Table V. Recovery of Steroid Fraction from Total Lipide Tissue Extracts"
Steroid fraction Weight, (CH :Ac : : 1:I), Wt., mg. mg. %
Lipide extract Ovary 180.3 4.3 2.4 Ovaryb 120.4 S.7 7.2 501.9 7.8 Placentac 6 , 4 2 8 . 4 Extracted by Goldzieher's method (3) using methylal-methanol mixture. * Contained large amount of nicotinamide. c Remaining extract after 80% ethanolpetroleum ether solvent pmtition so that much of the lipide has alrwdy been removed. The 7.8% therefore represents a further purification by means of the column. (1
about 9% tailed, mas run almost 1 year after the chromatogram which used pentane-ether as the fatty lipide eluent (in which about 5% was found in the second peak). Table V summarizes experiments in which gravimetic recovery of the steroid fraction from actual total lipide extracts was eiamined. Even in the presence of a large amount of a nonlipide contaminant-Le., nicotinamide-which is partially soluble in all solvent phases used, the resolution of the system is unaffected and more than 9057, of the total lipide weight is removed. A placental total lipide extract, purified by a commorily used extraction method ipetrolcum ether-80%
ethanol partition followed b y re-extraction of the 80% ethanol residue with ethyl acetate), was further purified b y means of the silicic acid column, resulting in less than 10% b y weight of the purified placental extract remaining in the final steroid fraction. -4lthough a great deal of data has been presented on model experiments rather than on actual extracts, the composition of a n y lipide mixture carries with i t phenomena of displacement and common solubility effects which distort any chromatographic separation. Therefore, particular attention was paid to the use of eluents which clearly defined the lipide classese.g., Table I, where repeated elutions still do not elute the lipides of the next class. The work was done to bring the utmost precision t o the separation of model systems and then to prove out experiments with recovery of steroids both in large and small quantities from lipide mixtures and actual tissue extracts. DISCUSSION AND CONCLUSIONS
Silicic acid column chromatography provides a relatively simple and rapid separation of the total steroid fraction from the major lipide contaminants. The collection of a specified volume of benzene or pentane-ether (150 ml.) removes the fatty lipides; and another definitive volume of acetone-chloroform (150 nil.) elutes the total steroid fraction, leaving little room for error
due to cutting of many fractions and changes in solvent composition, and eliminating entirely expensive or cumbersome fractionating equipment. The column can handle a wide range of mixtures of fatty lipides and steroids; and a proportionately larger column can be prepared for batch use. Although the standardization of silicic acid t o activity IIB requires some time of preparation, the material retains its activity (in a tightly stoppered bottle) for well over a year. The method has proved itself valuable in our laboratory for the purification of the steroids from the other lipides before separation of the individual steroids. LITERATURE CITED (1) Axelrod, L. R., J . Biol. Chem. 205, 173 (1953). (2) Axelrod, L. R., Pulliam, J. E., Arch. Bzochem. Bzophys. 89, 105 (1960).
( 3 ) Goldzieher, J. W., Baker, R . A., Riha, E.C . , J . Clin. Endocrznol. Metab. 21, 62 (1961). (4) Hernandez, R., Hernandez, R., Jr., Axelrod, L. R., ANAL.CHEM.33, 370 (1961). ( 5 ) Mattox, V. R., Mason, H. L., J . Biol. Chem. 223, 215 (1956). (6) Rousser, G., Marinetti, G., Berry, J. F., Federation Proc. 13, 286 (1954).
RECEIVEDfor review July 13, 1962. Accepted Sovember 14, 1962. Work supported by Grant No. A-3270 from the Sational Institute of Arthritis and Metabolic Diseases of the Xational Institutes of Health, Public Health Service, Bethesda, Md. This research constitutes the senior thesis of Raymond Hernandez, Jr., in partial fulfillment of the M.D. degree a t Tulane Medical School, Sew Orleans, La.
Automatic Column Chromatography of Ether and W a te r-Soluble 2,4- Dinitro phenyI-Derivatives of Amino Acids, Peptides, and Amines LEO KESNER, EDWARD MUNTWYLER, GRACE E. GRIFFIN, and JOAN ABRAMS Department of Biochemistry, State University of New York, Downstate Medical Center, Brooklyn, N. Y.
b The separation of over 35 derivatives of 2,4-dinitrofluorobenzene is accomplished with one column in 22 hours. A gradient elution system, composed of n-heptane, t-amyl alcohol, and methyl ethyl ketone, is deaerated, water-saturated, and pumped through a hydrated silica gel column. The effluent is passed through a flow cell from which the absorbance is continuously recorded. An identifiable peak is obtained with as little as 0.01 m o l e of DNP-amino acid. The technique is adaptable to the fractionation of mixtures of DNP-peptides; the constituent amino acids of an acid hydroly-
zate of a purified DNP-peptide can b e determined in a 5-hour analysis. Resolution is sensitive to the hydration of the silica gel and to the column temperature; advantage can be taken of changes in both of these variables for the separation of certain DNPderivatives.
s
ITS INTRODUCTION in 1945 b y Sanger (16), l-fluoro-2,4-dinitrobenzene has frequently been employed as an analytical reagent in studies involving proteins, peptides, amino acids, and amines. Numerous INCE
techniques for identifying the 2,4dinitrophenyl (DKP)-derivatives have been reported (I), including paper chromatography, column chromatography, paper electrophoresis, and countercurrent distribution. Column chromatography of the DNP-derivatives employing a variety of supporting materials, such as silica gel (1, 1 4 ) , Kieselguhr (IO), Celite (9, 11, I d ) , silicic acid-Celite (3, 16), ion exchange resin ( I S ) , and nylon powder (4, 20) has met with variable success. Xttempts t o use hydrated silica gel columns have led to a number of problems attributable to such factors as VOL. 35, NO. 1 , JANUARY 1963
83
variations in the properties of different silica gel preparations, frequent cracking of columns, the requirement of multiple short columns utilizing a variety of developing solvents, and the necessity of running, simultaneously, reference standards on equivalent columns for identification purposes. I n attempting to apply hydrated silica gel columns for the fractionation of organic acids and DXP-derivatives, the present authors (8) noted that the cracking and channeling of the columns could be eliminated if the eluting solvent were properly deaerated. As a result i t became possible to use hydrated silica gel columns of 100-cni. length, which were readily reproducible and free from disruption; this result led t o the development of the method herein described. EXPERIMENTAL
Reagents. 2,4-Dinitrofluorobenzene ( D X F B ) , 1.5% or 0.125M. Dilute 0.15 ml. of 2,4-dinitrofluorobenzene (Pierce Chemical Co., Box 117, Rockford, Ill.), with density of 1.537 grams per ml., to 10 ml. with absolute ethyl alcohol. This solution is prepared just before use. Silica Gel [Mallinckrodt, Acid Silicic A.R., 100-mesh (powd.). Suitable for chromatographic analysis by the method of Ramsey and Patterson]. The silica gel is transferred to a crystallizing dish and dried to constant weight a t 105" C. It is then transferred to an ovendried jar and securely closed with a desiccator-dried, plastic-lined screwcap. The jar is placed in a desiccator for cooling and storage. %-Heptane (H). n-Heptane (pure grade normal heptane, Special Products Sales, Phillips Petroleum Co., Bartlesville, Okla.) is redistilled in an all glass apparatus. ilcid-Washed Heptane. About 25 nil. 0.1N HzS04is Doured into a glassstoppered, 5001ml.Aseparatory finnel which has a Teflon plug or a Tefloncoated glass plug. ilpproximately 475 ml. of the redistilled n-heptane is added. It is inixed well and the aqueous and organic phases are allowed to separate. The organic phase is filtered through Whatman No. 40 filter paper and stored in a loosely stoppered glass bottle. Tertiary Amy1 Alcohol (t-AA) . Tertiary amyl alcohol [Practical grade tertamyl alcohol (or tcrt-pentyl alcohol), 100" to 103" C., Distillation Products Industries, Division of Eastmaii Kodak Co., Rochester 3, K,Y.] is redistilled in a n all glass apparatus. The distillate is collected over a 1" c. temperature range and stored in a siliconerubber stoppered (silicone-rubber stopDers are obtained from the West Co., Phoenixville, Pa.) brown glass bottle. Methyl E t h r l Ketone (MEK) (or 2-B;tanone, "79" to80" C., Distillation Products Industries) is redistilled in an all glass apparatus, collecting the 80% between 5% and 85% of 84
ANALYTICAL CHEMISTRY
the total volume. Beyond 85% collection, the absorbance of the distillate increases a t the wavelength used in the method. The reagent is stored in a brown glass bottle, silicone-rubber stoppered or closed with a metal foillined plastic screw cap. Water-Saturated Methyl Ethyl Ketone. About 75 ml. of distilled a a t e r is poured into a glass stoppered, 500ml. separatory funnel fitted with a Teflon plug. Approximately 425 mi. of redistilled methyl ethyl ketone is added. It is mixed well and allowed to separate into two phases. The organic phase is filtered through TS'hatman S o . 40 filter paper and stored in a silicone-rubber stoppered brown glass bottle. Organic Solvent Mixture. Mixtures of tertiarv amyl alcohol in heptane 3%, 18% (v. lv., respectively) and methyl ethyl ketone in heptane 50% (v./v.) are stored in silicone-rubber stoppered brown glass bottles. Ether, Anhydrous, Peroxide-free. Ether (ether, anhydrous, Baker Analyzed Reagent) is freed of peroxides with acidified ferrous sulfate.
1-
1
I
"
80RE
Figure 1.
D.
E. F. G.
H. 1. J.
PROCEDURE
Dinitrophenylation and Extraction of DNP-Derivatives. T h e dinitrophenylation reaction is carried out, ill Parafilm-covered silicone-coated glassware, as described by Schroeder and LeGette (17). After evaporation of thc alcohol, the residue is dissolved in 5 ml. of water and the solution is brought to a p H of 8.0. The solution is extracted six times with 10 ml. of ether to remove the excess D N F B and dinitroaniline. The aqueous phase, after adjusting to pH 1 to 2, is brought to dryness and is ready for transfer to thc column. If the reaction mixture contains protein, the DXP-amino acids and D N P peptides are extracted following ether extraction, at an acid p H with matersaturated methyl ethyl ketone. For this purpose, the 5-ml. aqueous phase after ether extraction is adjusted to pH 1 to 2 with constant boiling HC1. and extracted 6 times with 10-ml. quan-
m *BORE................
I
A. B. C.
,411 other reagents not specifically mentioned above are of reagent quality.
Column pouring apparatus
Stirrer with rheostat Button tip glass stirring rod, 6-mm. 0.d. Stirring rod guide m a d e of Teflon tubing, 3/s-inch i.d., fitted into silicone rubber stopper Kelly infusion bottle, 700 mi., 1 2 - m m . outlet bore attached to ball part (Scientiflc Glass Apparatus Co., Inc., Bloomfield, N. J.) Teflon stirring blade cut from I/a.inch Teflon sheet Ball and socket joint 28/12 Teflon tubing tail 0.034-inch i.d., 0.058-inch 0.d. Jacketed chromatographic column, 1 0-mm. i.d., 1 2 0 0 - m m . length (Scientiflc Glass Apparatus Co., Inc., Bloomfield, N. J.) Sintered glass disk Teflon stcpcack, bored for connection to flow cell
AIR
VENT
r-7
THEPMOYErfP
Figure 2. Two-chamber gradient apparatus employing solvent deaeration, water saturation, and continuous spectrophotometric recording
tities of M ater-saturated methyl ethyl ketone. The combined extracts are transferred to a porcelain evaporating dish and dricd in a rapid flow of air in the hood. Because DNP-derivatives are somewhat sensitive t o light (1, 2 ) . they should be shielded as much as possible. Column Pouring. T h e elution pattern of t h e DNP-derivative. is sensitive to t h e hydration of t h e silica gel. Consequently, individual laboratories .hould establish t h e ideal degree of hydration for the eluting system t o be employed, the batch of silica gel used, and the specific DSP-derii atires to be fractionated. On the other hand, the sensitivity t o the hydration of the silica gel can be put to practical advantage under the circumstances where two DNP-derivatkes are eluted together by employing a silica gel of a given degree of hydration. I n this case, better resolution of the two DXP-derivatives can usually be accomplished by rechromatography employing a silica gel of a different hydration. However, with constant conditions, the elution patterns are consistent and reproducible. Silica gels acidified in the proportions of 3.5 ml. of 0.5-VH2Qg4 for each 8 grams of silica gel-i.e., a 3.5 to 8 silica gel--to 5.2 ml. of 0 5-1- H2S04 for each 8 grams of silica gel-i,?., a 5.2 to 8 silica gel-have been used to advantage. Hence, the requisite quantity of the oven-dried silica gel (in the neighborhood of 45 grams for a 100em. X 1-em. column) is quickly weighed into a 600-ml. beaker. The evact quantity of 0.5A- H2SO4 is added im-
mediately and mixed well with a heavy stirring rod until the mixture is a freeflowing powder. The mixture is then transferred to a n appropriate size dr wide-mouth bottle containing a hen\ glass rod and mixed for 15 minutps on a ball mill. As illustrated in Figure 1, the modified Kelly infusion bottle is clamped by means of a ball and socket clamp to a dry column so that the Teflon tubing tail, attached to the stirrer blade, extends into the column several inches bdow the ball joint. The column stopcock is closed and the extra hole in it is plugged with a short piece of Teflon tubing into which a fine glass rod has been inserted. The system is filled with acid-washed heptane until about 50 ml. are in the bottle. After removing the acidified silica gel from the ball mill, 0.3-gram and 0.5gram quantities are weighed into individual 5-ml. beakers, to be used subsequently in transferring the aliquot to the column. The two beakers are covered securely with Parafilm or aluminum foil. The remainder of the acidified silica gel is transferred to the modified Kelly infusion bottle. Acid-washed heptane is added to wash don-n the walls and to bring the total volume near the 600-ml. mark on the bottle. Approximately 400 ml. of the acid-wished heptane are needed to pour a column. The rubber stopper containing the Teflon tube guide for the stirrer is inserted into the mouth of the infusion bottle. The motor speed is adjusted during the pouring t o maintain a layer of elear heptane above the suspended silica gel. The stopcock a t the bottom
of the column is opened to allo\v rapid settling of the gel by gravity. The automatic pouring of the column requires about 30 minutes, and should be terminated when the silica gel has settled below the ball and socket joint. The column stopcock is then closed, and with the aid of Teflon tubing attached t o a suction flask, excess heptane is withdrawn from the infusion bottle until the level is down to the ball and socket joint. The column is now ready t o be clamped into position to permit the connections t o the circulating water pump to be attached (see Figure 2). Occasionally there is a tendency for the silica gel t o pack against the wall of the column in the area stirred by the Teflon tail. T o avoid clogging at this point during the packing under pressure, any packed silica gel is loosened by moving a narrow glass rod back and forth through this area several times. Column Packing. T h e column is packed b y paqsing deaerated a n d acidwashed heptane through i t under pressure. For this purpose t h e latter part of the solvent system for column operation is used (see Figure 2). Thus, the heptane from reservoir D, heated t o 56" C. in mixing chamber 1 ( M C I ) , is passed through the acid-wash column and delivered directly t o the silica column. I n general, the same procedure is used when employing the Autograd (Technicon Corp.) to establish the gradient elution system. The socket area at the top of the column is freed from any particles of silica gel by filling it with warm, acidwashed heptane and stirring with a glass rod to allow any particles t o fall into the column proper. Heptane is inch below the siphoned off to about socket and the socket area is dried with a piece of filter paper. To prevent a leak a t the ball and socket joint, a gasket (18- x 21-mm. cut with cork borers from a 1/64-inch sheet of AID50 silicone rubber, manufactured b y RWay Synthetic Products, Div. Ronthor Reiss Corp., Little Falls, S . J.) is carefully centered in the socket area. It is important to avoid contact of the gasket with the heptane, which causes a rapid, marked swelling of the silicone rubber. The ball connection from the acid-washed heptane solvent system is carefully placed in the socket and the joint is secured with a ball and socket clamp (Size 28, Catalog KO. C5155, Scientific Glass Apparatus Co., Bloomfield, N. J.), The circulating constant temperature bath, set a t 35" C., is turned on to circulate water through the column jacket and mixing chamber 2 (;1ICJ. The plug a t the end of the column stopcock is rcplaced n i t h a Teflon tubing connection to the continual flow cell in the photometer and the stopcock to the flow cell is opened. The solvent pump is turned on and the necessary adjustments are made to give a flow rate of acid-washed heptane through the column of about 180 nil. per hour. A Milton Roy Chromatographic LIinipump C H M h I 1 - B 3 (Milton Roy Co., 1300 East Mermaid Lane. Philadelphia 18, Pa.) is convenient for propelling the solvent. The VOL. 35, NO. 1, JANUARY 1963
85
pump is connected t o the column ball joint b y Swagelok fittings (Crawford Fitting Co., Cleveland 10, Ohio) and '/&nch Teflon tubing. All other Teflon t o glass connections may be made b y use of l/le-inch wall Teflon tubing as previously described (8). The passage of 45 to 60 ml. of solvent in 15 to 20 minutes through the column should pack the silica gel from a n approximately 120- to a 100-cm. height. Occasionally there is a tendency for the silica gel to clog in the column during packing. The dislodging of the silica gel can be accomplished b y applying t o the column, or jacket, a cushioned vibrator. (Home Vibrator, Model E, Wall Clipper Corp., Sterling, Ill., can be readily adapted to this purpose.) When the packing of the column is completed, the solvent pump is stopped, the column stopcock is closed, and the ball and socket joint is opened for transfer of the aliquot. Transfer of Aliquot to Column. T o t h e d r y aliquot add 0.2 ml. of 0 . 5 N HzS04a n d use about 0.5 ml. of acetone for washing purposes. A heavy glass rod is used t o assist in bringing t h e DNP-derivatives into solution. T h e appropriate amount of oven-dried silica gel-e.g., 0.31 gram when the 5.2 to 8 silica gel is used, or, 0.36 gram if a 4.5 t o 8 silica gel is used-is immediately added t o the aliquot. The mixture is stirred with a rod and allowed t o evaporate to dryness, with occasional stirring, in the dark under the hood. The free-flowing powder is transferred to a small beaker, using a small thin stainless steel spatula to loosen any silica gel particles. The wall of the original container is washed down ivith about 0.5 ml. of acetone and the 0.3 gram of acidified silica gel reserved from column pouring is added. After mixing with a stirring rod, the mixture is again evaporated to dryness in the dark under the hood with occasional stirring. The free-floiving powder is transferred to the small beaker and mixed with the originally transferred silica gel. The heptane from the top of the column is siphoned off to a height of about 110 cm. and the top of the silica gel is leveled by means of a flat-bottomed glass stirring rod. The column is filled to mid-way in the socket with warm acid-washed heptane. The aliyuotsilica gel mixture is carefully and gradually transferred, tapping the beaker. The heptane in the socket area is stirred with a glass rod to permit any adhered silica gel particles to fall into the column proper. The top of the transferred silica gel is carefully lei-eled, if necessary. The sample area is covered by gradually pouring the 0.5 gram of acidified silica gel, reserved from column pouring, into the column. 9 10-mm. circle of Whatman 41 H filter paper is placed in the column and is gently positioned on top of the silica gel, with the aid of a glass rod. The column is refilled with acid-washed heptane t o about inch below the bottom of the socket. The socket nrea is dried with filter paper, the silicone-rubber gasket is inserted, and the ball and socket joint 86
ANALYTICAL CHEMISTRY
-A---
36
-I--t o 41
mm..
1 21 to 26
1
L
FROM COLUMN
11 TO WASTE BOTTLE OR FRACTION COLLECTOR
Figure 3. A. 8. C.
D.
Flow cell a d a p t e d to Beckman Model DU spectrophotometer
Teflon end from */,-inch rod '/le-inch hole to fit Tetlon tubing, 0.034-inch I.d., 0.058-inch 0.d. Glass tubing of Corning Vycor, No. 7910, 7-mm. i.d., 9-mm. 0.d. Removable plote in Beckman DU test tube compartment, made with holes for rubber stoppers
is connected, employing the precautions mentioned under column packing. The DNP-aliquot is protected from light by wrapping the column with aluminum foil. Solvent Systems. T h e gradient elution system employing two mixing chambers is illustrated in Figure 2. T h e mixing chambers are 500-ml., three-neck (T 24/40) jacketed distilling flasks (Cat. No. JF4590, Scient,ific Glass Apparatus Co., Inc., Bloomfield, N. J.). About 150 ml. of deaerated 0.1N H2SO4 is placed in the acidwash column. (The latter consists of a 12-mm. i.d. x 600-mm. tube fused t o a 150-ml. bulb, with a short tube attached to the top of the bulb.) For the chromatography of the DNPamino acids in a 1- X 100-cm. column, 450 ml. of heptane is placed in MC,,t o be heated to 56" C., and 450 ml. of heptane is placed in illC2, to be heated to 35" C. Reservoirs 8 , B, C, and D contain, respectively, 3% 1AA/H. 18% t-AAIH. MEK, and nheptane. S~milarlp,for the fractionation of DNP-peptides, 450 ml. of n-heptane is placed in both mixing chambers MCI and MC2, while reservoirs A , B , C, and D contain, respectively, 18% t-AA/II, 50% M E K / H , MEK, and n-heptane. Reservoir D is used only for column packing. Column Operation a n d Continuous Spectrophotometric Recording. T h e eluate is delivered t o a flow cell (Figure 3) adapted t o the Beckinan Model DU spectrophotometer, equipped with a n energy-recording attachment, AC-Power supply, and a 0- to 50-mv. strip chart recorder (a 6-inch per hour chart speed is convenient), or to the flow cell of the colorimeter (Phototube Colorimeter, Model 1, Technicon Instruments Corp., Chauncey, Ii.Y.) entering the arm directly over the light path, for continuous spectrophotometric recording. Upon delivery from the flow cell, the eluate can be collected in a fraction collector, if desired. Although the maximum absorption of most of the DNP-deriva-
tives in the eluate is a t 335-mp wavelength, 340 mp is used to reduce the absorption produced near the end of the run by the MEK solvent. For the separation of the D S P amino acids with the gradient elution system employing two mixing chambers (Figure 2 ) , the stopcocks are opened to permit the delivery of 3YQ t-AA/H from reservoir A to MC2,with the pump set to deliver 180 ml. per hour. After about 700 ml. of the solvent has passed through the column (or, after almost 4 hours) the DNP-phenylalanine is eluted. At this time the solvent is changed to 18% t-.4A/H by turning the upper stopcock manually so that the flow of solvent will be from reservoir B to MC2. The 18% t-AA/H is allowed to flow 4 hours (about 720 ml.) at which time an automatic device ( 7 ) opens t h e lower stopcock permitting the delivery of MEK from reservoir C to JIGS. The M E K is permitted to flow through the column until all DKP-amino acids have been eluted (about 2200 ml.). The same general procedure is employed for the fractionation of t h e DNP-peptides. Thus, the stopcock i s opened to permit the delivery of the 18% t-A-4," from reservoir A to MC,, with the pumg delivering 180 ml. per hour. After about 800 ml. of solvent have passed through the column (or. after about 4.5 hours) the solvent is changed t o 5070 M E K / H by turning the upper stopcock rnanually to permit the solvent from Reservoir B to flow t o &IC2. I n 4 hours (a flow of about 720 ml.) the automatic device turns the lower stopcock allorring the 100% JIEK to flow from Reservoir C to X C 2 . This is allowed t,o continue until no additional DNP-peptides are eluted. The separation of the DNPpeptides may be improved (if the peaks are close together) by altering the MEK gradient. Thus, the solvent system specified above should be used for the first or trial run. Also, the resolution of the DNP-peptides can be altered to advantage b y rechromatographing a given eluate employing a
silica gel of a different degree of hydration. Finally, some DNP-peptides may move slowly even though the gradient approaches 100% MEK. Additional elution can be accomplished by passing dimethyl formamide directly through the column, bypassing the acid-wash tube, and replacing the acidwashed MEK above the silica gel with the dimethyl formamide. Rapid Determination of DNPAmino Acids Derived from Protein Hydrolyzates. B y use of a modified nine-chamber variable gradient device (Technicon Corp., Chauncey, N. Y . )described by Peterson and Sober (IS), the solvent gradient may be conveniently produced and deaerated by heating the solvent in chamber one prior to its delivery to the watersaturating tube. A silicone rubber insulated heating tape is wrapped around the bottom third of this chamber and is held in place by Teflon adhesive tape. Because each chamber is already vented, no special precautions for the release of gases are required. Although many different combinations of silica gel hydration, column temperature, flow rate, and gradient will produce excellent separations, the following conditions for a column temperature of 45" C. and a flow rate of 100 to 150 ml. per hour are useful:
Figure
4.
Separation
of a
Chambers one through six each contains 100 ml. of 3, 3, 3, 10, 10, and 3% t-ilA/H, respectively, while each of the chambers seven through nine contains 84 ml. of MEK. On the other hand, a t a column temperature of 25" to 28" C. and a flow rate of 250 ml. per hour, chambers one through six each contains 200 ml. of 2 , 2 , 2 , 7 , 10, and 2% t-Ad,", respectively, and each of the chambers seven through nine contains lG8 ml. of MEK. The upper half of the bulb of the acid-wash column is filled with heptane. It is necessary to pass some additional SIEK through the system a t the end of a run to elute the final eDiYPlysine peak which appears after DXParginine. For mixtures containing both amino acids and peptides, preliminary experiments indicate that the gradient should be prepared in two parts. The first elutes all DNP-derivatives up to serine and consists of 2, 2, 2, 10, 10, and 15% t-AA4/H in chambers one through six. There are 150 ml. in each chamber and the column may be run a t room temperature a t I50 ml. per hour. The second gradient is designed to elute the water soluble DNP-derivatives and DNP-peptides. Chambers one and two contain 200 ml. each of 15y0 tAA/H, three and four contain 183 ml. of 50% AIEK,", and chambers five
known mixture
of
through nine contain 168 ml. of NEK. It may be necessary to vary this latter gradient to improve the separation of particular peptide mixtures. RESULTS AND DISCUSSION
Separation of DNP-Amino Acids. T h e column has a sintered disk and when used for the first time will adsorb some of the yellow color of the DNP-derivatives. After several runs with a dinitrophenylated mixture of amino acids, t h e disk appears t o become conditioned and this no longer is a problem. Water droplets may separate upon changing to the N E K solvent, if the temperature of the flow cell is below the column temperature. This becomes evidenced by blips on the recording chart, as the effluent flows through the cell. The formation of water droplets can be minimized by circulating 35' C. water through Beckman thermospacers when the Beckman Model D U spectrophotometer is used. Similarly, when the Technicon colorimeter is used, the temperature of the effluent is maintained a t 35" C. b y jacketing the Teflon tubing leading from the column to the flow cell.
DNP-derivatives of amino acids and related substances
Peaks representing unreacted DNFB and dinitrophenol a p p e a r before the DNP-amino acids and a r e not shown. scribed in the text, its p e a k appears in the alanine.proIine area
If dinitroaniline i s not removed, as d e -
VOL. 5 NO. 1, JANUARY 1963
87
Table I.
Reproducibility of the Method
-4cid hydrolyzate of oxidided ribonucleaseB _____ .-
.Iniiiio Acid
l~cyroducibility of known acids Relative S o . of std. dev., c, /C iietiis.
I'ht~iiylalanine Tyrosine
Leucine
Isolcuciiie \.aline .Ilanjne (;lycine Proline Glutamic acid Serine Threonine Methionine sulfone Aspartic acid Cysteic :kcid drginine Histidine Lysine Tryptophan
Cj-stine Methionined
3.3 7.6 0.0 2.4
2.8 4.7 5 .3 4.i A. 1 4.3 0.6 (3.4 *5. 4 3.3 7 2 4 5
8.9 6.8 5.5
.Inalyzer method, residues
DSP-method Residues fihloles prr
Itl1q""t
)
Per
Inole :3. O b 3.2 3.2
0.09 0.09
0.09 0.09
3 . 0" 3.0
0.17
0.16
5.5
0.20 0.36 0.14 0.16 0.31 0.35 0.25 0.09 0.41 0.21 0.15 0 10 0 34
U.2P 0.38 0.15 0.17 0.32 0.35 0.25 0.10 0.12 0.20 0.15 0 10 0 35
7.0 12.3
... ... ...
. .
.>- . - .> 6.0
10,s 4.7
4.S 5 . .5
4.S
10.5 11.7
s.3 :3 . 2
IS.$
6.8 .5 . 0 3 3 11 5
I
10.6 10.9 s 7 3.0 12 7 6,l 4.2 13 4 ...
. . ...
Per
molf~
... ...
a Discrepancies betweeii the relative molar ratios reported here and those found by Hirs, Moore, and Stein ( 6 )ma,v be related t o the purity of the preparation mid the conditions of oxidation and hydrolysis. Three residues per mole was assumed as a reference. Some error in the value attributable t o slight leucine peak overlap.
,' Calculated as met,liionineplus methionine sulfoxide.
Figure 4 presents the separation within 22 hours of a known mixture of 18 DSP-amino acids and DXP-derivatives of certain substances which may be !)resent in protein-free tissue extracts, cniploying a two-chamber elution system illustrated in Figure 2. DSPisoleucine and DKP-leucine are not well separated. Silica gel ( 5 . 2 t o 8) and a column temperature of 35' C . m r e employed for fractionation. Better resolution can be accomplished by rechroniatographing the conihined peaks with a silica gel of l o w r hydration, by increasing t,he column temperature, or by decreasing the flow rate. I n ot1ic.r experiments, the separation of t h r known niixture of 18 DSP-amino acids was observed ivith silica gc.1 columns of different degrees of hydration from 3.5 to 8 t o 5.2 to 8, and, with various column temperatures, while maintaining a uniform solvent system described earlier. In general. decreasing the silica gel hydration, or raising tlic tcniperature at which the column was run, had the effect of retarding the appearance of all DYP-amino acids. This indicates that a higher per cent slcohol was required to elute equivalent peaks under these conditions. I n addition, such substances as di-DKPtyrosine, -lysine, and -cystine appear to behave somewhat differently to these variables, resulting in some in88
ANALYTICAL CHEMISTRY
stances in an alteration of the order of elution. For example, di-DSPcxstine was eluted between DKPthreonine and DKP-serine when a 3.9 to 8 silica gel was used instead of between DKP-glutamic acid and D S P aspartic acid when a 5.2 to 8 silica gel was employed (Figure 4). Similarly, DKP-glycine was eluted between D S P tryptophan and di-DSP-tyrosine with the lonered hydration of the silica gel instead of het'ween di-DSP-tyrosine and di-DKP-lysine with the more hydrated silica gel (Figure 4). The quantities of thc knon.n DSPderivatives varied from 0.06 pniole to 0.2 pniole in carrying out the iractionation represented in Figurp 4. DSPamino acids in aniounts as lo^ as 0.01 pniolc yield reimduciblc, pen ks and can swvc for identification purpows. Consistent results are obtained with known niixt~ures of aniino acids, proT-ided the conditions for dinitrophenylation and for the separation of the DSP-derii-atives are held constant. The area of the peaks on the effluent curvrs is obtained by niultiplying tlie net height of a peak in absorbance units, by the width a t half t l i ~height (19). A calibration constant i. obtained for each amino acid by dil-iding the area thus obtained by the pmoles of standard amino acid required to producte it. With tlie column hydration
coniinonly twployed to separate the DSP-amino acids, the isoleucine and leucine peaks overlap to a degree, requiring the use of the area of the combined peaks. The latter is expressed as square centimeters (measured by a compensating polar planimeter, K and E, S o . 423611) multiplied by a factor of net height in ternis of absorbance divided by the net height, in ternis of per cent transmittance. VT;1ien automatic photometric recording is unavailablr, &ml. fractions of effluent may he collccted in opt'ically inatelied test tubes and the area may be calculated by addition of absorbance readings (1.9). I n Table I are presented data illust'rating the reproducibility of the procedure R S applied to knon-n inistures of amino acids in the range of 0.06 to 1 pmole per amino acid. The third column gives the relative standard deviation of the per cent recovery of the fimole of amino acid in the mixture. Performic acid-oxidized ribonuclease (Mann Research Laboratories, Inc., New York 6, N. Y., Lot KO. G-1056) !vas hydrolyzed in an evacuated tube with 6 N HCI a t 105' C. for 21 hours. The results of a duplicate analysis of the amino acids in t,he hydrolyzate, each aliquot representing 0.56 mg. of oxidized ribonuclease, by the DSPmethod are shown in columns four and five. An aliquot of the hydrolyzate, representing 1.75 nig. of oxidized ribonuclease, was also analyzed by nieans of the Phoenix Amino Acid .-lnalyzer. (The authors express appreciation to Alfred Strachcr for the latter data.) The amino acid residues per mole of protein obtained by the two methods are shown in columns six arid w1-m. Separation of DNP-Peptides. -hi inspection of Figure 4 will rrve:tl that with tlie solvent system rewniniendetl for t h e fracbionation of DSP-amino acid, DSP-glutathione (reduced), DKP-carnosine. and D?I;I-'-:+nscrine are eluted. The m i i e solvent system is employed n-here it is tlcsirctl t,o fractionate both DSP-amino acids and DKP-peptides. 1'0 separxte additional DhTP-peptides. it is iicccsaary to prolong tlie dc~liveryof 100% M 7 K to the coluinn. If tlir principal interest is ill the scparation of DSP-peptides, the t-.l.i gradieiit is stepped up permitting the AIEK gradient to start carlier. The elution of the DSP-amino acids is tlicwby coml)ressed and tlic elution of tlic DSP-peptidvs hcgins woner. The resolution of the latter can he altered by modifying the 1IEK gradient, changing the hydration of tlie silica gel, or using another column teniperat'ure. If two or more DNP-peptide peaks are fuscd under a given set of conditions, rcssolution can be improved by rechroniatographing the combined 1)eaks under
c
c
: 5
0
I
,
* s
I 1
