Sample introduction

To introduce the sample, the left-hand end of the capillary is dipped into a sample vial with several centimeters hydrostatic pressure for a fixed number of seconds. This will force a small volume of liquid sample into the end of the capillary. 

Another method of sample introduction is to dip the end of the capillary into the sample vial and turn on the power for a few seconds. Sample ions thus flow into the system by electrical migration. 

The method used to introduce the sample to the source of the spectrometer will depend on the method of ionisation used (see section 12.3), the physical form of the sample, and any requirement for separation techniques to be applied in line with the MS analysis. 

For many routine applications, a solid or liquid sample may be introduced to the source of the spectrometer by direct mechanical insertion, i.e. it is held on the end of metal probe which is inserted into the source via a system of air-locks. All of the sample is presented to the source simultaneously, allowing no separation of the components, and the resulting spectrum is a function of the whole of the sample. For gas-phase ionisation techniques an added level of discrimination may be obtained by selectively vaporising the components of the sample. Volatility discrimination is achieved by acquiring data as a function of time while the 

For most volatile or gaseous species more selectivity may be achieved by combining mass spectrometry with gas chromatography. This is applicable to some nonionic surfactants [1] and derivatives of anionics [2]. The combination of gas chromatography with mass spectrometry (GC-MS) is well established, and relatively cheap dedicated instruments are available for this application. 

When less thermally labile compounds require separation prior to analysis, in-line liquid chromatography-mass spectrometry (LC-MS) can be used. Even where liquid chromatographic separation is not required LC-MS interfaces may be used as a convenient method of sample introduction, e.g. for polar, ionic, thermally labile, involatile or high-molecular-weight species. There are several different types of LC-MS interface each with its own characteristics in terms of the solvent systems and sample types that it can handle and the appropriate solvent flow rates no one interface is applicable to all solvent-solute combinations. 

Three considerations determine how samples are introduced to the gas chromatograph. First, all constituents injected into the GC must be volatile. Second, the analytes must be present at an appropriate concentration. Finally, injecting the sample must not degrade the separation. 

Preparing a Volatile Sample Gas chromatography can be used to separate analytes in complex matrices. Not every sample that can potentially be analyzed by GG, however, can be injected directly into the instrument. To move through the column, the sample s constituents must be volatile. Solutes of low volatility may be retained by the column and continue to elute during the analysis of subsequent samples. Nonvolatile solutes condense on the column, degrading the column s performance. 

Volatile analytes can be separated from a nonvolatile matrix using any of the extraction techniques described in Ghapter 7. Fiquid-liquid extractions, in which analytes are extracted from an aqueous matrix into methylene chloride or other organic solvent, are commonly used. Solid-phase extractions also are used to remove unwanted matrix constituents. 

General structures of common stationary phases for gas chromatography. 

Fused-silica fiber coated with stationary phase 

Care must be taken to introduce the sample for pyrolysis into the furnace without admitting air, since the pyrolysis zone is already hot and degradation begins immediately. The heating rate of the sample is dependent on the sample material itself 

Another approach has been developed by Shin Tsuge at the University of Nagoya in Japan. His furnace pyrolyzer includes a cool chamber where samples are loaded into a small crucible above the hot zone. Once the sample is in place, the cup is rapidly lowered into the furnace for pyrolysis. 

In most cases, sample introduction on-chip is achieved using electrokinetic (EK) flow [3]. Two important EK injection modes, namely, pinched injection and gated injection, have been developed. Furthermore, some alternative injection methods are described. 

There are a number of methods for applying sample to the column which can be categorised as 

However, syringe injection is little used today. 

1 Valve injection. Valve injection of the sample is now the preferred and accepted technique. Sample application is rapid, the solvent flow from the pump does not have to be stopped and these systems are easy to use, readily adapted for automated injection and can operate at pressures up to 6000psi (41.4MPa) with reproducibility 0.2%. Six-port valves are commonly used, either fitted with an internal or an external sample loop and are an integral component of an HPLC system. 

2 Syringe injection. This was the first type of sample application system developed and closely resembled GC and involved syringe injection of the sample through a self-sealing septum. The greatest column efficiency is achieved by application of the sample via a syringe directly onto the column bed, due to 

The principal merits of such systems were the ability to vary sample size the efficiency of sample usage and that they were relatively inexpensive. However, on-column injection has a number of disadvantages principal amongst which is that they cannot be used at the high column back pressures associated with modern packing materials and thus this technique is simply of historical interest. 

For the purpose of sample introduction, any sample introduction system (also sample inlet system or inlet) suitable for the respective compound can be employed. Hence, direct probes, reservoir inlets, gas chromatographs and even liquid chromatographs can be attached to an El ion source. Which of these inlet systems is to be preferred depends on the type of sample going to be analyzed. Whatever type the inlet system may be, it has to manage the same basic task, i.e., the transfer of the analyte from atmospheric conditions into the high vacuum of the El ion source Table 5.1 provides an overview. 

Note The below-mentioned sample introduction systems (reservoir inlets, various direct insertion probes, and chromatographs) are of equal importance to other ionization methods. 

Given the variety of forms and the nature of possible samples, there are numerous methods of introducing samples, each with its own particular requirements, into mass spectrometers. The sample can be ionized either before or in the ion source of the spectrometer. 

For gases or volatile liquids a very small quantity of sample is introduced with a micro-syringe to a kind of reservoir linked to the ionization chamber via a very narrow channel. Under the effect of a high vacuum that is maintained in the reservoir, the compound is sucked up and vaporized. This procedure is known as a molecular leak or molecular pumping. For continuous monitoring of gases and volatile compounds in the vapour state or dissolved in a liquid such as water, the sample can be diffused through a porous membrane. 

Solid samples, able to have a sufficient vapour pressure at about 300 °C, are deposited on the tip of a metal support, which is inserted in the source of the instrument through a vacuum lock, then heated. For some other modes of ionization the solid sample is mixed into a matrix (e.g. glycerol or benzoic acid). 

Complex mixtures of compounds are often analysed by hyphenated techniques in which a separative method, as chromatography, is interfaced with a mass spectrometer. Thus, the various constituents of the sample can be separately analysed. Coupling of a gas chromatograph to a mass spectrometer (GC/MS) has become widespread. The outlet of the GC capillary column is directly inserted in the ionization chamber of the MS. In this way, compounds eluting from the column, mixed with the carrier gas, are introduced in the ionization chamber and then analysed in the order in which they exit the GC. As the flow rate never exceeds 1-2 mL/min, the pumping capacity of the spectrometer is sufficient for maintaining the high vacuum required for the analysis. 

Nowadays, interfacing of a liquid chromatograph or a capillary electrophoresis instrument with a mass spectrometer is used too, although technically more complex. The presence of water in elution solvents is an undesirable compound for the mass spectrometer. With HPLC, the use of micro-columns is desirable, to have very low flow rates. They are also well-suited with different ionization techniques for the analysis of high molecular-weight compounds. A rather old device, whose sensitivity is now judged to be very poor, is the particle beam interface 

Normally samples are introduced as solutions into the plasma, but the direct introduction of solids and gases is also possible. Hydride and cold vapour methods are also applied to plasma atomic emission spectrometry. In addition, plasmas can be used as detectors for gas and liquid chromatographs. 

The sample solutions are made into aerosols by a nebulizer system and transported into the plasma with a carrier gas. Pneumatic, ultrasonic, or grid nebulizers are used for nebulizing the sample. Water or organic solvents can be removed from the aerosol by a desolvation unit if the plasma source cannot take large quantities without interference. 

Since the mass spectrometer operates under high vacuum, the first important step is to transfer a sample analyte from atmospheric pressure to the high vacuum of the instrument. 

FIGURE 1.3 Direct insertion probe in vacuum lock. 

Matrix-assisted laser desorption ionization MALDI 

Many different techniques have been developed to effect the production of ions, the most common of which are shown in Table 1.4. Mass spectra that are generated by electron (El) are discussed in detail in Chapter 2. Some of the other ionization modes are discussed in Chapter 4. 

Ions may be positively charged by the removal of one or more electrons or negatively charged by the addition of one or more electrons. In addition, smaller mass ions are formed by various fragmentation processes discussed in the next chapter. 

With the exception of on-line trace enrichment and/or derivatisation systems, the form of injection is almost exclusively by use of a sample loop with rotary valve (Rheodyne or similar). Normally the size of sample loop is used to decide sample size rather than injection volume. As in other types of laboratory, septum type injection systems in environmental laboratories have long been superseded. 

The range of analytes now determined by HPLC in environmental samples is increasing all the time some of the established techniques used are as follows. 

Samples submitted for analysis can be introduced into the mass spectrometer in many forms. Probably the most common means are (1) direct introduction, (2) gas chromatography (GC), and (3) liquid chromatography (LC). 

In direct introduction the sample can be introduced via a sample probe or plate through a vacuum lock, and can subsequently be ionized via El, Cl or matrix-assisted laser desorption ionization (MALDI see Section 2.4). Alternatively, the sample can be introduced as a liquid stream into an ion source at atmospheric pressure, after which it is subjected to electrospray ionization (ESI see Section 2.3). Direct injection does not offer any form of sample separation. 

It is important to remember the few restrictions imposed by electrospray when considering an LC-MS analysis. Common solvents like methanol, water, acetonitrile and volatile salts (below 25 mM) like ammonium acetate and ammonium bicarbonate are acceptable in the mobile phase, whereas phosphate salts/buffers, mineral acids or other nonvolatile components cannot be used. Unfortunately, this conflicts with many of the routine mobile phases used for the analysis of phenolic compounds and anthocyanins, necessitating changes in methods when going from LC to LC-MS analyses. 

Many other analytical techniques can be coupled to mass spectrometers. These so-called hyphenated techniques, like GC-MS and LC-MS, include but are not limited to ICP-MS (inductively coupled argon plasma), SCF-MS (supercritical fluid), NMR-MS (nuclear magnetic resonance) and IR-MS (infrared). 

Chapter 3 examines one of the most critical areas of the instrument—the sample introduction systan. It discusses the fundamental principles of converting a liquid into a fine-droplet aerosol suitable for ionization in the plasma, and presents an overview of the different types of commercially available nebulizers and spray chambers. Although this chapter briefly touches upon some of the newer sampling components introduced in the past few years, the new breed of desolvating nebulizers and chilled spray chambers are specifically addressed in Chapter 17. 

When we examine each of these essential mass spectrometer functions in detail, we see that the mass spectrometer is somewhat more complex than just described. Before the ions can be formed, a stream of molecules must be introduced into the ion source (ionization chamber) where the ionization takes place. A sample inlet system provides this stream of molecules. 

With nonvolatile samples, other sample inlet systems must be used. A common one is the direct probe method. The sample is placed on a thin wire loop or pin on the tip of the probe, which is then inserted throngh a vacnnm lock into the ionization chamber. The sample probe is positioned close to the ion sonrce. The probe can be heated, thus causing vapor from the sample to be evolved in proximity to the ionizing beam of electrons. A system such as this can be used to study samples of molecules with vapor pressures lower than 10 mmHg at room temperature. 

In the last decade, a nnmber of open air sample introduction methods have been developed that essentially eliminate sample preparation. In various atmospheric pressure chemical ionization (APCI) techniqnes, the sample is placed in a stream of ionized gas (Section 3.3B) or solvent aerosol (Section 3.3D) between the ion source and the inlet to the mass analyzer. 

The most versatile sample inlet systems are constructed by connecting a chromatograph to the mass spectrometer. This sample introduction technique allows a complex mixture of components to 

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While some techniques require the absence of electroosmotic flow during the separation itself (capillary gel electrophoresis and capillary isoelectric focusing), most common techniques exploit electroosmotic flow for sample introduction and detection. Electrophoresis in buffer-filled capillaries uses electroosmotic flow in an analogous manner to a chromatographic mobile phase the flow is used to transport analyte from cathode to anode and separation occurs continuously between introduction and detection. 

Two introduction methods are commonly employed in capillary electrophoresis. Hydrodynamic injection is based on siphoning, or gravity feeding the sample into the anodic end of the capillary. The anodic end is removed from the buffer reservoir and placed in the sample solution. The capillary end is then raised so that the liquid level in the sample vial is at a height Ah above the level of the cathodic buffer, and is held in this position for a fixed time t. This process has been automated for reproducibility, and the hydrodynamic flow rate has been shown to obey Eq. 12.9  

Electrokinetic injection involves drawing sample ions into the capillary interior with an applied potential. The anodic end of the capillary is removed from the buffer reservoir and placed into a sample vial along with the anode. An injection 

Both injection techniques have been shown to suffer from the effects of analyte diffusion, specially when the clean capillary is initially introduced into the sample solution. Diffusion occurs across the boundary area between analyte and buffer, which is defined by the cross-sectional area of the capillary. Both techniques also suffer from the effects of inadvertent hydrodynamic flow that results from the reservoir liquid levels being at slightly different levels. While these effects are significant for the buffer-filled capillaries used in capillary zone electrophoresis, they are both much less important when the capillary is filled with a gel. 

Detectors used in the initial experiments with capillary electrophoresis were simple absorbance and fluorescence detectors that had been adapted from HPLC equipment. However, it soon became apparent that these instruments yielded poor 

Samples analyzed by El mass spectrometry must be converted to gas phase. For pure gases or volatile liquids the samples may be introduced directly through a small orifice that allows an appropriate amount of material into the vacuum chamber. A small amount of a solid sample can be placed in a melting point capillary tube and inserted into the mass spectrometer at the end of a metal rod, called a direct insertion probe (DIP).The temperature at the tip of the probe can be varied to promote sublimation of the sample. Another common method of sample introduction is gas chromatography, which is the ideal choice for samples that are impure. 

Sensitivity is another distinguishing feature of mass spectrometry. This sensitivity has allowed mass spectrometers to act as detectors for capillary columns, which can separate mixture containing hrmdreds of compounds, when less than a nanogram(10 g) of each compound is injected. 

Chromatographic resolution increases as a function of the square root of the column length, and the extraordinary length of capillary columns means that most simple mixtures are easily resolved on just a few types of stationary phases. One common nonpolar stationary phase is poly( methylsiloxane) (R = CH3) which can be made slightly more polar by the incorporation of phenyl groups (typically 5% phenyl) in place of methyl groups  

While samples may be directly injected onto a packed column, the small diameter of the capillary column presents a problem. In addition, it is easy to overload the capillary column with sample (Table 8.35). Two techniques for getting the sample into the column are split and split/splitless injection. 

TABLE 11.4 Some Species Measured by Mass Spectrometry in the Atmosphere up to about 1990  

Adapted from Viggiano (1993) see references therein for original literature. 

For example, if a compound (T) has proton affinity 170 kcal mol-1, transfer of a proton from H30+(H20) occurs  

Near the earth s surface, there is sufficient ammonia in air that it undergoes a reaction with H30+(H20) to form NH4(H20) , and this can also act as an 

In the negative ion mode, species with electron affinities greater than 44 kcal mol can accept an electron from 0J(H20) ions, forming a T ion with mass equal to the molecular weight, M  

Flow accuracy across a range of backpressures and solvent compositions can be evaluated by collecting and weighing timed aliquots of solvent. Pressure readings are plotted on a strip-chart recorder from a transducer signal output point. The recorded values should be within the manufacturer s specifications and as consistent and close to the programmed values as possible. 

Chapter 3 Instrumentation for High-Performance Liquid Chromatography 

The sample loop size can be varied depending on the volume of sample. Typical sample loops range from 5 /A to 5 ml, and each must be manually removed before another can be put in place. The replacement of one loop size for another is one of the major disadvantages of the injector design. The accuracy of this type of injector varies with sample loop size, from about 5% for a 2-ml loop to as much as 30% for a 5-pl loop. 

The need for unattended and precise sample injection for HPLC has lead to development of a wide variety of automated sample injection devices. Autosamplers function in essentially the same way as manual injectors, except that the sample is introduced automatically from a sample vial held in a carousel or an X-Y grid (Fig. 3.15). The carousel format provides a reliable and rapid means of moving samples past an injection station, whereas the XY grid format allows a convenient random access configuration. 

Sample is introduced into the sample loop in a variety of ways the vial may be pressurized to force the sample out, or a syringe may be used to draw the sample out. The syringe is usually controlled by a stepping motor so that different sample volumes can be injected reproducibly by partially filling the sample loop. Once the sample loop has been loaded, the valve is electrically actuated. Some autosamplers also include other features such as sample heating or cooling, and the ability to perform standard additions, thereby improving the precision of the analysis. 

Due to the very small volumes of the capillaries used, the capillary volume is 1 pi for a 50 pm ID X 50 cm capillary, the injection volume need to be small (low nanoliters) to avoid injection volume contribution to band broadening. The maximum plate number obtainable in an electrophoretic separation is N = Lp/lf, where Id is the capillary effective length and I is the length of the injection band. This means that the injection band should be 10nl to obtain 1000000plates in a 50 pm ID x 1 m capillary. 

Injection is performed mainly as hydrodynamic or electrokinetic, but can also be done by isotachophoresis, special loop techniques, and various stackingtechniques - the latter are not included in this book. For more information, refer to Ref. [3]. 

Foliovhng the injection, the inlet end of the capillary is moved to a vial with separation electrolyte solution, and the migration is started by applying the 

Commercial instruments are mostly available with a UV-Vis detector and some with a fluorescence detector. When polyimide oated fused silica capillaries are used, the polyimide coating needs to be removed in the detection area. Fiber optics are used in most detectors. 

The most commonly used detector is the U V detector. Unfortunately, the detection limits are not very good. This is mainly due to the short optical path length, which is identical to the diameter of the capillary. 

FIGURE 8.1 The components of a mass spectrometer. (From Gross, J. H., Mass Spectrometry A Textbook, Springer, Berlin, 2004. Reprinted by permission.) 

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Other methods of sample introduction that are commonly coupled to TOP mass spectrometers are MALDI, SIMS/PAB and molecular beams (see section (Bl.7.2)). In many ways, the ablation of sample from a surface simplifies the TOP mass spectrometer since all ions originate in a narrow space above the sample surface. 

Molecular beam sample introduction (described in section (Bl.7.2)). followed by the orthogonal extraction of ions, results in improved resolution in TOP instruments over eflfrisive sources. The particles in the molecular beam typically have translational temperatures orthogonal to the beam path of only a few Kelvin. Thus, there is less concern with both the initial velocity of the ions once they are generated and with where in the ion source they are fonned (since the particles are originally confined to the beam path). 

Frequently an analyst must select, from several instruments of different design, the one instrument best suited for a particular analysis. In this section we examine some of the different types of instruments used for molecular absorption spectroscopy, emphasizing their advantages and limitations. Methods of sample introduction are also covered in this section. 

The reduction of the yellow-colored Mo(VI) complex to the blue-colored Mo(V) complex is a slow reaction. In the standard spectrophotometric method, it is difficult to reprodudbly control the amount of time that reagents are allowed to react before measuring the absorbance. To achieve good precision, therefore, the reaction is allowed sufficient time to proceed to completion before measuring the absorbance. In the FIA method, the flow rate and the dimensions of the reaction coil determine the elapsed time between sample introduction and the measurement of absorbance (about 30 s in this configuration). Since this time is precisely controlled, the reaction time is the same for all standards and samples. 

Therefore, if a large quantity of sample is introduced into the flame over a short period of time, the flame temperature will fall, thus interfering with the basic processes leading to the formation and operation of the plasma. Consequently introduction of samples into a plasma flame needs to be controlled, and there is a need for special sample-introduction techniques to deal with different kinds of samples. The major problem with introducing material other than argon into the plasma flame is that the additives can interfere with the process of electron formation, a basic factor in keeping the flame self-sustaining. If electrons are removed from the plasma by... 

To examine a sample by inductively coupled plasma mass spectrometry (ICP/MS) or inductively coupled plasma atomic-emission spectroscopy (ICP/AES) the sample must be transported into the flame of a plasma torch. Once in the flame, sample molecules are literally ripped apart to form ions of their constituent elements. These fragmentation and ionization processes are described in Chapters 6 and 14. To introduce samples into the center of the (plasma) flame, they must be transported there as gases, as finely dispersed droplets of a solution, or as fine particulate matter. The various methods of sample introduction are described here in three parts — A, B, and C Chapters 15, 16, and 17 — to cover gases, solutions (liquids), and solids. Some types of sample inlets are multipurpose and can be used with gases and liquids or with liquids and solids, but others have been designed specifically for only one kind of analysis. However, the principles governing the operation of inlet systems fall into a small number of categories. This chapter discusses specifically substances that are normally liquids at ambient temperatures. This sort of inlet is the commonest in analytical work. 

In principle, DSI is the simplest method for sample introduction into a plasma torch since the sample is placed into the base of the flame, which then heats, evaporates, and ionizes the sample, all in one small region. Inherent sensitivity is high because the sample components are already in the flame. A diagrammatic representation of a DSI assembly is shown in Figure 17.4. 

Almost any kind of ion source could be used, but, again, in practice only a few types are used routinely and are often associated with the method used for sample introduction. Thus, a plasma torch is used most frequently for materials that can be vaporized (see Chapters 14-17 and 19). Chapter 7, Thermal Ionization, should be consulted for another popular method in accurate isotope ratio measurement. 

Sample introduction. The transfer of material to be analyzed into the ion source of a mass spectrometer before or during analysis. 

Sample introduction system. A system used to introduce sample to a mass spectrometer ion source. Sample introduction system, introduction system, sample inlet system, inlet system, and inlet are synonymous terms. 

Programmed-temperature vaporizers are flexible sample-introduction devices offering a variety of modes of operation such as spHt/sphtless, cool-sample introduction, and solvent elimination. Usually the sample is introduced onto a cool injection port liner so that no sample discrimination occurs as in hot injections. After injection, the temperature is increased to vaporize the sample. 

P. Sandra (Ed.), Sample Introduction in Capillary Gas Chromatography, Vol 1, Chromatographic Method Series, Huethig, Basel, 1985. 

Sample introduction into the ionizing plasma is normally carried out in the same manner as for ICP-OES. An aqueous soludon is nebulized and swept into the plasma. 

Approximately 70 different elements are routinely determined using ICP-OES. Detection limits are typically in the sub-part-per-billion (sub-ppb) to 0.1 part-per-million (ppm) range. ICP-OES is most commonly used for bulk analysis of liquid samples or solids dissolved in liquids. Special sample introduction techniques, such as spark discharge or laser ablation, allow the analysis of surfaces or thin films. Each element emits a characteristic spectrum in the ultraviolet and visible region. The light intensity at one of the characteristic wavelengths is proportional to the concentration of that element in the sample. 

An ICP-OES instrument consists of a sample introduction system, a plasma torch, a plasma power supply and impedance matcher, and an optical measurement system (Figure 1). The sample must be introduced into the plasma in a form that can be effectively vaporized and atomized (small droplets of solution, small particles of solid or vapor). The plasma torch confines the plasma to a diameter of about 18 mm. Atoms and ions produced in the plasma are excited and emit light. The intensity of light emitted at wavelengths characteristic of the particular elements of interest is measured and related to the concentration of each element via calibration curves. 

Table 1 Typical detection limits (ppb) for iCP-OES (using a pneumatic nabuiizer for sample introduction) of the most sensitive amission line betwean 175 nm and 850 nm for each element.

Detection limits for a particular sample depend on a number of parameters, including observation height in the plasma, applied power, gas flow rates, spectrometer resolution, integration time, the sample introduction system, and sample-induced background or spectral overlaps. ... 

Samples must be introduced into the plasma in an easily vaporized and atomized form. Typically this requires liquid aerosols with droplet diameters less than 10 pm, solid particles 1-5 pm in diameter, or vapors. The sample introduction method strongly influences precision, detection limits, and the sample size required. 

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