~~
Journal of Chromatography,
132 (1977) 267-276
C
Elsevier Scientific Publishing Company, Amsterdam
-
Printed in The Netherlands CHROM. 9653
THE VAN URK-SALKOWSKI REAGENT - A SENSITIVE AND SPECIFIC CHROMOGENIC REAGENT FOR SILICA GEL THIN-LAYER CHROMATOGRAPHIC DETECTION AND IDENTIFICATION OF INDOLE DERIVATIVES
AXEL EHMANN
Department 01 Botany and Plant Pathology, Michigan State University, East Lansing, Mich.
48824
(U.S.A.)
(Received July 14th, 1976) SUMMARY
The chromogenic reagent described has been tested with seventy-nine indole derivatives and found to be very sensitive and indole-specific. The lower limit of detection on silica gel thin-layer plates was between 25 and 5'0 ng for most indoles. Phenols and hydroxy-, and amino-benzoic acids, hydroxy-, and methoxy-cinnamic acids did not yield chromophores with the exception of p-amino-benzoic acid and p-l1\rtroxy-cinnamic acid which gave yellow and pink chromophores at concentrations g .: than I and 2 ,ug. Although many of the C-3 substituted indoles such as indole- 3-acetic acid and tryptamine had colors in the reddish-yiolet- blue color region, most exhibited sufficient color differentiation to allow their identification by thin-layer chromatography. The procedure was simple and required only 10 min from the time of spraying the thin-layer plate until full color development was reached. The colors had a wide spectral range from yellow of the indole-3-glyoxylamide chromophore to blue of the melatonin chromophore, and were extremely stable. INTRODUCTION
Silica gel thin-layer chromatography (TLC) has become a powerful technique in the purification, separation and possible identification of natural and synthetic indole derivatives
l-4
: The advantages over paper chromatography are short developing times, inertness of the silica gel layer towards corrosive spray reagents and minimal aone spreading of the chromatographing compounds, resulting in a l0-20-fold decrease of the detection limits
5
. The indole compounds have been visualized on TLC plates by one of the following chromogenic reagents: (a) Salkowski reagent
6-13
(strong mineral acid plus oxidant); (b) Ehrlich reagent
5,14-34
<p-:dimethylaminobenzaldehyde-HCl with or without oxidant); (c) van Urk reagent
35-42
(p-dimethylaminobenzaldehyde-H2SO4 and oxidant); (d) Renz and Loew reagent43-49 (p-dimethylaminocinnamaldehyde-HCl);
-
-
.
Journal Article No. 7746 from the Michigan Agricultural Experiment Station.
Page 268 (e) Adamkiewicz reagent50-57 (formaldehyde-HCI); (f) Maickel and Mille agent
58-61
(o-phthalaldehyde-HCI). , The last two reagents give strongly yellow fluorescing indole condensation products, which makes them the most sensitive reagents available. Their use is limited because extracts (especially from plant material) contain many non-indolytlic yellow fluorescing substances, and the visible yellow-orange colors are not diagnostic for indole derivatives. The Renz and Loew
43-47
reagent is claimed to be more sensitive for indoles on TLC
48,49,62
than the Ehrlich
14-21
or van Urk
35-37
reagents. A comparative study of the three reagents on TLC with a number of biologically important indoles, such.' indole-3-acetic acid (Iaa), tryptophan (Trp) and indole-3-acetyl esters, has shown
63
, that the p-dimethylaminocinnamaldehyde
(P-DMAC)
reagent is 3-8 times less sensitive for most of the indoles. In addition the
p-DMAC
reagent develops a yellow to red background within 12 h which makes the subsequent identification of the colored indole condensation products difficult. The Ehrlich and van Urk reagents are, to date, the most specific chromogenic reagents for indole derivatives., but color development is slow~ (3-8 h) and the colors are not stable, due to the mineral acid retained on the silica gel layer. We hay reported a modified van Urk spray reagent procedure25 which resulted in considerable color stability, but color development was slow (5-8 h). Color development with the Salkowski
6-13
reagent is rapid (15-30 min), but the colors change quickly to non-diagnostic brown tones. The sensitivity is about 10-fold less than for the Ehrlich and van Urk reagents, and has poor specificity for indoles except for Iaa and some Iaa derivatives.
A spray reagent has now been developed that has a high sensitivity and specificity for indole compounds, gives rapid color development and color stability of the indole condensation products.
EXPERIMENTAL
Materials
1
Sources of in doles.
The (indole-3-acetyl)-myo-inositols, di-O-, and tri-O-(indole-3-acetyl)-myo-inositols, (indole-3-acetyl)-myo-inositolglycosides, 2-0-, 4-0-, and 6-0-- (indole-3-acetyl)-D-glucopyranoses, N-(p-coumaryl)-tryptamine and N-ferulyltryptamine were isolated from sweet Corn kernels of
Zea mays25,29-31.
The 1-0-(indole-3 acetyl)-p-D-glucopyranoside was a gift from Dr. D. Keglevic (Institute "Ruder] Boskovic", Bijenicka 54, Zagreb, Yugoslavia). ; Other indoles were obtained from the following sources: 5-benzyloxy-6-' methoxyindole, 6-benzyloxy-5-methoxyindole, N-acetyl-indole, indole-3-acetonitrile; and 5-methoxytryptophol from Regis (Morton Grove, Ill., U.S.A.); l-methylindole, from Eastman-Kodak (Rochester, N.Y., U.S.A.); ethylindole-3-acetate, indole-3- propionic acid, indo~-3-butyric acid and indole-3-acetyl-L-aspartic acid from Cal- biochem (La Jolla, Calif., U.S.A.); tryptophol, gramine, tryptamine HCI and bufotenine from Sigma (St. Louis, Mo., U.S,A.). All other indoles were obtained from Aldrich (Milwaukee, Wisc., U.S,A.). '
Indole standards.
The indoles were dissolved in either absolute ethanol, 50%: ethanol, 2-propanol or chloroform to give 1- or 2-
µ
g/
µ
l solutions. From these stock solutions serial dilutions containing 10, 25, 100, 200 and 500 ng/
µ
l were prepared
Solvents and reagents.
Methanol, ethanol, 2-propanol, 2-butanone, ethylacetate and chloroform were reagent grade and further purified with activated charcoal and fractional distillation. The purity was monitored by UV. Water was distilled, deionzed, and redistilled- in an all-glass distillation apparatus. The reagents were made a follows. (A) van Urk
37
reagent: 1 g p-dimethylaminobenzaldehyde (Aldrich) decolorized with activated carbon and recrystallized from ethanol-water (m.p.
74.5°),
was dissolved in 50 ml conc. HCl (specific gravity 1.190) and 50 ml absolute ethanol was added; this reagent is stable for several months at room temperature when stored in a brown glass bottle. (B) Salkowski
6
reagent (as modified by Tang and Bonner
9
) 2.03 g FeCI3. 6 H2O were dissolved in 500 ml water and 300 ml conc. H2SO4 (specific gravity. 1.840); this reagent is stable indefinitely.
Spray
reagent.
The new TLC spray reagent used, was made up of reagent A and B (1 :3). The spray reagent may be kept at room temperature for several weeks
Silica gel TLC plates.
Precoated silica gel G TLC glass plates with or without fluorescent indicator and a layer thickness of 0.25 room were used throughout this study (E. Merck, Elmsford, N.Y., U.S.A.).
TLC solvent systems.
The indoles listed in Table I were chromatographed in one of the following solvent systems: (1), butanone-ethyl acetate-ethanol-water (3:5:1 :1); (2) propanol-water (8:2); (3), propanol-water-28 % ammonium hydroxide (8:1 :1); (4), chloroform-methanol-water (84:14:1).
Methods
Visualization of indoles.
After one-, or two-dimensional TLC of indole standards or partially purified extracts containing indolylic compounds in the appropriate. solvent system the plate was dried at
45°
until all traces of solvent had evaporated (5-10 min). The dry plate could then be examined under UV for fluorescing and/or quenching spots. Spraying of the TLC plate was done in a fume hood, using a glass atomizer such as a Desaga standard glass atomizer (100-ml capacity; Brinkman Westbury, N.Y., U.S.A.) connected to an air line. The plate was sprayed evenly in an upright position until the silica gel layer became transparent. If the plate was accidentally over sprayed the excess reagent on the silica gel layer could be removed with a paper towel. The plate was heated in a 100° oven for 5 min, then removed from the oven and allowed to cool to room temperature. Heating up to 10 min had no adverse effect on the indole condensation products, but heating for more than 10 min caused a graying of the silica gel background. The plate was immersed in distilled water (2-3 1 per 20 X 20-cm plate), agitated periodically for 1 min. The plate was washed two more times as before. Thorough washing of the TLC plate was necessary to assure the complete removal of acids since it was found that incomplete washing often resulted in yellowing of the silica gel within a few weeks. The plate was removed from the last water wash and blotted with a dry paper towel. At this time the colors of the indole condensation products were evaluate. (Table I; wet-plate color reading). The plate was then dried at
45°
(20-30 min). The colors of the indole condensation products were evaluated once more (Table I; dry-plate color reading). The colors of the indole condensation products are extremely stable and fade resistant. We have kept TLC plates at room temperature in the dark
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Substitution Name of compound Color region Color Name(page no.) Limits of Detection
(ng)
C-2,
C-3,C-5 5-Chloro-2-methylindole-3-acetic acid reddish violet Rose Purple (140) 75 reddish violet Pastel Mauve (127) 100 5-Methoxy-2-methylindole-3-acetic acid reddish violet Pansy Violet (116) 25 bluish violet Pastel Lavender (129) 25 EthyI2-ethoxy-5-hydroxy-indole-3- light umbra 50 Carboxylase yellowish green Pod Green (120) 25 2,3,5- Trimethylindole light brown rose 100 reddish orange Orient Pink (124) 100
C-
-.3, C-6 Reserpine green Cyprus Green (59) 300 Yellowish green Pod Green (120) 300 Rescinnamine bluish green Verdigris (88) 600 yellowish green Pod Green (120) 600
For more than two years with little or no fading of the original dry-plate colors. However the silica gel background tended to become slightly grey-yellow if the plates were stored for more than six months. This could be eliminated by covering the dry TLC plate with permanent, invisible mending tape (Highland-Brand No. 6200; 3M . 0., St. Paul, Minn., U.S.A.) or similar tape. TLC plates covered with this tape have been kept for more than one year with no detectable color changes of the indole condensation products, and have retained a white silica gel background;
Purity and identity of indole standards.
In order to evaluate the color reaction of each indole standard listed in Table I the indoles were chromatographed at 1-; 5-, 10- and
20-
υ
g
concentrations in one of the four solvent systems to assure a minimum migration of 6 cm of each indole from the origin (solvent front, 10 cm). After the plates were sprayed and processed as described above, they were examined for possible secondary chromogenic spots. In addition each indole standard was analyzed by gas-liquid chromatography and the identity confirmed by mass spectrometry (GLC-MS)
65
.
Colors of the indole condensation products:.
Each indole standard with the exception of the indole derivatives isolated from mature sweet corn karyopses (see
Sources of indoles)
was spotted at 1-, 5-, 10- and
20-
µ
g
concs. on a 5 x 10-cm TLC plate. The corresponding spot areas were 20, 30 and 60 mm2. The plate was sprayed and processed as described above. After the third water wash of the plate and removal of excess water the color of the indole condensation product was matched by eye with one of the 200 plates of 64 full hues, 60 tints, 38 shades and 38 grayed hues of the Horticultural Color Chart
66
. The 200 plates had been arranged according to color families, and the matching could be done routinely in less than 1 min (Table I; wet-plate color reading). After the plate had been dried the color was matched again (Table I; dry-plate color reading). In addition each color was rematched three more times after 6, 12, and 24 h to evaluate any color changes which might have occurred. The color evaluation of the indole condensation products of the indole derivatives isolated from mature sweet com of
Zea mays
was done the same way except that the indoles were first chromatographed on a 20 X 20-cm TLC plate at 1-, 5-, and
10
υ
g
concentrations in solvent 1.
274 TLC of Indole Compounds
Limits of detection.
The indoles were chromatographed at concentrations ranging from 10-1500 ng in the appropriate solvent systems and a minimum spot migration of 6 cm. The limit was determined as the smallest amount of indole to give. a 5-7mm
2
detectable color spot. RESULTS AND DISCUSSION The colors of 79 indole condensation products with p-DMAB on silica gel acid TLC and their limits of detection are listed in Table I. Most of the indoles can readily. be detected at the 25-50-ng level which makes this reagent a very sensitive and indole specific chromogenic reagent. Certain phenols and aromatic acids are known to give positive color reactions with p-DMAB-HCI (Ehrlich reagent)
67
. A number of hydroxy:. and aminobenzoic acids, hydroxy-, and methoxycinnamic acids were examined and found that only p-aminobenzoic acid (yellow) and p-coumaric acid (pink) give a positive color reaction. However the limit of detection for these compounds is about 40-80 times (1-2
µ
g)
higher than for most of the indoles tested. The indoles in Table I are arranged according to their substitution(s) on the indole ring system. It has been reported that condensation of the indole derivative with p-DMAB occurs at the free C-2 position and results in a violet-blue color product if C-3 has a -CH2-R group
68
. A structural analysis of a number of indole condensation products by low- and high-resolution MS has shown that condensation of p-DMAB can also occur at C-3, N-1, and to a limited extent on C-5 and C-6 of the. indole ring system (see Table I, I-methylindole-2-carboxylic acid, carbazole and 1I-phenylcarbazole)
69
. Although many of the C-3 substituted indoles such as IAA and its esters tryptamine and its derivatives have colors in the- reddish-violet-blue color region., most exhibit sufficient color differentiation to allow their identification on TLC. We have chosen the Horticultural Colour Chart to illustrate this color differentiation (Table I, color region and color name). Frequently it is possible to identify tentatively 18 endogenous indole derivatives from plant extracts by their colors and
RF
values on TLC, and the subsequent identification by GLC-MS confirms the great diagnostic' :~ value of the chromogenic spray reagent.
Another advantage of the spray reagent for TLC is the relatively simple procedure. It takes about 10 min from the time of spraying until the wet-plate colors can be evaluated, and less than 1 hr for the permanent color evaluation. The majority of: the colors of the indole condensation products are extremely stable. TLC plates of some 40 indole standards at 1- and
5-p.g
concentrations and developed in solvent systems (1) and (4) have been kept for more than two years with virtually no fading , or change of the original dry-plate colors. Some indoles, such as indole, I-methylindole, l-acetylindole, 2-methylindole and indole-3-acrylic acid exhibit a characteristic color change as the plate dries (see Table 1). It was found that this color shift and or fading is reversed to the original, wet-plate color by rewetting the plate. It is thus possible to evaluate the wet-colors of those indoles which in the dry- plate state are no longer diagnostic. The colors of some of the indoles have been reversed as often as ten times in one day with no apparent loss of color or color intensity. 3 An important aspect of this reagent is the finding that it can also be used as
TLC OF INDOLE DERIVATIVES 275
a colorimetric reagent with a sensitivity and specificity for many indoles equal to that on TLC. The use
of
this reagent for the colorimetric determination of indole derivatives will be published in a separate communication..
ACKNOWLEDGEMENTS
I thank Dr. Robert J. Moon for the generous gift
of
5-hydroxy-indole-3-acetic acid, N,N-dimethyl-5-hydroxy-tryptamine and N-acetyl-5-hydroxy-tryptamine, and Dr. Robert S. Bandurski for his interest in this work. This work was supported in part by the National Science Foundation (GB- 40821-X). REFERENCES ~ I E. Stahl,
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-------- End of Journal Article ------
In order to better use the information here, color photographs of tryptamines of interest are included below. These are not indicated in the document, so these are provided for reference. Color references are provided via common household items.
Psilocybin and 5meo-DMT 5 – HTP and d-Lysgeric Acid, Diethylamide (LSD-25)
5-Meo DMT, DiPropyl Tryptamine (DPT) 5-HTP , LSD –25
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