855
V O L U M E 28, NO. 5, M A Y 1 9 5 6 precipitant. I n preliminary esperinieiits nsing the overspotting technique, a l d l polution of (ethylenedinitri1o)tetraacetate in ammonium hydroxide was used as the prespotted precipitant ; in this inettinee, the 2-ketogluconic acid moved as a compact spot to I?, 0.28. Presumably, the acid moved as the ammonium salt, ivhich has a much loner mot)ility than the pyridinium salt. Overspotting the Lvater solution of calcium 2-ketogluconate on I pmole of barium acetate leads to complete retention of the acid in the rrgion from Rj0.14 to Ri 0.03, while the calcium moves freely to its normal popition. Correlation of Molecular Structure and Rj Value. 11:xnj- wnpirical generalizations can be derived from the data in Table I. Desosyaldohesoses have R J valurs f r C m0.75 to 0.83, aldopentoses from 0.70 to 0.76, ketohesoscs from 0.6‘3 to 0.70, aldohexoses from C.63 to 0.70. These ranges are probably too narrow to e the rarer sugars-e.g., the aldohexose idose n.ould include s ~ m of probably have an Rj of 0 . i 5 , hg conipariwn with the data gii-en by Ishenvood and Jermyn ( 7 ) for niovenicnt in an ethyl acet:itepyridine-water solvent. These authors have sho~vn th:it the R J values for cnrbohydratcs follow the wine sequence in :ill rhromatographic solvents (except phenol 1. Galxc*tose and its homomorphs (arabinose, fucose, dulcitol, g:tiarturonic arid) hxvc the lowest R i values Lvithin the group to ivhic*hthey hcloiig. Gliirose and its homomorphs (xylose, sorliitd, glucuronic a ( % ) have and ~ its homomorphs (Iysose, slightly higher R J values. 1 1 a r i n o ~ rh:ininow, nimnitol, mnnnuronir ;wid) h;ive still higher R,
values, near the median value for each structural group. The configuration of the hydroxyls within each homomorphic group apparently determines the relative water affinity and thereby the relative mobility of the molecule. Xonophosphoiic esters have R , values from 0.37 to 0.43 unit lower than those of parent carbohydrates such as glycerol, glucose, or adenosine. Carboxylic acids have R , values 0.20 t o 0.30 unit lower than the parent polyol, but usually also show a strong lactone spot with an Rj value 0.20 unit higher than the parent polyol. .4mino sugars (the only one tested, glucosamine) have an R J about 0.25 unit Ion-er than the corresponding hydrovyl foim. LITERATURE CITED
Buuhanan. J. G . , Dekker, C . .I..Long, A . G . , J . Chem. Soc. 1950, 3182. Cifonelli, J. .%., Smith, F.,A x a ~CHEM. . 26, 1 1 3 2 (1954). Dalgliesh, C . E., .l-atiue 166, 1076 (1950). Feigl, F., “Chemistry of Specific, Selective, and Sensitive Ileactions,” Academic Press, New York, 1949. Gordon, H. T..Hewel. C . .i..d s a ~CHEY. . 2 7 , 1 4 7 1 (1955). Isherwood, F. A., Brit. M e d . Bull. 10, 2 0 2 (1954). Isherwood, F. A . , Jermyn. 11..I., Biochem. J . 48, 515 (1951). Lederer, E., Lederer. 11.,“(‘hromatography, a Review of Prillciples and .Ipplications.” Elsevier, S e w York, 1953. Partridge, S. 11., Binchein. SOC.Symposia (Cambridge, E n o l . ) , S o . 3, p. 5 2 (1950). R E C L ILD \ for reyiew J u n e 1 4 , 1955
Accepted Deceinber 28, 1955.
Determination of Pipecolic Acid in Biological Materials 0 . 0.SILBERSTEIN’, R. M. ADJARIAN, and J. F. THOMPSON
u. S. Plant,
soil, end Nutrition ~!eborstory,Agricultural Research Service, khaca,
Because pipecolic acid has videspread occurrence in plants, a quantitative method for its determination in biological materials is desirable. A sensitive quantitative method utilizes the color formed between pipecolic acid and ninhydrin under acid conditions. The reaction is subject to interference by salts and alpha amino acids. These interfering materials are separated from pipecolic acid by chromatography on Dower 5O-Xl2 in the sodium form. The method has been applied successfidly to urine and to plant extracts.
P
IPEXOLIC acid has been isolated from and identified in a number of plants (3-5, 7 , 1 7 , 1 8 ) . I n both plants and animals ( 2 , 8 , 9 )pipecolic acid is formed from lysine. I n order t o study the possible utilization of pipecolic acid bv animals, a method for its determination in animal tissues, feres, and urine was developed and has also been applied t o plant tissues. Since the inception of this work, Schxeet ( I f ) has published a method that has been applied to the analysis of proteins, but in nonprotein fractions salts .and amino acids interfere. The method proposed here separates pipecolic acid from these interfering substances and is more sensitive than Schiveet’e. The first attempts t o measure pipecolic acid Tvere made bg the method for the quantitative determination of amino acids on paper ( I S , Id), b u t this reaction lacked sensitivity and reproducibility. The colorimetric method described here is based on the reaction of pipecolic acid in vitro with ninhydrin under acidic conditions. The pipecolic acid is first freed of interfering materials (amino acids and salts) by chromatography on ion exchange resins. This report is conveniently considered in two parts: the proPresent address, T h e Welch Grape Juice Co., W’estfield, N. Y.
N. Y.
duction of color between pipecolic acid and ninhydrin and the separation of pipecolic acid from interfering materials. PRODUCTIOY OF COLOR BETWEEh PIPECOLIC ACID ANI) NIYHYDRIV
Reagents. Phosphoric acid, 1 N . Distilled 1-butanol. Ethyl acetate; practical grade is satistactory. Sinhydrin solution, 4% (weight/volunie) solution of ninhydrin in a mixture of 9 parts of 1-butanol and 1 part of 1M phosphoric acid. This is prepared fresh just before use (Table I). Procedure. A sample containing 1 to 20 y of pipecolic acid is evaporated to dryness in a test tube (12 X 175 mm.) in flowing air a t room temperature. One milliliter of ninhydrin solution is added and the tube is capped and shaken. After being heated in boiling water for exactly 15 minutes, the tube is cooled rapidly and the solution is diluted to a convenient volume (5 to 10 ml.) with ethyl acetate. During and after heating, the tube must be kept out of intense light because light causes fading of the color. T h e color is measured a t 575 mp (Figure 1) within 1 hour. The quantity of pipecolic acid is obtained from a standard ciirve made from the pure acid treated in the same way. If proline is also present, the color at 510 mp (Figure 1) must also be measured, T h e quantities of proline and pipecolic acid can be calculated from the absorption coefficients of these tlvo acids a t the two wave lengths. D578 = APi BPr. Djl0 = CPi DPr. DST8 Dslo = density measurements a t 575 and 510 n u . respectively. Pi and P r are the micrograms of pipecolic and proline. -4 = density from 1 y of pipecolic acid at 575 mp. B = density from 1 y of proline at 575 mp. C = density from 1 y of pipecolic acid at 510 mp. D = density from 1 y of proline a t 510 mp. The blank color is negligible. -4ccurate determinations of pipecolic acid up to 100 y can be made by further dilution with ethyl acetate, but larger quantities produce an insoluble precipitate.
+
+ +
856
ANALYTICAL CHEMISTRY 7 ,600
Table I. Factors Affecting Amount of Color Produced in Reaction of Pipecolic Acid (10 7)with Ninhydrin (Conditions used are those rwommcnded, except as specifically stated) Relative Amount of Color Treatment Recommended procedure (see text) 1 00 Effect of water concentration on color development Complete absence of water-sample dry and ninhydrin solution prepared by adding concentrated phosphoric 0 90 acid directly to butanol Additional water-sample dissolved in 0.1 ml. of water 0 42 Drying procedure 0 85 Sample dried in oven at 105' C . Effect of various factors on efficacy of ninhydrin solution I N sulfuric acid used in ninhydrin solution instead of 1M phosphoric acid 0 3B 2M hydrochloric acid used in ninhydrin solution instead 0 21 of 1M phosphoric acid 6.M phosphoric acid used in ninhydrin solution instead 0 57 of 1 X phosphoric acid 0.1M phosphoric acid used in ninhydrin solution instead 0 98 of 1M phosphoric acid 0 98 2 ml. of ninhydrin solution used instead of 1 ml. 0 99 Ninhydrin concentration 8% instead of 4% 0 66 Three-day-old ninhydrin solution Effect of different substances 0 82 1 mg. of sodium chloride added to sample 0 95 0.1 mg. of sodium chloride added to sample 0 so 100 y of phenylalanine or leucine added to sample Ammonium phosphate equivalent in nitrogen content to 0 93 100 y of leucine added to sample 1 0') 100 y of proline added t o sample 1 01 100 y of hydroxyproline added to sample Color produced by other procedures Pipecolic acid reaction run by proline method of Chinard (1)
Pipecolic acid reaction run according to procedure of Schweet (11)
WLl
L coo
/
aw
am
480 W A Y 1 LIWOTW
BOO
so0
080
. YILLIYIOROWI
860
Figure 1. Spectral curves of compounds produced by reaction of ninhydrin with pipecolic acid and proline T o t a l v o l u m e of solution, 10 ml. 500
r
,450
,400
,350
.300 W
,250 4
m
0 11
a
'io
m
0
400
p
.zoo
4
. I50 DISCUSSION ,100
The described method is sensitive to water concentration (Table I ) , to drying procedure, t o the type of acid used, and to the age of the ninhydrin solution. It is relatively insensitive to volume and concentration of the ninhydrin solution. At the reaction temperature specified (100" C.), 15 minutes' heating time gives maximum color (Figure 2). Of a number of solvents tried (Table 11),ethyl acetate proved the most satisfactory both as to intensity and stability of color. Both salts and amino acids interfere with color production (Table I). However, the interference by amino acids (Table I ) is not entirely due to the ammonium salt produced in the ninhydrin reaction (IO). The error caused by proline (Table I ) can be corrected for as shown above. Hydroxyproline does not yield enough color t o interfere (Table I). Other methods for proline and pipecolic acid are less sensitive (Table I). SEPARATION OF PIPECOLIC ACID FROM IKTERFERING MATERIALS
I n order to make the above method applicable to samples of biological origin, i t is necessary to separate pipecolic acid from interfering substances such as amino acids and salts (Table I). Rothstein and Uiller (8, 9) used consecutive columns of ion exchange resins (IR-4 and IRC-50) followed by the formation of copper salts t o isolate pipecolic acid from rat urine. I n this case the quantities of pipecolic acid were considerable and their methods are not applicable to most biological materials. One-directional chromatography was unsuccessful because interfering substances were not removed satisfactorily. It was learned ( l a ) that in subjecting amino acids and salts t o the process of ion exclusion (16) by chromatographing them on an ion exchange resin, pipecolic acid is separated from all @-aminoacids
.050
0 0
IS S IO HEATING T I M E
-
20
25
30
MINUTES
Figure 2. Effect of time on development of color between pipecolic acid and ninhydrin
Table 11. Effect of Diluent on Intensity and Stability of Color Developed between Pipecolic Acid and Ninhydrin Diluent
Relatire Density a s
% of Standard
Ethyl formate Ethyl acetate Butanol-1M phosphoric acid, 9 t o 1 95% ethyl alcohol 9: 1 ethyl alcohol-acetic acid 1: 1 ethyl alcohol-acetic acid 1 : 1: 1 ethyl alcohol-acetic acid-water (3') Methanol Isoamyl alcohol 90y0 acetic acid
.
100.0 100.0 80.0 91.0 85.0 79.5 78.2 83.2
85.5 79.4
Relative Stability a s % of Initial Color after 1 Hour 100.0 100.0 47.7 92.7 90.2 88.9 90.9 95.0 92.1 95,7
and saltE (Figure 3). This separation is not accomplished in the presence of sugars and other nonionic materials, and these must be eliminated by first retaining ionic materials on ion exchange resins. Heavy metals which intefere with the ion-exclusion process are also removed in this step.
Preparation of Columns and Ion Exchange Resins. COLUMNS. Glass columns are required for two purposes-the separation of sugars from amino acids and the separation of pipecolic acid from the alpha amino acids and salts. Only in the latter case are the
V O L U M E 28, NO. 5, M A Y 1 9 5 6
857
dimensions of the column important. Glass tubes (0.9 cm. in inside diameter and 50 em. long) are prepared with an opening of 3 to 4 mm. at one end. RESIXSFOR SEPARATIOV OF SCGARS FROM AMINOACIDS. Ion exchange resin Dowex I-X2 (200-400 mesh) (Dow Chemical Co., Midland, RIich.) is prepared by treatment with excess carbonate-free 3iv sodium hydroxide overnight and the base is removed by washing with pure n-ater. ACID FROM SALTS AND RESINFOR SEPARATIONOF PIPECOLIC ~ ~ - A J I I SACIDS. O The preparation of this resin is the most critical part of the whole method. Resin Dowex 50-X12 is treated with excess 3AVhydrochloric acid on a steam bath for 24 hours. The excess hydrochloric acid is removed and the resin is neutralized with excess 3N sodium hydroxide on a steam bath for 24 hours. T h e excess base is removed by washing the resin with deionized mater until the p H of the wash water reaches 10 f 0.2. (Deionized water is prepared by passing distilled water through a mixture of strongly acidic and strongly basic ion exchange resin.) This resin is now in the sodium form.
S
10
IS
VOLUME OF EFFLUENT
Figure 3 .
-
20 ML.
2s
Pattern of elution of various substances
- -
From column (0.9 om. in diameter and 30 c m . in length) of Dowex 50 12% divinylbenzene in sodium form (200 400 mesh)
Table 111. Recovery of Pipecolic Acid Added to Rabbit Urine and Turnip Leaf Extracts Pipecolic Acid Recovery, AIaterial Formed % R a b b i t urine, 5 nil. 3 . 7 7 + 0.10" 0 , 8 0 0 101 2 ' ' 7 . 4 a Rabbit urine, 5 ml. 10 y of pipecolic acid 1 3 . 9 0.62 & 0.0 T u r n i p leaf extract Turnip leaf extract 10 y of pipecolic acid 10.73 =t1 , 0 2 & 101 2 ' 1 0 . 2 a a Standard deviation on three determinations.
+ +
+
PROCEDURE
Extraction of Samples. Fresh samples are ground with sufficient 95y0 alcohol t o make the resultant alcohol concentration not less than i570. The liquid is separated from the residue and the extraction is repeated n i t h 75% alcohol as many times as is necessary to extract all amino acids. Dry samples are treated similarly, using 75y0 alcohol from the start. The insoluble material is discarded, because pipecolic acid does not occur in this fraction (11). Samples containing carbonates should be acidified before application to resin columns, because carbon diovide released in the resin disrupts the column. Separation of Nonionic from Ionic Substances. Sample extracts are passed through a column of ion exchange resin, Don ex I, making sure that all amino acids are retained on the resins by testing for ninhydrin activity (6,15) in the effluent. The resins are washed thoroughly ivith deionized n ater until tests for sugar are negative and the washings are discarded. The amino acids are eluted from the resins with 1 S hydrochloric acid until the effluent is acid. The eluate is dried a t room temperature and dissolved in water, the p H is adjusted to between 9.5 and 10.5, and the solution is made t o a volume such that 1 ml. contains 1 to 20 y of pipecolic acid. Use of Dowex 50 for this purpose may result in difficulties with samples containing large amounts of urea, since this is retained on Dowex 50 and chromatographs with pipecolic acid (Figure 3). Chromatographic Separation of Pipecolic Acid from or-Amino Acids and Salts. B column of resin Dowex 50 -X12 in the sodium form is prepared by pouring a thick slurry of this resin into a glass column (0.9 em. in internal diameter) using a filter paper disk a t the bottom t o retain the resin. Sufficient resins should be poured in so that the height of the settled resin is 30 em. or more. Any resin above 30 cm. in height is removed.
A 1-ml. aliquot of extract from which nonionic materials have been removed is placed carefully on the top of the resin column and alloned to pass into the resins. The sides of the column are nashed down with two successive 1-ml. portions of pure water, each time allowing the water t o pass into the resin. Thirteen milliliters of pure water are placed on top of the resin and allowed t o move into the resin. The corresponding effluent from the column contains the or-amino acids and salts and is removed. The next portion is collected in a test tube after 10 ml. of pure water are added to the top of the resin. This fraction is dried a t room temperature and analyzed for pipecolic acid by the method given above. DISCUSSIOK
Separation of Nonionic from Ionic Materials. Konionic materials interfere n i t h the separation of pipecolic acid from the 01amino acids and salts on the column of sodium resin and must therefore be separated from the amino acids and salts with ion exchange resins. The procedure also serves as a convenient method of concentrating an extract. The amount of resin used is not critical, as long as all amino acids are retained on the resin. Separation of Pipecolic Acid from Other Materials. The preparation of the Dowex 50 -X12 in the sodium form is the most probable source of error in the whole procedure. If the acidic groups on the resin are not completely neutralized, pipecolic acid \$ill be retained by the resin. Because of the high cross linkage of this resin, the penetration and consequent neutralization by sodium hydroxide are slow and require heat for a relatively long time. Excessive n-ashing of the resin may result in subsequent retention of amino acids. Sample solutions of lorn p H nil1 act in the same manner. Insufficient removal of excess base from resin may result in incomplete separation of pipecolic acid. The samples free of nonionic substances should be applied in a volume no greater than 1 ml. in order t o ootain a sharp separation of amino acids in the effluent. The Dowex 50 -X12 column can be used only once, because impurities in the sample change the adsorption characteristics of the resin. One such test resulted in the loss of 44yo of added pipecolic acid. Ornithine, lysine, and hydroayl? sine which inteifere in Chinard's method ( 1 ) and citrulline nhich interferes n i t h Schmet's method (11) are retained by this resin. The procedure presented here has not been found applicable to quantitative proline anal) sis Tests of Methods. The above procedures have been tested uith pure pipecolic acid, urine, and nonprotein extracts of turnip leaves. K i t h standard pipecolic acid (15 y ) duplicates agree within 1%. The recovery of pipecolic acid added to aliquots of rabbit urine and turnip leaf extracts are presented in Table 111. The replicate analyses of iinsupplemented samples are good and the recovery of added pipecolic acid is within 10% of that added, LITERATURE CITED
Chinard, F. P., J . B i d . Chem. 199, 91 (1952). Grobbelaar, N., Steward, F. C., J . A m . Chem. SOC.75, 4341 (1953).
Ibid., 76, 2912 (1954). Harris, F., Pollock, J. R. A , J . Inst. Brewing 59, 29 (1953). Hulme, A. C., Arthinaton, W.. Nature 170. 659 (1952). Moore, S., Stein, W. H . , J . B i d . Chem. 176; 367 (1948j. Morrison, R. I., Biochem. J . 5 3 , 474 (1953). Rothstein, AI., Miller, L., J . Am. Chem. SOC.75, 4371 (1953). Ibid., 76, 1459 (1954). Schlenker, F. S., r l s a ~CHEM. . 19, 471 (1947). Schn-eet, R. S., J . Dzol. Chem. 208, 603 (1954). Thompson, J. F., AIoi-i-is, C. J., unpublished data. Thompson, J. F., Steward, F. C., Plant Physiol. 26, 421 (1951). Thompson, J. F., Zacharius, R. AI., Steward, F. C., Ibid., 26, 373 (1951).
Troll, W., Cannan. R. K., J . B i d . Chem. 200, 803 (1953). Wheaton, R. If.,Bauman, W. C., I n d . Eng. Chem. 4 5 , 2 8 8 (1953). Zacharius, R. AI., Thompson, J. F., Steward, F. C., J.Am. Chem.
SOC. 7 4 , 2 9 4 9 (1952). Ibid., 7 6 , 2 9 0 8 (1954). RECEIVED
for review July 23, 1955.
Accepted February 3 , 1956.
