic steroid abuse like gyneacomastia and reduced testosterone production or to prolong the anabolic activity of administered anabolic steroids by inhibiting the aromatase pathway. Mazzarino and colleagues (6) describe an LC–ESI-MS-MS screening method for 28 doping agents, in which five antiestrogenic drugs were included. Sample preparation consisted of a buffered liquid–liquid extraction at pH 9.2. Detection was performed in positive ionization mode and LODs varied between 5 and 30 ng/mL. Another method describes the detection of 15 antiestrogenic substances (18). After hydrolysis, liquid–liquid extraction at pH 9.2 was used for the sample preparation. LC-ESI-MS-MS analysis was performed in 13 min. The LODs were lower than 10 ng/mL except formestane (300 ng/mL) and aminoglutethimide (100 ng/mL). In this method, only parent drugs were included, whereas according to Mareck and colleagues (19, 20), several antiestrogens metabolites should be screened for. Another paper describes the detection of six antiestrogenic substances using LC–ESI-time-of-flight (TOF) MS after enzymatic hydrolysis and solid-phase extraction (21). For all substances, the LODs were 100 ng/mL. 

The metabolism and excretion of 6-OXO, a nutritional supplement sold on the internet as an aromatase inhibitor, was investigated in our laboratory (22,23). This supplement contains androst-4-ene-3,6,17-trione as active ingredient. Androst-4-ene-3,6,17-trione is metabolized to androst-4-ene-6α-ol-3,17-dione and androst-4-ene-6α,17β-diol-3-one. An LC–APCI ion-trap MS method was developed and validated for the quantification of androst-4-ene-3,6,17-trione and its metabolites in urine. Sample clean up consists of a liquid–liquid extraction at pH 9.2 with diethyl ether after enzymatic hydrolysis. Using this method, androst-4-ene-6α-ol-3,17-dione was identified as a major urinary metabolite and androst-4-ene-6α,17β-diol-3-one as a minor metabolite (Figure 2). 

Diuretics are used therapeutically in the treatment of fluid retention. In sports, they do not enhance performance, but could mask the intake of other drugs by diluting the urine (24). Diuretics also are used to lose weight rapidly prior to competition in sports in which weight limits are set, for example, wrestling, judo, and boxing. Diuretics include polar compounds with a wide variety of physico-chemical properties and great differences in their structure. As a consequence of these differences in molecular structure, differences in ionization are observed. Thiazide diuretics represent an important group of diuretics and form abundant deprotonated molecular ions in negative ionization. Canrenone (a diuretic with a steroid skeleton), amiloride and triamterene are exclusively detected in positive ionization. Hence, both polarities are necessary to detect them all. Because these compounds can coelute, scan-to-scan polarity switching is necessary to detect them in a single run. Initially, due to instrument limitations, scan-to-scan polarity switching was not possible and two consecutive runs were necessary (25). By the end of the 1990s, LC–MS instruments allowing reliable and fast polarity switching became available, and positively as well as negatively charged diuretics could be detected in a single chromatographic run. As a consequence, several doping laboratories have developed screening methods using scan-to-scan polarity-switching for the detection of diuretics (26–28) (Figure 3). 

When LC–MS screening for diuretics was introduced in our laboratory in 2001, the method contained 18 diuretics and probenecid. Because in this screening method full-scan MS in both polarities was applied throughout the entire chromatographic run, new compounds easily could be added without modifying the MS-method. Currently, this method is extended to 26 diuretics, 21 beta-blockers, some stimulants, and carboxy-finasteride (28). Carboxy-finasteride is the target compound to detect the abuse of finasteride a 5a-reductase inhibitor (29). This compound exhibits acidic properties and can be extracted easily at pH 5.2 used for the diuretics. 

 The most well known stimulants are amphetamines. In endurance sports requiring intense anaerobic exercise, amphetamines can prolong the tolerance to anaerobic metabolism. Most stimulants are prohibited at any concentration. However, for some ephedrine-type compounds, a threshold is applied (4). This group of substances traditionally is analyzed by GC with nitrogen–phosphorus detection or GC–MS with moderate to good sensitivity. Therefore, routine application of LC–MS for this class of substances is still limited to stimulants that are difficult to detect by GC. Such substances are modafinil, adrafinil, modafinil-acid (a metabolite for adrafinil and modafinil), ritalinic acid (methylphenidate metabolite), mesocarb-hydroxysulphate (mesocarb metabolite), and strychnine (Table I). In our laboratory, these substances are included in a full-scan ion-trap MS screening method for the diuretics. The extracted chromatograms of a urine sample in which modafinil and modafinil-acid were detected are presented in Figure 4. For modafinil, the ions at 

 167 are monitored in positive mode while for the acidic metabolite, the ions at 

 273 are monitored in negative ionization. 

 296 corresponds to the sodium adduct of modafinil wether the ion at 

 167 corresponds to a benzyltropylium originating from the neutral loss of the sulfonyl-side chain from modafinil. For modafinil-acid no sodium-adduct was observed in positive ionization, but a benzyltropylium also could be observed (Figure 4). In negative ionization, the [M–H]

  could be detected together with a phenyl-benzyl anion. This phenyl-benzyl anion also corresponds to an 

 of 167. The spontaneous fragmentation of modafinil and modafinil-acid into this kathion or anion, stabilized by the aromatic rings, could not be avoided at any temperature of the heated capillary. Another stimulant included in our LC–MS screening method for diuretics is ritalinic. Ritalinic acid is a metabolite of methylphenidate and can be detected much longer in urine than the parent compound (30). Ritalinic acid is screened for by full-scan MS

 84 (Figure 5). For confirmation purposes, additional diagnostic ions are generated in MS

  (Figure 5). Another application of LC–MS is the quantification of ephedrine and cathine (threshold level 10 and 5 μg/mL, respectively) The sample preparation is limited to filtration of the urine and subsequently injection into the LC–MS system (31).  

The comprehensive detection of 27 stimulants has been investigated in our laboratory (21,32). The method consisted of a liquid–liquid extraction with diethyl ether at pH 14 and analysis of the extracts with an ion-trap instrument with APCI in positive ionization mode. The total run time was 15 min. All compounds were analyzed in MS

. The LODs for all compounds were lower than 25 ng/mL except for chlorphentermine (detection limit: 250 ng/mL). For several substances, LODs of 1 ng/mL were obtained.  

 In sports, narcotics can be used by athletes to relieve moderate to severe pain, allowing athletes to perform while they actually are injured. 

 Although several screening methods in other fields have been published, routine LC–MS screening of narcotics for antidoping purposes has been described only recently by LC–ESI-TOFMS (21). In this approach, narcotics were detected after enzymatic hydrolysis and solid-phase clean up (Table I). 

The detection of narcotics by LC–ESI-MS-MS was investigated in our laboratory (33). Chromatography was performed on a C18 column, starting at a low percentage of organic modifier to improve the retention of morphine and hydromorphone. High percentages of organic modifier are necessary for the elution of hydrophobic compounds including buprenorphine and methadone. As internal standard nalorphine was used. Two extraction solvents were evaluated, methylene chloride (CH

) and diethyl ether. With diethyl ether LODs ranged between 0.5 and 20 ng/mL and with methylene chloride LODs ranged between 0.5 and 10 ng/mL.  

Morphine is the only narcotic with a threshold level of 1 μg/mL. A LC–ESI-MS-MS method has been described for its quantification by direct injection of the urine into the LC–MS system after filtration (31). In this way both morphine and morphine-glucuronides can be detected without the need for hydrolysis and extraction in GC–MS analysis.  

 Corticosteroids are very powerful anti-inflammatory agents used for the treatment of inflammatory diseases. Before the introduction of LC–MS, their detection was performed by GC–MS. Selectivity was sufficient, but a time-consuming derivatisation step was necessary due to the low volatility of most corticosteroids. In the second half of the 1990s, LC–MS research revealed that corticosteroids were ideal compounds for LC–MS. They can be detected with both ESI and APCI, in both polarities. In positive ionization mode corticosteroids can be detected by protonated molecular ions. Because corticosteroids have no protons with a gas-phase acidic behavior (34), no [M–H]

  can be observed in negative mode. Instead, they form very abundant adducts with acetic or formic acid, which are used frequently as mobile phase additives (Figure 6). This fact has been used to distinguish between endogenous corticosteroids and endogenous anabolic steroids, which do not form such adducts in negative mode (14). Because corticosteroids and anabolic steroids only differ in the presence of the C

 are involved in the adduct formation in negative ionization (Figure 6). 

In our initial corticosteroid LC–MS-MS screening method, negative ionization was preferred. LODs of this method ranged between 0.5 and 5 ng/mL. Currently, glucocorticosteroids are included in the LC–MS-MS screening method for anabolic steroids (nonpublished results). 

A corticosteroid frequently detected during routine analysis is 16α-hydroxyprednisolone. 16α-hydroxyprednisolone is the target metabolite for budesonide (35). Budesonide is a corticosteroid available in a metered dose inhaler and is prescribed frequently for the treatment of exercise-induced asthma. 

Several compounds described here were analyzed using full-scan ion-trap MS. Although full-scan MS spectra are useful for the identification of the analytes based upon their protonated or deprotonated molecular ions, they do not provide sufficient structural information for definite identification. Hence, tandem MS is used to obtain three diagnostic ions (36). APCI and ESI are widely accepted ionization interfaces in doping analysis. Despite the introduction of atmospheric pressure photo ionization (APPI), atmospheric pressure laser ionization (APLI) or heated electrospray ionization (HESI), these ionization techniques have not yet found routine applications in doping analysis. 

Currently, routine LC–MS applications are available for almost all classes of doping substances. Improvements in MS-technology with respect to scan speed, sensitivity as well as polarity switching allow for the combined detection of different classes of doping agents (Table I). Because of the expanded use of LC–MS, the number of manufacturers producing LC–MS systems has increased and consequently, the purchase costs are decreasing. Hence, LC–MS instruments, formerly too expensive, are becoming affordable. New techniques include particularly high-resolution LC–MS techniques. These high-resolution technologies are very promising in structure elucidation of unknown compounds due to accurate mass measurements. One of the high-resolution techniques is time-of-flight (TOF) MS, which allows for the detection of several hundreds of substances with high specificity and sensitivity (21,37). Because of the full mass range, new compounds can be added to the developed method easily. TOF-MS analyzers also provide a promising tool for peptide indetification due to their extended mass range.  

Postdoctoral grants by the Flemish Ministry of Culture, Youth, Sports and Brussels (PVE and KD) and the Spanish Ministerio de Educacion y Ciencia (OJP) are gratefully acknowledged. Technical assistance by Kris Roels was greatly appreciated. 

K. Deventer*, O.J. Pozo*† , P. Van Eenoo*, and F.T. Delbeke*

Articles

Article

In the last decade, research on the detection of all groups of doping agents has been investigated by LC-MS, and routine LC-MS screening applications are now available for almost all classes of doping agents.

Detection of Doping Agents by LC–MS and LC–MS-MS

Piccaro, Inc. announced an ultra-trace gas analyzer for precisely measuring isotopic CO

, and its origin. The instrument is based upon cavity ring down spectroscopy (CRDS) technology, exclusively licensed from Stanford University, Stanford, California and further enhanced at Piccaro.  

The CRDS analyzers deliver parts-per-billion to parts-per-trillion gas sensitivity at high speed and without interference. The isotopic CO

 application was developed for the Los Alamos National Laboratory, Los Alamos, New Mexico, where the instruments were used to study CO

 ratios and concentrations in plants and the atmosphere.  

 applications also can include the monitoring of carbon sequestration and measuring mud-gas samples collected during the natural gas exploration process. Carbon sequestration is a process of capturing carbon emissions from power plants, compressing them, and putting them underground instead of into the atmosphere. Mud-gas analysis is done to help energy service providers pinpoint the quality and potential quantity of natural gases. 

Departments

Peaks of Interest- Gas Analyzer Pinpoints Types and Origins of CO2 Emissions

Pittcon 2008 — the 59th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy — was held in New Orleans, Louisiana, March 1–7, 2008, the first time the Conference has returned there since the Katrina disaster. This year's event hosted more than 1150 instrument manufacturers and laboratory suppliers in more than 2500 booths. In addition to attending the exposition, the conferees listened to 1250 oral presentations, viewed more than 1000 posters, checked numerous company seminar rooms, or attended one of 114 short courses. The total attendance was around 19,500, down 10% from 2007. 

Undoubtedly, Pittcon still remains the most important yearly international analytical exhibition, where companies introduce their latest instruments, instrument accessories, software, columns, sample preparation, and other consumable products. Because many past attendees have purchased one or more new products within three months after attending the show, most exhibitors attempt to maximize their booth traffic to meet as many potential customers as possible. 

The purpose of this report is to provide information about many of the new separation consumables and accessory products that were displayed at Pittcon 2008. In some cases, products that were introduced during 2007 but after Pittcon 2007 (1,2) might be included for reasons of completeness. The information is based upon manufacturers' responses to a questionnaire mailed in December 2007. Because of space limitations and the fact that some manufacturers did not respond to the questionnaire, this report cannot be considered an exhaustive listing of all the new products that were introduced in New Orleans. However, over the years, these Pittcon introduction summaries have provided a good source of information that would be difficult for one individual to gather during the four days of the exhibition. In addition, the products introduced have shown definite correlations to current research, development, and application activity in the separation sciences. 

As in previous years, columns and other products recommended by their manufacturers primarily for biomolecule separations or sample preparation are denoted in the tables with the designation BIO. Some of these products can be used for general high performance liquid chromatography (HPLC) separations as well, but their main emphasis is for biological samples. This year there was a marked reduction in products devoted strictly to bioapplications.  

In this month's coverage, I will look at gas chromatography (GC) products, sample preparation products, hardware, and accessories and kits for chromatography and sample preparation. 

Although gas chromatography (GC) is considered to be a relatively mature separation technique, new GC columns continue to be introduced each year at Pittcon (Table I), 11 in all for this year. All GC columns were the wall-coated open tubular (WCOT) format with the exception of one packed column. An established company, Hamilton (Reno, Nevada), has entered a new market with a full line of GC capillary columns and a few packed columns while Agilent (Palo Alto, California) has introduced a new line of 0.18-mm i.d. capillary columns that provide high efficiency and high speed without requiring modification of the GC pressure system, which is sometimes required for 0.10-mm i.d. columns. 

In recent years, there has been a GC trend in the introduction of ultralow-bleed WCOT stationary phases that allowed better detection limits, less baseline drift during temperature programmed separations, and improved compatibility with GC–mass spectrometry (MS) analyses. This year at Pittcon, many more columns are of the low-bleed, low-activity variety. Several are the common nonpolar and medium-polarity phases but based upon the incorporation of an arylene bridges into the polysiloxane backbone. This modification provides enhanced thermal stability and coating stability compared to regular methylpolysiloxane phases. In most cases, the upper temperature limits have been increased. Sometimes there are some subtle changes in selectivity with the arylene–polysiloxane phases. 

Applications-specific columns are always popular introductions at the exposition. New columns for biodiesel separations and reformulated spark ignition fuels (a two-column set) were displayed by Restek . A fluorophase that shows strong affinity for analytes containing lone pair electrons such as ketone, aldehydes, and unsaturated compounds was highlighted by VICI (Valco). Figure 1 shows a typical chromatogram of acrylate impurities on the ValcoBond VB-Fluoro capillary column. The low-bleed characteristics make this phase well suited to MS and electron capture detection, and its high thermal stability allows its use as a complementary column for high-temperature applications that commonly use low-polarity stationary phases. 

As indicated in Table II, Pittcon 2008 was another productive year with 20 new sample preparation products or families of products displayed. Not counted were sample preparation accessories (Table III), which will be covered later. Among the more popular introductions were products to deal with increasingly smaller amounts of available sample. Many solid-phase extraction (SPE) products ranging from cartridges, to pipette tips, to 96-well plates and spin tubes were among the formats introduced. Just about every mode of SPE (reversed phase, strong anion and cation exchange, normal phase, complexation, affinity, mixed mode, molecular imprinting, and hydrophilic interaction chromatography [HILIC]) was displayed. With a continuing trend, polymeric SPE phases represented a majority of the SPE products seen this year. Water-wettable polymeric phases that can dry out during operation and still have good experimental reproducibility and recovery were available more widely.  

Table III: Chromatography and sample preparation accessories 

Products for supported liquid extraction (SLE) also were noted. The SLE technique is similar to liquid–liquid extraction (LLE) except one of the two immiscible phases, usually the aqueous solution, is sorbed onto diatomaceous earth packed into cartridges or well of a well plate. As depicted in Figure 2, the aqueous phase is allowed to wet the diatomaceous earth and spread out over its surface. Then, the organic solvent (the second phase) is passed slowly through the sorbent. Due to the widely dispersed aqueous phase, there is repeated intimate contact between the two phases, allowing for a very efficient liquid extraction without the fear of emulsion formation. The Isolute SLE+ product from Biotage is an SLE 96-well plate recommended for the extraction of drugs in biological fluids. An interesting variation introduced this year by Agela Technologies was a combined SLE–SPE cartridge that contains not only the diatomaceous earth but also a typical SPE sorbent such as silica, carbon or Florisil that allows SLE and SPE to take place in one operation. The product is recommended for pesticide residue analysis in juices and drinks. 

Another popular area was high-abundance protein-depletion products, mainly for human serum proteomic studies (3). The high-abundance proteins, human serum albumin, and immunoglobulins constitute as much as 75% of the protein content of human plasma and often mask small-concentration protein biomarkers in gel electrophoresis or high performance liquid chromatography (HPLC) studies. Immunoaffinity techniques are quite popular here due to their specificity. Two new product joined those already present on the market; one will deplete the top 14 high-abundance proteins from human serum and a second designed for the removal of the top two high-abundance proteins in human saliva. The latter product includes as part of a kit an ion-exchange column to separate the remaining low-abundance proteins.  

Molecular-imprinted phases (MIPs) are quite popular in Europe, less so in the U.S. These phases are very selective due to the presence of molecular imprints of a specific template on the surface of a polymer. The MIPs are prepared by adding a target template molecule to the monomer reaction mixture and forming a polymer around the template. The template is extracted away, leaving behind its imprint on the surface. When a mixture of compounds containing the target molecule is introduced to the MIP, usually packed into a column or cartridge, there is a stronger affinity for the target molecule by the imprinted site. This is viewed by some as a "lock and key" mechanism. So the process very much resembles affinity attraction. Usually MIPs are custom-synthesized but MIP Technologies has uncovered some high-volume applications and have developed off-the-shelf MIPs for specific pharmaceutical compounds. This year, they extended their product offering to include MIPs for amphetamines, clenbuterol and chloramphenicol. These products are marketed in the U.S. as SupelMIP by Sigma Aldrich/Supelco (Bellefonte, Pennsylvania). 

One of the more interesting new sample preparation techniques has an interesting name coined QuEChERS SPE (4–6). These letters are short for Quick, Easy, Cheap, Effective, Rugged, and Safe SPE. This method is an emerging sample preparation technique that is becoming increasingly popular in the area of multiresidue pesticide analysis in food and agricultural products. In QuEChERS, food–agricultural samples are first extracted with an aqueous miscible solvent (for example, acetonitrile) in the presence of high amounts of salts (for example, sodium chloride and magnesium sulfate) and buffering agents (for example, citrate) to induce liquid-phase separation and stabilize acid and base labile pesticides, respectively. Upon shaking and centrifugation, an aliquot of the organic phase is subjected to further clean up using SPE. Unlike traditional methods using SPE tubes, dispersive SPE cleanup is conducted by mixing bulk amounts of SPE sorbent with the extract. After sample clean up, the mixture is centrifuged and the resulting supernatant can be either analyzed directly or can be subjected to minor further treatment before analysis. One new QuEChERS introduction occurred at Pittcon this year (Table II). 

New products do not always have to be "high tech." Xenosep introduced prefolded, prestacked conical filters in a variety of diameters and retention ratings that are a convenience item for the busy laboratory. Pictured in Figure 3, the filters are designed to fit perfectly into a typical analytical glass funnel without any additional folding or prewetting to get them to stay in the funnel. 

Chromatography and Sample Preparation Accessories

Table III lists 34 new hardware products and accessories for HPLC, GC, capillary electrophoresis (CE), and sample preparation. This year, I have extended the columns and sample preparation accessories to include dedicated instruments that are designated to automate some of the sample preparation techniques reported in Table II. All of the products introduced were practical devices designed to make the chromatographer's life easier. I will cover each area individually. 

HPLC, LC–MS, and ion chromatography products:

 With the use of increased pressure in ultrahigh-pressure liquid chromatography (UHPLC), devices have sprung up to be compatible with the high pressures or for the ultraclean system needs to prevent clogging of low porosity frits and narrow connecting lines. For example, for pressures up to 20,000 psi, Optimize Technologies introduced a precolumn filter with 0.2-μm porosity with a built-in depth filter and Valco showed high-pressure fittings with special ferrules and nuts that will tightly hold fused-silica, PEEK, or other tubing used for columns and connectors. Supelco introduced high performance finger-tight fittings and connecting tubing with special sealing designed to work at 15,000 psi. Millipore introduced UHPLC sample preparation devices such as syringe filters and ultrapure water systems (Table II). Guard columns capable of 12,000-psi operation that were prepacked with sub-2-μm packing also were introduced. 

It has become quite fashionable to use higher temperatures in HPLC. High temperature lowers pressure, provides better efficiency, and decreases retention. Product introductions for higher temperature operation included a new oven that accommodates both analytical and preparative columns and sleeve heaters that can slip over small columns and mobile phase lines to prevent thermal mismatch of solvents.  

 With the world's supply of helium dwindling and becoming more expensive, users are looking for other carrier gas sources that can provide an inexpensive, safe, pure, and plentiful gas supply. Hydrogen gas has superior properties to helium in terms of diffusivity and viscosity but users often are concerned about having additional cylinders of hydrogen around the laboratory. Hydrogen generators can provide carrier gas without fear of safety because only a few tens of milliliters are actually present at any given time. Nitrogen and zero air generators also provide a source of pure gas needed for chromatography and MS applications. Gas purifiers ensure that the quality of carrier gas that makes it to the instrument is as pure as any source of gas can be. Gas flow meters are available to check on the reproducible delivery of the gases. Table III shows a number of gas generator systems and other components that were introduced at Pittcon 2008.  

Figure 4 shows another convenience product that should be of interest to gas chromatographers who must change columns on the run, the Cool-Lock Nut developed by Phenomenex (Torrance, California). This product was designed with an outer low-thermal-mass knurled installation wheel and an inner second securing nut. It allows a column to be changed before the oven has cooled because the knurled installation wheel cools down faster than a standard column nut. In addition, the second nut with a compression ferrule helps to hold the column at a constant length during installation. 

 Table II shows that manufacturers are serious about providing certified and high quality accessories like vials, syringes, and GC glass inlet liners that have become quite vogue as automation users desire to ensure that these consumables are of high purity and inert for trace analysis and have tight specifications for automated sample handling and autosampler systems. Autosampler innovations also were present at Pittcon 2008. Thermal extraction, automated weighing stations, automated liner exchange, and dynamic headspace were some of the features added to autosamplers to provide levels of automation physically close to the instrument.  

Devices such as homogenizers, evaporators, concentrators, and vortexers are designed to take away some of the drudgery of sample preparation. Such products always are found on the Pittcon floor, and I list some of those shown this year with improved features over previous models. 

I would like to thank the manufacturers and distributors that kindly furnished the requested information in advance of Pittcon 2008, thus, allowing a timely report on new product introductions. For those manufacturers who would like to be considered for inclusion into Pittcon '09 coverage, please send the name of the primary company contact, the mailing address, FAX and E-mail numbers to Patrick Kempf, Editor, LC/GC North America, c/o Advanstar Communications, 485 Rte. 1 South, Bldg. F, Iselin, NJ 08830, 

"Sample Prep Perspectives" Editor Ronald E. Majors is business development manager, Consumables and Accessories Business Unit, Agilent Technologies, Wilmington, Delaware, and is a member of LCGC's editorial advisory board. Direct correspondence about this column to "Sample Prep Perspectives," LCGC, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

(1) R.E. Majors, LCGC 25(3), 248–266 (2007). 

(2) R.E. Majors, LCGC 25(4), 340–358 (2007). 

(3) N. Zolotarjova, P. Mronzinski, and R.E. Majors, LCGC 25(2), 118–140 (2007). 

(4) S.J. Lehotay, K. Mastovska, and A.R. Lightfield, JAOAC Int., 88(2, Mar-Apr):615–629 (2005). 

(5) S.J. Lehotay, Proc, AOAC Annual Meeting, St. Louis, Missouri (2004). 

(6) M. Annastassiades, QuEChERS — A Mini-Multiresidue Method for the Analysis of Pesticide Residues in Low-Fat Products, 

Column Watch

This month's installment of "Column Watch" is the second part of a two-part series in which columnist Ron Majors examines the trends and highlights in columns and consumables at Pittcon 2008.

New Chromatography Columns and Accessories at Pittcon 2008: Part II

I started to study the history of the evolution of chromatography in the 1970s. However, at that time it was rather a hobby. In 1979, I edited (with Al Zlatkis' help) the compilation 

75 Years of Chromatography — a Historical Dialogue

 (1), with the life stories of three scores of prominent chromatographers. It was a particular pleasure to work on it, after all, I knew each contributor personally. However, my main occupation at that time was still the study of various aspects of chromatography and travel to various countries, lecturing on the newest developments. I started to deal more intensively with historical aspects around the end of the 1980s, particularly after I officially retired as a senior scientist at Perkin-Elmer and joined Yale University's Chemical Engineering Department as an adjunct professor. Soon I had some discussions with Jeffrey Schier, then the editor of 

, with the idea to establish a new column about various questions related to the evolution of chromatography. We agreed that I would periodically contribute such articles; however, I was reluctant to commit myself on a regular, scheduled basis. After all, I was not young anymore. 

In my proposal to Jeff I emphasized that "recently, historical aspects have been often presented distorted. This is partly due to the fact that the present generation did not go through the evolution like my colleagues and I did, and thus, it is ignorant of any literature more than 2–3 years old." Thus, my aim was to "set the record straight," clear up many misconceptions and give the proper credit to the pioneers of our technique. 

We selected the title "Milestones in Chromatography" for my column. By chance, the start of this series coincided with the 50-year anniversary of the Pittsburgh Conference, and for this occasion 

 planned a special issue, to which I already had agreed to contribute. This article, published in the February 1999 issue, was actually considered as the first installment of the column, but officially we launched the "Milestones" column in the June 1999 issue, with Jeff's editorial and a discussion of two chromatography conferences, one organized in 1946 by the New York Academy of Sciences, and the other organized in 1949 in London, by the (British) Faraday Society. These two symposia served as a kind of springboard for future development: without them the growth of chromatography would probably have advanced at a slower pace. 

From there on, I regularly contributed installments to this column, between three and five each year. These truly dealt with the "milestones" of chromatography development. I have discussed some early developments resembling chromatographic separation (even Moses enacted such wonders during his wandering in the wilderness!), then the circumstances that led M.S. Tswett 100 years ago to the discovery of chromatography. The column installments also dealt with the work of the pioneers who significantly contributed to the development, with the meteoric rise of the use of the technique starting in the 1930s, and with the development of the variants of chromatography. The column also discussed major instrument development and summarized the impact of a few key symposia reporting on the newest results, showing the way for future expansion. In the eight years I had a total of 35 installments in the "Milestones in Chromatography" column and these are listed in the table. 

It was great fun to write these columns: to search old publications and interview those who have been involved in the pioneering work. A number of times I could secure the co-authorship of colleagues who were more familiar with the particular subject than I, and in four cases, the column was actually written by an expert in that field. I would like to use this opportunity to thank them for their help. I also was very happy to receive comments from my readers who, years earlier, actually had been involved in the reported investigations: reading and answering their letters was like a nostalgic trip to the past. 

As mentioned, originally I was reluctant to make a commitment for a longer period, but then things went smoothly. However, as the German proverb says, "Alles gutes kommt zu ende" (everything good must end). On September 16, 2007, I passed my 85th year and this is a good occasion to say farewell to my readers: the "Milestones" column published in the January 2008 issue is my last. This does not necessarily mean that from now on, I will sit in a rocking chair, observing the shades on the ceiling, and hope that periodically, on the occasion of a major anniversary, I can contribute to our magazine. I am also finishing the manuscript of a book dealing with the history of the evolution of chromatography (2), discussing for our young colleagues the unique period when everything we did was new and interesting. This will hopefully be published this summer. I believe it was Winston Churchill who once said that those who do not know history will repeat its mistakes. By providing a more-or-less objective history, I will try to point out not only our achievements but also our errors. 

I hope that eventually some member of the present generation will pick up my pen and summarize the background of the newest developments. Don't forget that by then the present will be the past of the future: what you are doing today will be history for the next generation. 

After a 30-year association with the Perkin-Elmer Corporation, Dr. Ettre had been a member of Yale University's Chemical Engineering Department as an adjunct professor and then as a research fellow until 2004. He is a member of 

's editorial advisory board. Direct correspondence about this column to "Milestones in Chromatography," 

, Woodbridge Corporate Plaza, 485 Route One South, Building F, First Floor, Iselin, NJ 08830, e-mail 

75 Years of Chromatography; a Historical Dialogue

Chapters in the Evolution of Chromatography

, J.V. Hinshaw, Ed. (Imperial College Press, London – Singapore, 2008). 

Installments in the "Milestones in Chromatography" Column 1999–2007

Pittsburgh Conferences — a Personal Appraisal 

The 1949 Chromatography Conference of the Faraday Society 

 Development of Gas Chromatography Instruments at Shimadzu 

Yutaka Nagayanagi, Shingo Takimoto, and Hisashi Saito 

Preparative Liquid Chromatography and the Manhattan Project 

Two Symposia, When HPLC Was Young: the 1969 Las Vegas and the 1973 Interlaken Symposia 

A.A. Zhukhovitskii — A Russian Pioneer of Gas Chromatography 

Evolution of Capillary Columns for Gas Chromatography 

The Invention, Development, and Triumph of the Flame Ionization Detector 

Fifty Years of Gas Chromatography — The Pioneers I Knew; Part I 

Fifty Years of Gas Chromatography — The Pioneers I Knew; Part II 

Milestones In Chromatography

After nearly 10 years of writing "Milestones in Chromatography," resident historian Leslie Ettre will step down from his position as an LCGC columnist.

Farewell to "Milestones in Chromatography"

One of the most critical areas for obtaining the best results is instrument installation and setup. Without the right equipment and supplies, chromatographers run the risk of compromising an instrument's potential to deliver the required level of performance. Here are some important do's and don'ts for installation and setup. 

Do Obtain a Preinstallation Checklist From the Instrument Manufacturer and Follow It.

Instrument manufacturers are the best source of information about installation requirements. Following such guides is a very good habit to acquire. Detailed lists of the correct supplies and services that an instrument will require are readily available. For example, the electrical supply must be of the correct voltage and frequency, it must be properly grounded, and it might require an individual connection and circuit breaker. Additional considerations such as power filtering, surge suppressors, and uninterruptable power supplies (UPSs) are important for the data handling system associated with an instrument. Also, the laboratory temperature and humidity should fall within specified guidelines. An approximate British thermal unit (Btu) output rating for an instrument helps estimate the load on laboratory air-conditioning systems. Sometimes adding a number of new gas chromatographs requires an upgrade to existing heating, ventilating, and air conditioning (HVAC) systems. 

Do Pay Attention to Gas Delivery Requirements

The types of gases that the instrument requires depend upon the installed inlets and detectors. Capillary inlets and trace-level detectors need higher-grade gases than do packed inlets or thermal conductivity detectors. The installation checklist will specify additional items such as tubing, filters, and basic supplies. Appropriate gas-supplies should be on-hand before an instrument arrives in the laboratory. 

Many preinstallation checklists provide detailed plumbing diagrams for commonly configured combinations of inlets and detectors. Another good source of this information is available in Bulletin 898, "Gas Management Systems for GC," from Supelco (Bellefonte, Pennsylvania). This publication includes a wealth of general information on gas chromatograph installation as well as detailed diagrams of appropriate connections for many combinations of pneumatic components. It covers tanks, gas generators, regulators, tubing, on-off valves, fittings, multitank manifolds, gas filtering, leak detection, and multi-instrument installations. The brochure also contains ordering information for many recommended components. 

Don't Wrap Gas Fittings with Lots of Pipe-Sealing Tape

The logic behind this practice is that more is better. I have seen white tape where it will do no good, on high-pressure tank fitting threads that do not provide a seal in the first place, as well as on the nipple-and-cup sealing surfaces where the tape is more likely to cause a leak than prevent one. Sometimes sealing tape even shows up wrapped around the ferrules of ¼- or ⅛-in. swaged tubing fittings. The correct application of sealing tape is two layers applied to the threaded portion of pipe or tapered fittings before assembly. Remove all old tape and residue beforehand, and do not overtighten: one-half to one turn beyond finger tight is all that is required. Wrap the tape in a direction so that it doesn't unravel when the fitting is screwed in. Never apply tape to swaged or compression fittings. Instead, use new ferrules when required. 

Do Use the Correct Grade of Carrier and Detector Gas

Good, clean supplies of carrier and detector gases are well worth the expense. Get into the habit of using the right gas grade from a reliable source as the first requirement for establishing the best supply. Be sure that your gases are manufactured specifically for GC use. Select the gas grades specified by the instrument manufacturer for the exact inlet, column, and detector. Although a lower grade might work, you risk encountering problems later on as contaminants accumulate or the column degrades more rapidly than it should. 

Gas generators are an attractive alternative to tanks as air and hydrogen sources. The rising cost of helium carrier gas is making the switch to hydrogen more and more attractive: the cost of a high-purity hydrogen generator can be recovered after producing the equivalent of as few as eight tanks. Gas generator delivery capacities are limited, though. One generator will support a limited number of instruments, so be sure to determine in advance the total flow requirements from the number of carrier channels and detectors in use. 

Do Install High-Grade Pressure Regulators

It would be a shame to purchase expensive gases or gas generators only to contaminate their pristine output by using inexpensive pressure regulators and other less than top-grade gas-delivery devices. Those old single-stage cylinder gas regulators belong in the welding shop or party balloon store, and not in the laboratory. Develop a habit of using only high-purity dual-stage regulators rated for GC. These devices wet the gas stream with noncontaminating material, and they incorporate high-quality seals that prevent the influx of atmospheric oxygen. In the unlikely event of failure, most high-purity regulators include a safety vent that will direct the high-pressure gas to the atmosphere, and not into the GC system. 

Don't Vent the Gas Tank's High-Pressure Outlet Before Connecting a Gas Regulator

Myth: Venting high pressure tank gas to remove dirt particles from a tank's outlet fitting is required to obtain a good seal and keep dirt out of the pressure regulator. I've encountered this practice while visiting several different laboratories. The telltale roar of helium gas escaping at 2400 psi always startles me. The particles would seem to be a leak source if they end up on the tank fitting sealing surfaces; they also might enter the regulator and interfere with its operation. However, gas manufacturers ship high-purity GC gas tanks with a plastic cap on the tank fitting that keeps the dirt out in shipment and storage. And quality dual-stage GC gas regulators include a sintered metal frit in the entrance that stops any particles from entering the regulator. In fact, venting a high-pressure gas tank is potentially dangerous due to uncontrolled pressure release: the gas stream exits from the tank valve with a force exceeding one ton per square inch, strong enough to cause extremely serious injury. 

Do Use a Purge-Tee Fitting Downstream From the Regulator

It is necessary to vent a gas system upon installing a new tank; some air incursion into the lines will always occur. The best way to keep air away from active filters and the gas chromatograph itself is to install a purge tee with restrictor in-line. Then, after reconnecting the pressure regulator at the tank and turning on the tank pressure, open the purge valve and the regulator shut-off valve for one minute or so. This will vent the initial gas output away from the devices downstream. I once connected a helium line to a gas-sampling valve in a GC system with a molecular sieve column, as part of another experiment; I was surprised at how much gas is required to bring the air contamination down to under 50 ppm after disconnecting and reconnecting the regulator. 

Don't Check Newly Attached Gas Fittings for Leaks With a Liberal Application of Soap Solution

This one should be left to the plumbers and refrigerator service people. Almost every GC laboratory seems to have several bottles of soap solution with a paintbrush or dropper that enforces a liberal application. The problem is that a leak is a two-way street: gas goes out but soap, as well as atmospheric oxygen, can go in. Once inside, the water evaporates and leaves a soap film behind. If applied directly to pressure regulators and needle valves, the soap residue will interfere with their operation. Surprisingly, some soap solutions contain organic surfactants that have significant vapor pressures and can migrate along the gas lines into inlets and detectors. It doesn't take much: many GC detectors respond to sub-part-per-million levels of organics. Use isopropanol instead of soap solution, or better, acquire an electronic leak-checking device. 

Having just spent a substantial sum on high-quality gases and pressure regulators or on a gas generator, you will want to develop the habit of installing the best gas filters to maintain gas quality on its way into your GC system. Although filtering high-purity gases might seem redundant, in reality it is not. There are a number of unions in any gas supply setup, each of which is a potential leak. The number of connections, and therefore the chance for a leak, increases in proportion to the number of instruments in use. Gas-line tubing also presents a potential contamination source, especially if the tubing is reused by migration from one instrument setup to another. I once saw a laboratory with manifolded carrier gas supplies where they had done everything correctly, except for the cheap on-off toggle valves at each instrument; they risked contaminating the carrier with air at the last moment before going into the instrument. 

Ensure the best possible gas purity by placing a series of filters as close as possible to the instrument. Solely putting filters in line immediately after the gas cylinders does not help as much, especially if multiple instruments are connected to a single group of tanks. Some laboratories put high-capacity filters next to the tanks and include "final" filters at the instrument bulkheads. 

Select high-performance filters, and then change or regenerate them regularly. Replenishing filters after a few tanks worth of gas is good practice. For carrier-gas supplies, use a molecular sieve filter, then a large-capacity oxygen trap, and finally an indicating oxygen trap. Some analysts include a charcoal trap just before the molecular sieve trap. For detector supplies, a molecular sieve filter and possibly a charcoal trap are desirable — you will need an oxygen filter for an electron capture detector. Avoid using inexpensive moisture traps, as the adsorbent material might not remove all of the water. There are some very good combination filters and filter manifolds available that include built-in isolation valves for ease of filter element replacement. 

Periodic maintenance often gets neglected because instrument operators haven't set aside the time to perform such tasks regularly and are saving their energy for heroics when something does go wrong. It's far better to assume that there will be a problem at some point, and then take precautions that will delay or minimize the eventual difficulties. Here are a few of the more often-overlooked trouble spots. 

Do Use a New Ferrule Whenever Connecting a Column

The column–ferrule–fitting link is one of the weakest and most-often compromised connections in a GC pneumatic system. While reusing ferrules a few times is a tempting cost-saving habit, it's a very good idea to use a new ferrule whenever connecting capillary or packed glass columns to the inlet or detector. Although reusing a ferrule will succeed often, you risk the possibility of a leaking or contaminated connection. The exception is metal-packed columns with metal ferrules — they are not removable once in place, so be extra careful not to overtighten them. 

Often, chromatographers remove a capillary column from the instrument and store it on the shelf or in a box. A common habit calls for sealing the column ends to avoid contamination or degradation of the stationary phase, often by inserting the column ends into a piece of septum. This is good practice, but it immediately contaminates the column with septum particles. Upon reinstallation it's necessary to trim off 1 cm or more from the column end to remove them. Flame-sealed column ends require trimming as well, by as much as one full turn. In either case, once the ends have been trimmed it's necessary to reposition the ferrule so that the column rests the correct distance into the inlet or detector. This motion disturbs the seal between the ferrule and column surfaces; the seal cannot be reliably reestablished by tightening the fitting nut.  

Do Inspect and Change Autosampler Syringes Often

An autosampler syringe is subject to far more actuations than a manually operated syringe. Autosamplers relentlessly move the syringe plunger through preinjection sample washes, sample acquisition, and postinjection solvent washes. An autosampler syringe can experience twenty or more plunger actuations per injected sample. The presence of any nonvolatile or particulate residue in the sample will greatly exacerbate the eventual demise of the syringe through plunger wear, plugging, and residue buildup. 

You can avoid, or at least delay, these problems by making a habit of taking several precautions. First, inspect and test the syringe every 100 samples, or when adding a full sample tray. Remove the syringe from the sampler and manually actuate the plunger with the needle immersed in an appropriate solvent. The plunger should move up and down smoothly. You should observe liquid move up into the syringe, and see it squirt out of the needle. Check the needle and plunger for straightness. Carefully pull the syringe tip back over a clean piece of paper; any roughness or noticeable drag on the paper indicates the presence of burrs. Replace the syringe at the first sign of problems. The relatively small expense of a new syringe does not compare to the cost of lost samples. 

Inlet septa are a weak spot in a gas chromatograph's pneumatic system. The syringe needle punctures the septum repeatedly and gradually punches out a small void that eventually extends straight through the septum to the pressurized carrier gas inside. But long before catastrophic failure, the septum will leak enough — especially in the first few seconds after injection — to produce a significant response with an electronic leak detector. This kind of acute leak self-heals quickly at first, but older septa take longer to reseal than new ones. Operation at higher pressures and inlet temperatures also reduces septum life. 

Different types of septa are appropriate for different conditions and analyses. For routine packed column analysis, low-cost Teflon backed septa with a relatively soft silicone body work well. At higher temperatures, or for trace-level capillary work, high-temperature low-bleed septa are a necessity. The syringe needle pushes septum particles onto the inlet liner with each injection, and these particles release volatile components directly into the carrier-gas stream. Pieces of  high-temperature septa produce less background interference than other varieties. They are harder than the low-temperature septa, however, and may leak after fewer injections or at lower inlet pressures. 

Do Inspect and Change Inlet Liners Frequently

As a corollary to septum inspection, analysts also should pay close attention to the inlet liner. Any kind of contamination in the liner can have a dramatic effect on the chromatogram. Particles of dirt can cause peak tailing or even produce total adsorption. High-boiling sample residue can delay solute transfer into the column and produce peak tailing as well as solute loss with splitless sampling. Pieces of septum will bleed and introduce ghost peaks or silicone background peaks into the detector. Sometimes, syringe needle movement or large flow fluctuations gradually displace the liner packing downward, until the misplaced packing affects sample transfer from inlet to column. Periodic liner inspection identifies all of these problems. 

Because pulling out the liner requires inlet cooling and depressurization, it makes sense to check the inlet liner whenever inspecting or changing the septum. Handle the liner with contamination-free gloves, because any contamination will be exposed to the carrier gas at the inlet temperature. Be sure to check the internal liner seal as well. After installing a liner, allow the inlet system and column to purge with carrier gas for at least five minutes worth of flow, while making sure that the column has been purged for at least five times the unretained peak time. Otherwise you risk ruining a column by heating in the presence of residual air. 

Each GC system operates under a rule of cause and effect. Instrument performance depends directly upon the establishment of appropriate operating procedures, all of which combine to enhance day-to-day results quality. The list of good habits enumerated in this article represents a selection of some of the best procedures applied in a laboratory environment. Conversely, the "don't do this" items show how easy it is for chromatographers to go awry of good practices. Every step in installing, operating, and maintaining a gas chromatograph should be subject to critical examination.  

"GC Connections" editor John V. Hinshaw is senior staff engineer at Serveron Corp., Hillsboro, Oregon, and a member of 

s editorial advisory board. Direct correspondence about this column to "GC Connections," 

, Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

For an ongoing discussion of GC issues with John Hinshaw and other chromatographers, visit the Chromatography Forum discussion group at http://

GC Connections

This month, "GC Connections" presents a few of the best and worse laboratory habits.

Do's and Dont's

I remember reading a study on learning in the all-day short-course format. Because teaching liquid chromatography (LC) classes is a significant part of my work, my attention was captured. The writers claimed that in a 6–8 h class, only three points would be remembered. One of my LC troubleshooting classes has approximately 200 slides — what does this say about how effective a short course is at conveying critical knowledge? As the saying goes, I have tried to make lemonade out of these lemons, and use the "only three things" concept to help reinforce what I think are the key points. So, this month's "LC Troubleshooting" installment will use these points to form the core of a preventive maintenance program for your LC system. 

Mobile phase degassing is the single most effective way to avoid problems with an LC system. Liquid chromatographs and air just weren't meant to be together! LC pumps are very effective at pumping liquids, but introduce an air bubble and in the best circumstances you will observe a momentary reduction in the flow rate and a drop in the system back pressure. If the bubble is large enough, the pump will not deliver any solvent, and if the pressure drops below a preset low-pressure limit, the pump will stop. Some pump designs will pass bubbles fairly well, whereas other designs will fail to operate when a bubble is present.  

Once a bubble passes through the pump, it usually will stay in solution due to the system pressure as it passes through the column. But on arrival at the detector, the system pressure returns to atmospheric pressure and the bubble might reappear in the detector flow cell, causing spikes in the chromatogram. This problem can be minimized by the use of a back-pressure restrictor on the detector outlet to provide sufficient pressure to keep bubbles in solution until they exit the detector. Of course, care needs to be taken not to exceed the pressure limits of the flow cell, or detector damage can occur. 

Although noise spikes are the most common symptom of bubbles going through the flow cell, such as with UV detection, some detectors can be very sensitive to the presence of air. For example, dissolved oxygen has been reported to quench the fluorescence of some compounds when the fluorescence detector is used (1). In the reductive mode, the electrochemical detector is extremely sensitive to dissolved oxygen. Care must be taken to eliminate oxygen from the mobile phase and avoid oxygen-permeable tubing (such as PTFE) in the flow stream. 

All of these problems related to dissolved air in the mobile phase can be avoided if proper care is taken to degas the mobile phase before it is used. For many years, the gold standard for degassing has been helium sparging. This simply involves bubbling helium through a frit placed in the mobile phase reservoir. Helium sparging is the most effective way to remove dissolved air from the mobile phase, removing approximately 80% of the oxygen (2). With a well-distributed sparging stream, one volume of helium will remove almost all the gas that can be displaced from an equal volume of mobile phase (3). This means that 1 L of helium bubbled through 1 L of mobile phase will do the job. 

Helium is the only effective way to remove sufficient oxygen from the mobile phase to avoid problems specific to dissolved oxygen, such as the fluorescence quenching or electrochemical detector problems mentioned earlier. However, if the main objective is to remove sufficient dissolved air so that bubble formation is not a problem, vacuum degassing is also effective as a degassing technique. Most of today's LC systems come with an in-line vacuum degasser either as a standard feature or an optional one. In-line degassing is simple to use, trouble-free, and effective. I give it credit for the huge reduction in bubble-related complaints that I have heard in the last few years. 

Unless special precautions are taken, any particulate matter that enters the LC system will end up on the inlet frit of the column, eventually blocking the column, increasing the system back pressure, and distorting peaks in the chromatogram. As a consequence, any effort made to reduce the particulate load of the system will pay back in reliability. There are three major sources of particulate matter in the LC: the mobile phase, the sample, and the wear of internal components. 

If the mobile phase comprises only high performance liquid chromatography (HPLC)-grade solvents, there is no need to filter the mobile phase. This is because organic solvents, such as acetonitrile or methanol, are filtered through 0.2-μm porosity filters during the manufacturing process. Similarly, whether you buy HPLC-grade water or generate it in the laboratory with a purification system, the last step is filtration through a 0.2-μm filter. However, if there are any additives that were once solids, such as phosphate buffer, filtration of the mobile phase is a wise step to take. Although a buffer salt might be of high purity, it can contain particulate matter, such as bits of plastic generated when the lid of the bottle rubs on the edge of the bottle. In some cases, a solid additive might not dissolve completely, leaving bits of debris in the mobile phase. Any particulate matter from the mobile phase also can cause check valve leakage if it gets trapped in the check valves. Filtration of the mobile phase through a 0.5-μm porosity filter is an effective way to remove any particles from the mobile phase. 0.2-μm filters can be used, but they are not much more effective than 0.5 μm ones for this application and they filter much more slowly. Some laboratories write their mobile phase preparation standard operating procedures (SOPs) so that mobile phases prepared only from HPLC-grade liquids do not need to be filtered, whereas all other mobile phase compositions require filtration before use. It also is important to use a sinker frit at the inlet end of the tubing connecting the reservoir and pump. This frit typically is ≥ 10 μm porosity, so it is not a substitute for a mobile phase filtration step, but it does keep dust out of the system and it holds the tubing in the bottom of the reservoir for operational reliability. 

The sample is a second source of particulate matter in the LC system. Some laboratories filter all their samples through a 0.5-μm filter before loading them in the autosampler tray. This is an effective way to remove sample-related particles, but I have several concerns about this procedure that cause me to avoid sample filtration in most cases. First, it is expensive — $1 or more per filter — which can add a significant amount to sample processing costs. Also, for a validated method, you need to validate the filtration process and use filters for every sample, not just the ones you think need filtration. You never get 100% of the sample through the filter — there are always a few microliters left behind. Is there any adsorptive loss on the filter or contaminants leached from the filter? If there is loss, is it the same at all sample concentrations? If filtration is to be used, all of these issues must be addressed in the validation process, which can add work and expense to the validation procedure. I have found that an equally effective procedure for most samples is to centrifuge the sample in a bench-top centrifuge for a few minutes to settle out any particles, then transfer the supernatant to the autosampler tray.  

The final major source of particulate matter in the LC system is wear of pump seals and injection valve rotors. Pump seals generally will last six months to a year in a normal laboratory. I recommend replacing these on a semiannual or annual basis as part of a preventive maintenance session. The cost is low compared with the expense of a column blocked by pump seal particles. Some pumps have frits or screens in the flow path to trap wear debris from the pump seals before it works its way further downstream. Consult your pump operation manual to find the recommended cleaning or replacement intervals for such filters. Autosampler rotor seals also wear over time, but in my experience, it takes several years of intense use before the rotor seals wear out. If your system has a function that counts injection valve cycles, you might be able to set an alarm to notify you when a specific number of valve cycles have occurred. I have heard quotes that the injector should last for 20,000 cycles, but this is only 10,000 injections — not much of a lifetime for a laboratory involved in regular sample analysis. I think they last much longer — several years in my experience. Of course, pump seal and rotor seal wear will increase in the presence of more abrasive mobile phases. Thus, if you routinely run ion-exchange gradients, such as 0–1 M sodium chloride, I would expect the seals to wear more quickly than if you use reversed-phase conditions with 10 mM phosphate buffer. 

No matter what source of particulate matter I am trying to eliminate, I always use an in-line 0.5-μm porosity filter between the autosampler and the column on every system, even if a guard column is in use. This in-line filter will become blocked instead of the 2-μm filter at the head of the column, and it can be replaced in a few minutes with an inexpensive replacement frit. Just check the system pressure at the beginning of each batch of samples. When the pressure rises to a trigger point, such as a 25% or 500-psi increase, just replace the frit and you should be back in service in a few minutes.  

My third key practice for reliable LC system operation is to keep it clean. If you follow the flow path through the system, you will notice several areas that can benefit from regular flushing. First, the mobile phase reservoirs should be washed or replaced with each new batch of mobile phase. A dirty reservoir can contaminate an otherwise pure mobile phase. I don't like to use buffers longer than about a week and organic solvents for more than a month. Rather than refilling a reservoir, get in the habit of filling the reservoir, using the solvent, then replacing the reservoir when a new batch of solvent is made rather than topping it off. 

Next in line is the pump. I don't like to shut off a pump for more than a few minutes if it contains a nonvolatile buffer, such as phosphate. When mobile phase evaporates, such as on the back side of the piston seal, nonvolatile materials will leave a solid deposit. This is one of the major causes of pump seal wear. If buffered mobile phases are left in the system for extended periods, especially if acetonitrile is used, they can form precipitates, which can cause seal wear and check-valve leakage. So flush the pump with nonbuffered mobile phase before shutting it off for any extended period. 

The autosampler should be cleaned on a regular basis, too. I've never seen an autosampler that was not subject to leaks and drips. These can leave deposits of buffer or sample, contaminating the system. The wash solvent in the autosampler should be treated in the same manner as the mobile phase in terms of expiration dates and regular washing or replacement of the wash reservoir. 

Contaminants build up on the column over time, often being eluted as additional background noise in future chromatograms. This problem can be minimized by flushing the column with the strong solvent of the mobile phase (for example, methanol or acetonitrile) at the end of each batch of samples or whenever the column is removed from the system.  

In my experience, it is easier to damage the detector than to improve it with routine cleaning efforts. For this reason, I rely on the column and system flushing procedures to remove contaminants from the detector flow cell. Only if there is some compelling reason do I take specific action to clean the flow cell for detectors that operate in the liquid state, such as UV or fluorescence detectors. Evaporative detectors, such as evaporative light scattering detectors or mass spectrometers are a different story. These detectors eventually build up a film of nonvolatile contaminants and require cleaning on a regular basis. 

So there it is — degas, filter, and flush. Now you've received all the knowledge that you would have acquired in a one-day short course on preventive maintenance. Of course, it isn't quite that simple, but these three practices will go a long way toward more reliable LC system operation. Good luck! 

"LC Troubleshooting" Editor John  W. Dolan is Vice-President of LC Resources, Walnut Creek, California; and a member of 

s editorial advisory board. Direct correspondence about this column to "LC Troubleshooting," 

Woodbridge Corporate Plaza, 485 Route 1 South, Building F, First Floor, Iselin, NJ 08830, e-mail 

For an ongoing discussion of LC trouble-shooting with John Dolan and other chromatographers, visit the Chromatography Forum discussion group at http://

(1) S.R. Bakalyar, M.P.T. Bradley, and R. Honganen, 

(2) J.N. Brown, M. Hewins, J.H.M. van der Linden, and J.H.M. Lynch, 

LC Troubleshooting

After taking a course on learning that stated most people only remember three main points from an eight-hour training class, John Dolan decided to use three points to form the core of a preventive maintenance program for your LC system.

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