This article discusses a practical and reliable gas chromatographic procedure that has been successfully developed for the direct measurement of trace levels of lindane, aldrin, heptachlor epoxide, dieldrin, 

-DDD, and dichlorodiphenyltrichloroethane (DDT). With appropriate sample preparation, this analytical technique can be applied to matrices such as soil, consumer products, fruits, and vegetables.

Chlorinated organic pesticides are chemical substances that are widely used against household, garden, and plant pest diseases. The use of pesticides, however, demands great care and control as they can enter the environment and have a negative impact on human health. For instance, lindane is a persistent organic pollutant with a relatively long life in the environment, it is transported long distances by natural processes like global distillation, and can bioaccumulate in food chains. Dieldrin and aldrin are chemicals that were widely applied in agricultural areas throughout Canada and the world. Both chemicals are toxic and bioaccumulative. Aldrin does break down to dieldrin in living systems, but dieldrin is known to resist bacterial and chemical breakdown processes in the environment. Heptachlor epoxide is another commonly employed insecticide and is a well known persistent organic pollutant (POP). Dichlorodiphenyltrichloroethane (DDT) is one of the most well publicized synthetic insecticides, with a unique and controversial history especially with birds.  

The topic of analyzing ultratrace levels of chlorinated pesticides has been reported by researchers with many references in the open literature (1–5). Developed techniques include the incorporation of sample enrichment devices such as headspace, purge-and-trap, solid-phase extraction (SPE), solid-phase microextraction (SPME), and stir-bar sorptive enrichment with selective detection like electron capture or electrolytic conductivity detection, as well as with the employment of mass spectrometry (MS) (5–10). One of the issues commonly reported by researchers is the decomposition and irreversible adsorption of the organopesticides in the solute path from inlet liner to the column used and even inside the detectors, especially with mass spectrometers (5–15). Recently, highly inert quartz wool–filled liners (14), column technologies, and mass spectrometers have been innovated and commercialized. Highly inert equipment has been incorporated in developing a practical and reliable gas chromatography–mass spectrometry (GC–MS) method for the characterization of trace levels of chlorinated organic pesticides, such as lindane (CAS-58-89-9), aldrin (CAS-309-00-2), heptachlor epoxide (CAS-1024-57-3), dieldrin (CAS60-57-1), 

-DDD (CAS-53-19-10), and dichlorodiphenyltrichloroethane (DDT) (CAS-50-29-3). This article summarizes the results obtained. 

Two Agilent 7890 gas chromatographs (Agilent Technologies) were used for the development of this analytical technique:    

The first gas chromatograph was equipped with an Agilent G-4513A autosampler and an Agilent G-4514A tray, as well as a multimode inlet, a split–splitless inlet, and a flame ionization detector. A 30 m × 0.25 mm, 0.25-μm 

 DB-5ms Ultra-inert (Agilent Technologies) capillary column was used for all analyses. An Ultra-Inert split inlet liner from Agilent also was used.  

The second gas chromatograph was equipped with an Agilent G-4512A autosampler, an Agilent split–splitless inlet, and an Agilent 5975B Inert mass selective detector. A 30 m × 0.25 mm, 0.25-μm 

 DB-5ms Ultra-Inert (Agilent Technologies) capillary column was used for all analyses. An Ultra-Inert split inlet liner from Agilent also was used.  

Chromatographic data were collected using Agilent ChemStation B.03.01 Service Pack 1 and Agilent ChemStation G-1720A Mass Selective Detector software. Instrument operating conditions are reported in Table I.  

Table I: Gas chromatographic conditions for the analysis of chlorinated organic pesticides			

Chemicals used for testing were all obtained from Polyscience Inc. Cyclohexane was obtained from JT Baker. The limit of detection for the solutes of interest was calculated based on a signal-to-noise ratio of 3:1. 

When analyzing for chlorinated pesticides such as lindane, aldrin, heptachlor epoxide, dieldrin, 

-DDD, and DDT by GC, two main concerns must be addressed. The first is the decomposition of the compounds in the liner and the second is the adsorptivity of the solutes of interest by the GC system along the analyte's path. Both matters are addressed below. 

In general, GC inlet liners with quartz wool provide some distinct advantages compared to "wool-less" liners. The presence of wool aids in the vaporization of the liquid by providing extra surface and heat capacity for vaporization, retains nonvolatile sample residues, minimizes split discrimination with enhance mixing, and wipes the syringe needle upon sample dispensation. Until recently, the use of liners with quartz wool for pesticide analysis has been met with limited success because of the ineffectiveness of the manufacturers' deactivation process for both the wool and the inner surface of the liner. 

Various liners were investigated in an attempt to find one that would show less adsorptivity toward the solutes of interest with an acceptable reproducibility of less than 5% RSD. The liner described in the experimental section was found to deliver the best overall and sustained performance for the application described as illustrated in Figure 1. 

Figure 1: Chromatogram of 1.6 ppm (w/v) each of pesticides by GCâMS in SCAN mode. Peaks: 1 = octane, 2 = lindane, 3 = aldrin, 4 = heptachlor epoxide, 5 = dieldrin, 6 = o,p-DDD, and 7 = DDT.			

Among stationary phases, polydimethylsiloxane offers the highest degree of inertness to active solutes, high chromatographic column efficiency, and strong resistance to hydrolysis. A 5% phenyl arylene column was selected to improve surface wettability for aqueous matrix and maintaining a high degree of inertness for active solutes, such as those being analyzed. 

Figure 1 shows a chromatogram of 1.6 ppm (w/v) each of lindane, aldrin, heptachlor epoxide, dieldrin, 

-DDD, and DDT. Note the respectable peak symmetry obtained for the compounds mentioned, including those that are highly active such as lindane and DDT.  

Clearly, the inertness of the system selected contributed to the overall chromatographic performance of the methodology developed. 

Using the conditions outlined in Table I, the technique was evaluated for analytical performance. A complete analysis can be conducted in less than 20 min. Table II lists the retention and target mass fragments used for the solutes of interest.  

Table II: Retention times and target mass fragments used for chlorinated organic pesticides			

The practical detection limits obtained were 5 ppb (w/v) for all solutes involved, as depicted in Figure 2. This is quite respectable when compared to published results of 5 ppb (w/v) with GC–MS-MS (16) despite the fact that the detector used was a single-quadrupole mass spectrometer.  

Figure 2: Chromatogram of 8 ppb (w/v) of targeted solutes.			

A precision study was conducted for the compounds of interest. At a concentration of 16 ppb (w/v), the RSD at the 95% confidence level was less than 5% (

 = 10). The respectable reproducibility obtained substantiated the overall inertness of the analytical system with a minimal or nondetectable level of solute decomposition at the level studied. 

Linearity also was acceptable for all compounds. Linearity data were collected between 8 ppb (w/v) and 320 ppb (w/v) for all compounds. 

 values greater than 0.999 were obtained and the results are illustrated in Figure 3. It can be inferred that with the excellent correlation coefficients obtained for highly reactive solutes, such as lindane, aldrin, and DDT, if decomposition of solutes occurred it was at an insignificant level over a range of 5 ppb to 320 ppb (w/v) for the solutes cited.  

Figure 3: Linear curves for targeted solutes.			

With a proper sample preparation technique, this analytical method can be used to analyze for said components in different matrices. Figure 4 shows a chromatogram of 800 ppb (w/v) each of the solutes of interest spiked into a dried, and sieved bioremediated locally obtained soil and subsequently extracted with 5 mL of dichloromethane followed by 10 min of sonication. A recovery of greater than 92% for all analytes was obtained. Commercially available lice treatment shampoos can contain lindane as an active ingredient. A 1-mL sample of the medicated shampoo was extracted with 10 mL of water for surfactants removal, and the subsequent extract was exchanged with 2 mL of dichloromethane. Figure 5 shows a chromatogram obtained with GC–MS in SIM mode using 181 Da as the target ion, illustrating the presence of lindane in the extract. The same sample also was analyzed by GC coupled to flame ionization detection (FID). Figure 6 shows an overlay of a chromatogram of a standard with the sample of interest. The presence of lindane was identified by retention time along with other detectable components in the extract. The selectivity and sensitivity improvements obtained with GC–MS in SIM mode can be advantageous in screening for the presence of the targeted pesticides in various matrices.  

Figure 4: Chromatogram of 800 ppb (w/v) of targeted solutes spiked into a dried, sieved bioremediated soil. Peaks: 1 = lindane, 2 = aldrin, 3 = heptachlor epoxide, 4 = dieldrin, 5 = o,p-DDD, and 6 = DDT.			

The analysis of low-level chlorinated pesticides by GC has been challenging because of the adsorptivity of these compounds in the chromatographic system.  

Figure 5: Chromatogram of an extract of medicated shampoo with GCâMS in SIM mode.			

The practical and simple GC method described in this article combines an inert liner, a well-deactivated capillary column, and a mass-selective detector and enables the direct measurement of ultratrace parts-per-billion levels of lindane, aldrin, heptachlor epoxide, dieldrin, 

-DDD, and DDT. This technique has demonstrated a high degree of reliability and is sufficiently rugged for implementation in plant laboratory facilities.  

Figure 6: Overlay of a pesticide standard and an extract of medicated shampoo with GCâFID.			

Special thanks to Dan Martin and Dave Walter of the Dow Analytical Technology Center for their support and encouragement. Dr. Taylor Hayward is also acknowledged for the fruitful discussion on the topic of the impact of pesticides in environment. 

(1) R. Soniassy, P. Sandra, and C. Schlett, 

Water Analysis, Organic Micropollutants, 

Hewlett-Packard Co. 141 (HP part N 5962-6216E) (1994). 

 (Elsevier, Amsterdam, The Netherlands, 2003). 

(3) W. Jennings, E. Mittlefehldt, and P. Stremple, 

 (Academic Press, New York, New York, 2006). 

Applications of Gas Capillary Gas Chromatography

 (Marcel Dekker, New York, New York, 1981). 

(Publ. 5950-3562, Hewlett-Packard, Avondale, Pennsylvania, 1979). 

(6) K. Neuber, M. Gunter, and R. Randow, 

(8) L. Tudir, T. Harner, P. Blanchard, Y. Li, L. Poissant, D. Waite, C. Murphy, and W. Belzer, 

(9) M. Abb, J. Breuer, C. Zeitz, and W. Lorenz, 

(10) M. Cooke, K. Khalief, G. Nickless, and D. Roberts, 

(11) C. Bicchi, C. Brunelli, C. Cordero, P. Rubiolo, M. Galli, and A. Sironi, 

Articles

Article

A review of a gas chromatographic procedure developed for the direct measurement of trace levels of lindane, aldrin, heptachlor epoxide, dieldrin, o,p-DDD, and dichlorodiphenyltrichloroethane (DDT)

A Practical Method for Trace Level Analysis of Chlorinated Organic Pesticides by Gas Chromatography–Mass Spectrometry

Electron ionization (EI), (formerly known as e

[1]) is used in gas chromatography–mass spectrometry (GC–MS) to ionize and fragment analyte molecules before mass spectrometric analysis and detection. A GC column is connected via a transfer device to a mass spectrometer, the simplest and most popular of which will contain an electron ionization source with a single-quadrupole mass analyzer and a detector.  

A typical electron ionization source exposes the analyte, under vacuum, to a stream of thermionic electrons produced from a resistively heated filament. Close passage of highly energetic electrons to the neutral analyte molecule causes large fluctuations in the local electric field, which induces ionization via the removal of a single electron from the highest occupied molecular orbital (HOMO) of an atom within the molecule, forming a radical cation according to the general scheme (2):  

In general, the order of ease with which electrons are lost under electron ionization conditions is lone pair > π-bonded pair > σ-bonded pair.  

The cations formed within the ion source can then be electrostatically ejected from the ionization source using a repelling voltage applied to the rear element of the source. The analyte ions are collimated, focused, and accelerated through a series of electrostatic lenses and subsequently into the mass analyzing device (3).  

To induce analyte ionization, "standard" electron ionization uses 70 electron volt (eV) electrons, a level that is higher than the energy required for ionization of most common organic compounds (typically between 6 and 15 eV). Most of the excess energy imparted by the ionizing electron will be internalized and dissipated via vibrational and electronic excitation modes.  

The radical cation formed typically undergoes a single or multiple fragmentations or a rearrangement reaction, resulting in the elimination of a neutral species to achieve a more stable state. The degree of fragmentation or intermolecular rearrangement will depend on the analyte molecule ionization cross-sectional area and the ability of the molecule to stabilize charge (4). 

Fragmentation and rearrangement pathways are well characterized and predictable according to some fairly rudimentary principles. A typical mass spectrum produced from electron ionization is shown in Figure 1. The mass-to-charge ratio and intensity of the parent (pseudomolecular) and fragment ions act as a "fingerprint" that can be used to deduce analyte chemistry or entered into a spectral library for identification against thousands of library entries acquired under similar operating conditions.  

Figure 1: A typical electron ionization spectrum of the cocaine molecule. Note the large number of intense fragments created. 

Electron ionization is known as a "harsh" technique because of the degree of fragmentation induced. It is possible to reduce the energy of ionizing electrons to reduce the degree of fragmentation, however the sensitivity of the technique decreases markedly. Chemical ionization (CI) is a complementary technique that uses a reagent gas to create a plasma of charged gas molecules that then transfer charge to the analyte via a range of gas-phase chemical and charge transfer reactions.  

Electron ionization typically is the first method of choice when screening samples and is applicable to the vast majority of analytes that can be analyzed by gas chromatography. 

Compendium of Chemical Terminology, 2nd ed. 

(the "Gold Book") (1997). (Online corrected version: 2006–). 

(2) R.J. Anderson, D.J. Bendell, and P.W. Groundwater, 

 (The Royal Society of Chemistry, Cambridge, UK, 2004), pp 120–148. 

(3) G. Zschornack, V.P. Ovsyannikov, F. Grossmann, and O.K. Koulthachev, "Electron Ionization Ion Source," United States Patent No 6717155. 

(Brooks/Cole Publishing Company, Pacific Grove, California, 1992).

The Essentials

A look at the process of forming radical cations for analysis in GC–MS and the role of various ion source components

Electron Ionization for GC–MS

Part I of our yearly report on new products introduced at Pittcon. This month, columns are highlighted, covering all modes of HPLC, UHPLC, and liquid-phase separations.

Pittcon 2012 (more formally known as the 63rd Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy) returned to the Orange County Convention Center in Orlando, Florida, from March 11 to 15, 2012 after a one year absence. This year's event hosted 948 instrument manufacturers and laboratory suppliers in 1854 booths. In addition to attending the exposition, conferees listened to more than 2000 technical presentations (orals, posters, workshops, invited and contributed talks, and award symposia), checked on numerous company seminar rooms, or attended one of 95 short courses. 

Although the average attendance has been shrinking over the last several years, 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 2012. In some cases, products that were introduced during 2011 but after Pittcon 2011 (1,2) may be included for reasons of completeness. The information is based on manufacturers' responses to a questionnaire mailed in early 2012. 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 new products that were introduced since last year's event in Atlanta. 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 

. Some of these products may be used for general high performance liquid chromatography (HPLC) separations as well, but their main emphasis is for biological samples.  

In this month's coverage, I describe new column introductions in the areas of HPLC, reversed-phase chromatography, normal- and bonded-phase chromatography, flash chromatography, supercritical fluid chromatography (SFC), ion-exchange and ion chromatography, size-exclusion chromatography, hydrophilic interaction chromatography (HILIC), and specialty chromatography. Next month, I will look at gas chromatography (GC) columns, thin-layer chromatography, sample preparation products, and hardware, accessories and small tabletop instruments, mainly for sample preparation. 

 This year, I observed a continuing trend in high-throughput chromatography driven by dramatic increases in chromatographic efficiency, especially in HPLC and ultrahigh-pressure LC (UHPLC). But as chromatographic efficiency becomes exploited to the ultimate, more focus is now directed toward improved selectivity with mixed-mode separations, new bonded-phase development, and application-specific separation products. In addition, the introduction of more products with inert surfaces driven by increases in instrument sensitivity especially through increasing use of tandem mass spectrometry (MS) techniques in both GC and LC. The development of systems with more inert flow paths, newer deactivated packing materials, deactivated surfaces such as fused-silica columns, vials, and GC injection liners are all being addressed to improve recovery of very low levels of analytes. 

In HPLC, there was a further increase in columns and accessories designed to work with UHPLC. Many application-specific columns for HPLC and GC tested with "real world" samples before shipment were noted. Again at Pittcon, a considerable number of new accessories, hardware, and devices were introduced to make the chromatographic laboratory more productive. 

 Pittcon 2012 saw the introduction of more sub-2-µm columns for UHPLC, additional superficially porous particles (SPP, also known as core–shell particles) and the second generation of silica monolith columns, giving users alternative approaches for high-throughput and high-efficiency separations. UHPLC has established itself as a viable solution to today's separation problems; at least nine major instrument vendors now address this market with instruments capable of operation at pressures as high as 19,000 psi. This year, even more HILIC columns appeared for the separation of polar compounds weakly retained or unretained by reversed-phase HPLC. Still, reversed-phase HPLC maintained its dominance. Application-wise, chiral columns, protein (particularly monoclonal antibody) columns, and ion chromatography–ion exchange columns dominated. 

Column stability and lifetime issues are being addressed by newer UHPLC columns and column hardware, new approaches to chemical bonding, and more polymeric and hybrid columns with high-pH capability. 

 GC column introductions were dominated by high-temperature columns and columns with new levels of inertness. SFC has been getting more attention; this "intermediate" separation technique is catching on, especially in the pharmaceutical area. Packed SFC columns are more like HPLC columns but have some unique phases not found in a typical LC laboratory. 

 Sample preparation products for automation, such as 96-well filtration and solid-phase extraction (SPE) plates, seem to be in vogue. Higher stability phases with lower levels of bleed, particularly for mass spectrometric work, were prevalent. Mixed-mode phases are getting more popular in SPE and HPLC as well. Combinations of molecular interactions such as hydrophobic and ion exchange can often enhance analyte isolation. QuEChERS (quick, easy, cheap, effective, rugged, and safe) methods are still "catching on" as the favored sample preparation technique for pesticides in fruits and vegetables. Dried blood spotting products are beginning to appear as more pharmaceutical companies are investigating these devices to replace traditional blood handling. 

 More than 40 new hardware products and accessories for HPLC, UHPLC, GC, and sample preparation were noted. UHPLC-specific products with higher-pressure ratings and with decreased dead volumes were introduced. A number of automated sample preparation instruments or instrument accessories contribute to minimizing the labor-intensiveness and drudgery of manually preparing samples for chromatography. This year, a number of workstations that do some (but not all) of the sample preparation steps were evident. 

Each year, manufacturers introduce entire new families of HPLC columns at Pittcon. For the sake of brevity, I have elected to cover those series here instead of listing each column under individual headings in the tables. In some cases, the series are extensions of existing products but cover additional phases, whereas in other cases the families are entirely new to the market. 

New series of HPLC columns were shown by several companies (Table I). Because these series offer a wide range of particle sizes (from UHPLC to preparative) that are made by the same process and chemistry, seamless scaleup and scaledown within a family is possible. Method development can be performed on a short, fast analytical column and then can be scaled up to preparative dimensions with easy method transfer. Most introductions of new series included both sub-2-µm and larger particles. 

In ion chromatography, larger particles (5 µm and larger) and analytical–microbore columns have been the norm. This year, Dionex, now part of the Thermo Fisher Scientific company, introduced capillary ion chromatography columns with a 0.4-mm internal diameter packed with 4-µm ion-exchange particles. The capillary column format provides lower mobile-phase consumption because flow rates are reduced proportionally based on radius ratios squared and the mass sensitivity is increased (assuming the same sample mass is injected). The 4-µm particles give an improved efficiency over the larger particles. Figure 1 shows a 2.5-min separation of simple inorganic anions using a 4-µm IonPac AS18 cartridge column (150 mm × 0.4 mm) at a flow rate of 30 µL/min. Conductivity was used for detection. 

Figure 1 (Courtesy of Thermo Fisher Scientific.)  

It seems that UHPLC is here to stay. Most new column series will have at least one column with sub-2-µm particles. The obvious advantage of such small particles is better column efficiency so that shorter columns can be used to accomplish the same separation achieved on a longer column. The end result is that separation times are greatly decreased in proportion to the column length reduction. In addition, a shorter column with the same internal diameter that is run at the same flow rate saves a proportional amount of solvent, always important these days with the cost of everything on the rise.  

The introduction of SPP columns continues with the entry of new companies and more phases from existing suppliers. These columns have caught on not only with those who have older 6000-psi systems but also with those using UHPLC systems. The columns have the efficiency of the sub-2-µm columns with about half of the pressure drop when comparing the same dimensions. Three of the new companies with the addition of new SPP column product lines are ChromaNik Technologies of Osaka, Japan, PerkinElmer of Waltham, Massachusetts, and Thermo Fisher Scientific from Runcorn, United Kingdom, bringing the total number of companies with such columns to eight. 

As usual, many new reversed-phase chromatography columns were introduced at Pittcon 2012 (see Table II). If one also adds all the columns capable of reversed-phase interactions covered in the new series (Table I), in the HILIC category (Table III), and in specialty columns (Table IV), one can clearly see the dominance of this operational mode. This Pittcon added more than 69 new reversed-phase chromatography columns to the huge numbers that already existed from past introductions. Silica-based bonded phase columns dominated the introductions again this year (~90%) but polymer-based, organic–silica hybrids, and a silica hydride columns were also on the scene.  

Table II: Reversed-phase chromatography  

The C18 (octadecylsilane) functionality proved again to be the most popular with 30% of all reversed-phase chromatography columns being this alkyl phase. However, this phase isn't always the optimum for all separations and a great number of other hydrophobic stationary phases were introduced. Besides the normal C8, C4, and phenyl phases, C18 with mixed functionality (such as phenyl and diisopropyl sterically protecting groups), phenyl-hexyl, fluorophenyl, diphenyl, embedded polar, propyl pentafluorophenyl (PFP), cyanopropyl, cholesteryl, naphthylethyl, AQ (which tolerate highly acqueous mobile phases), and graphitized carbon phases were made available by various suppliers to present selectivity alternatives. An interesting mixed-mode phase consisting of PFP and C18 also was introduced. The HILIC columns that have some hydrophobic interaction possibilities are found in the "specialty column" section (Table III). 

Table III: Columns for HILIC separations  

In keeping with the trend noted in the last few Pittcons, Table II shows a number of introductions of small particle columns in the 2-µm and sub-2-µm range, often referred to as UHPLC columns. There are now more than 25 manufacturers of these high-efficiency porous particle columns. It is almost expected now that new columns should provide at least one sub-2-µm particle size in the portfolio. 

At Pittcon 2012, besides the families introduced (listed in Table I), additional SPP columns for the reversed-phase chromatography mode were displayed (Table II). As pointed out earlier, these columns show the same efficiency as the sub-2-µm columns but at half of the back pressure. To balance the shift to these columns, several companies introduced porous particle columns with particle size in the 2–3 µm range. These columns aren't as efficient as the sub-2-µm columns but are more efficient than the popular 3.0–3.5 µm columns. In addition, they show a lower pressure drop than the sub-2-µm columns but a higher drop than the 3.0–3.5 µm columns. 

In recent years, there has been a trend in introductions of bonded silica-based columns with wider pH limits. Most bonded phase silicas are recommended to be used in the pH 2–8 range. At low pH (<2), the bonded phase, especially those with short chain lengths, will hydrolyze, leaving behind bare silica. Consequently, retention times will generally change and peak tailing becomes a big problem. At high pH (>8), the silica gel itself will begin to solubilize, which is dependent on the solvent, type of buffer, and temperature. By bonding carbon phases with sterically protecting groups, with high coverage of long alkyl groups, or by coating the silica with polymeric material, the pH range can be extended. By using organo–silica hybrid chemistry, bidentate bonding, and polymer coatings or polymer encapsulation, the range can be further extended to pH 1–12. This year, columns with an extended range of pH stability using some of these principles were introduced. Of course, polymeric columns have an even wider range but still don't rival silica-based columns in terms of efficiency.  

Monolithic columns have shown promise for many years but have been unable to displace the traditional microparticulate columns. The silica monoliths have been around for over a decade but the introduction of the second generation monoliths may change their acceptance. Merck KgaA and its U.S. affiliate Millipore EMD have introduced the Chromolith High Resolution columns that display 50% higher efficiency than the first generation. Merck has accomplished this by further optimization of the mesopore/macropore ratio of the monolith at the price of a slightly higher pressure than the first generation monolith. However, because one of the attributes of the monoliths is their low pressure drop, this increased pressure in not much of a disadvantage. Figure 2 shows a comparison of the new Chromalith High Resolution C18 endcapped monolith column with one of the popular SPP columns using the same chromatographic conditions and column dimensions. Note that the pressure drop of the monolith is less than half of the pressure of the SPP column. An improvement in tailing factors over the first-generation version, especially with basic compounds, is also a positive attribute of the second-generation monolith columns. 

Normal-phase chromatography implies that a nonpolar mobile phase and a polar stationary phase are used for the separation. Although normal-phase chromatography is not used nearly as much as reversed-phase chromatography, there are instances when the technique can offer advantages. For example, for water-sensitive compounds the technique is ideal because totally organic solvents are used. Also, for certain geometric isomers and 

 isomers are well separated by normal phase but sometimes are more difficult to resolve by reversed-phase chromatography. This year, two companies introduced columns with functional groups well suited for normal-phase separations. The Waters Xselect HSS Cyano column can be used in normal-phase solvents as well as reversed-phase solvents. In reversed-phase mode, it behaves as a short-chain material with some moderate polar functionality. In our last column survey (3), this was the predominant use of cyano phases. On the other hand, it serves as a good normal-phase column when used with organic solvents. The cyano column is prepared with a monofunctional silane with diisopropyl groups surrounding the –CN group, which gives it better stability in acid solution. Columns are available with 1.8-, 2.5-, 3.5- and 5-µm particles with scaleability among all the particle sizes. Columns are available with 20–250 mm × 1.0, 2.1, 3.0, and 4.6 mm dimensions.  

The Merck KGaA/ Millipore EMD Chromolith NH2 is a second-generation silica monolith that provides column efficiency of a 3-µm porous particle column but with the pressure drop of a 5-µm column. The aminopropyl-modified column has polarity in between bare silica gel and reversed-phase chromatography silica. In acidic solution, the phase is protonated and can be used as a weak anion exchanger. It also can be used in reversed-phase mode in a similar manner to the cyano columns. Its major application, though, has been for the separation of carbohydrates where using water–acetonitrile mobile phases in a HILIC mode; mono- and disaccharides can be separated using an acetonitrile–water gradient (where water is the strong solvent) and the carbohydrates can be monitored at wavelengths below 200 nm. 

HILIC is a separation technique for highly polar analytes that gets around some of the problems associated with reversed-phase chromatography such as low retention or phase collapse (dewetting). HILIC uses a polar stationary phase such as bare silica gel, a polar bonded phase (such as diol), or certain mixed-mode or zwitterionic phases. Operating conditions usually require a high percentage of a nonpolar mobile phase, similar to the requirements for normal-phase chromatography. However, unlike normal-phase chromatography, which uses nonpolar solvents like hexane and methylene chloride and tries to exclude water from the mobile phase, HILIC requires some water in the mobile phase to maintain a stagnant enriched water layer on the packing surface into which analytes may selectively partition. In addition, water-miscible organic solvents are used. Under HILIC, polar analytes are well retained and are eluted in order of increasing hydrophilicity. HILIC is especially favored by mass spectroscopists because ionization efficiency often is enhanced in organic solvents and the presence of lower concentrations of volatile buffer salt compared to reversed-phase chromatography. The HILIC technique has been growing by leaps and bounds the last few years. Our recent survey (3) showed that the share of HILIC among all the LC modes has doubled in the past 4 years. 

This year, I decided to list the columns featured specifically for HILIC use in a separate table (Table III). Given that any column that has polar functional groups (such as silica, amino, cyano, or diol) can be used for HILIC, often these columns are found spread out in the new series (Table I), reversed-phase chromatography (Table II), and specialty columns (Table IV). If one adds up all the columns introduced at Pittcon 2012 that can be used for HILIC, the total is 19. Those listed in Table III were specifically identified as HILIC columns and possess functional groups such as cyano, penta alcohol, amide, and diol; two are zwitterionic (sulfobetaine = sulfonate + ammonium). 

This form of LC dates back to old-style separations where gravity feed was used and self-packed columns with large-particle silica gel or alumina were common. Its main use was the fractionation of synthesized organic compounds. The modern form of this technique is referred to as 

. In the modern form, columns come prepacked with adsorbents like silica gel, but also are available with reversed-phase packings and other bonded phases. Instruments are available that pump liquids through the columns at moderate pressures. Modern instruments run gradients, have various detector options, and are really a subset of HPLC separation instruments but work at lower pressures because the particles used are still rather large compared to HPLC and UHPLC columns. This year, three companies introduced new flash column products. Biotage, a leader in this field, showed their ZIP flash chromatography cartridges packed with 40–63 µm irregular silica gel particles. These cartridge columns are designed to fit their own flash instrument and instruments of other companies. Automated manufacturing allows them to provide a value-priced column with better reliability, reproducibility, and performance. Laser-welding on the inlet fitting provides leak-free operation and high pressure tolerance. Their SNAP Ultra flash chromatography cartridges are packed with 25-µm spherical silica gel with 40% more surface area, giving high performance with high capacity and lower pressure than other small-particle cartridge flash columns. 

Buchi Sepacore showed new flash columns with 40–63 µm normal- and reversed-phase packings in both column and cartridge formats. Available phases included silica gel, C18, amino, cyano, diol, and a cation exchanger. Their High-Performance (HP) silica particle size of 15–40 µm gives improved efficiency. They also have polypropylene cartridges that can be self-packed using a cartridge packer. Glass columns are also available. They also have a flash instrument. Grace Davison Discovery Sciences displayed its amino- and diol-flash cartridges designed for operation at pressures as high as 200 psi that are compatible with most flash instruments. 

Supercritical Fluid Chromatography Columns

Now in its third generation of instruments, SFC has been growing for the rapid separation of complex mixtures, especially for preparative work. With the introduction of modern SFC instruments, analytical separations can also be performed with smaller particles (below 2 µm) more rapidly than with HPLC or UHPLC. The most commonly used mobile phase, supercritical carbon dioxide, which is relatively nonpolar, somewhat limits the types of compounds that can be successfully separated by SFC, but with mobile-phase additives, more-polar analytes can be analyzed. Up to now, many SFC users have been forced to use normal-phase HPLC columns such as bare silica gel, diol, and cyano as stationary phases. More recently, companies have been developing stationary phases that are more suited to supercritical solvent mobile phases. Two such new products were unveiled at Pittcon 2012. ES Industries of West Berlin, New Jersey, has introduced new SFC phases and showed its GreenSep Ethyl Pyridine II, which is a unique stationary phase unknown for HPLC. The ethyl pyridine bonded phase, bonded to silica gel, is available in particle sizes ranging from 1.8 µm to 20 µm. It is recommended for the SFC separation of acidic compounds. Microbore, analytical, and semipreparative dimensions are available. Nacalai USA, a subsidiary of Nacalai Tesque, of Kyoto, Japan, introduced the Cosmosil 3-Hydroxyphenyl column, available in 150 mm × 4.6 mm dimensions. As can be seen in Figure 3, the new 3-hydroxylphenyl phase shows quite a different selectivity from the conventional 2-ethypyridine column for SFC. In fact, in this example, there was a reversal of elution order of these beta blockers. The 2- and 4-ethylpyridine phases have been found to be quite advantageous for SFC over the polar bonded phases borrowed from HPLC. So now an alternative selectivity is available. 

In the past few years, only a few new SEC columns have been introduced at Pittcon. That doesn't mean that SEC is not a highly practiced separation mode. It is a standard technique for the separation and characterization of organic polymers, both organic- and water-soluble. In addition, the mode is widely used for the separation of proteins and other biopolymers, sometimes known as 

. Only two companies (Waters and Sepax) introduced columns for SEC at Pittcon 2012. The majority of the columns were recommended for the separation of biomolecules, but some can be used for nonbiological water-soluble polymers too. 

The Waters offering was an aqueous SEC column based on their 1.7-µm bridged ethylene hybrid (BEH) particle Acquity UPLC BEH125 SEC technology. The pore size of this porous packing is 125 Å, making it suitable for most peptides and proteins from 1000 to 80,000 Da and thus complementing their BEH200 SEC product designed for high-molecular-weight proteins up to 450 kDa. For the surface of the packing to be biocompatible, a hydrophilic moiety must be introduced. Waters used a proprietary diol coating. An advantage of this hydrophilic surface is that it reduces the requirement of high salt concentrations in the mobile phase to cut down on undesired, secondary analyte–particle interactions. Analytical columns (150 mm × 4.6 mm) and a guard column (30 mm × 4.6 mm) are available. 

Sepax Technologies is a company that specializes in biocolumns. This year, they introduced two new column lines and a protein screening kit. Like Waters, one of their introductions was the sub-2-µm Unix SEC-200 aqueous SEC packing. The average particle diameter is 1.8 µm and the pore size is 200 Å. Three column dimensions (with stainless steel construction to hold up to the high pressure) were shown at Pittcon: 250 mm and 300 mm × 4.6 mm for analytical work, and a 150 mm × 7.8 mm size for semipreparative applications. The surface of their packing has a hydrophilic, neutral, nanometer-thick coating. This column is recommended for peptides and proteins with a molecular weight range from 1 kDa to 500 kDa. Their second column introduction (Zenix SEC-80) had a similar chemistry but the pore size was only 80 Å and the particle size is 3 µm. Both stainless steel and PEEK versions are available. Standard dimensions are 300 mm × 2.1, 4.6, and 7.8 mm; guard columns of 50 mm × 4.6 mm and 50 mm × 7.8 mm are available. Other dimensions can be custom ordered. The Zenix column is recommended for the size separation of peptides and small proteins (up to 20 kDa) and has also found use to fractionate complex peptide mixtures as the first-dimension column in a multidimensional column setup. The columns are compatible with a wide variety of mobile phases including trifluoroacetic acid–acetonitrile and methanol mixtures. 

The Sepax Screening Kit consists of two hydrophilic, neutral, nanometer-thick film columns, one called Zenix, a mechanically stabilized 3-µm silica with a "stand-up" bonded monolayer, and Zenix-C with a "lay-down" monolayer on porous 3-µm silica. The two columns are designed to complement each other and provide different secondary interactions. The SEC columns (300 mm × 7.8 mm) are recommended for the screening of monoclonal antibodies, hydrophobic proteins, pegylated peptides, proteins, oligonucleotides, bacteria, and viruses. 

Column Watch

A review of columns unveiled at Pittcon this year, including all modes of HPLC, UHPLC, and liquid-phase separations.

New Chromatography Columns and Accessories at Pittcon 2012, Part I

One of the world's premier conferences and expositions on laboratory science, the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy (Pittcon) is a showcase for the latest advances in laboratory instrumentation and technology, attracting all of the major high performance liquid chromatography (HPLC) vendors in addition to more than 900 exhibiting companies from around the world. Pittcon is a great place for vendors to introduce, and scientists to see, touch, evaluate, compare, and hear all about the latest in HPLC technology. This installment will highlight some of the new HPLC and related technology showcased both before and at this year's conference. 

This year, the Pittsburgh Conference on Analytical Chemistry & Applied Spectroscopy (Pittcon) was held on March 11–15 at the Orange County Convention Center in Orlando, Florida. The conference, which annually attracts more than 15,000 attendees from industry, academia, and government from 90 countries worldwide, is sponsored by the Spectroscopy Society of Pittsburgh and the Society for Analytical Chemists of Pittsburgh. Pittcon 2012 had more than 900 exhibitors registered to appear in more than 1800 booths. Upwards of 2000 technical presentations and nearly 100 short courses also were given. The conference provides a great opportunity for vendors to expose new high performance liquid chromatography (HPLC) products to both new and existing customers, and with this fact in mind, many HPLC vendors have coordinated their new product development cycle around Pittcon for years, introducing new technology at the conference for the first time. Although the proliferation of smaller specialized conferences has in recent years altered this approach, for example, many new mass spectrometry (MS) product introductions are now made at the annual Conference on Mass Spectrometry and Allied Topics (ASMS), Pittcon still remains a must-go-to meeting to see the latest and greatest in HPLC and related technology. However, this year marked the first time that at least one HPLC vendor decided to go completely virtual, and instead of attending Pittcon, chose to offer a series of local seminars and on-line web-based e-conferences instead. For the attendees, the conference provides a venue to evaluate the latest instrumentation, compare vendors, participate in product demonstrations, and speak with technical staff to resolve problems or investigate potential applications. It will be interesting to see how things play out in the virtual world. 

At this years' Pittcon, several vendors introduced new systems, software, and components, as well as product line extensions. In this installment, I'll review some of the new HPLC and related instrument technology shown virtually and physically at the conference. Table I lists the company introductions that are reviewed in this column. (New column and sample preparation technology will be reviewed by Ron Majors in the April and May installments of 

Table I: Summary of systems and components reviewed in this article  

Supercritical fluid chromatography (SFC) has been commercially available since about 1982, but more recently it has gained in popularity because of advances in technology and applications to several difficult separation challenges (for example, the separation of chiral compounds). SFC is a form of normal-phase chromatography that is used for the analysis and purification of low- to moderate-molecular-weight, thermally labile molecules. The theory and principles of SFC are similar to those of HPLC; however, SFC can be differentiated from other chromatographic techniques (gas chromatography [GC] and HPLC) by the use of a supercritical fluid as the mobile phase and, therefore, the entire chromatographic flow path must be pressurized. SFC has several advantages compared to GC and HPLC methods, including rapid separations without the use of organic solvents, reduced use of organic additive chemicals, and the use of carbon dioxide collected as a by-product of other chemical reactions (or collected directly from the atmosphere), thereby contributing no new chemicals to the environment and promoting "green" chemistry. In addition, SFC separations can be done faster than HPLC separations because the diffusion of solutes in supercritical fluids is about 10 times greater than that in liquids (and about three times less than in gases). The lower diffusion results in a decrease in resistance to mass transfer in the column and allows for fast, high-resolution separations. Compared to GC, capillary SFC can provide high-resolution chromatography at much lower temperatures. This allows for the fast analysis of thermolabile compounds. Many vendors have recognized these advantages and have started to offer SFC instruments on existing HPLC and ultrahigh-pressure liquid chromatography (UHPLC) platforms.   

Agilent offers three points of entry into SFC: the original 1260 Infinity Analytical SFC instrument; a Hybrid SFC/Ultra High Pressure Liquid Chromatography (UHPLC) system; and an upgrade path based on the Aurora Fusion A5 Evolution system that converts existing Agilent 1100 and 1200 HPLC systems to SFC. The Agilent hybrid SFC/UHPLC system is based on the 1260 Infinity Analytical SFC system, but adds both a quaternary or binary HPLC pump and a two-position 10-port valve to be able to switch between UHPLC and SFC and provide true orthogonal separations at pressures as high as 9000 psi. The hybrid system is capable of screening methods by switching between SFC and UHPLC in a single run sequence, significantly reducing cost (only one system to purchase), laboratory footprint, equilibration time, and instrument-to-instrument variability. Figure 1 shows an example of both an UHPLC and a SFC separation of a mixture of polycyclic aromatic hydrocarbons (PAHs) on the hybrid system. 

The new Aurora SFC Fusion A5 Evolution system is built on the original Aurora SFC Fusion A5 instrument and features a combination of optimized pumping technology with an ultralow-noise back-pressure regulator. The upgrade kit allows full conversion of an Agilent 1100 or 1200 series HPLC system to a full analytical SFC system that moves SFC smoothly into routine laboratory analyses with enhanced features for full qualification in a good laboratory practice (GLP) or good manufacturing practice (GMP) environment. With cost containment being the focus of many companies, conversion can reduce the overall cost of entry. Four orders of magnitude of dynamic range provide accurate quantitation of 0.01%-level impurities, high precision, HPLC-like sensitivity, and UHPLC speed using UHPLC columns. The system is fully compatible with the manufacturer's chromatography data system software. 

At past shows, Waters has showcased its SFC system, the Waters Acquity UPSFC system that is built on the Waters UPLC sub-2-µm particle chemistry platform. The UltraPerformance SFC (UPSFC) system is designed to incorporate all of the advantages of SFC, in addition to superior separation, resolution, and speed afforded by the sub-2-µm particle chemistry platform. This year, Waters introduced the UltraPerformance Convergence Chromatography Acquity UPC

  system, which gives scientists the ability to precisely vary mobile-phase strength, pressure, and temperature. With the ability to fine-tune the resolving power and selectivity of the system, scientists can exercise better control over the retention of analytes for separating, detecting, and quantifying structural analogs, isomers, and enantiomeric and diasteriomeric mixtures — all compounds that are often difficult to separate by any other means. Its key features include  

A 10-µL fixed-loop injector for partial-loop injections of between 0.5 µL and 10 µL, which preserves sample and eliminates the need to change sample loops.

Reduced system volume, which enables shorter run times, optimizes gradient performance, reduces band broadening, and enables the use of small-particle-size columns.

Cosolvent and column switching for more flexible method development, and faster solvent and column screening.

Gradient accuracy and precision for retention time reproducibility.

Improved optical detection and MS compatibility for conducting both qualitative and quantitative analyses.

Waters showcased its second-generation UHPLC instrument called the Acquity UPLC H-Class system at Pittcon in 2010. Last year, Waters introduced three offerings in this area, the Acquity UPLC H-Class Bio System, the nanoAcquity UPLC System with 2D Technology, and the nanoAcquity UPLC system with HDX Technology. New for 2012 is another platform extension, the Acquity UPLC I-Class system, which is a binary system designed for MS. The I-Class system features reduced system volume and carryover. Reduced system volume significantly decreases dispersion rates for reproducibly higher resolution and peak capacity, and the resulting small peak volumes extend the sensitivity of any mass spectrometer. Additionally, low dispersion allows reduced separation cycle times without impacting resolution even for gradients lasting less than 1 min. Depending on the application, users can choose either a high-precision, low-dispersion fixed-loop sample manager (injector), or a low-dispersion, variable-volume sample manager with a flow-through needle design, which is designed to deliver high-precision injections, excellent sample recovery, and low sample carryover to optimize the performance for mass detectors. The I-Class system offers the capability to switch between two columns with independent thermal zones that can be operated from 4 °C to 90 °C. The column manager can be configured with interchangeable valves for serial column runs when configured with a second Acquity pump to support two-dimensional separations. Figure 2 illustrates an example of a separation from a 

 monograph for the analysis of irbesartan that was scaled from HPLC to UHPLC, comparing the scaled separation on the I-Class system to a standard UHPLC system. While providing linearity over four orders of magnitude, an increased signal-to-noise ratio (S/N) is also observed for both the main component and a minor 0.05% related compound impurity. 

Eksigent, part of AB Sciex, introduced the new ekspert microLC 200 system, a dedicated splitless microflow UHPLC system. Optimized for integration with AB Sciex mass spectrometers with control by Analyst software (while still operating as an inlet for any vendors mass spectrometer), it offers increased sensitivity over analytical flow UHPLC, faster separations, and reduction in sample and mobile-phase consumption in a small footprint. The microLC binary gradient pumping system operates at 5–200 µL/min, at pressures up to 10,000 psi. Microfluidic Flow Control technology based on actual flow rate feedback to a rapidly adjustable pressure source reportedly delivers flow rate precision better than 5% RSD. Featured applications include peptide analysis from digested liver microsomes, and pharmaceutical and personal care products at trace levels in wastewater. 

Agilent introduced a new photodiode-array detection (DAD) module, in what the company has termed the 1200 Infinity Series High Dynamic Range (HDR) DAD system. The system takes advantage of Agilent's Optofluidic Waveguides Max-Light flow cells using total internal reflection in a noncoated fused-silica fiber detector cell. By combining the signals from two diode-array detectors with different pathlength Max-Light flow cells, the system allows detection and quantitation of all components in a sample in a single run — making it ideal for the analysis of mixtures with widely different concentration levels. Two model 1260 or 1290 Infinity diode-array detectors are deployed; one is fitted with a 3.7-mm pathlength flow cell, and the other has a longer 60-mm pathlength flow cell. The signal from the longer-pathlength cell is used to quantitate low concentrations, and the signal from the shorter pathlength cell is used for the high concentrations. Specially developed algorithms are designed to correct for the different retention times and compute these signals to expand the linear dynamic range by a factor of 30 and sensitivity by 8–10 times compared to earlier generation Agilent diode array detectors. An example of the use of the HDR DAD system is shown in Figure 3 for the analysis of a pharmaceutical formulation that has two active pharmaceutical ingredients (API). The concentrations of API 1 and API 2 are widely different and previously two separate runs with different injection volumes were necessary to quantitate both APIs and their respective impurities. With the HDR detection system both APIs and all impurities can be quantitated in one run, reducing sample turnaround time and boosting laboratory productivity. 

Shimadzu introduced the SIL-30ACMP UHPLC autosampler, which combines with the optional CTO-30AS column oven as a front end for liquid chromatography–mass spectrometry (LC–MS) and LC–MS-MS systems. The autosampler holds up to six plates for increased capacity and up to 2304 samples when using 384-well plates. It has an open-access design that permits users to access the sample racks at any time, except during the injection process. Low carryover is maintained with a modified rinse mechanism, minimizing sample contamination. With an injection operating time of 7 s and a cycle time of 14 s, Shimadzu claims it is the "world's fastest autosampler." The optional CTO-30AS column oven mounts directly on the side for close proximity to the autosampler and can be positioned from the vertical to the horizontal to minimize tubing lengths, which is important for reducing extracolumn band broadening effects in UHPLC applications. 

Waters introduced NuGenesis 8 software featuring Laboratory Execution (LE) Technologies, a comprehensive data management and workflow system designed to support the entire product life cycle from discovery through manufacturing. The user-centric platform comprises NuGenesis SDMS, a compliant-ready data repository, and NuGenesis ELN, a flexible electronic laboratory notebook. The software's sample management feature allows analytical laboratories to manage and track samples and test requests through their entire life cycle, including transferring sample information from a laboratory information management system (LIMS) or systems applications and products (SAP) automatically into the electronic laboratory execution (LE) methods along with the specifications for the product to be tested. Data retention and legal hold capabilities allow organizations to manage the entire scientific data life cycle based on the organization's record retention policies from beginning to end in a compliant, secure environment. For example, if the record retention policy at an organization is to keep a certain type of record for five years, then purge records beyond five years, the system can automatically manage this process. However, if any records are undergoing legal consideration, then a legal hold flag is placed on these records and they are retained until legal consideration has concluded. 

Electronic LE methods (predefined templates) that guide analysts through standard operating procedures (SOP) for pharmaceutical quality control (QC) testing also are a part of NuGenesis 8, providing direct interface to instruments and software platforms used for QC testing such as balances, pH meters, reagent databases, LIMS, enterprise resource planning (ERP) systems, and chromatography data systems. With instrument and consumable inventories you can ensure that analysts use only valid or calibrated instruments and consumables throughout a given analysis. Automated fit-for-use checks avoid the inadvertent use of expired chemicals or solutions as well as noncalibrated instruments and provide a single focal point for test result review and approval. NuGenesis 8 also manages data flow between laboratory instruments and information systems such as LIMS and ERP, increasing the efficiency of laboratory workflow and reducing waste and variability through information management standardization by  

providing quick and easy data accessibility for research and intellectual property management, 

facilitating product development and delivery by improving collaboration between functional groups and across multiple geographies, and 

serving as a long-term solution for data preservation and retention management.

The information in this review is partially based on manufacturers' responses to a preconference questionnaire mailed in late 2011. I used the information received in the questionnaires that were returned, as well as information from personal communication to try and make this review as comprehensive as possible. But keep in mind that because of the fact that some manufacturers did not respond to the questionnaire, or do not release preshow information, and the sheer size of the conference this report cannot be considered an exhaustive listing of all new products that were introduced in Orlando, Florida. I'm sure there were a lot of additional noteworthy items at Pittcon, but time, space, and the scope of this column prevents me from covering them all in detail. I'm bound to have missed a few items or details, and I apologize for any omissions. In 2013, Pittcon is scheduled to take place March 17–21 in Philadelphia, Pennsylvania. I hope to see you there! 

 "Innovations in HPLC" Editor Michael E. Swartz, PhD, is Principal Scientist in Analytical Development at Ariad Pharmaceuticals, Cambridge, Massachusetts, and a member of LCGC's editorial advisory board. Direct correspondence about this column to "Innovations in HPLC," at 

Innovations in HPLC

Highlights of some of the new HPLC and related technology showcased both before and at this year's conference

HPLC Systems and Components Introduced at Pittcon 2012: A Brief Review

A pioneer in high performance liquid chromatography reflects on his career and how "enlightened trial-and-error" can reduce the effort involved in method development.

Choosing conditions for a final separation ("method development") is often carried out by trial-and-error. With some experience and basic high performance liquid chromatography (HPLC) knowledge, this process can evolve into enlightened trial-and-error — in which fewer trials are required. Efficient method development aims at optimal results with minimal effort; it requires a quantitative understanding of how different experimental conditions affect the quality of a separation, and is best implemented with computer simulation. My own career, which emphasized improvements in method development, provides a background for the following short review. 

I was born in Sacramento, California, where in 1939 I received a chemistry set for Christmas. What proved fascinating was that stuff called compounds could be predictably assembled from something called atoms — nicely illustrated in the form of jigsaw cutouts (Figure 1).  

The concepts of valence and balanced equations were thus revealed, if in primitive form. This initial interest in chemistry grew over time with visits to the local library and later experiments (many dangerous!) in a home laboratory. When I entered the University of California at Berkeley in 1948, I was firmly committed to a career in chemistry.  

An important refinement of this goal came during graduate research at Berkeley under Professor Donald Noyce (1952–1954). My research topic was the mechanism of an aldol condensation (1), which introduced me to physical-organic chemistry and model building. This provided an important tool for the next six decades . . . the only thing missing was a research area in which to apply this tool. That would soon be supplied. 

My first job was at Shell Oil in Houston, under the direction of M.J. ("Jack") O'Neal. Jack received the ACS Award in Petroleum Chemistry in 1956 for his pioneering development of mass spectrometry (MS) for the analysis of high-molecular-weight petroleum samples. Al Zlatkis, a future "impresario of chromatography," was part of the group when I arrived. A little later we were given the job of constructing a gas chromatography (GC) system using information from the proofs for a book by Keulemans (my first exposure to chromatography).  

We began measuring retention times for various hydrocarbons, which led me to a connection between chromatography and model building in the form of the Martin equation:  

 is the retention factor for a substituted molecule (for example, 

 for the unsubstituted molecule (for example, benzene), and Δ

 is a constant for a given substitutent group (for example, methyl or ethyl). Later, we were asked to develop an assay of gasoline, which resulted in the development of "two-stage GC" (2) — an early form of column switching. By now, the power of chromatography, its connection to physical-organic chemistry, and the practical importance of method development were apparent. 

Adsorption Chromatography and Petroleum Analysis (1957–1971)

In 1957, I moved to the Union Oil Co. of California, where my starting assignment was the analysis of high-boiling petroleum samples such as lubricating oil. Pioneering work of this kind had been carried out in O'Neil's group, where the sample was initially separated by adsorption chromatography into saturated hydrocarbons, aromatic hydrocarbons, and nonhydrocarbons. This was followed by the MS analysis of the hydrocarbon fractions. My experience at Shell suggested that previous such separations were empirically derived and poorly understood — so they likely could be improved. George Lake, my new supervisor at Union Oil, agreed and gave me a month to study the problem. The month grew into a separate 14-year research program and a 1968 book (

 [3]). A simple equation summarized many of our findings,  

 is a solute retention factor, Ψ is the phase ratio, 

 is the relative area of the solute molecule, and ε measures mobile-phase polarity or "solvent strength." 

 is the adsorption energy of the solute, values of which can be estimated as a function of solute molecular structure via equation 1 — in turn enabling predictions of 

 for different compounds and separation conditions. 

My 1968 book would not be needed in today's HPLC laboratory, as information of present interest has since migrated into more recent books and publications. But it did describe a previously unappreciated phenomenon: 

. Localization, the noncovalent attachment of polar molecules to specific adsorbent sites, was further studied over the next four decades (4) and found to largely determine solvent selectivity for adsorption chromatography (5) — an important contribution to method development for separations using silica or other adsorbents. 

Petroleum consists mainly of hydrocarbons (alkanes, cycloalkanes, alkylbenzenes, and so on) plus lesser concentrations of related sulfur compounds (alkyl sulfides and thiophenes), and still smaller amounts of nitrogen- and oxygen-containing ("hetero") compounds. Our work on adsorption chromatography quickly led to improved compound-type separations of hydrocarbons plus sulfur compounds, for their subsequent MS analysis, as at Shell-Houston. This was accompanied by the development of complementary procedures for assaying certain other compound types in petroleum by so-called "linear elution adsorption chromatography" (6). 

By 1966 we had completed work on the total analysis of hydrocarbons and sulfur compounds in any petroleum distillate, but our knowledge of the hetero compounds in such samples was incomplete. These oxygen- and nitrogen-containing molecules include several different compound types (such as, substituted pyridines, indoles, and furans), many of which were yet to be identified. Consequently, a complete analysis of these petroleum hetero compounds represented a major challenge. Our planned approach was the separation of different hetero-compound types from each other and from interfering hydrocarbons and sulfur compounds, followed by the spectral analysis of individual hetero fractions.  

A multistep separation (7) made sequential use of three different adsorbents (alumina, silica, and charcoal) plus cation and anion exchange to provide about 50 hetero fractions. We could predict which compounds might be present in each fraction (equation 2), and this information with corresponding MS, UV, and infrared (IR) data resulted in the first comprehensive analysis of the N- and O-containing compounds in a petroleum distillate (8,9).   

So, here we have an approach to pre-HPLC method development that relied on a quantitative knowledge of separation as a function of conditions for different possible compounds present in petroleum. This anticipated "efficient" method development, but is not applicable to HPLC, which deals with the more challenging separation of individual 

The arrival of HPLC in the mid-1960s has been described (for example, see reference 10). In 1966, I began packing columns with alumina for use with a crude, homemade HPLC system (11). An example of its subsequent use in our laboratory is shown in Figure 2 for the analysis of hydrogenated quinoline samples. An initial gradient separation (Figure 2a) revealed major peaks 1–5 that were eluted between 10 and 25 min. This suggested the routine isocratic separation of Figure 2b, where an additional peak (peak 3a) is seen.  

By 1970, the potential of HPLC had become obvious, but clearly its application within the petroleum industry would be limited. This realization — combined with the completion of our original petroleum-analysis project — led me in 1971 to join the Technicon Corporation (Tarrytown, New York). At that time Technicon was the largest manufacturer of automated-analysis equipment for use in clinical laboratories. 

It's worth recalling what HPLC looked like in 1971, as summarized in an early HPLC book (13). Commercial equipment and columns were available, and some of what had been learned about GC was being applied to LC. For example, the quality of a separation could be described by the resolution 

 of the two, poorest separated peaks in a chromatogram; 

 can be related (indirectly) to experimental conditions via retention factors 

, separation factors α (selectivity), and the column plate number 

In the beginning, HPLC method development emphasized the improvement of resolution by predictable changes in conditions that affect values of 

 this might involve varying column length or flow rate (for example, see reference 15). The selection of preferred values of 

 ≤ 10) could be achieved by changing the proportions of a binary-solvent mobile phase; for reversed-phase chromatography an increase of organic solvent in the mobile phase by 10%v provides a predictable, 2–3-fold decrease in 

The use of reversed-phase chromatography and gradient elution was quite limited in 1971; most HPLC separations were carried out isocratically by partition, adsorption, or ion exchange. Little was known of how best to change selectivity α, which is the most important parameter for controlling resolution. As in GC, random changes in the column (for example, stationary phase) were commonly tried at first. HPLC method development by "enlightened" trial-and-error was not often practiced, and a basis for "efficient" method development did not exist. 

From 1972 to 1977, I was VP of Research and later Clinical Chemistry at Technicon, with HPLC being downgraded to a "hobby interest" — however, the hobby was pursued with vigor. In 1971, Jack Kirkland and I presented the first ACS short course on HPLC ("Introduction to Modern Liquid Chromatography"), a two-day tutorial that continued during the next three decades with the later participation of John Dolan and Joe Glajch as co-instructors. Three editions of a book with the same title resulted (1974, 1979, and 2010 [17]), as well as two editions of a related book on method development (1987 and 1997 [18]). Both the course and the books proved popular, with >6000 total attendees and 50,000 books sold. 

 (Wiley) by Karger, Snyder, and Horváth also appeared in 1973. 

In 1977, I reduced my management responsibilities at Technicon and returned to hands-on R&D. Our immediate goal was an integrated HPLC system for the automated analysis of pharmaceutical drugs in serum — including sample preparation. The final equipment could be regarded as a technical success (19), but a market failure; automated immunoassay proved better suited for routine use. However, we maintained an active HPLC research program from 1977 until I left Technicon in 1982. Our group included John Dolan, Sjoerd van der Waal, Steve Bannister, and Russell Gant, aided by collaborations with Nobuo Tanaka and others from Barry Karger's group at Northeastern University.  

Lloyd R. Snyder, Inc., was founded in 1982 as a one-man company based on HPLC. For the next seven years I also served as a consultant for DuPont Instruments in Wilmington, Delaware, which included supervising HPLC projects of my choice for four PhD students from nearby universities: Marilyn Stadalius (Delaware), Mary Ann Quarry and Julie Eble (Villanova), and Barbara Ghrist (Pennsylvania). In 1984, Lloyd R. Snyder, Inc., morphed into LC Resources with John Dolan as cofounder and headquarters in California; Tom Jupille joined us in 1987 as a third partner. Method development formed a significant part of our business from the beginning, with LC Resources growing to 30 employees by 2002. I also served as an editor of the 

 from 1987 to 2000; Figure 3 shows several colleagues at my 2001 retirement party.  

In 2002, LC Resources was split and sold to Rheodyne and Bioanalytical Systems. A few years later, the company was resurrected with the original three-man staff and an emphasis on short courses and collaborative research. From 1982 to present day, John, Tom and I taught HPLC to more than 10,000 individuals (apart from the ACS course), developed and marketed DryLab software (see below), and managed a method-development contract laboratory. 

By 1977, reversed-phase chromatography had become the primary HPLC procedure, and the use of gradient elution was becoming more common. However, further improvements in method development would require a better understanding of 

 (equation 3) as a function of separation conditions, for both isocratic and gradient elution. "Efficient" method development (EMD) would not be possible until these relationships were made quantitative, so as to allow predictions (and optimization) of separation by a computer. A systematic approach to method development would also be needed. 

Over time, a better understanding developed of which separation conditions have the greatest impact on selectivity (values of α). The importance and use of mobile-phase pH was well understood (for example, see reference 21), but only ionizable compounds are affected by pH. Less was known about other conditions that determine selectivity. EMD requires a choice of which separation conditions are best varied for an improvement in resolution. This choice must then be narrowed to specific solvents or columns, followed by preliminary separations. Finally, optimal conditions can be selected by some combination of further separations, experience, and computer simulation.   

Work that eventually enabled EMD will be discussed next: advances in our understanding of selectivity as a function of separation conditions; a practical model of gradient elution; quantitative predictions of separation as a function of either isocratic or gradient conditions (computer simulation); and a method-development strategy. While many workers have contributed to our present knowledge, the following summary emphasizes those projects in which my group was involved. 

The solvent-selectivity-triangle was introduced in 1973 (Figure 4), based on GC data by Rohrschneider for six test compounds and 80 solvents. This allowed the characterization of each solvent in terms of three possible interactions with the sample: hydrogen bonding (donor or acceptor) and "polar" (22).  

Three generalizations follow from Figure 4. First, as expected, solvents of similar functionality, for example, alcohols or ethers, fall within the same part of the triangle (grouped within circles). Second, a change in selectivity requires the use of a solvent from a different part of the triangle. Finally, it should be possible with mixtures of just three, well chosen solvents to approximately match the selectivity of any other solvent, for example, a reduction in the number of solvent choices from the original 80 to just three. The latter observation contributed in 1980 to the widely used four-solvent mapping system of Glajch and Kirkland for reversed-phase chromatography (23). Seven experiments allowed resolution to be predicted as a function of mobile-phase composition (mixtures with water of methanol, acetonitrile, and tetrahydrofuran), followed by the computer-selection of a "best" mobile phase. Based on this approach, the DuPont Sentinel system for "automatic" method development was introduced a few years later. Sentinel represented a big step toward "efficient" method development.  

While the solvent triangle had its virtues, its derivation from data for pure solvents made it at best a qualitative model for the aqueous mobile phases of reversed-phase chromatography — as noted over the years (24). Indeed its ininitial application was intended for nonaqueous liquid–liquid chromatography, for which it was better suited. Other studies at about the same time also contributed to the selection of preferred solvents for controlling selectivity in reversed-phase chromatography (25,26). Today, methanol or acetonitrile is usually a first choice, with solvent selectivity optimized by varying their proportions in the mobile phase. 

Solvent-Strength and Temperature Selectivity

Before 1985 it was widely assumed that controlling selectivity required changes in either the organic solvent, mobile-phase pH, or the column. That is, changes in either solvent strength (that is, the organic-solvent concentration, %B) or temperature were regarded as ineffective. In the mid-1980s, however, two different studies (27,28) showed that a change in isocratic %B or (equivalently) gradient steepness could in some cases provide marked changes in selectivity and successful separations. While isolated examples of temperature selectivity were known from the 1950s, its potential usefulness in reversed-phase chromatography method development was widely ignored. Some key experiments suggested by Bill Hancock in 1993 demonstrated that a change in temperature could be of great value for the separation of peptides (29). A few years later, we undertook a comprehensive study of the contribution of both %B and temperature to reversed-phase selectivity for various nonpeptide samples (30). Simultaneous changes in these two conditions were found to provide a convenient and generally effective control over selectivity (31), and for many laboratories this is now a preferred first step in method development. See the example of Figure 5 in a following section on computer simulation.  

While a change of stationary phase can be an effective way of varying selectivity, at the present time there are >1000 different reversed-phase columns from which to choose. A number of procedures for characterizing column selectivity were reported prior to 2000 (for example, see references 32–34), but with certain limitations (35). In 1998, John Dolan and I organized an exploratory meeting with Jack Kirkland, Pete Carr, John Dorsey, and Uwe Neu

LCGC North America-04-01-2012

A Practical Method for Trace Level Analysis of Chlorinated Organic Pesticides by Gas Chromatography–Mass Spectrometry

Electron Ionization for GC–MS

New Chromatography Columns and Accessories at Pittcon 2012, Part I

HPLC Systems and Components Introduced at Pittcon 2012: A Brief Review

Efficient HPLC Method Development and Personal Reflections

Retention Time Changes

Vol 30 No 4 LCGC North America April 2012 Regular Issue PDF