Meticulous temperature control is essential for the highest precision. The allowable temperature fluctuation will depend on the thermal coefficient of expansion of the material and on the required accuracy of the measurement. The utmost care must be taken to avoid air bubbles and inclusions. In those techniques that require immersion of the solid in a liquid, it is assumed that no chemical or physical interaction occurs between the liquid and the solid, and that the volume of the liquid displaced represents the true volume of the solid. For most hard crystalline materials, liquids can easily be found for which these assumptions are valid. However, for amorphous powders, porous structures such as zeolites, crystalline proteins, and natural and synthetic fibres, the measured `density' may depend markedly on the particular liquid chosen and on the details of the method applied. In these cases, penetration or swelling of the solidDensity measurementpenetration or swelling of solid will depend on a variety of factors such as interfacial tension, the relation of pore size to molecular dimensions, adsorption, and electrostrictive forces. The structural unit to which the measured density applies may be very difficult to specify. Even with materials not subject to these difficulties, variability in the measured density is frequently found. Such variations may arise from differences in trace impurities or in the previous history of the sample (Johnston & Adams, 1912).

This technique is simple, versatile, and capable of the greatest sensitivity. It is the method of choice except in those cases where immersion liquids with an appropriate density and chemical inertness cannot be found.

Originally devised by Linderstrom-Lang (Linderstrom-Lang, 1937; Linderstrom-Lang & Lanz, 1938) for the determination of the density of aqueous solutions, the procedure has been adapted for the measurement of crystal densities by Low & Richards (1952a). For the original solution measurements, a precision of 0.000001 g ml⁻¹ was obtained, although no attempt has been made to attain that precision with solids. This technique was apparently developed and used quite independently in the sugar-cane industry [see, for example, Guo & White (1983) and earlier references contained therein].

3.2.2.1.2. Suitable substances for columns

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Suitable substances for columns

Some representative liquids are listed in Table 3.2.2.1; all are readily available. For further information, see Meyrowitz, Cuttitta & Hickling (1959), and for very heavy liquids Sullivan (1927). Standardized solutions or mixtures from one list in Table 3.2.2.1 may be used as calibrating drops in gradients made from those of the other.

Table 3.2.2.1| top | pdf | Possible substances for use as gradient-column components Hydrophobic components Hydrophilic components Liquid Approximate density at 298 K (g ml−1) α Solute Approximate maximum density of concentrated aqueous solution at 298 K (g ml−1) Isooctane (2-methylheptane) 0.69 −0.8 Sodium chloride 1.20 Kerosene 0.79 −0.5 −0.8 Potassium chloride 1.40 m-Xylene 0.86 −0.85 Potassium iodide 1.63 Chlorobenzene 1.10 −1.1 Iron(III) sulfate 1.80 Bromobenzene 1.49 −1.3 Zinc bromide 2.00 Carbon tetrachloride 1.60 −1.9 Zinc iodide 2.39 Methyl iodide 2.28 −2.7 Thallium(I) formate 3.5 Bromoform 2.89 −0.9 Thallium(I) formate–malonate 4.3 s-Tetrabromoethane 2.96 −2.2     Methylene iodide 3.32 −2.6 Ficoll† (60% w/w in water) 1.25 The density at temperature T K can be computed by substituting the values of the density at 298 K and α in the formula dT = d298 + 10−3α(T − 298). †Trade name for a synthetic high-molecular-weight polysaccharide derivative.

Possible substances for use as gradient-column components Possible substances for use as gradient-column components

For rapid preparation of mixtures from stock solutions of the basic compounds, a nomogram is very useful, such as is given in Fig. 3.2.2.1

Figure 3.2.2.1 | top | pdf | Nomogram for the preparation of bromobenzene–xylene gradient column components at room temperature. From the desired component density and total volume, the required amount of bromobenzene is read from the chart, the volume difference being made up with xylene. To adapt this chart to any other pair of liquids, it is only necessary to change the component density scale. A uniform scale is drawn up such that the density of the heavy liquid lies at the point A while that of the light liquid is at B. The volume scales may be multiplied by any constant factor in order to change their range.

Nomogram for the preparation of bromobenzene–xylene gradient column components at room temperature. From the desired component density and total volume, the required amount of bromobenzene is read from the chart, the volume difference being made up with xylene. To adapt this chart to any other pair of liquids, it is only necessary to change the component density scale. A uniform scale is drawn up such that the density of the heavy liquid lies at the point A while that of the light liquid is at B. The volume scales may be multiplied by any constant factor in order to change their range.

Nomogram for the preparation of bromobenzene–xylene gradient column components at room temperature

for the system bromobenzene–xylene at room temperature. In the construction of the nomogram, it has been assumed that the volumes of the liquids are additive. In general, this assumption is not valid, but it is a sufficiently good approximation for the purpose.

Although historically used much earlier, this technique is essentially an approximation to the gradient-tube method. The specimen is immersed in a liquid, and a denser or less dense liquid miscible with the first is added until the sample neither rises nor sinks in the solution (Wulff & Heigl, 1931).The density of the immersion medium is then determined immediately by standard techniques such as pycnometry, by the Westphal balance, or by refractive index (Midgley, 1951). The method is reported as capable of a probable accuracy as great as 0.02%.

The compounds listed in Table 3.2.2.1 are also useful in this method. With slurries or with specimens smaller than 1 mm³, a centrifuge must be used to achieve a reasonable rate of settling. As little as 0.05 mg of material has been used with good results (Bernal & Crowfoot, 1934). A modification of this method has been described in which the density of the immersion medium is varied by altering the temperature (Reilly & Rae, 1954; Wunderlich, 1957).

This is one of the most demanding of the available techniques. A previously calibrated pycnometer containing the sample is weighed. A liquid of known density is then introduced, air bubbles are removed by reducing the pressure, and the filled bottle is reweighed. The volume of the sample and its mass may thus be determined. With care, a probable accuracy of 0.02% may be achieved (Johnston & Adams, 1912). Contrary to many published statements, the accuracy of this technique is not dependent to any significant extent on the use of immersion media of high density.

Liquids with low surface tension will facilitate the removal of air bubbles. In some cases, it is advantageous to fill the bottle with the mother liquor from which the crystal grew. Powders or many small crystals may be used as well as large single specimens. There is no restriction on the density of the materials for which this technique is suitable.

A micropycnometer for use with samples of total volume as small as 0.01 ml has been described (Syromyatnikov, 1935). An accuracy of better than 1% has been achieved with this instrument.

The specimen is weighed in air and again in a liquid of accurately known density. From the apparent loss of weight the volume is computed, and thence the density (Reilly & Rae, 1954). The technique requires little special equipment and is capable of great accuracy when used with large, well formed crystals. The accuracy is maximized by using immersion liquids of density as close to that of the crystals as possible. For precise work, correction must be made for the interfacial tension between the supporting wire and the upper surface of the suspending medium.

A torsion microbalance has been adapted to the determination of crystals as small as 25 mg (Berman, 1939). A probable accuracy of better than 1% may be achieved with this micro-method.

A densitometer based on Archimedes principle with control of the composition of the gas phase and a wide temperature range has been described by Graubner (1986). The method is not suitable for finely divided materials.

Some crystals, such as those of globular proteins grown from alcohol–water mixtures, rapidly change their composition, and thus their density, when removed from the mother liquor in which they were grown. The density may then be computed from the weight of the crystal immersed in its mother liquor, the density of the latter, and the volume of the crystal (Low & Richards, 1952b, 1954; Richards, 1954).

A horizontal quartz fibre, free at one end, is mounted in a glass case that can be filled with liquid. After calibration, the deflection of the fibre gives the weight of an immersed crystal suspended on the free end. The volume is computed from the crystal dimensions as determined from two photomicrographs of the immersed crystal taken at right angles to each other. The density of the mother liquor is measured by one of the standard techniques for liquids.

The method is suitable for single, well formed crystals having a volume of about 0.1 mm³ or greater. The accuracy is related inversely to the difference in density between the crystal and its mother liquor.

This is the only technique not requiring immersion of the sample in a liquid medium. The technique is therefore used in instances where the specimen would be attacked by the customary immersion media, or where one wishes to work over a temperature range where liquid media would be inappropriate.

The gas-pressure change caused by altering the volume of a calibrated vessel by a given amount is determined when the vessel is empty, and again after the weighed specimen has been introduced (Reilly & Rae, 1954).

Any gas inert to the crystal may be used. Powders and crystal fragments may be employed. A probable accuracy as great as 0.1% may be attained. Samples with an aggregate volume as low as 0.01 ml have been measured with a probable accuracy of 1% (Hauptmann & Schulze, 1934).

A novel procedure that may be useful in special circumstances is based on measuring the frequency of a vibrating stringDensity measurementvibrating-string methodVibrating-string method for density measurement of the material in question. If the length of the string is fixed and the transverse deformation is small, the various harmonic frequencies of vibration will be related inversely to the square root of the density for small oscillations. The potential accuracy of frequency measurements makes this useful for following density changes in the sample while altering the temperature or pressure; see Rabukhin (1982).