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Tuatara: Volume 5, Issue 3, March 1955
Paper Chromatography *
Paper Chromatography *
Sixty years ago the chemist's contribution to botany and agriculture was limited to the analysis of soils and manures. He advised farmers concerning the fertility of their soils and what manures should be applied to obtain the highest crop production. In these early studies the main emphasis was placed on nitrogen, phosphates, potash, and perhaps lime. The other so-called minor elements such as boron, magnesium, manganese, zinc, copper, etc., that are now known to be essential for plant growth were not then considered. This work led naturally to the analysis of plants, particularly of the mineral contents that remained after plant material had been carefully burnt. Thus attempts were made to correlate the uptake of minerals by crops with the amounts of those minerals in the soil.
But nitrogen requirements of plants had intrigued chemists from the start, first as to whether plants could draw on atmospheric nitrogen; and later when it was recognised that most plants drew their nitrogen from the soil, the problem of explaining the stimulus to plant growth that resulted from additions of small quantities of nitrates (20 Ib. per acre) to soils that already contained several thousand pounds of nitrogen per acre. It soon became apparent that the total amounts of nutrients in the soil, whether minerals or nitrogen, were not available to plants but only a portion that was soluble in the soil water. Nitrogen was of course driven off during the combustion of plant material, and could not be isolated from the ash. In the early days it was measured as total nitrogen by techniques in which it was removed from its chemical compounds as ammonia. The compounds in which it occurred in the plant were not identified, but were hypothesised as ‘proteins’ and ‘non-proteins’ (amino acids, etc.).
Though some attempt had been made to study physiologic processes such as photosynthesis prior to 1920, the major consideration of plant chemistry during the 1920's was directed towards an evaluation of the nutritive value of plant products in terms of carbohydrates, proteins, fibre, and later minerals. The techniques often ignored the actual identity of the proteins and sugars concerned and aimed only at estimating the total food value under general headings such as protein, fat, fibre, ash, and by subtraction, carbohydrates. A natural corollary of this work resulted in studies of the utilization of foodstuffs by animals, their digestibility and page 101 eventually of their incorporation into the various fats and proteins of the animal body. Such studies involved the elucidation of various physiological processes, but attempts to understand such processes were handicapped by the difficulties of identifying the products of synthesis (anabolism) from those of degradation (katabolism). Animals offered a better field of investigation since they added nothing to the food materials that they consumed, but digested them, and converted them into new compounds, discarding waste materials which could be analysed in excreta and urine. In plants such synthetic and destructive processes proceed simultaneously and the identification of any compound as a product of photosynthesis or respiration is rather difficult. Understanding these processes rested largely on hypothesising a possible course that photosynthesis might follow and attempting to isolate the hypothesised compounds from plants during photosynthesis. If such compounds were found to be absent during the hours of darkness one might reasonably assume that they were derivatives of photosynthetic activity.
By such means of trial and error parts of the photosynthetic and of the respiratory processes were located, but their significance in the physiology of the plant or animal was not fully established until we had means of labelling individual atoms and molecules, and tracing their movement through various chemical compounds that constitute the organic body. For such studies the chromatographic techniques are ideal in that they require very little labour and permit analysis of minute quantities of material, and the separation and identification of individual compounds even though these may be represented by only a few molecules of each. Once the compounds have been separated those that are labelled with radio-active elements can be readily recognised. As the process proceeds for longer periods more and more compounds acquire radio-active elements and so the sequence in which compounds are formed can be determined. If radio-active carbon which has an atomic weight of 14 and is usually represented by C14 is brought into contact as C14O2 with the actively photosynthesising plant, the first stable product in which the radio-active carbon accumulates is phosphoglyceric acid (probably 2-phospho-d-glyceric acid), a compound with three carbon atoms in the molecule. In this the radio-active carbon is located in the carboxyl (C14 OOH) group of the acid. In photosynthesis the radio-active carbon appears early in phosphopyruvates and pyruvic acid besides other compounds, and shortly afterwards in glucose. The position of the C14 atoms in the first formed glucose suggests that this substance is derived by a chemical fusion of two molecules of glyceric acid in such a way that two carboxyl (C14 OOH) groups condense and the two carbon atoms become the two central atoms of the six carbon atom chain which forms the backbone of the glucose molecule.
Such information could scarcely have been obtained by the older methods of chemical analysis — and the outline given above is but a very small part of what has been achieved by paper chromatographic techniques in the study of photosynthesis.
page 102Chromatography has been freely used in many fields of physiologic research, both plant and animal, and can be adapted to study any compounds that occur in plants or animals provided the molecular structure permits their movement with the solvent. The method has been adapted also to the isolation and identification of inorganic compounds.
In New Zealand the techniques have been used for the past fourteen or fifteen years — notably at the Plant Chemistry Laboratory at Palmerston North, the Medical Research Council Laboratories, the Wallaceville and Ruakura Animal Research Stations, the Dominion Laboratory, the Fats Research Laboratory, and other laboratories.
In other fields it is adapted to the study of vitamins, hormones, growth substances (auxins), nucleic acids, respiratory processes, physiologic differences between resistant and susceptible plants, the study of physiologic differences of closely-related genotypes, hydrolytic products, enzymes and their products, bacterial physiology and many other types of the research. The methods of developing and identifying the compounds which are isolated are just as intriguing. Radio-active isotopes are autophotographic and may be identified by the fogging they produce on a photographic plate. Other substances are fluorescent or may be made so, while the commonest method is probably to develop the paper by treatment with a reagent that brings about a colour change of the compound. This may require heat to bring out the colour. Vitamins may be identified by biological assay — i.e. the adding of a culture medium which lacks a given vitamin or other food material to the paper, and inoculation with a specific organism (bacterium or fungus) that will grow only when the vitamin or other substance being studied is present. Thus when the chromatogram is incubated, growth occurs only on the spots in which the vitamin is located. Occasionally two compounds may move on the paper as one, but require different reagents for their development. The scope and possibilities of the techniques are numerous and offer a wide scope for ingenuity and imagination.
Last year Dr. Harvey prepared the following schedules for the Botany course as a practical introduction to the techniques. The procedure was simplified so that initial separations could be made with ordinary laboratory equipment, e.g. corked test-tubes, filter paper and about four or six solvents and reagents. After the first separation of known compounds in prepared solutions had been made, the class found considerable interest in identifying the amino-acids and sugars in several fruits and vegetables. The technique is easily mastered and is adaptable to many projects that could be carried out in post-primary school laboratories such as the changes that take place in the ripening of different fruits, seasonal changes in the amino acid and carbohydrate compounds of growing and/or dormant plants, the changes in seeds during germination, or during their formation, and many other projects. The advantage of the method is that only one drop of liquid extract is necessary for most analyses. The main precautions are; (1) the drop applied to the paper must be allowed to dry before the paper is placed in the solvent to be run; (2) the drop must not be so near the page 103 bottom of the paper that it is immersed in the solvent; (3) the paper must not touch the sides of the test-tube except at its four corners. This is achieved by cutting the paper to 2 or 3 mm. less than the diameter of the tube and folding it longitudinally; (4) the tube must be corked so that the atmosphere about the paper is saturated with the solvents used and no evaporation of solvent from the paper occurs during the actual running. Finally the solvent should not be allowed to reach the top of the paper.
The technique of paper chromatography, although of comparatively recent origin, has assumed such importance in the last few years that it is now no longer a novelty and every worker in the fields of chemistry and the biological sciences should have some knowledge of the scope and limitations of the method. The aim of this article is to give an introduction to the subject, sufficiently detailed to enable the non-specialist to make use of the technique in at least its simpler forms.
When Consden, Gordon and Martin, in 1944, published their experiments with paper chromatograms, several branches of pure and applied chemistry were almost revolutionised overnight. Problems which up to that time had seemed virtually incapable of solution became amenable to attack and such was the simplicity of the equipment required and the techniques involved that interest in the method spread rapidly and its use is now commonplace in laboratories throughout the world.
Paper chromatography is essentially an analytical tool for separating chemical compounds, and in this respect is comparable with distillation, crystallisation, precipitation, etc. The latter methods all depend on differences in volatility or solubility of the substances involved, whereas partition chromatography in general, and paper chromatography in particular, rely on differences in another physical property — namely the partition coefficient. If a pure substance is shaken up with two immiscible solvents the ratio of the concentrations of the substance in the two liquid layers is known as the partition coefficient. For a given solute and solvents at constant temperature the partition coefficient is a constant, and, in particular, is independant of the actual concentration of solute in the liquid phases. Partition coefficients vary from 0 to 1 and it is the small differences in the partition coefficients of various substances which are utilised for their separation.
Paper chromatographic methods possess several advantages compared with the more conventional methods of separation. Perhaps the most important of these is the facility with which substances which are extremely similar chemically can be separated. The classical example of this kind is the separation of the amino-acids and of the carbohydrates. So great are the difficulties associated with the separation of members of either of these important groups of substances by methods other than chromatographic ones, that the analysis by conventional methods of even comparatively page 104 simple mixtures was a time-consuming and intricate process requiring considerable amounts of material. Accurate quantitative analysis was in general impossible. Paper chromatography has made possible the analysis of mixtures of 18-20 amino-acids in a time of the order of 36 hours.
Another major asset is the small amount of material required. Qualitative analyses can be carried out on amounts which may be as small as a few micrograms of each component, and only slightly larger quantities are often sufficient for approximate quantitative results. Few other analytical procedures can be adapted to this minute scale, yet in the case of material of biological origin, amounts greater than a few milligrams may be almost unobtainable.
The wide versatility of paper chromatographic methods is illustrated by the fact that they may be successfully employed for separations in such diverse series as the amino-acids, sugars, plant pigments, phenolic constituents of wood, etc., volatile fatty acids, sugar phosphates, and related compounds, nucleic acid degradation products, compounds such as adenosine triphosphate (A.T.P.) and adenosine diphosphate (A.D.P.), metallic ions, etc.
In view of this wide range of application and the fact that the equipment required is simple and the technical skill required not great, it is not surprising that the methods of paper chromatography have assumed such importance in a short space of time.
Partition of a solute or solutes between two immiscible liquids was utilised by Craig in his elegant counter current distribution technique which is essentially a method of carring out a series of extractions of one liquid phase with another in a definite order. However counter current distribution studies often require considerable amounts of material, may require expensive precision equipment and are frequently time-consuming. Martin and Synge conceived the idea of keeping one of the liquid phases stationary and allowing the other liquid to pass continuously over it, thus essentially having an extremely large number of extremely small-scale extractions. If one of the liquid phases is water it may be held stationary by absorbing it on the surface of a material such as silica gel, diatomaceous earth, starch or cellulose. This idea led to the development of partition chromatography using columns of materials such as these saturated with water, with solvents such as chloroform or butanol flowing slowly down the column. This technique is of great importance and is used extensively for the separation of substances in amounts of the order of 5-10 mg. upwards.
For smaller quantities Consden, Gordon and Martin showed that a sheet of filter paper serves admirably to hold the stationary phase (water) and the moving phase may be made to travel slowly up or down the paper by capillary action and/or gravity: thus paper chromatography came into being.
In practice the mixture to be separated is applied in the form of a small spot a few millimetres in diameter near one end of a strip of filter paper which is then hung in an atmosphere saturated with both water and the page 105 solvent to be employed. The strip may be suspended with the spot near the top from a trough containing the solvent (descending technique) or alternatively hung upside down dipping in a trough or dish of solvent (ascending technique). The mobile phase is allowed to move down or up the paper for a time which may vary from minutes or hours to several days. During this time the material originally placed on the paper will have moved a greater or lesser distance in the direction of flow of the solvent and in the case of a mixture under ideal conditions the original spot should be resolved into several spots distributed along the length of the strip. If the paper is then removed from the solvent and dried it should be possible to reveal the position of the spots by spraying the paper with a suitable reagent or examining it in ultra-violet light, etc.
The ratio Distance travelled by substance/Distance travelled by solvent, known as the RF value, is a constant which, for a given solvent at a given temperature, is characteristic for the substance, and thus ‘unknown’ substances may be identified with some degree of certainty by measuring their RF values preferably in several different solvents. RF values should be reproducible, but sometimes careful control of conditions is necessary for this to be the case, and it is normal practice to run standard spots simultaneously alongside the ‘unknowns’.
A number of factors influence the ease with which paper chromatograms can be successfully prepared, and of these brief mention may be made of the design of the equipment, the type of paper, the nature of the solvent, and the method of detecting the spots.
(a) Equipment
The original apparatus used by Consden, Gordon and Martin consisted of a drain pipe closed at the lower end by immersion in a dish containing the aqueous phase and at the upper end by a sheet of glass. A small glass trough resting on the shoulder near the top of the pipe contained the solvent and the paper, held in the trough by means of a glass rod or sheet, passed over a glass rod and hung down inside the pipe. If the paper was hung directly over the rim of the trough difficulty was experienced with solvent syphoning out between the paper and the trough. It is convenient to be able to follow the progress of the solvent down the paper visually and a tall glass jar is a more satisfactory container. The trough must then be supported on some type of stand preferably of glass (to resist the attack of solvents). Metal troughs have the great advantage of not being fragile but are often unsatisfactory with strongly acidic or basic solvents. It is an advantage, for reasons which will appear later, if the solvent can be run into the trough through a small hole in the top cover so as to avoid opening the jar completely to the atmosphere.
The ascending technique requires less complicated apparatus and any container in which the paper can be hung so that it dips into a pool of solvent at the bottom may be employed. Again a glass container is page 106 desirable so that the movement of the solvent front can be followed visually, although this is by no means essential. Pieces of wide bore glass tubing closed at the top with a cork carrying some support may be used, or alternatively bottles such as milk bottles, or large test-tubes. For accurate work it is desirable that the solvent front may be able to move 30-40 cms., but for demonstration purposes, especially when time is an important factor, shorter distances may be adequate.
In general the descending method is preferred for accurate work, although the ascending technique is somewhat simpler and requires less complicated apparatus. The former method has the advantage that the solvent may be allowed to run off the bottom of the paper and, although RF values can not then be determined, substances with very low RF values can be separated more completely. As mentioned above, standard spots are usually run alongside the ‘unknown’ so that the fact that RF values are not obtainable is not of major importance. The ascending method, on the other hand, has the advantage that the solvent cannot ‘run off’ the paper and thus the chromatogram needs less attention.
A combination of the two methods in which the paper dips into a trough, runs up over a glass rod and then hangs down is useful especially when handling large sheets of paper as it obviates the use of a large tall glass container and vessels such as glass aquarium tanks may be employed Whatever the type of equipment used it is most desirable that it can be sealed as completely as possible.
(b) Type of Paper
A large variety of types of paper have been tried, some of which possess advantages for specialised work. Whatman No. 1 filter paper is most widely used and will give satisfactory results in the majority of cases. Thicker papers such as blotting paper are not usually suitable.
(c) Solvents
The choice of solvent depends on the type of substances to be separated, the most common ones being alcohols such as butanol with or without the addition of acids such as acetic acid or bases such as ammonia or amines, phenol, hydrocarbon solvents, collidine, etc. The solvent phase is saturated with water except in certain cases where the solvent is actually miscible with water. (Although it would appear at first sight that such a solvent would not give any separation this is, for reasons which will not be discussed here, not always the case.) It is perhaps unfortunate that the more common solvents are often unpleasant to handle and toxic, and care should be taken to avoid unduly prolonged exposure to their vapours.
(d) Detection of the Spots
The lower limit to the quantities of substances which can be separated usually depends on the sensitivity of the method used for detecting the resulting spots. Detection is usually carried out by spraying the dried page 107 paper with a suitable reagent which will develop colours with the substances present, often on gentle warming. A small atomiser or throat spray or some such device is convenient for applying the detecting reagent. In certain cases the paper may be dipped into a solution of the reagent instead of sprayed with it. Although spraying is the most common method of detecting the spots, many ingenious methods have been used as alternatives. Fluorescence in ultra-violet light and absorption of ultra-violet light, have been utilised as have biological methods of detection (for example for antibiotics) and radio-autographs (as for example in the analysis of compounds present in the thyroid gland after the administration of radio-active iodine) to mention but two.
General
As a general rule the spots applied to the paper should be small, preferably not more than a few millimetres in diameter and the concentration of the individual substances should not be too high or ‘tailing’ and excessive spreading of the spots will take place. In most cases a single small drop — say 5 microlitres — of a 1% solution is about the amount of substance required. For quantitative work an accurately known amount must be applied to the paper and for this a micro-pipette is required, but this elaboration is unnecessary for qualitative studies.
It is often desirable though not essential that the paper after the application of the material be allowed to stand in an atmosphere saturated with respect to both the aqueous and mobile phases before the chromatography is commenced. For this reason it is desirable that the solvent can be added to the trough or, in the case of ascending chromatograms, the paper may be lowered into the solvent, with the least possible dismantling of the apparatus.
The paper must almost always be dried before spraying and this is best accomplished either in a hot air oven or in a current of warm air such as that provided by a hair drier. Because of the toxic nature of the solvents drying should be carried out in a fume cupboard, or failing that in a well-ventilated place. Overheating of the papers should be avoided.
The spray should be applied lightly so that the paper becomes merely damp and not saturated in order to avoid undue migration and diffusion of the spots. For the same reason most spraying reagents are made up in non-aqueous solution.
With complex mixtures it is often impossible to obtain complete resolution with any single solvent and in such cases two-dimensional chromatograms may be resorted to. This may lead to a very high degree of resolution of complex mixtures but it is not possible to run standard substances simultaneously.
Quantitative paper chromatography may involve measurement of spot sizes, intensities of coloured compounds developed by sprays or elution of page 108 the spots from the chromatogram and subsequent determination by standard micro-chemical methods, to give some examples. However, the techniques employed are often specialised and outside the scope of this article.
The following practical directions should enable the beginner in the field to become familiar with the general methods and the suggested materials for investigation should provide a guide to those interested in further studies.
Ascending Chromatograms in Test Tubes
Whatman No. 1 filter paper is cut into strips which will fit easily into an ordinary test-tube and the strips are creased lengthwise to form a V-shaped trough. A line is drawn lightly across the strip about 2 cm. from the end, and by means of a fine glass capillary tube or a match-stick, a small spot of the sample (about 2 mm. in diameter) is placed on the line in the centre of each half of the V. The spots are allowed to dry then the paper strip is placed, spotted end down, in a test-tube containing 1-2 cc. of solvent in such a way that the strip touches the side of the test-tube only at the outside top corners. It is essential that the starting line marked on the strip be above the solvent surface, and the paper strips will stay in position readily if the tube is placed in a slightly slanting position. The test-tube is corked and set aside undisturbed until the solvent front rises almost to the top of the strip. This may take up to about one hour, depending on the temperature and the nature of the solvent. The strip is removed, the position of the solvent is marked, and the solvent is evaporated either by allowing the strip to stand in the air or, preferably, in a hot air oven or in a current of warm air. The chromatogram is sprayed lightly with the detecting reagent and heated if necessary, the position of the spots is marked, and their RF values determined. For this purpose the ‘distance moved by the solvent front’ is the distance between the starting line and the final position of the solvent front. The ‘distance moved by the substance’ is usually taken as the distance from the starting line to the ‘centre of gravity’ of the spot.
Ascending Chromatograms in Bottles
Wide-necked bottles such as milk-bottles are most satisfactory and the paper can be suspended from a glass or wire rod passed through a cork or rubber stopper. If the support can be moved vertically it is possible to allow the paper strip to hang in the atmosphere inside the bottle for up to 24 hours before it is lowered into the solvent, and if this is done more regular spots may be obtained and there may be less tailing. The additional width of the paper strip allows several samples to be run side by side. If this is done the individual spots should be spaced at intervals of 1.5-2 cm. along the starting line.
Ascending Chromatograms with Large Sheets of Paper
When a large number of samples must be run side by side or for two-dimensional chromatograms with sheets of paper up to 40-50 cms. page 109 square or larger it is often convenient to clip the sheet in the form of a cylinder which can then be stood in a dish of the solvent. In such cases a comparatively large container is required, or alternatively the chromatography may be carried out under a large bell jar. With large-scale chromatograms it is desirable to place in the container a dish containing water saturated with the solvent so that the atmosphere remains saturated with respect to both liquid phases. The rate of movement of the solvent front decreases with time and large-scale chromatograms may be conveniently allowed to run overnight.
Descending Chromatograms
The spots are placed on a line 6-8 cm. from the end of the paper strip which should be about 40-50 cm. long. The paper is arranged in the trough so that the starting line is well clear of the edge of the trough, a dish of water saturated with solvent is placed in the bottom of the container, the solvent is added to the trough and the container is closed. Once again, if possible, it may be desirable to allow the paper to equilibrate in an atmosphere saturated with both liquid phases before the solvent is added to the trough. The chromatogram may be allowed to develop as long as desired and if necessary the solvent may be allowed to run off the lower edge of the paper. If this is to be done the lower edge of the paper should be serrated to facilitate an even flow of the solvent. When development is complete the paper is removed, dried and sprayed in the usual way.
Two-Dimensional Chromatograms
When complex mixtures have to be analysed two-dimensional chromatography is a valuable modification because of the large increase in resolving power. A single spot of the mixture is placed near one corner of a sheet of paper 30-50 cm. square and the chromatogram is run in the usual way using either the descending or the ascending technique. At this stage the mixture will have partially separated into a series of spots distributed along a line near one edge of the sheet. The paper is removed and dried, then run in the direction at right angles to the original with a different solvent, then dried and sprayed in the usual way. The resulting chromatograms will show a series of spots distributed over the paper, and although standard substances cannot be run simultaneously, the identification of a spot is simplified by the fact that its position depends on a combination of two RF values. ‘Maps’ showing the positions which substances will appear in, with various solvent combinations have been published or can easily be constructed from tables of RF values. For two-dimensional chromatograms the amount of material placed on the original spot should be rather greater than for single dimensional ones, and the first solvent should be removed as completely as possible before running in the second direction. It is frequently found that there is a preferred order in which the two solvent combinations should be used.
Materials
For demonstration purposes amino-acids are the most satisfactory although some sugars can also be separated reasonably well in a comparatively short time.
The amino-acids can be applied as 0.2-1% solutions and mixtures are most readily obtained by applying the individual amino-acids successively to the same spot, the paper being allowed to dry between each application. For small-scale chromatograms in test-tubes or bottles, 80% phenol water (W/W) is a suitable solvent (this is rather cheaper to use than phenol saturated with water) and combinations of amino acids such as valine, glycine and aspartic acid or leucine, threonine and glutamic acid separate readily.
0.2% Ninhydrin (triketohydrindene hydrate) in water-saturated butanol is the spraying reagent, and the sprayed papers must be heated briefly at about 110° to develop the (usually blue) spots.
Sugars and related substances may be separated using a solvent mixture containing 5 volumes of acetic acid, 25 volumes of water and 110 volumes of butanol, although RF values are low and complete resolution of mixtures is difficult on a small scale. However, mixtures of monosaccharides and disaccharides such as glucose and lactose separate comparatively readily. The solvent mixture given above approximates in composition the organic (upper) layer obtained by shaking together butanol, acetic acid and water in the volume ratios 4: 1: 5 — a solvent which is very widely used in chromatographic work. This solvent mixture slowly changes in composition due to esterification and should not be kept indefinitely.
As spraying reagent, ammoniacal silver nitrate or 2% aniline hydrogen phthalate (made from 0.93 g. of aniline and 1.60 g. of phthalic acid in 100 ml. of water-saturated butanol) may be used. With either spray the paper must be heated to 105-110° to develop the spots.
* At our request Dr. J. G. Gibbs has written an introductory note indicating the importance to Biology of the techniques described by Dr. Harvey.