Quantitative Qetermination of Amino Acids on Filter Paper Chromatograms by Direct Photometry EARL F. MCFARREN A N D J A M E S A. M I L L S N a t i o n a l Dairy Research Laboratories, Inc., Oakdale, L. I.,
N. Y.
Ldequilte c h e m i c a l m e t h o d s have not b e e n available for d e t e r m i n i n g a l l the amino a c i d s and t h e available methods have been t e d i o u s and time-consuming. The miembiological assay method has been used w i t h considerable B U D E ~ S S , h u t occasionally e r r a t i c and w n f u s i n g results h a v e been o b t a i n e d w h e n it has been applied to c e r t a i n materials of biological origin. The q u a n t i t a t i v e analysis of beta-laotoglobulin b y paper c h r o m a t o g r a p h y has given remilts w m p a r a h l e to those o b t a i n e d by other methods. E x p e r i m e n t s n o w i n progress i n d i c a t e that the amino acid composition of t w o protein h y d m l y z a t e s or other u n k n o w n solut i o n s m a y be compared b y r u n n i n g the t w o u n k n o w n s o l u t i o n s alongside s t a n d ard s o l u t i o n s on the same c h r o m a t o g r a m .
HE methods daiescribed in this paper are concerned with the by a series of oneT s e p a r a i i o n of a mixture ,f dimensional buffered ( I 0)chromatograms and their quantitative determination bv direct ohotometrv on the moer. The buffered paper techniquhhas been employed t o separate the amino acids in a bydrolvzate of 8-lactodobulin. T h e seurtrattted amino acids have theri been quantitatively determined by the procedure en ployed fa,r the quantitative det.ermination of sugars ( 1 1 ) in a mixture. ,.. .... . _ .. . . ~, . Block ( 4 ) , ~ n , el, al, (7), anti ,toelil~nndand Uunn ( I J , have published somewhat similar methods for quantitatively de%termining the amino acids by etry, but many of techniques reported here arc t.
~.
EXPERIMI
The method of preparing vent and paper hss been des< o i a chamber with its glass li filter paper liner.
..
,
.
I
i!: en
un Na
;e. SI $8 111 .8 :
he
,.
'rn. '9 I
de th
P
91t
? 1
iic 3Ig
le on ei
iec
ar
1%
)E f
ed ;hi
t< ,he 650
centration of the standard and unknown adjusted t o a pH of 6.5 is limited by the solubility of tyrosine, it is necessary t o build UP the Concentration on the paper by repeated spotting in 5-pi. quantities, with drping between applications. Thus, two 5-ul. andieations of the standard will eiv-a eoncentmtion of0.25 micro&m, iour 5wI. appllcations a concentration of 0.50 microgram,
et%he unknown cB.n be concentrat,ed On the paper by making 1, 2, 4, or 8 applications in 5-al. quantities,, giving a total of 5, 10, 20, or 40 pi. of the unknavn solution applied a t a n y one point. Drying between applications can be hastened byplacing the chromatograms under an infrared lamp. T h e sheet is t b a i placed in the chromatographic chamber and tlllorved to remain the aonronriste t,ime for the solvent used. After re-
V O L U M E 24, NO. 4, A P R I L 1 9 5 2 Table 1.
6%
S u m m a r y of Q u a n t i t a t i v e Paper C h r o m a t o g r a p h y Procedure
Sol>-e"t
pH
Molarity
Phenol
~- 0
0.067
Amino Aoids Determined Aspartio Glutamic Serine
Running Time
Drying Time,
% AeeJic Acid I"
Ninhydrin
24
Mia. 30
40
60
2
40
30
2
..
30
2%
acid 2 4% in isatin 2
Hour;
4
Gl,-Oi"e
Threonine AIiin i n e Tyroeiine Histidine \'dim hletliionine
4
0.067
Luti'line
6.2
0.022
Lys!ne Argl"i"e
Phenol
1.0
0.2
csstine
IX.CNS"
o-Crerol
6.2
0.067
BenaylbutJ-I
8.4
0.067
Phenylalanine Proline Isolei?Ci"e
24 40
BO
Trlntoiilran
24
30
30
1,eWX"e
Collidine
9.0
0.067
The solvent, the pH, the molarity of the buffer, the running time, thedrying time a t 6 0 " C., thepercent scetic soidusedinthe developing reagent, and the amino acids determined in each solvent are summnrized in Table I.
triethylamine, no
Because of the faint yellow color produced with ninhydrin, proline is best developed with 0.4% isstin (1) in I-butanol containing 4% acetic acid. After dipping the chromatogram, t h e color is developed by heating in the meohanical convection oven a t 100" C. for 10 minutes. Proline is determined on the same chromatogram as leucine and isoleucine. T h e chromat.ogram is cut in approximately the middle before color development and t h e upper portion is developed with isatin. T h e lower portion can then be developed separately with ninhydrin for the determination of leucine and isoleucine. The dipping of the ohromatograma in either ninhydrin or isatin is accomplished by a single passage of the chromatogram through the developing reagent contained in a 6 X 10 inch borosilicate ghss baking dish. I n the sample for which analysis is reported here, hydrolysis was carried out essentially QS out,lined by Block (3).
Tryptophan was rlet.ermined after hydrolysis with 14% barium hydroxide. Because the introduction of any great quantity of acid or salts into the hydrolyzate may subsequently interfere with the chromatographic sepiaration theaddition of a n excess of sulfuric soid to prec$itate the barium should he avoided and only a minimum of acetic acid should be "Sed t o wash the precipitate. Cystine W&8 determined after hydrolysis with a 1 t o 1 mixture of ,v h3,droehloric acid and formic After hydrolysis the solution was taken t o dryness under vacnum or under a current of warm air from a hair dryer a t 60' C. T h e residue wa8 then taken up in 10% isopropyl alcohol to the desired volume and chromatogmmmed. All t h e other amino acids were determined after hydrolysis with 6 11' hydrochloric acid for 24 hours under reflux,. The hydrolyzate w a ~then taken up in water and extracted three times (or until neutral) with a 5% solution of di-2ethylhexylamine (purchased from Carbide and Carbon Chemicals Corp.) in chloroform (14). The solution was then extracted several times with chloroform t o remove t r m e s o i the amine. Theextracted material was again taken to dryness t o break u p any emulsion t h a t may have formed and to free the solution of chloroform. T h e residue was taken u p in 10% isopropyl alcohol (3) t o the desired volume for chromatogramming. This is t.he only method that has proved satisfactory, as other methods either leave residual hydrochloric acid or introduce salt which interferes, oarticularly on t h e buifered phenol and cresol chnimatogwms 4
w%
I
by Gotfred Haugaard of these laboratories and was one of the samples analyzed by B r m d et al. (5)and by Stein and Moore (15). The amino acid composition of 0-lactoglobulin as calculated from onedimensional quantitative buffered paper chromaiograms is summarized in Table 11. For convenience in evaluating this method, the results itre compared lrith those obtained b r other workers using other methods. No correction has been made of the data obtained by paper chromatography for the decomposition of serine and threonine during acid hydrolysis. The value given for histidine is questionable, since difficulty was experienced in determining this amino acid owing t o the small amount present and the low sensitivity of the ninhydrin reaction with histidine. The value calculated far leucine seems perticulady low in the light of the other data, but no explanation can he offered a t the present time for this low value. Figure 2 is a photocopy of one of the quantitative chromrttograms used Ear the determination of the amino acid camposit,ion of B-lact,oglobulin. ~~
~
DlSCUSSIOA
R, Values. It has been previously reported ( 1 0 ) that lysine and arginine could be best separated using lut,idine (Figure 3)
ANALYTICAL CHEMISTRY
652 Table 11.
Amino i c i d Composition of Beta-Lactoglobulin
0 .Amino hcid 100 G. Protein Aspartic Glutamic Lysine Arginine Histidine Csqtine herine Glycine Threonine Alanine Tyrosine Valine Methionine Tryptophan Leucine Isoleucine Phenylalanine Proline
Paper Chromatographs 11.16 19 6 2 11.25 2.38 I
.o
3.1 3.36 1.38 4 . .58 6 29 3 92 4.47 2.94
1 84 10.i5 3 12 3 41 4 Y8
Lewis et al. (9) 11.3 18.4 11.2 2.88 1.66
...
3.8 1.38 5.2 5.8 3.87 6.2 3.12
...
15.2 I .33 3.63 5.0
Stein a n d Moore (16) 11,:2 19.08 12.58 2.91 1 6.3 3 40 3 96 1.30 i.92 I ,00 3.64 5 62
Brand el ai. (6)
11.4 14.5 11.4 2 88 1 58 3 40 5 .0 1.40 5.85 6 2 9 .i 8 .\ 83 8 22 1.94 15 6
...
13:io 5.86 3 78 5 14
8 4
3 .54 4.1 -~
Iiuffered n i t h a 0.022 JI buffel (not 0.22 as Frioiieously printed) of p H G.2. The separation of lysine and arginine has been accomplished at a molarity of 0.022 but not at higher molarities. I n n similar manner, cystine as well as lysine and arginine can be separated at a pH of 4.0 in lutidine with a 0.022 M buffer. Some separation of cystine occurs at other p H values, but decomposition and streaking usually result, particularly at more alkaline p H values. For quantitative determinations cystine (Figure 3) is even better separated and produces a more compact spot when phenol is buffered at p H 1 with a 0.2 ;li buffer. Color Development. The objective has been to develop the maximum color intensity possible for a given amino acid concentration without a t the same time causing destruction of the amino acids. -4 number of articles have appeared recently which indicate t h a t amino acids are destroyed by heatdrying on paper chromatograms. Patton et al. ( l e ) have shown t h a t amino acids dried on filter paper at 105" C. will fluorcsce when placed under a n ultraviolet light. A similar solution of amino acids dried on a glass plate under t h e same conditions did not fluoresce. FOKden and Penny (8) report that the recovery of certain amino acids was decreased by heat-drying of paper chromatograms. Brush et al. (6) concluded from the s t u d r of radioautographs of alanine-2-Cld run in phenol t h a t chromatograms run in this solvent should not be heated at all. Berry and Cain ( 2 ) conclude that the intensity of the ninhydrin color reaction does not decrease appreciably if the chromatograms are not heated over 85" C. for longer than 8 to 10 minutes.
I t is apparent that the t h e and temperature of drying and color development are of importance. I n order t o determine the time and temperature of mssiniuiii color development, four chromatogranis were prepared hy spotting each with a solution of all of the amino acids a t a concentration of 0.5 microgram of amino nitrogen per each amino acid. T h e chromatograms were run iii phenol buffered a t a pH of 12 for 20 hours, removed, and air-chied for 2 hours. Each v-as sprayed once nith a solution of 0.4 gi:ini of ninhydrin in 90 grams of butanol, and 10 grams of phenol ( 7 ) t o which had been added sufficient glacial acetic acid to make R 1% solut,ion of the acid. T h e first chromatogram was hrated for 15 minutes, the second for 30 minutes, the third for 45 minutes. and t,he fourth for 60 iiiiiiutes. One such swies n-:is developed at 80" C., another :it 60" C., and a final seiiw at 40" c'.
a4
I
a5
e6
0 7
%* 4 9
@ B
Figure 3. Photocopies of Additional Useful Buffered Chromatograms A . Lutidine saturated with 6.2 pH buffer of 0.022 molarity, paper buffered a t pH 6 . 2 1. Lysine 2. Arginine 3. Valine B. Phenol saturated with pH 1.0 buffer of 0.2 molarity, paper buffered a t pH 1.0 4. Cystine 5. Alanine 6. Hydroxyproline 7. Tyrosine 8. Valine 9. Methionine
From the data in Table I11 it, vas coiicluded that the masiinurn density of the spots \v-a~obtained by developing the c h r o m a t e gram at 60" C. for 15 minutes. Similar studies \vith other solvents has shown that this is true,, regardless of the solvent used to obtain the chromatographic aeptration. The air-drying of chromatograms is time-consuming and is unfavorable, as ammonia absorption may subsequently causc a hlue background color to develop. I n order t o determine vhether any apparent destruct,ion of the amino acids occurs on heating, and t o determine the minimuni time for solvent removal, three chromatograms were prepared and run for 20 hours in phenol (pH 12.0). The first chromatogram was air-dried for 2 hours, t h e second was oven-dried for 15 minutes a t GO" C. (t,he minimum time required t o make the sheet feel dry t o the touch), and the third \vas oven-dried a t GO" C. for 30 niiiiutes. Each chromatogram {vas sprayed once with t h e reagent used 1)y Bull (?) cont,aining 4% acetic acid and t,he color was developed a t GO" C:. for 15 minutes. The highest density readings were obtained when the chromatograms nere dried a t GO" C. for 30 minutes (Table I T ) . Similar studies were made for each solvent, and the preferred drying periods are summarized in '!hide I. The data in the first column of Tablc IT are not similar to thoie i n column five of Table 111,although the time and temperature of drying and developing were the same in both cases. Such a coniparison cannot be made, for densities cannot be reproduced from one chromatogram to anot>hw,paiticularly if the chromatograms are run on different days for longer or shorter periods of tirne. or from different sheets of paprbr. Densities may also vary with the relat,ive humidity of thc room a t thc time of drying and developing the chromatograms, This phenomenon has also been notcvl in developing sugar chrom:ttogrnme Kith ammoniacal ,silver nitrate. Addition of Acid to Developing Reagent. I n using buffrrcd filt'erpaper and solvent the amino acids d o not develop vie11 with ninhydrin unless the buffer is neutralized. This has been a(*complished by adding glacial acetic acid to the spray. The amount of acid necessary t o olitrtin maximum color developmmt is established by the data in T a h l ~V. I n this case, three chromatograms w r e prepared on the saine sheet of paper by spotting n mixture of all t h e amino acids a t three different points along the line 7.5 cni. from t h e origin. These chromatograms were run in phenol buffered a t a pH of 12.0 and air-di~ied. The sheet vas then cut into t,hree separate chromatograms and the first chromatogram was sprayed Tvith t h e reagent used by Bull, t h e second n-ith the same reagent containing 27, acetic acid, and the third with t.he reagent containing 4yo acetic acid. Each sheet was sprayed on the front and back with 0.5-hour drying bet,ween sprayings. These three sheets n'ere then developed together in the oven. T h e highest density reatlings (Table V) ivere obtained b>- using 4yo acid in t h e spray. Similar dat,a were collected for each solvent system studied The results for the systems used for quantitative determination are summarized in Table I. Spraying of Chromatograms. Bull ( 7 ) has suggested that the chromatograms be sprayed twice and Rockland and Dunn ( I S ) have sprayed chromatograms for quantitative development as many as seven times. However, if time is allowed to elapse be-
V O L U M E 2 4 , NO. 4, A P R I L 1 9 5 2
__ Table 111.
40
*1
30
15
GO
45
0.22 0 70 0 . 3 3 0 . 1 6 0 . 3 2 0.50 0.60
.\>jiartic Glritaiuic Serine Glycine Threonine .\lanine
:,::!:$,2; :,:i ::i
O:$3 o 11 0 0 88 0.'09 0 . 4 1 0 . ( 4 0 . 6 4
2
215-
ez
g:: a 0
4 L
8: 7 -
6
5 4 -
2
E
3 -
Y
2 -
2
liutanol containing 0.4'; ninliydriii is a more satisfactory reagent, in that higher densities are obtaitied. Care needs to he cse Teniperature, C. ___~_ cess solvent is drained from the elironlatograni 80 2~ 1 60 i 1 after dipping, if ninhydrin in water-saturated butaTime, l l i n u t e s no1 is used. If color starts to develop while excess 13 ;30 4 5 60 15 30 45 GOs o l v c ~ is ~ tpresent on the paper, the blue pigment 0 96 0 . 2 8 0 . 2 6 0 . 2 0 0 . 2 7 0 . 2 9 0 . 2 4 0 ?$I 0 . 3 6 0 66 0 . 5 2 0 . 5 4 0 . 5 5 0 . 7 6 0.51 0 4 3 formed may streak, as occurs in spraying the ::?," ,"::; clinomatogrsmsmoretliiino~ice.Thisphenomenori o :0 0 . 3 1 0 . 2 0 0 . 3 3 0 . 4 1 0 . 4 3 0 . 4 2 0 20 has not been investigated further and it is not 0.86 0.63 0 . 5 6 0.71 0.88 0 . 7 7 0.79 0 . 6 2 known whether t h e use of t h e butanol-phenol sol__ vent mixture or other solveuts Ivould eliminate thiq 1mcautiori. Since moisture seeins t,o bc necessary for hest color tlevelopmerit, other solvents n o u l d have to he a compromisil. Filters and Densitometer. In colorimetric procedures it is cuetomarv to use a colored filtrr \vhicah ivill give a wave length of maximum light absorption. Hull ( 7 ) has found a broad minimuni irithe optic.tl transmittance of amino acids on papor developed with ninhydrin at 570 mp. On the other. hand, Block ( 3 )suggests that a 570 filter may lie used, hut it, i:: riot riecrssary. The (lata plotted in Figure 1 indicate t h a t the 570 filtrr gives thc. maximum absorytion and tlic highest densit!- readings:.
\-ariation of Density with Time and Temperature of Color De\-elopment
______~__
'A
653,
::ti ;0:
I
0
0
IO
20
30
50
40
60
70
80
90
g;:
ii
/ 100
Talde \-I.
DENSITY
Figure 4.
\ ariation of Densit>- with .\[ethotl of Applying Developing Reagents
1 ariation of Densit: \+it11 Filter Used i n 1)ensi to In e t e L'
t\\.c.czn sprayirigs, tht, i ~ o l o rstart-: to tl~veloliarid the colored pig-
niciiit is caused t o diffuw or run b!~ subwqurrit sprayinpe. 111 vi(.\\- of t,hesc clifficultic+ dipping of thc cliromatograms was at tempted. Tv-o strips were prepared by spotting a solution of all thc amino acids at a concentration of 0.5 microgram of amino nitrogen aiid running for 40 hours in vi-cresol buffered at a pH of 8.4. Tht. two strips were oven-dried for 60 minutes a t 60" C. T h e re:igrnt used by Bull containing 2% acetic acid was sprayed heavily on one sheet and the second was dipped into this same re:rgent. Both strips were developed a t 60" C. for 15 minutes.
In :d1 cases the 11igh:zr tlc~iiritic~s n-ei'c o1)titiried (Table V I ) n.1ie.n the chromatogr:inis ivere dipped rathci, than sprayed, and streakiiig or spreading of the amino acids i~oultinot lie noted. Choice of Developing Reagent. Sinhydrin (O.1y0)in Lvatersatwated butanol has been used most commonly for developing aniiiio acid chromatogram^. Bull ( 7 ) has clai~nedbetter results uping 0.4 gram of ninh>-di,inin 90 gram.. of butanol and 10 grams of phtlnol. The data i n Tal)le VI1 indic~atc~ that water-saturatd
Sp r age d
Dipped
0 31 0.08 0.08 0.32 0.12 0.40
0.69 0 10 0.14 0.31 0.12 L52 0.30
llanirie .\rginine Ilydroxyproline Tyrosine Histidine Valine llethionine
Table 1 11.
0.13
Comparison of Densities Obtained [-sing \ arious Developing Reagents
(Concentration, 0 i y ) -Reaeent" .- . 0 . 4 ( 5 ninhydrin in O.iyoninhydrin in O.l'.& ninhydrin in water- n.ater-*aturated 90 g. butanol a n d saturated butanol butanol 10 g. phenol ( 7 ) Aspartic 0.30 0.54 0.33 1 02 Glutamir 0.66 0.97 0.92 1.06 Serine 0.67 Glysine 0.63 0
