In liquid chromatography (LC), the syringe functions primarily as a pipette or liquid-transfer device that loads a sample loop. The syringe generally does not take an active role in injection, which occurs only after sample has been displaced from the syringe. The same is true of most gas chromatography (GC) gas-sampling valves — the time at which the gaseous sample is injected into the column is separate from the moment that it is transferred into the injection system. In GC analysis of liquid samples, however, the syringe becomes an integral part of the inlet during injection: sample, in liquid or gaseous form, starts to enter the column as soon as the syringe enters the inlet. 

In GC inlet systems for liquids, the injection technique, choice of syringe and inlet operating conditions all play a crucial role in the injection process. Two principal sample-transfer mechanisms move sample from the syringe into the inlet while the syringe is in the inlet. First, liquid-sample transfer takes place as the syringe plunger is depressed and liquid is expelled from the syringe tip. In cold injection, where the inlet temperature is not high enough to produce significant solvent vaporization, this pipette-like action is the only major sample-transfer mechanism. However, a competing process occurs in a hot injector. Within a few tenths of a second after the needle enters a hot inlet, sample begins to evaporate inside the needle. Bubble formation and concomitant increased internal pressures force some liquid out along with the vapour, so that part or all of the sample contained in the syringe needle volume is introduced into the inlet as a mixture of liquid and vapour. As the plunger is depressed, additional room-temperature liquid sample is forced from the syringe through the needle, which cools the needle and suppresses but does not entirely stop in-needle evaporation. The needle heats up again once the syringe plunger motion ceases, which causes additional sample vaporization from the needle into the inlet. All of these processes take place in a matter of seconds. The total amount of sample that is actually injected into the inlet depends strongly upon these two processes, their timing, the volumes involved and the inlet conditions. Along with judicious injection condition control, a good understanding of the role of the syringe in these processes will help gas chromatographers obtain better injections. 

Syringe-needle effects influence not only the injected sample volume; they can also modify the relative amounts of individual sample components that enter the inlet. To understand this secondary effect, consider that not all sample components have the same vapour pressures at a given temperature. Lower molecular weight compounds have higher vapour pressures, and conversely, heavier molecules have lower vapour pressures. This differentiation forms the basis for simple thermal fractionation of a mixture of compounds — in a distillation column, for example.  

Unfortunately, for many gas chromatographers, the same kind of thermal fractionation process can occur in the syringe needle during injection. When in-needle vaporization occurs, the lighter components vaporize first and leave the syringe needle quickly. Heavier compounds take longer to evaporate and leave the syringe needle more slowly. The effect of initial needle heating during injection is remediated largely by subsequent bulk liquid transfer through the needle: straggling heavy compounds will be rinsed out. At the end of injection, however, if the needle is withdrawn from the inlet before complete transfer of all compounds can occur, then the sample that enters the inlet system will contain more of the volatile components and less of the heavier components than were present in the sample before injection. This effect is called "mass discrimination" because it tilts the sample composition according to components' vapour pressures, which are related largely to their masses.  

Two other thermal side-effects go hand-in-hand with needle fractionation. Many compounds are sensitive to thermal stress, especially in the presence of a hot steel syringe needle, and decompose rapidly when subjected to high inlet temperatures: high molecular weight glycerols are one good example. Polar compounds can become strongly adsorbed on the needle surface. Many of the polar polyaromatic hydrocarbons (PAHs) as well as a host of chlorinated pesticides suffer from this problem. In both instances, the use of nickel syringe needles, or needles that have been deactivated by advanced surface treatments, can help tremendously, as can deactivation of inlet liners and injector inner surfaces exposed to the sample. In these situations, thermally mild injection techniques such as cold on-column injection or programmed temperature vaporization usually produce results superior to what can be obtained by careful deactivation of needles and other hot injection materials, but the specialized inlets needed for these somewhat more complex techniques might not be available.  

Judicious data-system calibration and the use of multiple internal standards can compensate for inaccuracies caused by mass discrimination, decomposition and adsorption, as well as for a host of other injection problems. However, the precision and repeatability of multiple analyses are affected adversely when the relative amounts of individual components strongly depend upon injection conditions. Heavier components — against which injection strongly discriminates — or any other components that do not come through the injection process at 100%, will exhibit degraded lower detection limits (LDLs) as well. Degradation of precision, repeatability and LDL are significant effects even for samples that do not span a wide range of volatilities and do not suffer from mass discrimination 

When this type of injection problem does arise, its effects must be either suppressed or controlled by applying special syringe-handling techniques or by using cold injection if possible. The "sandwich" technique is a good example. The syringe is first filled with 1 μL or so of pure liquid solvent followed by a small air plug. The sample is pulled into the syringe, then another plug of air, and finally some more solvent. The entire liquid array is pulled up into the syringe so that the needle is largely empty. Upon injection, only a little solvent is in the needle as it enters the inlet. Sample passes through the needle as the syringe plunger is depressed, followed by a plug of solvent that forces any remaining sample into the inlet. Only pure solvent is left in the needle at the end of injection, so needle fractionation does not occur. The drawback of the sandwich technique is that it significantly increases the amount of solvent that enters the inlet, which might overload the inlet with solvent vapour or broaden the solvent peak and interfere with early-eluted analytes. 

A high-speed autosampler can also remediate needle fractionation. By injecting the sample in a very short time, typically less than 500 ms and certainly much faster than a human can achieve, the syringe needle residence time in the inlet is insufficient for significant needle heating to occur, and so needle fractionation is eliminated. Another benefit of a fast injection is the suppression of sensitive-component thermal decomposition or adsorption on hot steel needle surfaces, again as a result of reduced contact times. Most modern GC autosamplers employ high-speed actions by default. 

In-needle fractionation and other thermal effects are some of the syringe-related processes that interfere with injection accuracy and repeatability. There are many other effects that occur before, during and after sample has entered the inlet system: see the book by Grob for a comprehensive discussion.

The choice of syringe type plays an important role in obtaining the best performance from a gas chromatograph. There are two basic kinds of manual syringes: plunger-in-barrel [Figure 1(a)] and plunger-in-needle [Figure 1(b)]. In the first type, sample is drawn up into a calibrated glass barrel; in the second, the entire sample is contained in the needle, and a thin wire plunger fitted into the bore of the needle forces liquid out during injection.  

In this type of syringe, sample is contained in the glass barrel [see Figure 1(a)]. Since you can see the sample, it is easy to observe the presence of liquid sample and determine if the syringe needle is obstructed. In use, the syringe is usually overfilled with liquid and the extra is then ejected. A final clinging droplet can be removed from the needle tip with a clean laboratory wipe or by rinsing with solvent. To gauge the injected volume, consider that the amount displaced from the markings on the syringe barrel does not include sample vaporized from the needle and is only an approximation. The actual amount injected is closer to the needle volume (about 0.7 μL for a 10 μL syringe) plus the amount displaced. Another way to determine injected volume is to load the syringe with sample, pull the plunger back and read the total volume of the liquid plug. After injection, pull the plunger back slightly, read the remaining liquid volume, and subtract it from the original amount. In this way, sample evaporated from the needle is included in the total amount. Plunger-in-barrel syringes can be used for most routine applications. The common 10 μL syringe can reliably inject liquid volumes ranging from 1 μL to 10 μL.  

Plunger-in-barrel syringes can be cleaned in several ways. Pumping solvent in and out of the syringe is effective and can be a good idea between injections to remove leftover sample before it evaporates and leaves a film of residue behind. Eventually, a layer of nonvolatile residue and some particles will contaminate a syringe. The slightest amount of particulate matter inside the syringe barrel will immediately begin to degrade the precision fit between the plunger and the inner glass barrel bore, so cleanliness is crucial for the longest syringe life. This is even more important for plunger-in-barrel autosampler syringes, in which the plunger is cycled up and down many more times than for manual injection. For best results with slightly contaminated samples, use a syringe that has a polymer seal on the plunger tip. 

A commercial syringe cleaner heats the syringe needle to as high as or above the injection temperatures under a mild vacuum. This will remove some residual material, but it will not remove nonvolatile residues and particulates, nor will it clean the barrel and plunger. In most syringes of this type, the plunger is removed easily for solvent washing. With the plunger removed, the solvent can be drawn through the syringe body from the back with mild suction. Be careful, however, that particles inside the upper end of the barrel are not drawn into the needle where they could block flow. If the needle does become blocked, insert a fine cleaning wire — usually supplied with the needle — to remove the blockage. Whatever cleaning method you use, make sure you do not use abrasives because they will destroy the precision fit between.  

The plunger-in-needle syringe is useful for injection volumes < 5.0 μL; the 1.0 and 0.5 μL sizes are more common. In this type of syringe, the needle contains the entire sample [see Figure 1(b)]. Sample-distortion effects that are as a result of partial sample displacement are reduced because the plunger extends the length of the needle, but these effects are not eliminated entirely. The injected amount can be read directly off the barrel. But these syringes are more complex in construction, with a removable needle and seal assembly. You cannot see liquid sample in the needle, so it is difficult to tell if the needle is obstructed.  

Cleaning plunger-in-needle syringes is more difficult because the plunger should not be removed except for major repairs (such as replacing the needle). Nevertheless, you can get good results by pumping solvent into the syringe to remove soluble residue, followed by heating the needle under vacuum. Note that for any GC syringe, only the needle should be heated above 50 °C. Plungers can stick, glass barrels can crack and syringe seals can develop leaks if the entire assembly is overheated.  

Needles come in fixed and removable styles, with various tip shapes. The fixed-needle syringes are the most economical, but if the needle is damaged, the syringe cannot be repaired. Removable-needle syringes will last longer, but must be maintained by checking that the needle-to-barrel seal remains intact as the moving parts wear. Never use a Luer-lock (medical) type of removable syringe needle for GC liquid injections because these have an excessively large dead volume. Instead, select one of the types specifically designed for GC and designated as a "microsyringe" with a zero-dead-volume seal.  

Needle tip shape can affect septum life. Most manufacturers produce a standard tip with a 20° bevel that is optimized for penetrating GC septa. The 14° medical bevel is not recommended. Plunger-in-needle syringes have blunt, nonbevelled tips. Of these, the dome-shaped and the electropolished taper types are the best for the septum. The correct needle tip minimizes septum coring and the deposition of septum pieces inside the inlet, which otherwise can produce "ghost" peaks. In some situations, a needle with a side hole can be advantageous. 

Syringe needles also come in a variety of dimensions. An outer diameter of 0.47 mm (26 gauge) is fairly standard and permits the needle to penetrate a 0.53 mm i.d. wide-bore fused-silica column in an on-column style inlet. The somewhat stronger 0.7 mm (22 gauge) o.d. needle is often used if bent needles are a problem. However, this larger needle can shorten septum life. Syringe needle lengths are generally either 5 or 7 cm. The length required is usually a function of the inlet design, and the manufacturer's recommendations should be followed.  

Several types of specialized syringes are available. The most familiar is the autosampler syringe. Original equipment manufacturer supplied syringes are best, but compatible syringes are available from syringe manufacturers that might have some features not otherwise available, such as specific needle tips or plunger seals. 

There are two types of autosampler syringes: front-loading and flow-through. The front-loading type, found on essentially all modern GC autosamplers, mimics a human operator's motions (except much more quickly and precisely). With the flow-through type, found on older autosamplers, sample is pushed from a pressurized sample vial through the syringe and out to a waste receptacle. Once the autosampler is loaded with sample, injection occurs in the normal fashion. These syringes tend to be more tightly integrated with the autosampler mechanics and are harder to replace. As mentioned previously, and worth repeating, is the fact that autosampler syringes need better care than manual syringes because they are subjected to greater mechanical stresses and more operating cycles.  

Another familiar speciality syringe is the cold on-column syringe. This is generally a plunger-in-barrel type with a special removable needle made of narrow-bore steel or fused-silica tubing. The needle is designed to penetrate a narrow-bore (0.20 or 0.25 mm i.d.) capillary column to accomplish on-column injection. Such syringes are not interchangeable across different injector designs; the manufacturer's specifications must be followed. On-column syringes can be more susceptible to needle blockage because of their smaller inner diameters. Many on-column injectors use a precolumn or retention gap made of uncoated 0.53 mm i.d. tubing, which accepts a standard 26 gauge syringe needle. 

Many syringe manufacturers offer syringe repair kits, needle replacements and repair service. Most removable needles can be replaced in the field if the rest of the syringe is in good shape. For plunger-in-needle syringes, needle damage is often accompanied by plunger damage, and in such instances, it is necessary to replace both as a matched pair.  

Syringe plungers are generally not field-replaceable (or interchangeable) because of the tight matching tolerances between plunger and barrel. One exception is microlitre gas-tight syringes with PTFE-tipped plungers, which will accept replacement plungers if the barrel is not otherwise damaged.  

Is syringe repair worth the cost? In the case of plunger-in-needle syringes, the answer is a definite "yes". Repair kits of matched plungers and needles are a quarter to a third of the cost of a new syringe. For the less-expensive plunger-in-barrel types, replacement needles are as much as half the cost of a new unit, and instead it might be worthwhile to attempt to reduce syringe damage and extend syringe life through better handling and cleaning procedures.  

A few syringe and sampling accessories are especially useful. First, a syringe rack is a good way to keep syringes in one place, rather than on top of the gas chromatograph or the bench, where they can be pushed off and broken easily. Syringe labels are also handy to keep track of which syringe is used for which type of sample.  

Another useful item is one of the various repeating adapters that permit the plunger to be returned to the same position each time, which can improve the repeatability of multiple injections. Also, repeating adapters act as guides and prevent plunger bending or accidental withdrawal from the barrel, a feature that is important on plunger-in-needle syringes. Several manufacturers also make syringes with extended or reinforced barrels and plungers that prevent damage from a heavy hand. 

When viewed as an active part of an inlet, the syringe takes on new importance. While injection technique is crucial, the proper care and cleaning of the syringe can contribute significantly to improved accuracy and precision. Choosing the correct syringe to meet sample and injector requirements, and then applying and maintaining it correctly, can go a long way toward producing better and more reliable analytical results. 

 is senior staff engineer at Serveron Corp., Hillsboro, Oregon, USA and a member of the Editorial Advisory Board of 

. Direct correspondence about this column to "GC Connections", 

, Advanstar House, Park West, Sealand Road, Chester CH1 4RN, UK, e-mail: 

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

Split and Splitless Injection for Quantitative Gas Chromatography: Concepts, Processes, Practical Guidelines, Sources of Error

, 4th, Completely Revised Edition (Wiley, Hoboken, New Jersey, USA 2001). 

Articles

GC Connections

Most modern GC autosamplers employ high-speed actions by default.

Syringes for Gas Chromatography

Bio-Rad Laboratories' Informatics Division has received a gold award for its KnowItAll Informatics System spectroscopy software and a silver award for its KnowItAll Cheminformatics Edition. The company was chosen by readers of 

 and the readers select favourite products in a number of different categories based on quality, reliability, ease-of-use, technical support and value. The company has been recognized for its products in the spectroscopy software category for nine consecutive years. 

The company's KnowItAll Informatics System is a fully integrated software and database package for multiple spectroscopic techniques, with tools for database building, management, search, analysis, prediction, reporting and spectral reference databases within a single user interface. Additionally, the software offers a series of solutions for chemistry, chemometrics, ADME/Tox and metabolomics. The Cheminformatics Edition is a complete unified system used to draw, modify, store, search and retrieve chemical structures and other information.  

For more information visit the company's website at 

Cohesive Technologies has announced that it has entered into a collaborative agreement with Mayo Clinic to increase patient sample throughput and improve data quality. The turbulent flow chromatography (TurboFlow technology) systems from Cohesive will be employed for clinical assays using tandem mass spectrometry (MS–MS). 

This collaboration is aimed at accelerating the development of clinical assays in areas including endocrinology, toxicology, drug monitoring and neonatal screening. The companies will share in the development of the TurboFlow and mass spectrometry methods to improve the quality and efficiency in diagnostics for adults, children and newborns. 

For more information visit Cohesive's website at 

The Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy has announced that the ACS Division of Analytical Chemistry (DAC) will host four invited symposia, four contributed sessions and an undergraduate poster session at Pittcon 2007. This venture is an experimental one-year trial and is expected to benefit all the parties concerned. 

In 2007, the ACS meeting and Pittcon will be held within three weeks of each other in Chicago (Illinois, USA) as the DAC committee has decided that this would be a good time to partner up. The DAC executive committee believes that its members would be better served if the DAC hosted sessions at Pittcon instead of the 233rd ACS National Meeting and Exposition. 

				Pittcon's publicity chairman, Adrian Michael, stated, "The benefit of this partnership is it will allow analytical chemists to attend Pittcon and the ASC-DAC Meeting at once."			

For more information visit the organization's website at 

German publication for separation scientists

In response to demand from German-speaking readers of 

 a German publication will be launched this month. 

 (LCGC AdS) will be an electronic magazine featuring the same turning-page format as seen in 

. It will be published entirely in German on a quarterly basis and distributed to 6000 German-speaking separation scientists in Germany, Austria and Switzerland.  

Editorial content will feature application-based technical articles that have not previously been published in 

 as well as translated popular columns, such as "LC Troubleshooting", "GC Connections" and "Sample Preparation Perspectives". The launch focuses on sample preparation and liquid chromatography–mass spectrometry. 

If you would like to subscribe to LCGC AdS please visit the website at 

News

Bio-Rad Laboratories' Informatics Division has received a gold award for its KnowItAll Informatics System spectroscopy software and a silver award for its KnowItAll Cheminformatics Edition. The company was chosen by readers of Scientific Computing in the 2006 Readers' Choice Awards.

Readers' choice

Hewlett-Packard (HP) has announced that its researchers have developed a miniature wireless data chip that could provide broad access to digital content in the physical world.  

Said to have no equal in terms of its combination of size, memory capacity and data access speed, the tiny chip could be stuck on or embedded in almost any object and make information and content available that is now found mostly on electronic devices or the Internet.  

Some of the potential applications include storing medical records on a hospital patient's wristband, providing audiovisual supplements to postcards and photos, helping to fight counterfeiting in the pharmaceutical industry, adding security to identity cards and passports, providing perfect photocopies and supplying additional information for printed documents: The chip can include a history of all the corrections and additions made to the text, as well as voice notes and graphical images. 

The experimental chip, developed by the "Memory Spot" research team at HP Labs, is a memory device based on CMOS (a widely used, low-power integrated circuit design) and about the size of a grain of rice or smaller (2–4 mm square), with a built-in antenna. The chips could be embedded in a sheet of paper or stuck to any surface, and could eventually be available in a booklet as self-adhesive dots.  

"The Memory Spot chip frees digital content from the electronic world of the PC and the Internet and arranges it all around us in our physical world," said Ed McDonnell, Memory Spot project manager, HP Labs. 

The chip has a 10 megabits-per-second data transfer rate — 10 times faster than Bluetooth wireless technology and comparable to Wi-Fi speeds — effectively giving users instant retrieval of information in audio, video, photo or document form. With a storage capacity ranging from 256 kilobits to 4 megabits in working prototypes, it could store a very short video clip, several images or dozens of pages of text. Future versions could have larger capacities. 

Information can be accessed by a read-write device that could be incorporated into a cell phone, PDA, camera, printer or other implement. To access information, the read-write device is positioned closely over the chip, which is then powered so that the stored data is transferred instantly to the display of the phone, camera or PDA or printed out by the printer. Users could also add information to the chip using the various devices. 

"We are actively exploring a range of exciting new applications for Memory Spot chips and believe the technology could have a significant impact on our consumer businesses, from printing to imaging, as well as providing solutions in a number of vertical markets," said Howard Taub, HP vice president and associate director, HP Labs. 

The chip incorporates a built-in antenna and is completely self-contained, with no need for a battery or external electronics. It receives power through inductive coupling from a special read-write device, which can then extract content from the memory on the chip. Inductive coupling is the transfer of energy from one circuit component to another through a shared electromagnetic field. A change in current flow through one device induces current flow in the other device. 

New Technologies

Hewlett-Packard (HP) has announced that its researchers have developed a miniature wireless data chip that could provide broad access to digital content in the physical world.

Mini data chip

This article describes two methods for analysing chelating agents found in nuclear waste: 									

ion-suppression chromatography using an anion exchange stationary phase and mobile phase consisting of a nitric acid solution and pure water gradient. UV detection was performed at 330 nm after the reaction with a postcolumn reagent composed of iron nitrate in perchloric acid. 

ion-pair chromatography with a mobile phase consisting of a mixture of nitric acid, tetrabutylammonium hydrogenosulphate, tetrabutylammonium hydroxide and iron chloride. A reversed-phase material was used as a stationary phase and detection was performed by direct measurement of the UV absorption at 260 nm. 

The quantification limits were lower for ion-pair chromatography than for ion-suppression chromatography.  

Both methods were easy to implement and allow a multi-element separation in less than 30 min with low detection limits. 

Nuclear waste regulations are becoming increasingly stringent and aminopolycarboxylic acids must be analysed in nuclear waste packages. Indeed, these species form water-soluble complexes with most heavy metals, which may result in the contamination of soils by radioactive elements. For example, studies at Oak Ridge National Laboratory (Tennessee, USA) have demonstrated that ethylenediaminetetracetic acid (EDTA) caused the low-level migration of 

Co from intermediate-level liquid waste disposal pits and trenches.

Thus, chelating agents such as nitrilotriacetic acid (NTA), ethylenediaminetetracetic acid, cyclohexanediaminetetracetic acid (CDTA) and diethylenetriaminepentacetic acid (DTPA) are considered by ANDRA (the French national agency for radioactive waste management) as compounds to be investigated because they may enhance the release of radioactive isotopes in the environment.

Ringbom explained that the chelating agents exhibit strong complexing power because of their carboxylic functions and their nitrogen and oxygen atoms.

  The complexation constants of iron (III) with chelates shows that these complexes are particularly stable.

Buchberger and Mülleder demonstrated that the stability of the complexes depends on the pH.

  When the pH increases, the chelating agents are more and more deprotonated and exhibit their complexing power (but obviously, at increasing pH metal ions may precipitate, which decreases the apparent stability). 

The chelating agents, such as EDTA, are largely used for the detection of metals by UV detection. For this reason metal (iron) complexes are generally used to perform the detection of these aminopolycarboxylic acids. Different analytical techniques have been used to perform the determination of chelating agents. 

Owens et al. studied EDTA and NTA with different metallic ions by capillary electrophoresis (CE), the determination limit for the iron–EDTA complex was 4 mg/L.

  Padarauskas et al. also separated EDTA, CDTA and DTPA by CE as complexes with metallic ions.

 The determination limits for the complex iron–DTPA was 0.9 mg/L. Laamanen et al., determined DTPA, EDTA and NTA by CE after complexation with Cu ions.

Collins et al. exploited ion chromatography/mass spectrometry to quantify metal–EDTA complexes in soil solution; with this method it was possible to identify and quantify the metal–EDTA complexes in environmental samples.

  Nevertheless, iron (III)–EDTA, Ca–EDTA, Mg–EDTA and EDTA itself were not detected. Miller et al. analysed EDTA in dried bloodstains by ion chromatography and electrospray liquid chromatography/mass spectrometry/mass spectrometry (LC–MS–MS).

 Nevertheless, the determination limit was only 5 mg/L. Dodi et al. used electrospray LC–MS to investigate EDTA traces in nuclear wastes. Quantification limits of 2 μg/L (for 20 μL injected) were achieved.

  A German standard, dealing with the analysis of water, wastewater and sludge, describes the determination of NTA, EDTA and DTPA through LC; determination limits were 70 μg/L, 40 μg/L and 120 μg/L, respectively.

  A similar work was published as an application note from Macherey Nagel to investigate EDTA, NTA, DTPA.

A French standard was published for the determination of chelating agents (EDTA, HEDTA and DTPA) in fertilizers using ion chromatography.

 Moreover, an application note from Dionex proposed the quantification of EDTA, NTA and phosphates by ion-suppression chromatography.

The determination of these chelating agents by gas chromatography has also been reported

  but the main drawback of this technique is the need to perform a derivatization step prior to injection. All samples must be treated with an esterification reagent prior to analysis but the yield of the reaction may depend on the sample matrix. 

Nowack and co-authors have reported works in ion chromatography coupled with ICP/MS to separate organometallic complexes with a mobile phase composed of NH

  The quantification limit for EDTA is between 0.5 and 1.0 μM.  

The aim of this article is to propose and to compare two ion chromatographic methods (ion-suppression and ion-pair chromatography) for the simultaneous determination of four aminopolycarboxylic compounds: NTA, EDTA, CDTA, DTPA (Figure 1) with low detection limits.  

For all eluent and standard preparations, deionized (DI) water was provided by a point-of-use water-purification system from Millipore (St Quentin en Yvelines, France). Milli-Q gradient A10 was linked to an Elix and Rios system. The eluents and DI water reservoirs were purged with helium before performing the separation. The standard solutions were prepared from EDTA (Titriplex III), NTA (Titriplex I), CDTA (Titriplex IV) and DTPA (Titriplex V) purchased from Merck (Darmstadt, Germany). 

 A nitric acid solution (100 mM) was prepared from a 69% solution purchased from Merck. This solution was poured into a 1 L flask and a second flask was filled with DI water. The postcolumn reagent was prepared in a 1 L flask with 1 g of iron (III) nitrate nonahydrate purchased from Prolabo (Paris, France) and 20 mL of 70–72% perchloric acid solution from Merck. 

 Preparation of the eluent required a diluted nitric acid solution (prepared from a commercial 69% solution purchased from Merck), tetra-n-butylammonium hydroxide (prepared from a commercial 40% solution) from VWR (Fontenay sous bois, France), tetra-n-butylammonium hydrogenosulphate from Merck and anhydrous iron (III) chloride from VWR. 

On both systems, a Dionex (Sunnyvale, California, USA) DX 500 ion chromatograph equipped with a manual degassing system, an automatic sampler, a quaternary gradient pump and an AD 20 absorbance detector was used.  

The sample loop size was 500 μL on both methods. Samples were introduced into the instrument via an AS40 automated sampler, using 5 mL PolyVials with pain caps. All instrument modules were purchased from Dionex. Instrument control and data collection were performed with a PC and Peaknet software (Dionex). 

For ion-suppression chromatography an AS7 column (4 × 250 mm) equipped with an AG7 guard column (4 × 50 mm from Dionex) was implemented. The mean particle size of the resin was 10 μm (data given by the manufacturer). A GP 40 gradient pump mixed the eluent constituents (water and 100 mM HNO

) for the gradient program used. The flow-rate was adjusted to 1.0 mL/min, and the UV/visible module was set at a wavelength of 330 nm. With this system, a postcolumn reagent was mixed with the mobile phase through a mixture chamber, just before detection. 

In ion-pair chromatography the stationary phase tested was an RP Nucleodur C

 Pyramid (4 × 250 mm) from Macherey Nagel (5 μm mean particle diameter). The GP 40 gradient pump was also used but no gradient program was established. The flow-rate was adjusted during the separation. 

The flow-rate was 0.8 mL/min for 15 min, after which it was set at 1.10 mL/min to reduce the elution time of the last peak (CDTA). Detection was performed with an absorbance detector (AD 20) at 260 nm. 

Eppendorf (Hamburg, Germany) research pro pipettes were used to prepare standard and sample solutions. A stock solution (500 mg/L) of NTA, EDTA, DTPA and (100 mg/L) CDTA was prepared by dissolving the commercially available product in deionized water. Diluted solutions for analysis were freshly prepared everyday of analysis. 

This ion exchanger (purchased from Dionex) permits the retention of cationic metals and was used to perform the tests dealing with the influence of metals.  

Statistical calculations were performed using the formula given below (from reference 19):  

 is the standard deviation measured with a low level standard and 

1 is the slope of the calibration curve. 

 Usually, chromatography of anions is performed with a basic mobile phase. In the instance of aminopolycarboxylic compounds, these anions would be too strongly retained on the resin with a basic media; for this reason it was necessary to proceed by ion-suppression chromatography with an acidic mobile phase. 

The eluent was composed of a gradient between water and a 100 mM nitric acid solution ([HNO

] = 15 mM from 0–7 min, to reach 20 mM at 17 min). The flow-rate was adjusted to 1 mL/min and the observed pressure was about 7.6 MPa (1100 psi). The chromatogram displayed in Figure 2 shows that the first eluted compound is CDTA followed by EDTA, then DTPA and NTA.  

This order of elution may be explained if we consider the pKa

 of the chelating agents (Table 1). The pH of the mobile phase was 2.6; subsequently, the higher the pKa

, the lower the concentration of the deprotonated form (A

 ) and thus the species is less retained on the resin. The pKa

 ]/[AH] calculated ratio) indicated in Table 1 allow us to explain the observed elution order.  

Table 1: pKa and [A-]/[AH] ratio (calculated at pH 2.6) for the two first acidities of the aminopolycarboxylates.			

Linearity was obtained up to 1 mg/L, according to the XP T 90–210 AFNOR standard. The least-squares equations and the correlation coefficients for each compound are reported in Table 2.  

Table 2: Calibration curves parameters, detection limits and quantification limits calculated for the ion suppression chromatography method.			

Then the limits of detection (LD) and quantification (LQ) were calculated with the formula given earlier, according to XP T 90–210 AFNOR standard; the results are shown in Table 2. The mean noise observed with this method was 65 μAU. 

 The mobile phase was composed of 0.6 mM HNO

, 2.6 mM tetrabutylammonium hydroxide, 7.53 mM tetrabutylammonium hydrogenosulphate and 37 μM anhydrous iron (III) chloride (from reference 13). The pH of this mobile phase was 2.33. 

The tetrabutylammonium groups of the ion pairing reagents are maintained close to the C18 alkyl chains by hydrophobic interaction and the cationic ammonium moiety allows the reverse phase to have a similar behaviour as a dynamic anion exchanger.  

The flow-rate was adjusted to 0.8 mL/min and the observed pressure was 11 MPa (1600 psi); when the flow-rate was increased at 1.1 mL/min, the pressure reached values of approximately 17.6 MPa (2550 psi). 

Ion Chromatography

Chelating agents such as NTA, CDTA and DTPA are considered by ANDRA (the French national agency for radioactive waste management) as compounds to be investigated because they may enhance the release of radioactive isotopes in the environment.

Simultaneous Determination of Chelating Agents by Ion-Suppression and Ion-Pair Chromatography in Wastewater

Despite the well-considered physics and engineering involved in mass spectrometer technology — that is, in creating ions and passing them through a series of lenses — it nonetheless seems that the quality of one's results is as much the product of art as it is of science. 

Years ago, an acquaintance of mine was pursuing graduate studies involving the physics of the vibrating string. I do not know what came of his efforts, but I do remember observing with interest that the mathematical precision of the science used to predict the string's behaviour and measure outcomes often went unappreciated by the end-users — the musicians. 

The term "intonation", when applied to a musical instrument, denotes how well the instrument plays in tune with itself. For example, as long as you immobilize a string at a specific point along its length, it should theoretically produce the same note each time. Yet on a finely crafted guitar, striking a harmonic at the 12th fret by dampening its vibration with your finger versus fretting the string (binding it behind the fret) to form the note can produce inconsistent results. In some positions along the length of the strings, one chord can sound perfect and another out of tune. These phenomena arise from the guitar's system of intonation; what musicians call its "temperament". 

An acoustic guitar's frets maintain a fixed tuning. The guitar's 12th fret is used to gauge whether the instrument's "equal temperament" (a system that divides the chromatic scale into 12 equal half-steps) produces the desired intonation. So for any guitar, what frustrates some refined ears is that the intervals between notes are not always perfect, or "pure", making it hard to accept idiosyncrasies in an instrument designed for precision. So, each guitar maker tries to create its version of optimal performance knowing well the physics that underlie the vibrating string, but knowing also that their instruments' particular brand of idiosyncrasy will play well in the hands of some musicians but not all. 

Like musical instruments, mass spectrometers operate with the same constancy of physics. Source and ion optics interface an atmospheric ionization inlet with a mass spectrometer, delivering sensitivity and economy of function. Producing ions from analytes must obey the same physical laws, such as the (still-debated) formation of electrospray ions, gas dynamics and desolvation to produce ions in the gas phase and conserve them via the source and ion interfaces. This is true regardless of whether those interfaces are attached to an ion trap, quadrupole or time-of-flight instrument. Yet results often differ when parallel experiments are attempted using different instrument designs. Or worse, the same experiment is attempted on different days using the same instrument. So you might ask: How can this be when electrospray ions created from the same analyte are identical?  

Apart from differences between instruments themselves, artefacts occur such as sample degradation and other vagaries of the sample handling process. Even more significant in terms of guaranteeing unequal results are the idiosyncrasies of practitioners. People can differ in their approach to calibration and tuning their instruments, and to some degree an assessment of a mass spectrometer's "tone" can be subjective. Producers of mass spectrometers design their instruments to work well within specific parameters. The constraints vary among manufacturers — otherwise all four major instrument manufacturers would offer the same design. Assessing the common points as well as the differences, real and alleged, can appear to be quite an art. 

Most in-service liquid chromatography–mass spectrometry (LC–MS) courses fail to address specific operational problems, and retaining consultants can be financially prohibitive. So one source that practitioners often turn to for advice is on-line discussion postings. These are perhaps the best way to appreciate that the rest of the MS world shares problems similar to yours. Yet, it is often a bit vexing to find the answers to those problems are not so clear-cut. With that in mind, I want to focus on sources to which you can go to find help, on helpful hints, and some anecdotes on spectral interpretation in upcoming columns. 

One well established source (since 1995) is the discussion group (SciTechniques), maintained by moderators David Bostwick (Georgia Institute of Technology) and Sarah Shealy (Georgia State University) at http://

. John Bartmess (University of Tennessee) offers a (more or less) weekly reprise of the discussions at https://

. Perusing the discussion thread can be useful and sometimes even amusing. Perhaps, best of all, it is free. 

I edited some samples of the exchanges, hoping to improve their coherence and also to ensure against unintended emphasis on either products or individuals. The exchanges lie buried as discussion threads, and digging them out can prove daunting. But it is simple enough to subscribe to the mailings, and have them show up periodically in your inbox. The helpful hints range from the obvious to what appear to be the beginnings of an advanced-degree thesis. 

Modifier, Linearity and Response Problems

This plea for help is by a chromatographer with a modifier problem: 

I have several positively charged (amine) compounds, which, for chromatographic reasons, must be analysed using a basic (triethylamine) buffer mobile phase. Because the mobile phase is only 0.1% triethylamine and about 50% acetonitrile, I thought I could simply acidify the effluent with a postcolumn infusion of formic acid (10:1 effluent–formic acid) via a tee. Unfortunately, I cannot see anything, even at high sample concentrations. Where am I going wrong? 

Have you ever had an LC–MS signal from your substances? If you have, was triethylamine then included in the mobile phase?  

(Comment: Triethylamine is highly ionic and can compete unfavourably with your analyte, especially if its concentration is much higher than the analyte's. You might find some additives such as triethylamine work better in negative rather than positive mode. Triethylamine can also present a persistent [m/z 102] background ion. Surprisingly, many do not first try to omit modifiers from their chromatography when transferring to the gas-phase environment of LC–MS from the condensed phase world of UV-only. Sometimes uncharted changes in equilibrium can and do take place in the transition. Sometimes it can be impractical — for instance, when it would require showing equivalence with an existing method based upon an accepted new drug application — but it can nevertheless provide a simplified comparison.)

Are you sure you are introducing the intended percentage of formic acid? 

Do the interface and postcolumn device work?  

(Comment: Any time a novel — that is, a less-than-satisfactory — response is encountered, operators must verify that the system is working correctly. An easy way to do this is to include a small amount of "known good" analyte to the postcolumn infusion. At the very least, every practitioner should have a sample that gives a well-documented response at hand.)

This spectrometrist has a linearity problem: 

I have problems with the linearity of some compounds while using LC–MS in negative ion mode. The calibration curves are very nonlinear. The concentration is in the same range as that of other linear compounds. The compound is an acid, such as other compounds, but others do not exhibit this problem. I have tried several eluent compositions but without success.  

(Comment: The distracted analyst comes to various seemingly reasonable solutions, such as considering what eluent additives he or she could try that would improve the method. Is acetic acid better than formic acid? Is methanol or acetonitrile better? Yet the solution could be much easier.)

This could be caused by adsorption (compound-dependent or analyte-dependent). Changing from stainless steel to PEEK might improve performance. 

I had similar problems when I worked with a [triple quadrupole]. We could not find the problem [replacing solvent type–acid, and so forth] and an engineer suggested we lower the temperature of the source. We were running at 180 °C and lowered it to 150 °C. That solved the nonlinearity problem. Sometimes solutions are very simple.  

(Comment: Amen. And sometimes they are not. Indeed, not all linearity problems can be resolved in the discussion group format.)

This chromatographer missed both linearity and response: 

I am hoping that someone can help me. I am developing a method to analyse alkylphenol ethoxylates by electrospray LC–MS, and I ran into a problem. The analytes in my method give exclusively (M+Na)

  ions, so I am monitoring those. During a sequence of three injections the chromatogram baseline remains stable for a period of time. Then it drops for a few minutes only to rise again. Any peaks during the baseline drop are much smaller than where the baseline does not drop. This plays havoc with my quantification, especially when the internal standards are eluted during this time. 

The baseline suppression does not happen at the same time during each of the LC runs. It migrates all over the place, showing no discernable pattern. Because the depression of the peak height is much greater than the magnitude of the baseline suppression, I am assuming that what is happening is a disruption in the ionization process, leading to low and irregular ion formation and sampling efficiency. The first thing I thought was that this was perhaps a competition effect from coeluted interferences. So, I put 50 μM of sodium acetate in the mobile phase. All this did was clog up the spectrum with a bunch of clusters and reduce the overall sensitivity (10 μM seemed to be okay, though). In any case, the baseline problem persisted. My next thought was that it might be a problem with really non-polar stuff sticking to the column and carrying over into the next run (my runs are short, about 15 min) because I was running isocratic. No, I inserted a 15 min flushing–equilibration step, and this still did not get rid of it. 

The problem seems to occur most often with samples. But when it happens with standards, it really ruins quantification. It does not happen in negative mode; I get a nice baseline. Does anyone have an idea about what is going on here? Surely I am not the only person who has ever seen this problem. 

(Comment: In this case the submitter's reasoning was absolutely on target. The problem is widely recognized as the bane of quantitative MS in complex mixtures, and numerous publications are available for guidance. If I were to recommend one, it would be "Ion Suppression: A Major Concern in Mass Spectrometry," a tutorial written by Lori Lee Jessome and Dietrich Volmer, which appears in an upcoming issue of LCGC North America. It serves as not only a very comprehensive overview of what painstakingly has been collected in recent practice, but as a collection of references to pursue as needed.)

We will examine this topic in detail soon, in another column. For the moment, though, consider that atmospheric ionization work can often create adducts that range from the common to the exotic, making spectral interpretation and quantification difficult. 

A case of mystery ions — common sodium in a not-so-common mode: 

Could anybody give me some clues about the mechanism of sodium adduct formation under atmospheric pressure chemical ionization (APCI) so that I can control it? The Na

  adduct is commonly formed in solution in electrospray ionization (ESI). Is it possible the Na

  adduct can form in the gas phase by APCI? 

(Comment: The issue remained unresolved after a long but interesting and informative exchange. In the ensuing exchange, which I summarize here, the consensus was that sodium adducts form in solution requiring a considerably higher voltage rather than forming in the gas phase.)

Perhaps because of APCI's higher probe temperatures, certain analytes can be desorbed into the gas phase, resulting in a mixed electrospray and APCI spectrum. (A mixed spectrum is not uncommon and is in fact being exploited in various mixed or multimode ionization source designs.) If it is of interest to determine which mechanism predominates, a high-speed switching design such as the ESCi multimode ionization source (which comes standard on all Premier products from Waters Corporation, Milford, Massachusetts, USA) can be employed to characterize the response between the two mechanisms with good, reproducible fidelity. The Turbo V design (MDS Sciex, South San Francisco, California, USA) offers similar common source abilities. If your purpose is simply to ionize the majority of molecules in a solution, a simultaneous design such as the Agilent (Santa Clara, California, USA) multimode will suffice. All of these devices are, of course, limited by each vendor for use with their particular instrument line. 

You can minimize or avoid sodiation by using plasticware instead of glassware. Glass surfaces produce infinite amounts of sodium, which seems to affect some compounds readily. Also, you might see in the sample history that sodium buffers were used in the prep. 

Because the concentration of sodium in solution is not fixed, using sodium-adducts for quantification can present problems. One reader suggests adding 1 mM sodium acetate to the eluent and then running in ESI mode rather than APCI mode. Even though APCI is preferred, studies indicate it is less susceptible to suppression. This suggestion is not so extreme these days: 

Z-spray and similar designs minimize contamination and clogging. 

You might find that expending a bit of effort to hold contamination to a minimum improves downstream interpretation and response. Two major sources of contamination in reagent prep are the containers used for preparing solvents and the transfer devices. Detergents and surfactants can increase propagation of ESI ions and, therefore, sensitivity. Unfortunately, the increased sensitivity is for the surfactant contaminants, not the analytes. So, as an old hand at electrochemistry once told me, do not use anything in your preparative practice that you would not want to see in your spectral background. Segregate LC–MS containers and measuring devices for LC–MS use only. Do not use detergents, and rinse containers repeatedly before setting them aside to dry. Good practice also dictates preventing acids and plastics from coming together. 

Readers' remarks, though they sometimes fail to offer technical help, always remind us that we do not face these problems alone — cold comfort, perhaps. So if your problem seems instrument-specific, you likely will find someone who can sympathize. Just remember that LC–MS practice can be quite a complex orchestration of sample preparation, instrument condition, and suitability for the intended use, not to mention your own level of understanding — all of which are evolving at different rates. Clearly, if there is one benefit to point out from among LC–MS courses, reference literature, and other self-help sources, it is the decision you make before purchasing your instrument. Weigh the accessibility and history of your service and sales contacts well in the equation. Unless you are blessed with a colleague who is an experienced operator — which aside from major pharma sites can be rare — these folks are your best allies. 

 is principal scientist, LC–MS technology development at Waters Corp. (Milford, Massachusetts, USA); an adjunct professor and visiting scientist at Roger Williams University (Bristol, Rhode Island, USA); and a member of 

 Editorial Advisory Board. Direct correspondence about this column to "MS in Practice", 

, Advanstar House, Park West, Sealand Road, Chester CH1 4RN, UK or e-mail: 

MS in Practice

Producers of mass spectrometers design their instruments to work well within specific parameters.

2D polymer liquid chromatography is a powerful tool for the deformulation of complex samples; however, it is considered to be very specialized and time-consuming. This article shows how recent hardware and software improvements have led to the technique becoming a method for routine analysis.

The demands on chromatography, regarding the information density of the results, increase with the complexity of the samples under investigation. Every single separation method suffers from limited chromatographic resolution (peak purity,  limited peak capacity, determination of multiple property distributions etc.). Many attempts have been made to overcome the intrinsic limitations of the chromatographic process.  

				A powerful approach is to combine different separation techniques into a single experiment (multidimensional chromatography; also known as 2D chromatography, orthogonal chromatography and cross-fractionation).			

The combination of independent separation techniques improves the chromatographic resolution because of increased peak capacity, which is not the sum but the product of the peak capacities of each technique:

				The separation "angle" between two techniques that separate strictly according to different properties is considered as 90° and the separation is referred to as "orthogonal". 			

An advantage of chromatographic hyphenation over performing two isolated separation methods is the ability to correlate and identify the properties of different sample components. 

The mapping of four 2D experiments is illustrated in Figure 1 — the separation according to one property is plotted versus the separation according to a second property. As can be seen, projections to the axes of each "contour plot" are the same for all examples. The traces of those projections would also result from isolated, one-dimensional separations. Therefore, any traditional separation optimization strategies would fail in all of these situations. Nevertheless, the direct combination of both separation techniques can easily show the sample differences. The 2D separation is achieved by a consecutive separation of fractions eluting from the 'first dimension' using the 'second dimension' technique. The resulting contour plots can be read in a similar way to a geographical map that shows the first dimension on the y-axis and the second dimension on the x-axis.  

This type of separation is extremely useful for any kind of complex samples, which are difficult to separate, identify comprehensively or fully deformulate. This is most often the situation for synthetic, natural and biopolymers (monodisperse proteins are one well-known exception), which possess coexisting multiple property distributions [such as molar mass distribution, functionality type distribution, chemical composition distribution, molecular architecture (shape) distribution, to name just a few]. 

This article will focus on comprehensive 2D polymer liquid chromatography (LC) coupling a liquid adsorption mode technique (LAC) with size-exclusion chromatography/gel permeation chromatography (SEC/GPC), and on the options to use them as a high-throughput application. 

Since the 1980s, several groups have tried to implement this technique for polymer analysis. However, it was seen as a very time-consuming technique that could take days for a single experiment — depending on the degree of automation (off-line or stopped flow). In the last decade of the twentieth century, the first fully automated 2D LC experiments were reported that could further reduce the time consumption to less than one day. This led to an increased use of 2D techniques in R&D laboratories. 

More information on other aspects of 2D chromatography may be taken from a review about 2D chromatography of synthetic polymers.

  Multidimensional separations are also performed in other areas, such as in a current review that focuses on biomedical and pharmaceutical applications.

The implementation of 2D chromatography is straightforward if robust separation methods are available for each dimension. Two existing chromatography systems (consisting of a minimum of two pumps, two columns, one injector and one detector) can be used for the separation. A transfer valve for automated fractionation and appropriate software are also required. 

Comprehensive 2D work employs complete transfer of injected mass from the first to the second dimension. This ensures that the complete sample is analysed and detected in the second separation method. The advantages and limitations of different approaches will be discussed.

Full automation is a basic requirement for short analysis times. However, in the last couple of years it has become possible to reduce the total analysis time for a 2D experiment, including data evaluation, to one or two hours and in some situations to even shorter times. The following considerations are based on the most commonly used set-up for 2D chromatography in polymer analysis with a dual loop transfer injection valve (8-port, 2-position valve), an HPLC separation in the first dimension and a SEC/GPC (size-exclusion chromatography) separation in the second.

  A schematic of this set-up is shown in Figure 2.  

The effluent of the first dimension (HPLC column) is collected in a loop of the transfer valve and then injected automatically into the second dimension system (GPC column). The two loops are filled and emptied into the second dimension alternately. The interval between two valve switchings is determined by the time needed for one second-dimension analysis. To keep the cycle times as short as possible, the second dimension flow-rate must be high. The first-dimension flow-rate is usually very low and can easily be calculated, dividing the loop volume through the above mentioned time interval (for 100% sample transfer). Using this procedure, the sample eluting from the first dimension will consecutively be fractionated and analysed completely in the second dimension. 

There are three important technical improvements that can be used to "speed up" 2D chromatography: column technology, detection options and chromatography data systems. 

 It is important to keep the volume that is going to be fractionated as low as possible, so this should be considered during the development of the first dimension separation method. Recent introductions of short HPLC columns with particles smaller than 2 μm might help to transfer existing methods with long columns and bigger particles to those that will reduce the volume consumption but retain the resolution. 

The approach to speed up GPC separations is a different one. The size-exclusion separation is based on the accessible pore volume and if the porosity of the stationary phase is increased above a critical value, the matrix will no longer be stable. Therefore, the column volume should not be changed if a comparable resolution is to be achieved. In 2D chromatography, SEC is usually used as the second dimension technique that must be very fast to have a short interval between the transfer of two fractions. Normal analytical SEC columns (300 mm × 8 mm) are used with an eluent flow-rate of 1 mL/min, which leads to an analysis time of about 12 min. This requires a very low flow-rate in the first dimension (and, therefore, a very long analysis time for the complete experiment), since the storage loop of the transfer valve must not be overfilled before the next transfer to the second dimension takes place. This interval could be shortened by increasing the flow-rate of the SEC, but normal analytical columns have an optimum flow-rate of about 1 to 1.5 mL/min. Newer HighSpeed SEC columns can allow the flow-rate to be increased up to 10 mL/min, resulting in associated decreases in analysis time, but without a loss in resolution. This could be achieved by changing column dimensions to 50 mm × 20 mm and further optimization of the stationary phase. As the total column volume is similar, the quality of the separation is maintained.

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