Quantitative Spectrographic Analysis of Biological Material--II

Quantitative Spectrographic Analysis of Biological Material--IIAuthor(s): J. S. Foster and C. A. HortonSource: Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol. 123, No.833 (Aug. 17, 1937), pp. 422-430Published by: The Royal SocietyStable URL: http://www.jstor.org/stable/82071 .

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545.823 :58I.I92

Quantitative Spectrographic Analysis of Biological Material-II

BY J. S. FOSTER, F.R.S., Macdonald Professor of Physics, AND

C. A. HORTON, Ph.D., Demonstrator in Physics; McGill University, Montreal

(Received 31 March 1937)

[Plate 16]

INTRODUCTION

It is well known that several metallic elements in small concentrations are essential to plant growth and normal development. The relative importance of a given element and the concentrations necessary or most beneficial vary with the plant. Quantitative spectrographic methods permit one to determine the distribution of the element throughout a single plant and in certain cases follow the distributions through the life of an individual specimen. Such methods may therefore be expected to aid in the solution of many problems now fully appreciated by biologists and to suggest further studies.

For many years it has been known that boron deficiency has disastrous effects on many plants of economic value such as sugar beets, tomatoes, turnips, tobacco, and celery. Chemical analysis as applied to studies of remedial treatments or of a purely scientific nature is relatively long, requires much material, is less sensitive, and is such as to risk loss of boron before the analysis is completed.

The present development of suitable spectrographic analysis of plants for boron has been made with the following aims: (1) use of small samples, (2) minimum preliminary treatment, (3) reproducibility, and (4) speed. It has been found that 200 mg. of fresh material is ample for quantitative analysis. There is no ashing or chemical treatment. Results rarely vary as much as 10% from the mean in a long series, and the time required per analysis is about 1 hr. There is a somewhat greater sensitivity than is required in practice.

It was first established as a fact that when a sample containing boron is excited in a suitable spark discharge, the intensities of the sensitive boron

[ 422 ]



Spectrographic Analysis of Biological Material 423

lines 2496 and 2497 A are proportional to the number of atoms present over the range of concentrations normally found to be of practical interest (fig. 1). As an internal standard, gold is introduced into duplicate samples. After the further addition of a known amount of boron to one of them, the two samples are sparked. From the relative intensities of the boron to gold lines in the two spectra, the amount of boron originally present in the sample is determined.

700

50 _

40 -

460 -

~~~~~~~~~30~~~~~~~~~~~~~~1

210 -.

00 I l 0 20 4 0 60 80 100 120

FIG. 1-Concentration of added boron x 106 g./c.c.

In the application of this method to plant tissue some modifications of technique are required to deal with certain features not met in the analysis of fluids (Foster, Langstroth and McRae I935). Since there are variations in the distribution of the element over small regions, the weighed sample from a fresh plant is made representative of the material to be investigated, yet not too bulky on the electrode. The internal standard and boron solution are thoroughly mixed with the sample, crushed and disintegrated in a small copper mortar. Before the sample is spread on a small plane electrode, a binding material is added so that the sample dries in a thin compact layer in good contact with the metal. This is necessary, since otherwise the explosive force of the first few sparks would cause too great loss of material. By this treatment one also prevents the appearance of loose flakes of the sample which would mask one section of the spark and



424 J. S. Foster and C. A. Horton

thus cause a change in the relative intensities of the lines under investi- gation.

EXPERIMENTAL PROCEDURE

Before starting a quantitative analysis of a new biological material for a given element, an exposure must be taken to determine qualitatively what other elements are present. The most suitable persistent spectral line of the element in question is then chosen with reference to its position in the spectrum and to possible interference from lines due to the air or to other elements in the sample. The internal standard is then selected subject to the same conditions, and the additional requirements (1) that it be not present in variable amounts in the sample, (2) that plate sensitivity does not vary appreciably toward the lines under comparison, and (3) that the lines arise from transitions between similar atomic states so that small differences in the excitation should not change appreciably the ratio of the intensities. The boron line 2pI,_2S (A 2497-7) and the gold line 2P,_2S (A 2427.9) have been found to satisfy these conditions.

At the beginning of an analysis two samples of 100 mg. each are con- veniently weighed out on a torsion balance. A duralumin arm is balanced at the centre of a violin E string 8 in. long and carries a mounting for a cover-glass to receive the sample. The material is ground to a fine pulp in the copper mortars shown in fig. 2, Plate 16. Copper was chosen as the material for the mortars due to its freedom from boron and ease of cleaning. By means of a capillary pipette graduated to deliver 0 040 c.c. of solution, 4 x 10-6 g. of gold from a gold chloride solution is added to each sample as an internal standard. In the same solution enough lithium tartrate (as binder) is included to give 2 mg. of this salt per sample. With a second pipette 0*050 c.c. of sodium tetraborate solution, giving 5 x 10-7 g. of boron, is added to one mixture. These solutions are thoroughly mixed with the pulp, and then poured out one on each electrode. If the solutions are thoroughly mixed through the pulp, no error arises from small but variable amounts of the sample adhering to the inside of the mortar.

The electrodes are rectangular aluminium plates 2-5 x 1-3 x 041 cm. The load is placed in the central portion of the plate (approximately 1P7 cm.2) marked out by a pointed piece of paraffin to keep the liquid from spreading. Before they are used, electrodes and all glassware are cleaned with chromic acid, hot nitric acid, and distilled water. Tests failed to reveal any boron in the gold or lithium solutions, in the paraffin, or in the aluminium.

The electrodes are put in a desiccator and allowed to dry slowly under reduced pressure provided by a Harrington-Cenco aspirator. If the




pressure is taken down to the boiling-point, the sample does not dry in a coherent layer but tends to form a hollow crust. Boron will be lost if the drying proceeds at much higher temperatures. For example, samples dried at 800 C. by means of electrical heating when conapared with like samples dried slowly reveal a loss of boron up to 5000 of the amount originally present. When properly dried, each sample is (a tough compact film firmly adhering to the electrode. A photograph of an electrode before and after sparking is reproduced in fig. 7, Plate 16.

The automatic source, fig. 3, Plate 16, is carefully aligned with respect to the spectrograph; for this purpose the lamp is useful. The pointed aluminium anode is set by a spacer and clamped with its tip 4 mm. above the position of the load. It may be turned back out of the way until the loaded cathode is fitted into its mounting, and then returned to position for operation. Both electrodes are insulated from the base of the apparatus, one by a section of glass rod, the other by an ebonite fitting attached to the movable platform which supports the central column. Through a worm drive the motor operates a cam designed to move the platform uniformly back and forth with a stroke equal to the width of the load. By means of a 21: 2 reducing gear, a duplicate cam moves the platform slowly back and forth in a direction at right angles to the first motion. The short period is about 1-5 sec. Thus after the source has been in operation for 2 min., a few hundred sparks have passed uniformly over the load, and each portion may be said to have had the same probability of excitation. To avoid possible contamination, a fresh anode is used for each sample.

In most experiments no condensing lens was employed. The centre of the source was placed on the axis of the collimator and 50 cm. from the slit. By tests it was found that the slit jaws were parallel and that the slit was evenly illuminated.

The above conditions are necessary for the wedge method of photometry which is employed. Almost in contact with the slit is a wedge of aluminium film formed by evaporation on quartz. An example of the resulting spectrum taken by a Hilger E2 quartz spectrograph is reproduced in fig. 4, Plate 16.

The high potential D.C. source has already been described (Foster and others I935).

PHOTOMETER FOR WEDGE SPECTROGRAMS,

The relative intensity of two neighbouring lines is found from the difference in the lengths of wedge penetrated, and the wedge density. The




method has been well developed by Merton (Nicholson and Merton I9I6)

who worked in the visible region with a neutral glass wedge corrected for dispersion by a wedge of clear glass. Assuming that the wedge has the same coefficient of absorption, a, for the two radiations of intensities I1, I2, and that the photographic plate is equally sensitive to their actions, the intensity of radiation which under the conditions of exposure and development just affects the plate is given by

_a a

11e m = M2e

where 11, 12 are observed lengths of the spectral lines, and m is the magnification within the spectrograph. The relative intensity is found from

loge,II= a1 (11 -12)- I2 M

Following Merton, it is convenient to define DA, the density of the wedge

at the wave-length under consideration, as logoII , where it is understood '2

that I1 penetrates the wedge to an excess length of 1 mm. Then in general

II = antilog,ODA (11- 12)/m. I2

In any application, the factors of chief interest are obviously the density of the wedge and the difference in the lengths of the lines. There is some simplification in the attention given to each factor in the present in- vestigation.

More recently it has been found that the absorption of certain metallic films evaporated on quartz is nearly constant through the ultra-violet region. The density of an aluminium film so deposited as to form a wedge is so nearly constant from 4000 to 2000 A that corrections are normally unnecessary. Such a wedge has been supplied to us through the kindness of Professor B. O'Brien, University of Rochester, whose careful investi- gation of methods of manufacture and properties of aluminium wedges has drawn attention to their usefulness. As is well known, a film of trans- parent oxide protects the wedge and even permits washing when necessary. The density of the present wedge as determined by Professor O'Brien at 4000 A is 0 168. Thus, using this value of DA, the relative intensity, R, of the boron radiation to that of gold is given by




since the magnification may be taken as unity; there then remains only the measurement of (1,-12). The selection of the end of a line, however, is uncertain. Merton used a fine process screen in the preparation of "half-tone" enlargements and then chose as the "end" the last dot at the top of each image. The present method is rather more direct, and places less emphasis on the density of the photographic impression at a particular point. In fact, the problem of finding the end of the line is entirely avoided.

Under the above conditions it is apparent that the light intensity is the same for each line not only at the "ends" but at all points equidistant from the "ends ". It is therefore possible to use independent optical trains for the formation of adjacent images of the two lines, and then move one image until the lines appear to taper off together. The amount of this motion is a quantitative measure of (1 - 12).

e

FIG. 5.

There are many ways of doing this. The first form of a photometer which is undergoing development is shown in fig. 5. Two beams of light from the evenly illuminated plate are reflected by stainless steel mirrors, M1, M2,

and made parallel by the lenses, L1, L2. After passing through the trapezoid prisms, the beams are focused in a horizontal plane through the middle of the special prism, P, and the images viewed through an eyepiece. If the magnification is not the same by each path, the lenses, L1, L1', may be adjusted to give equal magnification. After preliminary adjustments are completed, the plate is so placed that the image of one of the lines (reflected from M1) is in correct position for viewing, and the other mirror, MU2, together with the lens, L2, is moved as a rigid unit until the other line is picked up. By manual adjustment the plate may now be moved slightly if necessary until the field presents the appearance of fig. 6a. The breaks at the bases of the lines permit an initial matching, and are- made by a fine fibre across the slit near the thin end of the wedge. Finally the lens L2' is moved by a screw until the tips of the lines match, fig. 6b, and the relative intensity R is read directly from a specially prepared scale on the




drum. In routine work the measurements are completed very quickly. Unusual latitude is permitted in exposures, and since it is a null method, no calibration of the plate is required. Owing to a margin of sensitivity, no background appears on the plates in the region of the lines under comparison.

a / b

FIG. 6.

If Rl, R2 respectively are the ratios of the boron line to the gold line in samples with and without added boron, b, then the amount of boron, B, in the original sample of mass AL is given by

B = R2b

while the amount per gram of fresh material is B/M. If the results for one plant are to be compared with those for another, it is important to weigh out samples from fresh plants each time. Following the analysis of an entire plant on the above basis, it is sometimes helpful to know the boron per gram of dry tissue in the different structures. This is easily obtained by drying independent samples, or by weighing the initial samples (and electrodes) before and after drying.

JUSTIFICATION OF PROCEDURE

The following data chosen at random from numerous tests illustrate (1) the relation of the relative intensity of the boron and gold lines to the concentration of boron, (2) the reproducibility of analysis, (3) the effect of poor grinding, (4) the effect of poor mixing of the internal standard with the pulp.

Each curve in fig. 1 is drawn from data obtained by adding known amounts of boron to like samples of material. It is found that for concentrations of boron within the range encountered in practice the relation is linear. At higher concentrations, the density of boron atoms in the spark is sufficient to cause noticeable absorption. The change in slopes and



Foster and Horton Proc. Roy. Soc., B, vol. 123, Plate 16

FIG. 3

* : t ,; - 't- .i! ;1| F

a b FIG. 2 FIG. 4 FIG. 7

(Facing p. 4 28)




intercepts fronm one curve to another is of course due to the use of different concentrations of gold, and to varying amounts of boron originally present in the samples. Such a calibration curve is always drawn before routine analysis for a new element is undertaken.

TABLE I-REPRODUCIBILITY OF BORON DETERMINATIONS. THE AMOUNTS OF BORON ARE MULTIPLIED BY 106 AND ARE IN

GRAMS OF BORON PER GRAM OF GREEN TISSUE Determination ... 1 2 3 4 5 6 Mean Turnip leaf under standard 4.2 3.9 4.2 3.7 4 3 4-1 4.1

treatment 0 deviation from mean 2.4 4-9 2-4 9.9 4 9 0.0 4 0

Samples from same leaf, 2.4 3-9 4-1 poorly ground 0 deviation from accepted 41 4.9 0.0 mean

Like sainples well ground, but 3-2

6*3 3.7

7-1 MeanL

boron content 4-2 gold solution added after dryingX

% deviation from accepted 22 54 9-9 73 meaii

The first section of Table I shows the results of six determinations made under conditions adopted as standard. From this test and many others, it may be concluded that in practice one is justified in assuming that the error in a single determination is not in excess of 10%. It is important, however, to know what errors may be introduced by some departure from the standard conditions. These are shown in succeeding sections of the table, where the procedure has been varied in the most inadvisable manner. Some of the errors are explained by a simple consideration of one aspect of the discharge. The aluminium disk stuck on the anode, fig. 3, Plate 16, collects a surprising amount of material. The colour and other properties of the material suggest that most of it has never been excited at all, but has been driven from the electrode by the heat and mechanical action of the sparks. When the material is very poorly ground, it is not uniformly bound to the electrode, and in particular some appreciable portions of the sample are driven bodily from the source with consequent relative weaken- ing of the boron line.

The effect of poor mixing of the internal standard with the pulp is illustrated in the last section of the table, where it will be Ioticed that the values on the average are high. The electrodes were loaded as usual but no gold was added. After the samples were thoroughly dry the gold solution was put on top and allowed to dry. It was observed that much of the liquid flowed round to the edges of the load and upon drying left the gold in

Vol. CXXIII-B. 2G




that region which, as may be seen from fig. 7 a, Plate 16, receives the least excitation. This has led apparently to high values for boron.

This method of analysis has also been adapted to the determination of traces of copper, aluminium and iron in raw paper pulp. Carbon cylinders free from these elements were bored axially and cut in sections. A layer of collodion was spread over the surface. The thick paper was cut in strips, weighed, wrapped around the cylinder and fastened down with collodion. The internal standard and known solutions were added and evenly dis- tributed by capillary action. After the material had dried the cylinder was slipped on the end of a threaded shaft, so mounted on the source that it could be rotated under the pointed anode and at the same time be advanced in a direction parallel to the axis. Thus the sample was uniformly excited and the analysis from that point was carried out as in the above boron determinations.

We express our best thanks to Dr. J. M. Swaine, Director of Research, Dr. E. Archibald, Director of the Dominion Experimental Farms, and other members of the Department of Agriculture, Ottawa, for hearty co- operation. An account of co-operative studies still in progress will be given in a later paper. Our grateful thanks are further extended to the Rockefeller Foundation for the benefits of a grant to the University for the development of quantitative spectrographic methods under one of us.

REFERENCES

Foster, J. S., Langstroth, G. 0. and McRae, D. R. 1935 Proc. Roy. Soc. A, 153, 141-52.

Nicholson, J. W. and Merton, T. R. I9I6 Philos. Trans. A, 216, 459-88.



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Quantitative Spectrographic Analysis of Biological Material--II