~n:ixiniuiii absorption :lt 327 m t i 292 mp a t coiiceiitrations of 1 x and 1 x 10-3 mg. per cubic centiineter. llolur :ibsorptivitiw were 2600 and 14800 for solvent benzene, and 3740 and 23200 for solvent hexane. 13eer's la^ \vas obeyed froin couceiitrations of 1 X to 1 x mg. per cubic. centimeter, :it 4170 ,1.(small deviations 1%ere found on studies ronclucted at dl50 .I.). Conce~itr;itieiiisgreater th:m 1 X mg. lwr cubic centimeter deviate :is rvould be expected, but c m be determined by progressive dilution. These can be further ch:irnctcrized by gently heating the coniplex n-hich beconics bright green. Extreme care niust be taken tc avoid degradation. Also, decomposition of the complex ~vit'lisubsequent formation of 20-methylcho1:mthreiie (ni.11, 178" t,o 180" C.) can be effected bj. dropwise addition of water a t approximately 10" C. The results of bests on several cnrciiiogens and steroids (Table 1) indicate, in general, different chromogenic reactions than that of 20-methylcholanthrene. As shown in Table 11, all animals given 20-methylcholanthrerie reacted positively to the sulfuric acid test. All controls were negative. All gave spectrophotoniet'ric curves identical with those of the benzene extracts of solid 20-niethylcliolantlirene, and there n a s no uppreciahle background. This would indicate that no substance in normal body tissue interferes with the test, possibly because the substances were destroj-ed by acid, or inappreciable quantities were extracted. The above evidence indicates the feasibility of the sulfuric acid test for the of 20-niet~hylcholandetermination threne.
Table II.
Mouse
So.
1 2
Results of Test for Determination of Methylcholanthrene in Normal Body Tissue Reaction with Concentrated Sulfuric Acidc Observation Period Type* Lung Liver Brain Kidney 1 day E 1 daya E 1 daya E; 1 daya E; 1 daya E; 2 daysa E; 2 days E t 2 days E 2 days E 2 days E 1 day c 2 days c
++ ++
++ ++ ++ ++ ++ +
3
4 5
6
7 8 9
+ ++ +
++ ++ ++ ++ ++
10 11 to 16 10: to 20 Those wses determined spectrophoto~~ietrically.rill gave characteristic niet,hylcholanthrenc~~~sulfuric acid complex curve. Observation period was period intervening between time of injection and time of test. E = experimental mice (those given hICA injections), C = control mice. c = Deep orange color, = orange color, - = negative test.
++
+
ACKNOWLEDGMENT
The author is indebted to Lloj-d Old, Donald Clarke, and David Fukushinia of the Sloan-Kettering Institute, David Kilsoii and Robert dutrey of the University of Rochester chemistry department, and David Lavie of the Keitzniann Institute for all their invaluable advice. LITERATURE CITED
(1) Bandow, F., Biochem. Z . 301, 37
(1939).
( 2 ) Buu-Hoi, S.,Jacquignon, P., Cottzpt. rend. 234, 1056 (193%).
1 3 ) Doniach. 0.. Brit. J . Exvtl. Pathol. 23, 227 (1939). ( 4 ) Fieser, L., Campbell, IT. P., J . .Im. Chenz. Soc. 60, 1142 (1938).
(5) Goldzieher, J. IT., Bodenchuk, J. AI., Solan P., J . Bid. Chetti. 199,621 (1952).
Jones. S . . Cancer ReseaTch 2 , 237 (1942). ( T ) Kuschner, AI., Laskin, 6 . Cristofano,
16)
E,, Selson, S . , Proc. 3rd .Tatl. C'mcer Conj. 3 , 485 (1957). (8) Lakowski, 11. E., Barber, L). G., Arccrone, IT. C., ANAL. CHEXI.2 5 ,
1400 (1953).
( 9 ) Lisle, E. B., J . Chetn. Soc. (1942) 548. (10) Pietzech, .1., Pharniazze 1 2 , 2 4 (1957). ( 1 1j Yan icki, E., Elbert, IT., Stanley,
1. ]I7,. Hauser. T. R., Fox, F. T.,
(12) Umber&r. E.' J., Curtis, J )I., J . Bzol. C h e w 178, 265 (1949). (13) JTaivzonek, L., J . Am. Chenk. Soc. 64, 2366 (1942). (14) Zaflaroni, A,, Ibid., 72, 3828 (1950). S o R R l A S ~I\I\IER?I.kN
Ilepartnient of Chemistry Cniversity of Rochester Rochester 20, 5 . Y-. RECEIVEDfor review July 23, 1961. Resubmitted Koveniber 20, 1961. ACcepted March 12, 1062.
Desalting of Amino Acid Solutions by an Ion Retardation Resin SIR: Desalting of proteins, polyptytides, sinino acids, and similar materials is often a necessary analytical step. Dialysis, used in desalting proteins, is not suitable for desalting amino acid,? and polyprptides. Ot'her methods involving ion exchange techniques linvc h e n U S C ~TI-ith varying success. C'ohn and 13ollnm ( 1 ) eliminated untlcsirnhle anions (chloride, formate, etc.) froni nnclrotidc~ by sorption of the mixture o n Daws 1 and chromatogriiphic elution \vith SE-14HC03. Ion cschaiigc resins ( 2 ) were uscd to remove sotliuni citrate nntl p1ioq)linte from the effluent'of iori-esch:ingc chromatograms. The ncutr:~land aridic nmino acids were absor1)ed on Don-cx 2 :lnd eluted with
XcOH, and the basic amino acids and t,ryptophaiie were absorbed on Dowex 50 and eluted n-it'h HC1 t o yield salt free solut,ioiis. A cation cschange resin (3) has b c m iiscd to exchange atniiioniuiii ions for cations. T h e salts were then rcinovctl 1)y evaporation. Anion (xschangcrs (4) in t,lie OH- form hare h i w i used t o ahsorh amino acids nhile passing cations. The amino acids \yere eluted with 1 S HC1, and the chloride was rem o w d with the bicnrboiiatc, form of the resin. Sr1)hader. a cross-linkctl polysncclinritle. lins hcrn u s r d for t h r partial tlrs:iltina of high n i o l c ~ ~ u l niwiglit r lirati& (.ij.
.Ir;l!l:,!
l,Ilc,-.tl
usin% ;?(; 1 1 .'h
1'his is a nen. type of ion ri.t:irtlutioa rmin suppiicd by 13io-Rad L:il)oratories, Ilichixond, Calif., from m:itcri:il nxinufaeturetl hy the Dow Chcniicd Co. This resin, being more ncarly iieutr:il, h u l d not cause the intcrfcring cheliiic:il changes in proteins :iiid other matclrial that :ire caused by ronvc~nt~ional
c d bp poly I iicrixing a n acrylic (n-enkly acidic cation cschanger) insidc Doivt:s 1, thereby !.ielding both cation and anion esciinngc sites. l'hcsc sites are in intimatt, contact and tvntl to 1iriitr:ilizc~enc*li other; lion-ever, thr sitw still I1:ive a n attraction for J l i i l f > :illions :iud cations :ind ran 1, l ~ ~ l ' t l l ( oi' ' ~ ltll > < ~ ~ t i l l g 11.1- I ~ ~ i i~i 1 . t~ i i .oc,i:itci \\.itli tliem to sonie extent. VOI. 3 4 , NO. 6 , M A Y 1 9 6 2
71 1
The result is that the resin will absorb both anions and cations from solutions with which i t comes in contact. It is interesting to note that the resin must absorb equivalent amounts of both anions and cations simultaneously. The absorbed ions, except for hydrogen ions, are held so weakly, however, that they can be eluted from the resin with water, returning the resin to the selfneutralized form. AG l l A 8 shows a high preference for retarding chloride and its accompanying cation and does not retard amino acids, peptides, and proteins. AG l l A 8 is suitable for removing NH4C1 as well as XaC1, but is not suitable for removing citrates and acetates. Hydrochloric acid is absorbed irreversibly by the resin and cannot be eluted with water. After the resin has absorbed hydrochloric acid i t can be regenerated with dilute sodium hydroxide followed by a water rinse to remove the resulting sodium chloride.
- 1%
w
ALANINE
u
50.8-
m
$0.6
Ai
-
20.40.2 0
IO 20 30 40 50 60 70 80 90
EFFLUENT Figure 2.
with water from a separatory funnel connected to the top of the column. The effluent was tested qualitatively for amino acids with ninhydrin and for chloride with silver nitrate. Method of Test. Amino Acid. 1 ml. of effluent with 0.5 ml. of 0.1% ninhydrin was heated i n a boiling water b a t h for 15 minutes, diluted to 10 ml. and absorbance measured a t 570 m!J. Chloride. 1 ml. of effluent with 1 ml. of 0.llM sodium chromate was titrated in a porcelain crucible with 0.1M silver nitrate.
Thirty grams of AG l l A 8 ion retardation resin, 50 to 100 mesh, self-neutralized form as supplied b y Bio-Rad Laboratories, was slurried in water and poured into an ion exchange column 1.14 x 41 cm. The column length was doubled by placing two such columns in series. A partially hydrolyzed gelatin solution was prepared by boiling for two hours 10 grams of gelatin, 50 grams of KaC1, and 10 ml. of concentrated HCI in 1 liter of water. This solution was neutralized with YaOH before separation experiments. Water was drained from the column until the level reached the resin surface. Five or 10 ml. of the solution to be separated was then carefully pipetted onto the resin surface and a t the same time the effluent was collected. 1 Small portions of water were added and allowed to drain to the resin level, then elution was continued
RESULTS
ITe Fvere able to separate alanine and monosodium glutamate, as well as gelatin and gelatin hydrolyzate, from the bulk of the added sodium chloride. Results are shown in Figures 1 and 2. The resin shows high efficiency in separation of salt from amino acids even a t high salt concentration. S o t e that in the separation of sodium chloride from sodium glutamate the sodium glutamate is not rendered sodium free, but only the added sodium chloride is removed.
t
590 (1961).
E00.4 2 -
( 2 ) Drkze, A,, Moore, S., Bigwood, E., Anal. Chim. Acta 11, 554 (1954).
v)
,!,L, IO
20
I 30
,
,
,
40 50 60
EFFLUENT
, 70
ML.
80 90 100
Figure 1 . Gelatin hydrolyzate desalted with A G 11A8 a t near optimum conditions of low volume of load and low flow rate: sodium chloride separated with complete recovery of amino acids Load. 5 ml. (1 2% of bed volume) of 1 % gelatin hydrolyzate plus 5% NaCl
712
Flow Rate. As the flow rate increases, the peak of t h e salt moves closer t o t h e peak of t h e amino acid, thus cutting down the amount of completely desalted amino acid. F l o ~ rate seemed t o be the most important single factor affecting desalting with most complete desalting a t low flom rates (below 1 ml. em.-* minutes-’). Loading. T h e volume per cent of the material in relation to t h e resin bed volume determines the column load. Loads of 127, and 25% b y volume were studied. Increased load widens the amino acid band to the extent of the additional volume added and t h u s extends the amino acid band into t h e salt band. T h e total amount of materials to be separated can be increased b y increasing the concentration in the load or by increasing the volume of the resin bed so that the load is less than 23”7 by volunic of the resin. Column Height. Good separations were effected with a 41-em. and 82-cm. column a t 127, loading and 1- to 2-ml. cm.-* minutes-’ flow rate. There was little difference between the two column heights. A 10-cm. column a t the same percentage loading gave nearly complete desalting, but the flow rate had to be reduced to 0.2 ml. minutes-’. AG l l A 8 should find wide use in removing HC1 and desalting amino acids (1) Cohn, TTaldo E., Bollum, F. J., Biochim. et Biophys. Acta. 48, 588-
-
0
alanine with 1% NaCl
LITERATURE CITED
~ 0 .- 8 0
20.2-
Effect of change in salt concentration
Load. 5 ml. (12% of bed volume) of 1% and 1 % alanine with 12% NaCl
EXPERIMENTAL
50.6
ML.
ANALYTICAL CHEMISTRY
(3) Hirs, C. H. W., Moore, S., Stein, W. H., J . Bid. Chem. 219, 623 (1956). (4)Piez, K. A,, Tooper, E. B., Fosdick, L. S., Ibid., 194,669 (1952). (5) Porath, J., Flodin, P., -Tuture 183, 1657-59 (1959). CHARLES ROLLISS LARRYJEXSES ALICE ?rT. SCHWARTZ Bio-Rad Laboratories 32nd St. and Griffin hve. Richmond, Calif.
