The Sample Solution

The sample solution may contain up to 0.5 m moles of salt for a column volume of 110 ml, for example, for the LKB 8100-10. If necessary, dialyze with several changes against either a small volume of a 0.5% solution of carrier ampholytes, or against a 1% solution of glycine. Because of the focusing character of the method, the volume of the protein solution to be applied to the column is not very critical. In a 110 ml column up to 85 ml of protein solution can be applied. Several factors determine the total load of protein allowable. 

The capacity of the density gradient to carry a focused protein zone is limited. As the voltage increases, the zones focus more sharply. This in turn raises the density in the zone to a point at which droplet formation may start. 

Consequently the total load of sample depends on the number of zones obtained in an experiment (that is, on the heterogeneity of the sample), the pH range used for the experiment and the charge condition for the protein in question in the neighborhood of its isoelectric point. 

The upper electrode (32) consists of a coil at the top of the separation compartment (17). It is connected to a terminal (30). The central electrode is wound around a Teflon rod (20) which lies within the central tube (19). This electrode is also connected to a terminal (29). The Teflon rod has two functions it supports the electrodes and it operates the valve (26). This valve makes it possible to shut the central tube (19) off from the electrofocusing compartment (17). This operation is controlled by the setting device (25), which has two positions—open and closed. 

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The sample solution to Problem 17 16(a) showed the prepara tion of 3 methyl 3 heptene by a Wittig reaction involving the ylide shown Write equations showing the formation of this ylide beginning with 2 bromobutane... 

One synthesis of phenobarbital begins with ethyl phenylacetate and diethyl car bonate Using these starting materials and any necessary organic or inorganic reagents devise a synthesis of phenobarbital (Hint See the sample solution to Problem 21 3a )... 

Direct Titrations. The most convenient and simplest manner is the measured addition of a standard chelon solution to the sample solution (brought to the proper conditions of pH, buffer, etc.) until the metal ion is stoichiometrically chelated. Auxiliary complexing agents such as citrate, tartrate, or triethanolamine are added, if necessary, to prevent the precipitation of metal hydroxides or basic salts at the optimum pH for titration. Eor example, tartrate is added in the direct titration of lead. If a pH range of 9 to 10 is suitable, a buffer of ammonia and ammonium chloride is often added in relatively concentrated form, both to adjust the pH and to supply ammonia as an auxiliary complexing agent for those metal ions which form ammine complexes. A few metals, notably iron(III), bismuth, and thorium, are titrated in acid solution. 

In resolving complex metal-ion mixtures, more than one masking or demasking process may be utilized with various aliquots of the sample solution, or applied simultaneously or stepwise with a single aliquot. In favorable cases, even four or five metals can be determined in a mixture by the application of direct and indirect masking processes. Of course, not all components of the mixture need be determined by chelometric titrations. For example, redox titrimetry may be applied to the determination of one or more of the metals present. 

Illustration of a dialysis membrane in action. In (a) the sample solution is placed in the dialysis tube and submerged in the solvent, (b) Smaller particles pass through the membrane, but larger particles remain within the dialysis tube. 

This analysis is an example of a concentration technique. Once the original sample is brought to volume in the 100-mL volumetric flask, any portion of the sample solution, even that obtained on filtering, may be used for the analysis. 

In many applications in mass spectrometry (MS), the sample to be analyzed is present as a solution in a solvent, such as methanol or acetonitrile, or an aqueous one, as with body fluids. The solution may be an effluent from a liquid chromatography (LC) column. In any case, a solution flows into the front end of a mass spectrometer, but before it can provide a mass spectrum, the bulk of the solvent must be removed without losing the sample (solute). If the solvent is not removed, then its vaporization as it enters the ion source would produce a large increase in pressure and stop the spectrometer from working. At the same time that the solvent is removed, the dissolved sample must be retained so that its mass spectrum can be measured. There are several means of effecting this differentiation between carrier solvent and the solute of interest, and thermospray is just one of them. Plasmaspray is a variant of thermospray in which the basic method of solvent removal is the same, but the number of ions obtained is enhanced (see below). 

The nebulization concept has been known for many years and is commonly used in hair and paint spays and similar devices. Greater control is needed to introduce a sample to an ICP instrument. For example, if the highest sensitivities of detection are to be maintained, most of the sample solution should enter the flame and not be lost beforehand. The range of droplet sizes should be as small as possible, preferably on the order of a few micrometers in diameter. Large droplets contain a lot of solvent that, if evaporated inside the plasma itself, leads to instability in the flame, with concomitant variations in instrument sensitivity. Sometimes the flame can even be snuffed out by the amount of solvent present because of interference with the basic mechanism of flame propagation. For these reasons, nebulizers for use in ICP mass spectrometry usually combine a means of desolvating the initial spray of droplets so that they shrink to a smaller, more uniform size or sometimes even into small particles of solid matter (particulates). 

Solutions can be examined by ICP/MS by (a) removing the solvent (direct and electrothermal methods) and then vaporizing residual sample solute or (b) nebulizing the sample solution into a spray of droplets that is swept into the plasma flame after passing through a desolvation chamber, where excess solvent is removed. The direct and electrothermal methods are not as convenient as the nebulization inlets for multiple samples, but the former are generally much more efficient in transferring samples into the flame for analysis. 

D = diffusilivity of the solvent vapour from the sample solution (cm s ) o = surface tension of sample solution (erg-cm ) p = vapour pressure of sample solution (mm Hg X 1-359 = g cm )... 

The drop in pressure when a stream of gas or liquid flows over a surface can be estimated from the given approximate formula if viscosity effects are ignored. The example calculation reveals that, with the sorts of gas flows common in a concentric-tube nebulizer, the liquid (the sample solution) at the end of the innermost tube is subjected to a partial vacuum of about 0.3 atm. This vacuum causes the liquid to lift out of the capillary, where it meets the flowing gas stream and is broken into an aerosol. For cross-flow nebulizers, the vacuum created depends critically on the alignment of the gas and liquid flows but, as a maximum, it can be estimated from the given formula. 

From the example of Figure 19.1, a flow of argon can cause a pressure drop of 0.3 atmosphere (3x10 dyn cm ). Let the viscosity of the sample solution be the same as that of water 0.01 poise), the radius be 0.01 cm and the length of capillary be 10 cm. 

Using Poiseuille s formula, the calculation shows that for concentric-tube nebulizers, with dimension.s similar to those in use for ICP/MS, the reduced pressure arising from the relative linear velocity of gas and liquid causes the sample solution to be pulled from the end of the inner capillary tube. It can be estimated that the rate at which a sample passes through the inner capillary will be about 0.7 ml/min. For cross-flow nebulizers, the flows are similar once the gas and liquid stream intersection has been optimized. 

This formula for estimating droplet size was determined experimentally. Of the various terms, the first is the most important for small values of V. As V becomes small, the second term gains in importance. Unless the density or viscosity of the sample solution changes markedly from the values for water, mean droplet size can be estimated approximately by using the corresponding values for water, as shown. 

Figure 19.7 shows a typical construction of a concentric-tube nebulizer. The sample (analyte) solution is placed in the innermost of two concentric capillary tubes and a flow of argon is forced down the annular space between the two tubes. As it emerges, the fast-flowing gas stream causes a partial vacuum at the end of the inner tube (Figure 19.4), and the sample solution lifts out (Figure 19.5). Where the emerging solution meets the fast-flowing gas, it is broken into an aerosol (Figure 19.7), which is swept along with the gas and eventually reaches the plasma flame. Uptake of sample solution is commonly a few milliliters per minute.

In a concentric-tube nebulizer, the sample solution is drawn through the inner capillary by the vacuum created when the argon gas stream flows over the end (nozzle) at high linear velocity. As the solution is drawn out, the edges of the liquid forming a film over the end of the inner capillary are blown away as a spray of droplets and solvent vapor. This aerosol may pass through spray and desolvation chambers before reaching the plasma flame. 

The flows of gas and liquid need not be concentric for aerosol formation and, indeed, the two flows could meet at any angle. In the cross-flow nebulizers, the flows of gas and sample solution are approximately at right angles to each other. In the simplest arrangement (Figure 19.11), a vertical capillary tube carries the sample solution. A stream of gas from a second capillary is blown across this vertical tube and creates a partial vacuum, so some sample solution lifts above the top of the capillary. There, the fast-flowing gas stream breaks down the thin film of sample... 

Another variant (the cone spray) allows the sample solution to flow down the sides of an inverted cone and through a hole at the bottom of which flows a fast stream of argon gas. As the liquid film meets the gas, it is ripped apart into a finely dispersed aerosol (Figure 19.15). 

The sample solution flows onto a piece of fritted glass through which argon gas flows. The flow of argon is broken down into narrow parallel streams of high linear velocity, which meet the thin film of liquid percolating into the pores of the frit. At the interfaces, an aerosol is formed and is blown from the top of the frit. 

To accommodate smaller liquid flows of about 10 pl/min, micro-ultrasonic nebulizers have been designed. Although basically similar in operation to standard ultrasonic nebulizers, in these micro varieties, the end of a very-small-diameter capillary, through which is pumped the sample solution, is in contact with the surface of the transducer. This arrangement produces a thin stream of solution that runs down and across the center of the face of the transducer. The stream of sample... 

In one sense, the thermospray nebulizer could be considered a pneumatic device, in which a fastflowing argon gas stream is replaced by a very rapidly vaporizing flow of solvent from the sample solution. A typical arrangement of a thermospray device is shown in Figure 19.18. 

The sample solution is pumped along a narrow capillary tube, the end of which becomes the nozzle of the nebulizer. On the outside of the capillary near its nozzle end, an electrical heater rapidly... 

The sample solution is pumped (e.g., from the end of a liquid chromatographic column) through a capillary tube, near the end of which it is heated strongly. Over a short length of tube, some of the solvent is vaporized and expands rapidly. The remaining liquid and the expanding vapor mix and spray out the end of the tube as an aerosol. A flow of argon carries the aerosol into the plasma flame. 

A typical loop injector showing the sampling position with pressurized solvent flowing through one loop onto the column and the sample solution placed in the other loop at atmospheric pressure. Rotation of the loop carrier through 180° puts the sample into the liquid flow at high pressure with only momentary change in pressure in the system. 

FAB has evolved, and fast atoms are being replaced by fast ions, such as cesium (Cs+). This variation is called liquid secondary ion mass spectrometry (LSIMS) because the sample solution affords the secondary ion beam while the bombarding ions constitute the primary beam. Spectra... 

Therefore, the sample solution, which may or may not come from a liquid chromatographic column, is passed along a narrow capillary tube, the end of which is maintained at a high positive or negative potential. 

Thermodynamically, the activity of a single ionic species is an inexact quantity, and a conventional pH scale has been adopted that is defined by reference to specific solutions with assigned pH(5) values. These reference solutions, in conjunction with equation 3, define the pH( of the sample solution. 

The three-electrode system serves two important purposes. Because the reference electrode carries no current, but merely measures a potential relative to the working electrode, its stabiUty is not unduly influenced by the electrolysis. Furthermore, because it is placed close to the working electrode the measured potential difference is more nearly representative of the tme potential difference between the working electrode and the sample solution. This latter is the significant quantity in electro analysis. 

Procedure To an aliquot of the sample solution containing 12.5 - 305 p.g of platinum(IV) were added 5 ml of hydrochloric acid - sodium acetate buffer of pH 2.1, 1 ml of O.IM Cu(II) sulphate solution, and 3.0 ml of 0.5% propericiazine solution. The solution was diluted to 25 ml with distilled water, mixed thoroughly, and the absorbance measured at 520 nm against a reagent blank solution after 10 min. The platinum concentration of the sample solution was determined using a standar d calibration curve. 

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