MINERAL ELEMENTS IN PLANT TISSUES
Hollo, J., Koch, L., “Leaf Protein: Its agronomy, preparation, quality and use,” Pirie, N. W., Ed., Blackwell Scientific Publications, London, 1971, pp 63-68. Horigome, T., Kandatsu, M., Nippon Nagei Kagaku Kaishi 40, .. 449 (1966). Huang, K. H., Tao, M. C., Boulet, M., Riel, R. E,Julien, J. P., Brisson, G. J., Can. Inst. Food Technol. J. 4,85 (1971). Kinsella, J. E., Chem. Ind. ILondon) 550 (1970) Knuckles, B. E., Bickoff, E. M., Kohler, G . O., J. Agr. Food Chem. 20,1055 (1972). Kohler, G. O., Bickoff, E. M., “Leaf Protein: Its agronomy, preparation, quality and use, Pirie, N. W., Ed., Blackwell Scientific Publications, London, 1971, pp 69-77. Lexander, K., Carlsson, R., Schalen, V., Simonsson, A., Lundborg, T., Ann. Appl. Bid. 66, 193 (1970). Loomis, W. D., Battaile, J., Phytochemistry 5,423 (1966). Lu, P. S., Kinsella, J . E., J. Food Sci. 37,94 (1972). Mahler, H. R., Cordes, E. H., “Biological Chemistry,” Harper and Row, New York, N. Y., 1966, pp 53-54. McCown, B. H., Beck, G. E., Hall, T. C., Plant Physiol. 43, 578 (1968). McKenzie, H. A,, Wallace, H. S., Aust. J. Chem. 7,55 (1954). Mukhopadhyay, S., Millikan, D. F., Phytopathology 57, 853 (1967).
Nazir, M., Shah, F. H., Pak. J. Sci. Ind. Res. 9,235 (1966) Oelschlegel, F. J., Jr., Schroeder, J. R., Stahmann, M. A., J. Agr. Food Chem. 17,791 (1969). Pirie, N. W., “Leaf Protein: Its agronomy, preparation, quality and use,” Pirie, N. W., Ed., Blackwell Scientific Publications, London, 1971, pp 53-62. Pirie, N. W., “Proteins as Human Food,” Lawrie, R. A., Ed., AVI Publishing Co., Westport, Conn., 1970, pp 46-61. Poppe, J., Tobback, P. P., Maes, E., Lebensm. Wiss. Technol. 3, 79 (1970). Singh,N.,’J. Food Sci. Technol. 1,37 (1964). Stahmann, M. A., Econ. Bot. 22,73 (1968). Stahmann, M. A., Ann. Reu. Plant Physiol. 14,137 (1963) Stavely, J. R., Hanson, E. W., Phytopathology %?,482 (1967). Subba Rau, B. H., Singh, N., Ind.,J. Exp. Biol. 8, 34 (1970). P. K., J . Sci. E‘ood Valli Devi, A., Rao, N. A. N., Vilayaraehavan, _ . Agr. 16, 116 (1965). ~~~~~I
Received for review July 5 , 1972. Accepted September 26, 1972. Reference to a company and/or product named by the Department is only for purposes of information and does not imply approval or reccommendation of the product to the exclusion of others which may also be suitable.
Determination of Mineral Elements in Plant Tissues Using Trichloroacetic Acid Extraction Glen E . Leggett* and Dale T. Westermann
An extraction method is described for quantitatively determining Mg, K, Na, Zn, and M n in plant tiwues. The tissue is extracted with 2% trichloroacetic acid and the elements are determined on the filtrate by atomic absorption spectrophotometry. Results compare favorably with
The mineral content of plant samples is usually determined by analyzing the liquid phase after dry ashing or wet ashing ground dried plant materials. The procedures and t h e problems of fume disposal and special laboratory equipment required for the methods have been discussed (Johnson and Ulrich, 1959). Nicholas (1951) surveyed t h e literature on extraction methods for tissue analysis in determining t h e nutrient status of various crops. Many reports indicate good correlations between nutrients extracted and fertilizer applications, b u t only a few compare t h e extracted values to the total content. Nicholas (1948a,b) extracted plant tissue with acetate, citrate, malonate, and succinate buffers ( p H 4.8). The concentrations determined by extraction were correlated with t h e total concentrations in the tissue, but t h e two sets of values were not directly compared. Greweling (1962) a n d Baker and Greweling (1967) analyzed t h e EDTA extracts from a variety of plant tissues for several mineral elements and compared the results with those obtained after dry ashing. In most cases the extracted and ashed values agreed closely. Analysis of acetic acid extracts of plant tissues for Zn and M n indicated t h a t extraction of these elements was essentially complete from most but not all plant materials. Consequently, a stronger acid. trichloroacetic, was used and t h e number of elements determined extended to United States Department of Agriculture, Agricultural Research Service, Kimberly, Idaho 83341.
those determined after wet ashing with nitric a n d perchloric acids. In addition, trichloroacetic acid extracts the same fraction of P as does acetic acid and quantitatively extracts total Ca from plant tissues not high in oxalate.
include Ca, Mg, K, N a , Cu, Fe, and P. This report deals with the use of trichloroacetic acid for extracting t h e total contents of several mineral elements from various plant tissues and a study of some of the factors affecting its use. METHODS AND MATERIALS Plant Samples. Forty-two samples representing 20 different crops (Table I) were analyzed for their total content of several mineral elements by wet ashing and for the amount of t h e various constituents extracted in various solutions. All samples were washed in demineralized water, dried at about 60”, and ground in an all-steel Wiley mill to pass a 40-mesh stainless steel sieve. The ground material was redried for a t least 2 hr a t 60” before weighing out samples for analysis. Extracting Solutions. Trichloroacetic Acid (TCA) Solution. A 2% (w/v) solution was prepared by dissolving 20 g of reagent grade TCA in distilled demineralized water and diluting to 1 1. Acetic Acid (HOAc) Solution. A 2% (v/v) solution was prepared by diluting 20 ml of reagent grade HOAc to 1 1. with distilled demineralized water. Ammonium EDTA Solution. A 1.0 M stock solution was prepared according to the procedure of Baker and Greweling (1967). This solution was diluted with distilled demineralized water to make 0.1 M ammonium EDTA as needed. Extraction of Plant Tissue. Samples (0.5 g) were weighed into wide-mouthed 125-m1 linear polyethylene J. Agr. FoodChern., Vol. 21, No. 1, 1973
65
LEGGETT, WESTERMANN Table I . Plant Samples Used for Analysis
Vines (1)
Wet Ashing of Plant Tissue. Samples (0.5 g) of dried ground plant material were weighed into 100-ml Kjeldahl flasks containing a glass bead. The samples were predigested with 5 ml of H N 0 3 , cooled, and digested to fumes of HC104 after adding 5 ml of a mixture of H N 0 3 and HC104 (3 + 1). After digestion, about 40 ml of distilled demineralized water was added to each flask and the contents were brought to near boiling to ensure complete dissolution of sample except for silica. Samples were cooled, diluted to 50 ml, and filtered through Whatman No. 50 paper. Chemical Determinations. The metal cations in the extracts and digests were determined by atomic absorption spectrophotometry. Cu, Zn, Mn, and Fe were determined directly on the filtrates, whereas Ca, Mg, K , and N a were determined after diluting the filtrate (1 + 24) with a solution containing 1% La and 5% HC1. P was determined on a suitable aliquot of the undiluted filtrate by the vanadomolybdate method (Kitson and Mellon, 1944).
Tops (3)
RESULTS AND DISCUSSIOS
Plant part and no. of samples
Crop
Alfalfa (Medicago sativa) Asparagus (Asparagus officinalis) Beans (Phaseolus vulgaris) Garden beet (Beta vulgaris) Sugarbeet (Beta vulgaris L.) Brussels sprout (Brassica oleracea gemmifera) Cabbage (Brassica oleracea capitata) Corn (Zea mays) Hops (Humulus lupulus) Lettuce (Lactuca sativa L.) Peppermint (Mentha piperita L.) Spearmint (Mentha spicata) Parsley (Petroselinum crispum latifolium) Pea (Pisum sativum) Potato (Solanum tuberosum) Rhubarb (Rheum rhaponticum) Sorghum (Sorghum vulgare) Spinach (Spinacia oleracea) Swiss chard (Beta vulgaris cicla) Wheat (Triticum aestivum)
Stems and leaves Fern (1 ) Tops (2) Leaf blades (2) Leaf blades (3) Heads (1)
(3)
Heads (1) Leaves ( 4 ) , tops ( 3 ) Leaves ( 3 ) , cones (1) Leaves ( 1) Shoots (3), stems (1) Stems (1) Tops (1)
Leaf blades ( 1) Tops (3) Leaves (1) Leaf stalks (1) Ripe straw (1 )
bottles fitted with polyethylene-lined caps. Each sample received one spoonful (approximately 1 g) of activated carbon and 50 ml of extracting solution. Samples were shaken on a reciprocating shaker for 16 hr a t 2 5 O , or for prescribed periods of time. Some samples were heated at 60" in a bath of running t a p water; these samples were shaken intermittently by hand. The bottles containing the heated samples were fitted with polypropylene caps to withstand the pressure and to prevent evaporation. All samples were filtered through Whatman No. 50 paper. T o avoid having carbon appear in the filtrate the samples were shaken vigorously immediately before pouring them rapidly onto the filter.
Mineral concentrations determined after wet ashing and TCA extraction (16 hr, 25') are compared in Figure 1. Extraction of Mg, K, Na, Zn, and M n is quantitative for all 42 samples. The extraction of Ca was complete for most crops; but from beet leaves, rhubarb leaves, spinach, and Swiss chard it averaged 65% and tended to be low from potato tops (8770) and hop leaves (94%). Almost no Ca was extracted from beets, rhubarb, spinach, and chard by HOAc, and increasing the TCA concentration to 3% increased the Ca extracted from these materials to only 80% of the total. These crops contain large amounts of oxalate, thus it was assumed t h a t Ca was immobilized as the oxalate either before or during the extraction. Except for these eight samples, the agreement between Ca extracted with TCA and total Ca is good. The values reported for Cu should be considered tentative. The Cu concentrations in most samples analyzed approached the limits of determination for the techniques used here. Moreover. a variable blank for Cu was encoun-
Table II. The Effect of Time and Temperature on the Extraction of Mineral Elements from Plant Tissue by TCA Solution Mineral concentrations determined b y various Drocedures" Mg, %
Ca, % TCA
Hop leaves Corn leaves Alfalfa Bean tops Sorghum tops Potato tops Sugarbeet leaves
TCA
TCA
Total
TCA
-H
Total
TCA
-
H
Total
TCA
-H
4.18 0.90 1.51 2.62 0.46 1.98 1.47
4.02 0.91 1.35 2.55 0.46 1.70 0.56
4.10 0.94 1.43 2.66 0.47 1.84 0.92
1.58 0.64 0.43 0.49 0.21 0.71 0.91
1.56 0.63 0.42 0.48 0.21 0.70 0.89
1.51 0.67 0.45
1.16 2.46 3.61 3.62 1.62 5.40 2.65
1.24 2.45 3.52 3.53 1.63 5.37 2.52
1.19 2.47 3.62 3.66 1.66 5.52 2.59
0.50 0.20 0.71 0.91
Mn. ppm TCA
Hop leaves Corn leaves
Alfalfa Bean tops Sorghum tops Potato tops Sugarbeet leaves
P, %
K, %
- H
Totai
TCA
-H
22 13 23 25 34 56 62
20 12 25 25 32
21 13 24 24 31 57 60
84 236 40 84 44 58 102
82 234 40 81 40 57 101
81 234 38 81 39 54 97
J. Agr. Food Chem., Vol. 21, No. 1, 1973
0.118 0.169 0.162 0.113 0.173 0.319 0.313
TCA
-H
Total
9 31 12 8
6 22 9
5
3 6 12
7 26 9 6 4 7 13
410 203 80 458 470 282 422
10 15
0.111 0.192 0.169 0.118 0.175 0.315 0.312
TCA
Total
5
- H
-
"Total determined by wet-ashing, TCA by extracting for 16 hr at 25'. and TCA - H by extracting for 1 h r at 60"
66
0.276 0.323 0.390 0.324 0.376 0.574 0.435
TCA
TCA
TCA
62
TCA
cu, ppm
Total
55
TCA Total
TCA 51
70 42 115
- H
82 104 26 45 75 64 137
MISERAL ELEMENTS I N PLANT TISSUES
>
n U C .-
-Eb m
0
C m
._
-2 -4
c
U
0
I
.-C
I
J. Agr. Food Chem., Vol. 21, No. 1, 1973
67
LEGGETT, WESTERMANN TCA-SOL. Mg
TU-SOL. Co
-
b -0.983 r2
TCA-SOL. No
TCA-SOL K
b
rz
0.9973
-
0.983 = 0.9989
r2
6.
-
0.9989
/
I
TOTAL Co
-X
TOTAL Mg
- 'A
TOTAL K
m. n -42 I6O.
/
b -0987
rz -09975
120.
r2
fi
c
. 3 0
Table IV. The Range and Mean Concentrations for Several Elements Extracted by TCA from Seven Samples and the Coefficients of Variation Calculated from Analysis of Four Replications Range in values Low
Ca
High
Mean
Yo
Coefficient of variation, Yo
0.59 0.41 1.20 0.101
4.26 1.47 5.24 0.293 PPrn
1.70 0.67 2.84 0.183
1.4 1.4 2.9 1.4
Zn
10
Mn
40 4
56 256 24
30 94 10
2.5 1.5 7.3
Mg K P
cu
Samples consisted of one each of sugarbeet leaves, potato tops, sorghum tops, hop leaves, alfalfa, bean tops, and corn leaves
68
J. Agr. FoodChern., Vol. 21,No. I , 1973
TOTAL Na
-%
