shown in Table I, the anions tested being NOa-, COS+, C1-, and CzHsOn-. Cations that complex with EDTA would interfere, and would have to be separated from the solution (by ion exchange or other techniques) before amine analysis, if present in sufficient amount to affect the copper-EDTA complex. Tolerances for these cations might be large in some cases and small in others, depending upon the strength of the foreign metal-EDTA complex. For most foreign metals, no trouble should arise if the foreign metal concentration in the solution examined is less than 0.0016MJ or one-tenth the molar concentration of the copperEDTA complex in the solution examined. In developing this analytical method the authors have purposely used equimolar amounts of C U + and ~ EDTA to ensure the highest degree of reproducibility. Small excesses of C U + ~ or EDTA could most likely be tolerated without seriously affecting the results. However, a considerable excess of C U + ~ would tend to give low results because of coordination between Cu+z and primary amine. On the other hand, a large excess of EDTA would be expected to yield high results because
of its tendency to decrease the degree of polymerization of the Cu-EDTA polymer. The use of a controlled excess of EDTA to eliminate certain cationic interferences is a definite possibility which will be investigated. Data are also included (Figure 3) to show that this method is generally applicable to all primary amines. However, except in cases of very similar primary amines, such as CaHsNHz and n-CsH,NHn which have the same slope for the curve of absorbance vs. [primary amine]/ [(CuY) the amine being analyzed should be used in making up the standard solutions. Figure 3 shows that the limits of linearity for the curves of absorbance vs. [primary amine]/ [(CuY) -21 for the various amines tested are about the same. For 0.016M [(CUY)-~]the ratio [primary amine/ [(CUY)-~]should not exceed 0.75 to ensure results within the limits of reliability presented here. Among the primary amines tested were two amine alcohols, 2-aminoethanol, and 3-aminoI-propanol. From the slopes of the curves for these two compounds in Figure 3 it is evident that the -OH functional group in HgN(CH2)zOH interferes with the coordination of the amine to the Cu-EDTA complex to a greater
extent than in HzrYT(CH2)30H where it is further removed from the amino group. Other correlations between the kinetics of amine coordinations to CuEDTA and the structures of the amines might be possible by thorough study of more absorbance curves of this type. ACKNOWLEDGMENT
The authors are indebted to Harold Weinberger, Chairman of the Department of Chemistry, to Kathleen Hillers, and to the faculty of the Department of Chemistry a t Fairleigh Dickinson University for their advice and encouragement during this research. LITERATURE CITED
(1) Citron, I., Anal. Chim. Acta 26, 44657 (1962). ( 2 ) Day,. R . A,, Underwood, A. L., “Quantitative Analysis,” pp. 88-9, Prentice-Hall, Englewood Cliffs, N. J., 1958. (3) Erspamer, V., Progr. Drug Res. 3,152367 (1961). (4) Fritz, J. S., “Acid-Base Titrations in Non-Aqueous Solvents,” G. F. Smith Chemical Co., Columbus, Ohio, 1952. (5) Kirson, B., Citron, I., Bull. SOC. Chzm. France 1959,365-9.
RECEIVED for review July 9, 1963. Accepted October 24, 1963.
Quantitative Isolation and Improved Nephelometric Microdetermination of Lupine Alkaloids from Plant Tissues DONALD M. GRAHAM and MARY SPENCER Departments o f Biochemistry and Plant Science, Universify o f Alberta, Edmonton, Canada
b A simple method is described for quantitative isolation from plants of submicromole quantities of lupine alkaloids and for their purification, separation, and estimation. The method involves extraction with water, removal of the alkaloids from the extract with a phosphonic acid cation exchanger, followed by elution with acid, pH adjustment, solvent extraction, and low-pressure removal of solvent to yield the alkaloid salts in crystalline form. Separation is achieved by descending paper chromatography using a solvent system based on acetic acid, tertiary amyl alcohol, and water. The alkaloids are determined nephelometrically after elution from the paper. This procedure can be used successfully when the tissue investigated contains as little as lo-* mole of any one alkaloid.
0
NE OF THE CHIEF DIFFICULTIEB
in biochemical analysis of plant alkaloids is that most of the classical extraction procedures, even those de210
0
ANALYTICAL CHEMISTRY
scribed as quantitative, do not ensure complete extraction of alkaloids from the tissue; purification procedures are usually tedious and require large quantities of materials; transfer losses occur readily; and conventional analytical methods are often difficult to use with minute quantities of material (4). Some attempts, notably by Lee (6), Mattocks (7), and Tompsett (IO), have been made to use ion exchange resins for isolation of alkaloids from plant tissues and to a lesser extent for separation of alkaloids from one another, but these methodfi are of limited usefulness, and in particular, none is suitable for lupine alkaloids. Lee’s method, originally designed for the fractionation of opium, necessitates beginning with fairly large (gram) quantities of alkaloid that have already been isolated in crude form; Mattocks’ procedure is useful only for very large quantities and all three employ Dowex 50, which we have found to bind lupine alkaloids irreversibly. Acid cannot be used for elution, even a t negative p H
values, because this resin absorbs alkaloid from such solutions, and basic solutions are unsuitable since several of the alkaloids are unstable or insoluble, or both, in the unionized form. We discovered also that eshaustive extraction of the plant tissue with various solvents in common use for this purpose was usually not in fact quantitative. Furthermore, solvent partition methods, when used alone, are unsatisfactory for the quantitative isolation of alkaloids from plant tissues because they tend to be tedious, and involve large transfer losses as well as difficulties due to the formation of troublesome emulsions. The present method, on the other hand, permits of high extraction efficiency, gives a crystalline product, allows clean separations, and can be used with minute quantities of alkaloida (small fractions of a micromole). WPERIMENTAL
Apparatus. Beckman Model B Spectrophotometer with 1-cm. matched Pyrocell quartz cells.
Reagents. Duolite C-63 phosphonic acid resin (from the Chemical Process Co., Redwcod City, Calif., or the California Corp. for Biochemical Research, Los Angeles) is dryscreened t o 20- t o 40-mesh and washed continuously in a ccdumn with twice the volume of 6 N HC1 required t o discharge the yellow color, followed by demineralized water until the effluent gives no reaction with silver nitrate solution. The resin is removed from the column and allowed to dry in air (this does not damage the resin). All buffers used are potassium phosphate buffers, pH 6.5, either 0.1M or 0.21ZI. Stock alkaloid s o h tions are made to 0.01Jf in 0.04117 HCl. Alkaloids were obtained from Mann Research Laboratories, Inc., New York. The reagent for nephelometric microdetermination of alkaloids, based on the method of Reifer and Niziolek (9), is made up as follows: 23.2 grams of KI and 19.2 grams of KE;r are dissolved in about 100 ml. of watls; 22.0 grams of crystalline Iz is now added with continuous stirring using a magnetic stirrer and the total volume made up t o 240 ml. with water, then t o 400 ml. with 1N HCl. A slight precipitate remains and the supernatant is decanted off and stored in the refrigerator until required. This method of preparing the reagent is faster than that described by the originators. and avoids the use of elemental bromine. The solvent system for chromatography is glacial acetic acid-watertertiary amyl alcohol (1:4:7). The lower (aqueous) phase is used for equilibration and the upper phase for development. The chromatograms are sprayed with 1% 1, in CHClr to reveal the alkaloid spots without chang ng them chemically. Procedure. About 3 grams of fresh or frozen tissue is ground t o a uniform consistency with enough water t o bring the total volume t o 5 ml., in a 12-ml. heavy-duty graduated clinical centrifuge tube, using a glass rod as a pestle. Lupine Idssue is so friable that very thorough disintegration is readily achieved by this means. The residue is centrifuged down and the supernatant filtered through paper to remove any floating debris. The residue is resuspended in the same volume of water as before and the process repeated for a total of four extractions. The final residue is discarded and the aqueous extract mixed with its own volume of 0.2-11 buffer. If necessary, the pH is adjusted to exactly 6.5 with the appropriate 0.1M buffer component. About 2.5 grams of dry resin which has been equilibrated with 0.1M buffer is placed in a 1-cm. diameter glass column. The buffered extract, centrifuged to remove debris which may have precipitated on standing, is now passed through the column a t a flow rate not The exceeding 1 ml. per minute. effluent, which has thus been cleared of alkaloid, is discardl3d and the resin bed washed with about 25 ml. of water to remove residual plant material.
The alkaloids are eluted from the column n~ith50 ml. of 6N HCl (in which both the resin and the alkaloid were found to be stable), also a t a rate of 1 ml. per minute. To this acid eluate is added 1 to 2 ml. of 55f Ca(NO&, which precipitates any emulsifying agents which may have been carried along this far with the alkaloids. The eluate is chilled to just above its freezing point and mixed with a slight excess of 10N NaOH, also close to freezing; the maximum temperature on mixing is between 35" and 40" C. Any precipitate present a t this stage is removed by centrifugation. The basic solution is now extracted quickly with seven 15-ml. aliquots of chloroform in a separatory funnel fitted with a Teflon stopcock, and the extracts are added to 3 ml. of glacial acetic acid, miscible with chloroform (1). This converts the alkaloids into their acetates, which are much more stable in chloroform than are the free alkaloids. The chloroform and acid are evaporated off under reduced pressure to leave the alkaloid salts in the form of a white crystalline deposit, weighing a few milligrams. Haste is nevertheless necessary a t this stage because prolonged (several hours) contact with chloroform can cause irreversible changes even in the ionized alkaloids. Acetic acid does not partially decompose the alkaloids during evaporation to dryness, a difficulty encountered by Birecka et al. (8) with hydrochloric acid. The last traces of impurity are removed by washing the crystals in petroleum ether, in which they are insoluble, dissolving them in 1 ml. of water and filtering. Aliquots of this solution (or the whole sample if so desired) are spotted on Whatman No. 3MM chromatography paper, and the chromatograms are equilibrated and developed in darkness a t room temperature. The photosensitivity of the alkaloids if3 not great, and admission of light during the brief early stages of the extraction, prior to chromatography, has no detectable effect. After development the papers are dried and stained. The alkaloid spots are excised and cut into narrow strips for elution with 1N HCl. The eluates are made up to a volume of exactly 5 ml. each with this solvent for estimation of the alkaloid content and are centrifuged to remove filter paper fibers, which interfere with the nephelometric estimation technique. Three milliliters of mivture is pipetted from each tube into a reaction vessel in an ice If the concentration should bath. prove to be appreciably above the range most suitable for estimation, sufficient solution is left to permit dilution to a more suitable concentration range. Two milliliters of the iodine reagent, which forms a colloidal complex with the alkaloid salts, is pipetted into each sample and into a 1N HC1 blank. After 30 minutes the samples are transferred t o the spectrophotometer cells and the absorbance is measured at 820 mp. This is the wavelength a t which the iodine reagent was found t o have
Table 1. Efficiency of Extraction of Alkaloids from Plant Tissues by Different Solvents Alkaloid in CHCh extract of Solvent Efficiency residue 0.1N HC1 78% Present 0.1M HaPo, 73% Present 1.OM HPPOi 37% Present 0.1M Phosphate 89% Present buffer, pH 6 . 5 89% Present 0.1M KH2P04 100% Absent Water In each case 3 grams of tissue that did not already contain alkaloid, with 10 pmoles of added alkaloid, was extracted with four 5-ml. aliquots of solvent. maximum transmittance, and waa chosen in preference to the broader spectrum transmitted by the Leitz Uter B suggested by the originators (9) of the method, to yield maximum sensitivity for nephelometric measurements. Comparison with standard calibration curves yields the concentration of alkaloid in each sample, from which the total quantity of each alkaloid in the plant tissue may be calculated. RESULTS AND DISCUSSION
The efficiency of various commonly used extracting solvents was tested a follows: known quantities of alkaloids were mixed with plant tissue that did not contain alkaloid and re-extraction with the solvent in question was carried out, followed by quantitative estimation of the alkaloid extracted. In each case, after extraction, the tissue was dried, the residue extracted with chloroform, and this extract was tested qualitatively for alkaloid. The results are shown in Table I. The same solvents were tested, as was methanol, another commonly used solvent, on actual lupine samples and in each case a chloroform extract of the dried residue contained alkaloid, except when water was the original solvent. This is contrary to the observations of Birecka and Nalborczyk ( I ) , who achieved equally efficient extractions with methanol and with water. The reason for the increased efficiency of water over acid solvents is unknown, but a possible explanation is that the acid solvents may precipitate some tissue protein, trapping alkaloid in the coagulum. Since the cell contents are slightly acid to begin with, the alkaloids are therefore in the water-soluble salt form, so that they can be readily extracted with ordinary water. Chloroform itself is unsuitable as an extracting solvent because some lupine alkaloids, when ionized, are insoluble in chloroform, and when unionized, are unstable. The R, values of the alkaloids, shown in Table 11, indicate the good separations that are possible using the solvent system described. VOL 36, NO. 1, JANUARY 1964
21 1
Key:
- - _.o- _ _ _ -0-
-a-
1
Weight Lu pi ni n e Lupa nine
1.5
3
Age (days)
Figure 1. Variation of weight and alkaloid composition of lupine seedlings with age
The molar absorptivities of the reaction products of the alkaloids with this reagent are shown in Table I1 dong with the linear range of determination for each. Since the calibration curves are all sigmoid in form, the extrapolated linear portions do not pass through the origin; consequently the points at which the projected lines cut the abscissa are also included in the table. The figures are expressed in
molar unit. rather than in parts per million to render more meaningful the comparisons between absolute quantities of different alkaloids coexisting within the plant. Reproducibility is high with all alkaloids except osolupanine and gramine-not major alkaloids. They can nevertheless be detected qualitatively. Losses during processing were estimated by mixing k n o m quantities of individual alkaloids, and of mixtures,
Table 11. R, Values of Lupine Alkaloids in Acetic Acid-Water-Tertiary Amyl Alcohol (1 :4:7), and Characteristics of Calibration Curves for Their Nephelometric Determination
Intersection
of projected
E
of
reaction product
linear portion of calibration curve with Minimum linear range abscissa of determination (molarity) (molarity)
R, Alkaloid arteine 0.06 17,500 5.0 X 10-a2.0 X 2.2 x 10-6 ydrqxylupanine 0.18 11,600 1 . 5 X lO+-l.O X lod4 6.0 X Lupinine 0.30 8,400 1 . 5 X 10-5-S.5 X i.5 X Lupanine 0.52 17,500 'i5 X 10-"2.0 X lo-' 3 . 5 X lo-' Grrtmine 0.58 9,000 2 . 5 x 10-5-1.0 x 1 0 - 4 1 . 3 X lo-' Oxolupanine 0.86 3,200 2 . 5 X 10-6-1.5 X 1 . 0 x 10-6 Descending chromatography was performed at room temperature ( 2 i " C.) on Whatman S o . 3MM paper.
2 b
Table Ill. Characteristics of Lupine Seedlings over Period of Germination
Fresh wt.. Designation A
B
c
D E F G H
212
Developmental stage Seed swollen Root tip visible Germination complete Cotyledons green Cotyledons open Primary leaves open Secondary leaves open Tertiary leaves open
ANALYTICAL CHEMISTRY
Age, days 0 . 0 (defined) 1.o
1.9 4.1 6.8 9.3 14.4 20.3
(grams 1 0.32 0.31 0.35 0.53 0.60 1.08 1.17 1.37
with ground plant tissue that did not already contain alkaloid, processing the mixture in the usual manner and estimating the alkaloids recovered. With the exception of oxolupanine, some 55 to 75'30 of which is lost, all recoveries mere in the range 97 to 102%. The case of oxolupanine was investigated further and it was found that the losses were caused by inefficiency in both its absorption on the resin and its elution from it. To check the possibility that qualitative changes might occur in the alkaloids during processing, a plant specimen was extracted in the usual manner. The extract, kept at room temperature (27" C.), was sampled 15 minutes after the extraction was started, and a t intervals thereafter for 10 hours. A11 samples were chromatographed and estimated. No differences were detected among any of the samples thus taken. The method described overcomes most of the difficulties formerly encountered in microanalysis of lupine alkaloids and could probably be modified for use with other groups of alkaloids also. The precision is high with all the main lupine alkaloids. I t makes possible serial analyses of plants at different stages of development from both the qualitative and the quantitative points of view. By this means it is possible to locate developmental stages at which alkaloid metabolism is most active, so that these stages can be isolated for closer study. Lupinus Zuteus seedlings at various stages of development were analyzed by this method and the results are shown in Figure 1. The designations of the various developmental stages analyzed, together with the average age and weight at each stage, are shown in Table 111. [The alkaloid contents are expressed as micromoles per plant rather than as micromoles per gram of tissue for reasons discussed at length by Damson ( 5 ) ; the chief of these is that the weight of plant tissues is determined mainly by the quantities of cellulose, lignin, and protein present, as well as the mass of n-ater that happens to be in the tissues at the time of sampling. These factors bear little or no relation to the alkaloid content.] These results indicate that in the initial stages of germination and development of the plant, when pronounced physiological changes are taking place, both the total quantity of alkaloid and the ratios between different alkaloids undergo drastic changes. Mothes (8) cites some other observations that alkaloid metabolism is at its highest rate in rapidly growing tissue. When germination is complete and the plant has started its steady growth the content of various alkaloids appears to increase steadily with no marked changes of the type observed in the very young seedlings
taking place n-ithin the growth period examined. The present obsei*vations challenge the conventional idea that alkaloids are mere end-products of metabolism. Birecka et al. (3) obtained similar results for older plants a t flowering and seedpod formation, where high physiological activity might be esoected. Both sets of observations indicate that the most rapid alkaloid metabolism takes place when the plant is physiologically most active, and suggest that alkaloids may play a more active role in the physiology of the plant than Iias hitherto been suspected.
ACKNOWLEDGMENT
( 5 ) L)awson, K. F.,Lkhm.b ’ m ~ t t i o l . 8,
The authors are grateful to Hugh A. White for assistance in the analyses.
(6) Lee, K.-T., Nature 188,65 (1960). (7) Mattocks, A. R., Ibid., 191, 1251
LITERATURE CITED
( 8 ) Mothes, K., Ann. Rev. Plant Physiol. 6 , 393 (1955).
(1) Birecka, H., Kalborczyk, E., Bull. Acad. Polon. Sci. Sdr. Sci. Biol. 9, 401 (1961). (2) Birecka, H., Rybicka, H., SciborMarchocka, A., Bcta Biochim. Polon. 6 , 2 5 (1959). (3) Birecka, H., Szgmahka, A., SciborMarchocka, A., Acta SOC.Boian. Polon. 29,369 (1960). (4) Cromwell, B. T., “Modern Methods of Plant Analysis,” Paech, Tracey, eds., p. 367, Springer-Verlag, Berlin, 1955.
203 (1948). (1961).
(9) Reifer, I., Niziokek, S., Bull. Acad. Polon. Sci. Sdr. Sci. Biol. 7, 485 (1959). (10) Tompsett, S. L., ! c i a Pharmacol. Toxicol. 18, 414 (1961,. RECEIVEDfor review August 2, 1963. Accepted October 8, 1963. Part of this paper was presented to the meeting of the Canadian Society of Plant Physiologists, Winnipeg, June 1963. A grant-inaid of this research from the National Research Council of Canada is gratefully acknowledged.
Characterization of Lignosulfonates by Ultraviolet Spectrometry Direct and Difference Spectrograms ARTHUR S. WEXLER Dewey and Almy Division, W. R. Grace
b The ultraviolet dkect and difference spectrograms of alkaline and neutral (or acid) solutions of lignosulfonates are valuable in interpreting the chemistry of these Substances. The use of an ultraviolet double-beam recording spectrophotometer to obtain recorded spectral charts of both the direct and differential spectrograms greatly facilitates analysis and study of these and related substances. The direct spectrograms are useful in establishing the gross features of the material. The differential s(:lectrogram is of value in determination of the aromatic hydroxyl content and is also a characteristic physicochemical property of the material.
spec$,rometry has been extensively employed in the study of the chemistry and molecular structure of the lignins and 1 gnosulfonates (12, 16-18). The absorption maximum near 280 mp of aqueous or alcoholic solutions of these substances has been frequently cited as supporting evidence of the presence of guaiac.jl, syringyl, and related structures mi th a predominance of aromatic methoxyl over aromatic hydroxyl groups (1, 4, 16, 17). More recently, the differential absorption obtained by manually plotting the difference between the absorptivities of the ionized (in alkaline solution) and nonionized (neutral or acid solution) forms of lignins and ,ignosulfonates has been utilized in studies of molecular structure (2, 3, 8) anil in determination of aromatic hydroxyl content ( 7 ) . LTRAVIOLET
Co., Cambridge, Mass. Discrepancies betrreen the spectroscopically determined aromatic hydroxyl content and the values found by chemical methods hare been reported (6J
Practically all the reported investigations employing ultraviolet spectrometry have been based on single-beam, manually operated, nonrecording instruments. Much progress could be made in spectrophotometric studies of these and related substance by use of a high quality recording ultraviolet doublebeam spectrophotometer to obtain direct and differential spectrograms for analysis and interpretation. The interpretation of recorded direct and differential ultraviolet spectrograms of softwood lignosulfonates and the technique of obtaining such recorded data are the major topics of this paper. Both the direct and differential spectrograms can be recorded on the same spectral chart for ready comparison, analysis, and interpretation. The aromatic hydroxyl content can be determined using a base line technique in the differential spectrograms. Certain definitions are helpful in presentation of data. The differential spectrograms of lignosulfonates display a strong peak at about 250 mp and a weak peak at about 300 mp. Minima are found near 229 and 278 mp. The following symbols are used in this paper : A 250 followed by absorptivity or by mp. Strong peak maximum a t about 250 mp in the differential spectrogram. 4 300. Keaker peak maximum a t about 300 mp in the differential spec-
trogram. This peak is 0.4 to 0.6 as strong as the A 250 peak. A mp. Spacing in millimicrons between the two peak maxima in differential spectrogram. This value is about 50 mp in softwood lignosulfonates. EXPERIMENTAL
Procedure. A Beckman DK-2 ultraviolet recording double-beam spectrophotometer was employed in the linear absorbance mode as follows: Scan speed 160 mp, in 6 minutes Gain 140 Slit at 280 mp 0.06 mm. Time constant 0.2 Peak maxima and minima were spot-checked with a Beckman DU single-beam spectrophotometer. Results agreed within 1 to 2% of the measured absorbance. Wavelengths were checked and calibrated with a mercury lamp. Reagents. Solutions of lignosulfonates were prepared as follows for ultraviolet measurements: Exactly 0.2000 gram of sample was transferred to a 100-ml. volumetric flask, dissolved in distilled water, and adjusted to volume a t room temperature (usually about 23’ C.). Second dilutions were made by pipetting exactly 5 ml. with a delivery pipet into each of two volumetric flasks. To one flask was added 10 ml. of 1.ON potassium hydroxide; to the other, 10 ml. of 1.ON hydrochloric acid solution. Adjustments to final volumes of 100 ml. were made with distilled water. In this way identical concentrations of 100 p.p.m. in 0.1N alkali and in 0.1N acid were prepared for ultraviolet measurement. Matched 1-cm. silica cells were VOL. 36, NO. 1 , JANUARY 1964
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