So far, the most widely used reagent for detection of amino acids and peptides onpaperand~hln-layercbromatograms hzsbeen nirrhydrin. Kowever,in manycases, such zs the assessment of the purity of syritbetic peptides, a more sensitive reagent is desirable. The recently introducedz4 f&oresca_mine :4_phenylspiroCfu~n_2(3H), I’-phtbaIan]-3,Sdione) procedure, which allows detection of most amino acids at the %pmole level, meets the requirement of increased sensiti+y, Lut the high cost of this versatile reagent precciudes its everyday use in many laboratories. o-Phthziaidehyde, ia the presence of 2 strong reducing agent such as Zmercap-
toethanol, was shown by Roth5 to produce highly fluorescent compounds with most amim acids. This reaction ms later used for amino acid analysis6 and was recently extended to the pmole ra-ange ‘. The work presented in this paper describes its application to detection of amirLo acids and peptides on chromatograms by the _zcent finding’that, in soIutioR,thismethodwasmoresensitive
and was proinpted thanthefluores-
carnine procedure. Indeed, o-phthalaldehyde turned out to be as convenient and sensitive as fiuorescamine where used as B spray reagent.
iWff%?r~&S o-Phthalaldehyde and 2-mercaptoethanol were purchased from Fluka (Buchs, Swi’zerland) and thin-layer plates precoated with sika geefor cellulose and acetone from E. Merck (Darmstadt, G.F.R.). Triethylamine, also obtained from E. -Merck, was dried with potassium hydroxide and re-distilled. Bradykinin and arginine-vasopressin were synthetic prepararions from this laboratory.
AfiqROtS (i-@) cQRtaiRiRg 5, IO, 25, 50, 100, 250, 508 and loo0 pmoies, respectively, of amino acid or peptide in 0.01 _M HCT were spotted OR precoated plates, dried .zt 100” for 30 min and allowed to cool to room temperature. -&e plates were
then treated according to the staining procedure below. The detection knits in Table 1 refer to the sn;aikst amount of the respective compound giving a visible reaction.
.
+m
lco-i-Ef3
limit (prmles per pt)
Delecri~r:
Siiice g2!
C2llUlOS2 IOmir;
Ai25iSX Ai_j#ine
ASF~R~~FZ Aspartic acid Cystine Ghmmine Glumnic acid Glycine Histidine Iso!eucir?e
Leilcine Lysic-
Merhionine Phenyfakninc PrOli~~
Serine Thrconine Tryptop’nan Tyiosine Vdifie Gfycyl-giycine Bradykinin Arginine-vasoprsin
I ir
2k
250 10 250 100 10 lco ICI) 100 25 1GO
250 10 250 loo 10 10 100 103 50 100
250 25 250 IO0 10 !UO 100 It33 50 !OQ
la0
iGQ
100
25 la0 50 -
25 If30 50 -
25 103 53 -
1OO 100
ioD IO3
NO IOO
25 25 25 250 50 100
25 25 25 250 50 100
25 25 25 250 40 100
k_22r
Ii;, 100'
IOnrti ik
250 25 250 100 10 100 100 10 50 200 IQQ
50 10 25 25 109 25 x
250
5m
50
MO 250
500 500
103 250 100 1oi)
loo 250
1033
25 25 50
250
50
500 ml
I O-90 103 500
25 100 53 250 100 100 50 50 50 Z50 50 5OO
25 50 50
100 1OO 250
-
10 2; 50 50 50 I@3 50 2.50
too
150
-
250 250 100 500 500 500 500 Im3
tk
250 250
503
250 250 500 250 250 100 500 1m ia 10% 1m
Ik, 100" 1003
TLC IO nzirr
___.
too 25 50
ia
IO0
250
1GOO
50 100 103 25 100 100 50 100 103 250’ 25 SO 250 100 ieu
loo0 100 1033
Lm3 IO00 2.50
iQi3Q IQ09 1Qo
5t.lO 350 250 ICO 5clO 1wJ
* After heating at 1Kb’ for 1 h.
Tine @&es were sprayed generously wftfr_ a solution of 0.1 :/ o-phthalaldehyde and 0.1 ‘jO kmcaptoethano! in acetone followed 5 min later by 1 oA triethylamine in acetone. After 10 tin the plates were viewed under a long-wave (350 nm) ultraviolet lamp. The spray reagent was stable for several days when kept at room temperature in a closed boti!e protected from !ight but deteriorated rapidly upon addition of bzse (1% triethylmine). XEXJLTs
AND DISXSSION
The detection k&s and stability of the fluorescent spots given in Table 1 denonstrate that the o-~hthalaldehyde procedure allows detection of as little as 5X?-100 pmoles of many amino acids. In most cz~es the sensititity ki 2-S times higher on silica gel, but the aromatic anino acids and, esp ecially, cyst&e are more easily detected
441
NOTES
OE c&&se. The diifferenee in stability oz the two sorbbentsis most striking. On silica gel the spots decay already within a few minutes at room temperature whlie on ceIkE0s.ethey ale essentially resistant even 50 heating_ Profine, as expected, shows no reaction kmediateiy after spraying. However, heating at iCE3”for I h makes spots containing 2.50 pmoles clearly visible. Since this treatment causes the disappcmmce of most other spats on silica gel these need to be marked in advance. Amino acids substituted with labile N-protecting groups (~eerf.-buty:oxy~rbonyl, 2-pheoylisopropyloxycarbonyl, am! p-methoxybenzyloxycarbanyi) give no reaction when the plate is dried at room temperature but are detected at the SO-ptote level whe= heated at 2 h prior to spraying. Lowering the concentration of a-phthakidehyde in the spray reagent to 0.01% caused a sfight decrease in sensitivity whereas increasiizg above 0. f oT had SO effect. The optima! concentration, of 2-mercaptoethanol was found to be l-10 times that of the aldehyde. The composition of the triethylamine spray was not critical, but high concentrations of this reagent (LO %) increased background f?uorescence. The best resufts were obtained when the base was applied after the phthaIaIdehyde-mercaptoethanol solution or was mixed with it immediately before spraying_ Pre-treatment of the plates with 1% tiethyEaminP*” did not improve sensitivity. The stability was improved by substitution of triethanoiamine for triethylamine, however with somewhat reduced sensitivity. The use of sodium hydroxide 2s the base (1 part of an alcoholic solution con’Sking 0.1 T< o-phthaIaIdehyde and 0.1 T/d2-mercaptoethanol mixed with 1 part of 10 ok sodium hydroxide in 60 ok ethanol) which aRorded essentially the same sensitivity as the recommeoded formula did not lead to increased stability. In conclusion, the o-phthalaldehyde reagent is comparable to fiuorescamine in sensitivity and convenience and h.w the additional advantage of being considerab!y less expensive. ia?”
for
ACKNOWLEDGEMENTS
The author is indebted to DF. UIf RZ~~X~FSSO~ i”o~ helpful suggestions during this investigation and to The Swedish Natura! Science Research Council for financial
support. REFERENCES 1 S. Udenfriend,
S. Stein, P. BijHen
2 S. Udmfi-iend,
(Em)
and W.
hfeienfiafer (Editor), Chemisfry 1972, p_ 655. S. Stein, P. B%Ierz, W. Dz+imzm, W. Leimgruber uid M. Weigele, Scieme,
Bioiog~ oJPep.riries,
Am
Arbor
Scl. fib!.,
Da&-man,
in
5.
Ann Arbor, h&h.,
871.
3 A. M. Felix and M. H. limenez, J. Chrormtogr., 89 (1974) 361. 4 E. Mendez znd C. V_ Lai, Am!. Biochem., 65 (197.5) 281. 5 M. Roth, Am!. Chem., 43 (1971) 8SO.
6 M. Roffi and A. Hmqxzi, J. Chrommgr., 7 f_ R. J.Za.wn 2nd P. E H2re. Prac. Nat.
83 (1973) 353. kfCQd_
Sci.
U.S.,
72 (1975) 619.
and 17s
toethanol, was shown by Roth5 to produce highly fluorescent compounds with most amim acids. This reaction ms later used for amino acid analysis6 and was recently extended to the pmole ra-ange ‘. The work presented in this paper describes its application to detection of amirLo acids and peptides on chromatograms by the _zcent finding’that, in soIutioR,thismethodwasmoresensitive
and was proinpted thanthefluores-
carnine procedure. Indeed, o-phthalaldehyde turned out to be as convenient and sensitive as fiuorescamine where used as B spray reagent.
iWff%?r~&S o-Phthalaldehyde and 2-mercaptoethanol were purchased from Fluka (Buchs, Swi’zerland) and thin-layer plates precoated with sika geefor cellulose and acetone from E. Merck (Darmstadt, G.F.R.). Triethylamine, also obtained from E. -Merck, was dried with potassium hydroxide and re-distilled. Bradykinin and arginine-vasopressin were synthetic prepararions from this laboratory.
AfiqROtS (i-@) cQRtaiRiRg 5, IO, 25, 50, 100, 250, 508 and loo0 pmoies, respectively, of amino acid or peptide in 0.01 _M HCT were spotted OR precoated plates, dried .zt 100” for 30 min and allowed to cool to room temperature. -&e plates were
then treated according to the staining procedure below. The detection knits in Table 1 refer to the sn;aikst amount of the respective compound giving a visible reaction.
.
+m
lco-i-Ef3
limit (prmles per pt)
Delecri~r:
Siiice g2!
C2llUlOS2 IOmir;
Ai25iSX Ai_j#ine
ASF~R~~FZ Aspartic acid Cystine Ghmmine Glumnic acid Glycine Histidine Iso!eucir?e
Leilcine Lysic-
Merhionine Phenyfakninc PrOli~~
Serine Thrconine Tryptop’nan Tyiosine Vdifie Gfycyl-giycine Bradykinin Arginine-vasoprsin
I ir
2k
250 10 250 100 10 lco ICI) 100 25 1GO
250 10 250 loo 10 10 100 103 50 100
250 25 250 IO0 10 !UO 100 It33 50 !OQ
la0
iGQ
100
25 la0 50 -
25 If30 50 -
25 103 53 -
1OO 100
ioD IO3
NO IOO
25 25 25 250 50 100
25 25 25 250 50 100
25 25 25 250 40 100
k_22r
Ii;, 100'
IOnrti ik
250 25 250 100 10 100 100 10 50 200 IQQ
50 10 25 25 109 25 x
250
5m
50
MO 250
500 500
103 250 100 1oi)
loo 250
1033
25 25 50
250
50
500 ml
I O-90 103 500
25 100 53 250 100 100 50 50 50 Z50 50 5OO
25 50 50
100 1OO 250
-
10 2; 50 50 50 I@3 50 2.50
too
150
-
250 250 100 500 500 500 500 Im3
tk
250 250
503
250 250 500 250 250 100 500 1m ia 10% 1m
Ik, 100" 1003
TLC IO nzirr
___.
too 25 50
ia
IO0
250
1GOO
50 100 103 25 100 100 50 100 103 250’ 25 SO 250 100 ieu
loo0 100 1033
Lm3 IO00 2.50
iQi3Q IQ09 1Qo
5t.lO 350 250 ICO 5clO 1wJ
* After heating at 1Kb’ for 1 h.
Tine @&es were sprayed generously wftfr_ a solution of 0.1 :/ o-phthalaldehyde and 0.1 ‘jO kmcaptoethano! in acetone followed 5 min later by 1 oA triethylamine in acetone. After 10 tin the plates were viewed under a long-wave (350 nm) ultraviolet lamp. The spray reagent was stable for several days when kept at room temperature in a closed boti!e protected from !ight but deteriorated rapidly upon addition of bzse (1% triethylmine). XEXJLTs
AND DISXSSION
The detection k&s and stability of the fluorescent spots given in Table 1 denonstrate that the o-~hthalaldehyde procedure allows detection of as little as 5X?-100 pmoles of many amino acids. In most cz~es the sensititity ki 2-S times higher on silica gel, but the aromatic anino acids and, esp ecially, cyst&e are more easily detected
441
NOTES
OE c&&se. The diifferenee in stability oz the two sorbbentsis most striking. On silica gel the spots decay already within a few minutes at room temperature whlie on ceIkE0s.ethey ale essentially resistant even 50 heating_ Profine, as expected, shows no reaction kmediateiy after spraying. However, heating at iCE3”for I h makes spots containing 2.50 pmoles clearly visible. Since this treatment causes the disappcmmce of most other spats on silica gel these need to be marked in advance. Amino acids substituted with labile N-protecting groups (~eerf.-buty:oxy~rbonyl, 2-pheoylisopropyloxycarbonyl, am! p-methoxybenzyloxycarbanyi) give no reaction when the plate is dried at room temperature but are detected at the SO-ptote level whe= heated at 2 h prior to spraying. Lowering the concentration of a-phthakidehyde in the spray reagent to 0.01% caused a sfight decrease in sensitivity whereas increasiizg above 0. f oT had SO effect. The optima! concentration, of 2-mercaptoethanol was found to be l-10 times that of the aldehyde. The composition of the triethylamine spray was not critical, but high concentrations of this reagent (LO %) increased background f?uorescence. The best resufts were obtained when the base was applied after the phthaIaIdehyde-mercaptoethanol solution or was mixed with it immediately before spraying_ Pre-treatment of the plates with 1% tiethyEaminP*” did not improve sensitivity. The stability was improved by substitution of triethanoiamine for triethylamine, however with somewhat reduced sensitivity. The use of sodium hydroxide 2s the base (1 part of an alcoholic solution con’Sking 0.1 T< o-phthaIaIdehyde and 0.1 T/d2-mercaptoethanol mixed with 1 part of 10 ok sodium hydroxide in 60 ok ethanol) which aRorded essentially the same sensitivity as the recommeoded formula did not lead to increased stability. In conclusion, the o-phthalaldehyde reagent is comparable to fiuorescamine in sensitivity and convenience and h.w the additional advantage of being considerab!y less expensive. ia?”
for
ACKNOWLEDGEMENTS
The author is indebted to DF. UIf RZ~~X~FSSO~ i”o~ helpful suggestions during this investigation and to The Swedish Natura! Science Research Council for financial
support. REFERENCES 1 S. Udenfriend,
S. Stein, P. BijHen
2 S. Udmfi-iend,
(Em)
and W.
hfeienfiafer (Editor), Chemisfry 1972, p_ 655. S. Stein, P. B%Ierz, W. Dz+imzm, W. Leimgruber uid M. Weigele, Scieme,
Bioiog~ oJPep.riries,
Am
Arbor
Scl. fib!.,
Da&-man,
in
5.
Ann Arbor, h&h.,
871.
3 A. M. Felix and M. H. limenez, J. Chrormtogr., 89 (1974) 361. 4 E. Mendez znd C. V_ Lai, Am!. Biochem., 65 (197.5) 281. 5 M. Roth, Am!. Chem., 43 (1971) 8SO.
6 M. Roffi and A. Hmqxzi, J. Chrommgr., 7 f_ R. J.Za.wn 2nd P. E H2re. Prac. Nat.
83 (1973) 353. kfCQd_
Sci.
U.S.,
72 (1975) 619.
and 17s
