Injection sample

Manual injection into a liquid chromatograph is usually performed with the aid of a rotary valve (generally one manufactured by Rheodyne or Valeo), similar to those used in FIA, but more sophisticated and expensive on account of their stricter pressure requirements. The injection operation can be readily automated by coupling a synchronous motor alternately switching the valves between their two positions. This can be controlled via a button or the microprocessor This alternative is of limited versatility as it does not allow programming of the injection valve, because this depends on the loop used, so that any change can only be introduced by manually replacing the loop. 

In this configuration, the aspirated sample volume is swept to the chromatographic column. The syringe is activated to pour its contents to waste, thereby avoiding carry-over between samples. 

The automation of the introduction of liquid samples in gas chromatography does not differ substantially from that described above for liquid chromatography, although the nature of the mobile phase calls for a slightly different instrumental design. There are several commercially available models, the commonest of which have no sample turntable. 

only small quantities of sample can be introduced onto the capillary if the high efficiencies characteristic of the technique are to be maintained, as discussed in connection with electromigration dispersion in Section 4.3.4. In general, the sample length should be less than 2% of the total capillary length. Although this can be an advantage for applications with limited volumes of sample, it can be a problem from the point of view of detection. 

The injection port is designed to introduce samples quickly and efficiently. Most GC work involves the separation of volatile liquid mixtures. In this case, the injection port must be heated to a rather high temperature in order to flash vaporize small amounts of such samples so that the entire amount is immediately carried to the head of the column by the flowing helium. The most familiar design consists 

FIGURE 12.4 A sample gas chromatogram of a four-component mixture. 

TABLE 12.2 Typical Injection Volumes for Various Column Diameters 

As implied above, the appropriate range of sample injection volume depends on column diameter. As we will see in the next section, column diameters vary from capillary size (0.2 to 0.3 mm) to i/8 and i/4 in. Table 12.2 gives the typical injection volumes suggested for these column diameters. The capillary columns are those in which the overloading problem mentioned above is most relevant. Injectors preceding the 1/8 in. or larger columns are not split. 

The accuracy of the injection volume measurement can be very important for quantitation, since the amount of analyte measured by the detector depends on the concentration of the analyte in the sample as well as the amount injected. In Section 12.8, a technique known as the internal standard technique will be discussed. Use of this technique negates the need for superior accuracy with the injection volume, as we will see. However, the internal standard is not always used. Very careful measurement of the volume with the syringe in that case is paramount for accurate quantitation. Of course, if a procedure calls only for identification (Section 12.7), then accuracy of injection volume is less important. See Workplace Scene 12.1 for an example of a purge-and-trap procedure for injecting a GC sample. 

The introduction of samples into capillaries by means of differential pressure (hydrostatic injection) has become the most popular method in capillary electrophoresis. For sample introduction, the sample vial is raised to a defined level above the detection reservoir for a specified time frequently about 5 s. To terminate the injection, the end of the capillary is removed from the sample and replaced into the vial containing the background electrolyte. 

Electrokinetic injection of the sample may also be used. The capillary tip is dipped into the sample, potential is applied for a few seconds, and the capillary tip is replaced into the electrolyte reservoir. 

Electrostacking is a technique for injection that produces an unusually sharp, concentrated band of analyte ions in the front section of the capillary. The dependence of migration velocity (v) on the electric field (E) is  

For trace analysis, sample introduction without splitting part of the sample to a vent is the preferred mode for obtaining maximum sensitivity. This can be achieved with splitless or on-column injection techniques Schomburg, 1987). 

In the splitless mode, full efficiency of the colmnn is realized by concentrating the sample components in a narrow band near the front end of the column prior to analysis, either by utilizing the solvent effect or by condensation of the solutes. The latter mechanism operates effectively for compounds boiling about 150 °C above the column temperature. Compounds 

Finally, the position of the column in the insert is critical for column performance and for the amount of sample that can be transferred to the column in, e.g., 30 s. It must be determined experimentally in a series of repeated injections of the same solution with varying column positions until maximum detector response is attained. 

In the on-column technique the sample is introduced directly into the colunrn. The sample must be injected into a cold injector (or any other specific device, e.g., a programmable temperature evaporator), so that the evaporated solvent is retained in the colunrn quantitatively. The injection must be slow enough (say in 10 s) to avoid a (short-term) large solvent vapour carrier gas ratio. The needle of the syringe must reach far enough into the column to avoid escape of sample components into the injector block. As the presence of the needle causes a volume reduction to 0.5 mL, the pressure of the carrier gas must be high enough to enable the gas to enter the column. 

Experience obtedned over several years has shown that during slow and careful on-col-umn injection the solvent and sample components are distributed over a distance of = 40 mm in the coliunn before they evaporate completely. To utilize the full efficiency of the column, the sample components are transferred to a narrow band at a cold spot of the column after flash evaporation. 

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Retention Behavior. On a chromatogram the distance on the time axis from the point of sample injection to the peak of an eluted component is called the uncorrected retention time The corresponding retention volume is the product of retention time and flow rate, expressed as volume of mobile phase per unit time ... 

This, of course, is the concentration of fluoranthene in the sample injected into the HPLC. The concentration of fluoranthene in the soil is... 

Hydrodynamic injection uses pressure to force a small portion of the sample into the capillary tubing. To inject a sample hydrodynamically a difference in pressure is applied across the capillary by either pressurizing the sample vial or by applying a vacuum to the destination reservoir. The volume of sample injected, in liters, is given by the following equation... 

Source Adapted from Baker, D. R. Capillary Electrophoresis. Wiley-Interscience New York, 1995. "Concentration depends on the volume of sample injected. 

Because of the large number of samples and repetitive nature of environmental analysis, automation is very important. Autosamplers are used for sample injection with gc and Ic systems, and data analysis is often handled automatically by user-defined macros in the data system. The high demand for the analysis of environmental samples has led to the estabUshment of contract laboratories which are supported purely by profits from the analysis. On-site monitoring of pollutants is also possible using small quadmpole ms systems fitted into mobile laboratories. 

Atomic absorption spectroscopy is an alternative to the colorimetric method. Arsine is stiU generated but is purged into a heated open-end tube furnace or an argon—hydrogen flame for atomi2ation of the arsenic and measurement. Arsenic can also be measured by direct sample injection into the graphite furnace. The detection limit with the air—acetylene flame is too high to be useful for most water analysis. 

In the sphtless mode, the vent is turned off and everything injected goes onto the column. After a short period, the vent is opened and any residual solvent is vented. The spHdess mode is found particularly in trace analytical schemes (see Trace and residue analysis). Sphtless sample injection is an art, and it requites practice to ensure reproducible introduction of sample onto the column. This type of injection is usually used for quahtative analysis. 

Cool on-column injection is used for trace analysis. Ah. of the sample is introduced without vaporization by inserting the needle of the syringe at a place where the column has been previously stripped of hquid phase. The injection temperature must be at or below the boiling point of the solvent carrying the sample. Injection must be rapid and no more than a very few, usuahy no more than two, microliters may be injected. Cool on-column injection is the most accurate and reproducible injection technique for capihary chromatography, but it is the most difficult to automate. 

Another dynamic measurement is the LCEC technique which can be thought of, simpHsticaHy, as EIA using a chromatographic column positioned between the sample injection port and the detector. Bioanalytical systems (BAS) of West Lafayette, Indiana, specializes in instmmentation for LCEC. Their catalogs come with extensive bibhographies covering a variety of appHcations. 

Electroosmotic flow in a capillary also makes it possible to analyze both cations and anions in the same sample. The only requirement is that the electroosmotic flow downstream is of a greater magnitude than electrophoresis of the oppositely charged ions upstream. Electro osmosis is the preferred method of generating flow in the capillary, because the variation in the flow profile occurs within a fraction of Kr from the wall (49). When electro osmosis is used for sample injection, differing amounts of analyte can be found between the sample in the capillary and the uninjected sample, because of different electrophoretic mobilities of analytes (50). Two other methods of generating flow are with gravity or with a pump. 

Fast concentration and sample injection are considered with the use of a theory of vibrational relaxation. A possibility to reduce a detection limit for trinitrotoluene to 10 g/cnf in less than 1 min is shown. Such a detection limit can by obtained using selective ionization combined with ion drift spectrometry. The time of detection in this case is 1- 3 s. A detection technique based on fluorescent reinforcing polymers, when the target molecules strongly quench fluorescence, holds much promise for developing fast detectors. 

Let the volume of sample injected be (Vi) ml, then the charge measured in plate... 

Equation (3) allows the calculation of the distance traveled axially by a solute band before the radial standard deviation of the sample is numerically equal to the column radius. Consider a sample injected precisely at the center of a 4 mm diameter LC column. Now, radial equilibrium will be achieved when (o), the radial standard deviation of the band, is numerically equal to the radius, i.e., o = 0.2 cm. 

The effective use of column volume overload for preparative separations was experimentally demonstrated by Scott and Kucera [1]. These authors used a column 25 cm long, 4.6 mm I.D. packed with Partisil silica gel 10 mm particle diameter and employed n-heptane as the mobile phase. The total mass of sample injected was kept constant at 176 mg, 8 mg and 0.3 mg of benzene, naphthalene and anthracene, respectively, but the sample volumes used which contained the same mixture of solutes were 1 pi, 1 ml, 2 ml and 3 ml. The chromatograms of each separation are... 

The simplest mode of IGC is the infinite dilution mode , effected when the adsorbing species is present at very low concentration in a non-adsorbing carrier gas. Under such conditions, the adsorption may be assumed to be sub-monolayer, and if one assumes in addition that the surface is energetically homogeneous with respect to the adsorption (often an acceptable assumption for dispersion-force-only adsorbates), the isotherm will be linear (Henry s Law), i.e. the amount adsorbed will be linearly dependent on the partial saturation of the gas. The proportionality factor is the adsorption equilibrium constant, which is the ratio of the volume of gas adsorbed per unit area of solid to its relative saturation in the carrier. The quantity measured experimentally is the relative retention volume, Vn, for a gas sample injected into the column. It is the volume of carrier gas required to completely elute the sample, relative to the amount required to elute a non-adsorbing probe, i.e. 

Analyze the sample. Inject the dissolved sample onto the column. 

For best results, sample injection volume should not exceed... 

FIGURE 4.47 Dependence of HETP on sample volume. Column Toyopearl HW-55F, 22 mm X 30 cm. Sample 0.1% myoglobin. Elution 14 mM Tris-HCI, pH 7.9, in 0.3 M NaCI. Flow rate 52 cm/ hr. Detection UV at 220 nm. Legend to. sample injection time Z, column length u. linear velocity. 

SEC measurements were made using a Waters Alliance 2690 separation module with a 410 differential refractometer. Typical chromatographic conditions were 30°C, a 0.5-ml/min flow rate, and a detector sensitivity at 4 with a sample injection volume of 80 fil, respectively, for a sample concentration of 0.075%. All or a combination of PEO standards at 0.05% concentration each were used to generate a linear first-order polynomial fit for each run throughout this work. Polymer Laboratories Caliber GPC/SEC software version 6.0 was used for all SEC collection, analysis, and molecular weight distribution overlays. 

Following the backflush of the primary column and separation of the analytes on the second column, the system can then be returned to its original prefractionation position, ready for the next sample injection. 

Figure 13.7 Selectivity effected by employing different step gradients in the coupled-column RPLC analysis of a surface water containing 0.40 p-g 1 bentazone, by using direct sample injection (2.00 ml). Clean-up volumes, (a), (c) and (d) 4.65 ml of M-1, and (b) 3.75 ml of M-1 transfer volumes, (a), (c) and (d), 0.50 ml of M-1, and (b), 0.40 ml of M-1. The displayed cliromatograms start after clean-up on the first column. Reprinted from Journal of Chromatography, A 644, E. A. Hogendoom et al, Coupled-column reversed-phase liquid chromatography-UV analyser for the determination of polar pesticides in water , pp. 307-314, copyright 1993, with permission from Elsevier Science.

Figure 13.10 LC-LC chromatogram of a surface water sample spiked at 2 p.g 1 with ati azine, and its metabolites (registered at 220 nm). Conditions volume of sample injected, 2 ml clean-up time, 2.60 min ti ansfer time, 4.2 min The blank was subtracted. Peak identification is as follows 1, DIA 2, HA 3, DEA 4, atrazine. Reprinted from Journal of Chromatography, A 778, F. Hernandez et al, New method for the rapid detemiination of triazine herbicides and some of thek main metabolites in water by using coupled-column liquid cliromatography and large volume injection , pp. 171-181, copyright 1997, with permission from Elsevier Science.

Table 4 Processing Condition and Mechanical Properties of PC-TLCP Composite Samples Injection Molded...

Figure 22 Distribution of fiber aspect ratio l/d and fiber number N versus fiber length class for skin and transition layer of the four groups of samples injection molded.

Table 5 compares the tensile properties of Vectra A950 in the form of dispersed fibers and droplets in the matrix by injection molding, microfibril by extrusion and drawing [28], injection molded pure thick sample and pure thin sample, and the pure drawn strand [28]. As exhibited, our calculated fiber modulus with its average of 24 GPa is much higher than that of the thick and thin pure TLCP samples injection molded. It can be explained that in cases of pure TLCP samples the material may only be fibrillated in a very thin skin layer owing to the excellent flow behavior in comparison with that in the blends. However, this modulus value is lower than that of the extruded and drawn pure strand. This can be... 

A flow scheme for the basic form of ion chromatography is shown in Fig. 7.3, which illustrates the requirements for simple anion analysis. The instrumentation used in IC does not differ significantly from that used in HPLC and the reader is referred to Chapter 8 for details of the types of pump and sample injection system employed. A brief account is given here, however, of the nature of the separator and suppressor columns and of the detectors used in ion chromatography. 

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