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The Circular Ring Chromatographic Method:


General:

The capillary absorption property of specifically selected filter paper for chromatography (on Whatman #1) is used in order to separate different and specific fractions of organic fluids, extracting fluids, etc. Filter paper discs 146 mm. in diameter are used.

The discs are placed on a petri dish with a wick in the center; a hole is cut into the center' the wick extends downward into a small crystallization dish holding the extracted solution (see sketch); the solution is sucked upward, by way of capillarity, through the wick and expands into the horizontally placed disc.

The wick is 20 mm. in length and 2 mm. in diameter. It is important that all wicks are of the same dimensions. They are made by tightly rolling up a filter paper strip of 20 mm. width and 20 mm. length.

The discs need "preparation" or "development" with suitable reagents in order to bring out the properties and chemical reactions.

Specific Method in this Case of Comparing "untreated" and "Treated" Samples:

1. The first step is the preparation of the dishes. A 0.5% solution of nitrate of silver (AgNo3) is placed in the lower dish and migrates through the wick into the horizontal disc. It is allowed to penetrate until the diameter of the absorbed area is 40 mm. This will show up as a moist, almost colorless zone. It is to be done in darkness or mild diffused light, not in bright daylight or sunlight.

2. The absorption is interrupted by removing the solution and the discs are allowed to dry.

3. The prepared dry discs are placed with another wick over the solution to be tested and the absorption is allowed to continue until the expansion of the outer zone has reached 60 mm.

4. The wick is removed and the discs are allowed to dry.

5. The discs should not be exposed to bright daylight or sunlight.

6. For keeping, place between non-absorbent paper layers; avoid moist room. Never inspect in bright light.

The discs, which are attached to this report, were exposed to bright light, until we learned the hard way that they are light sensitive.

The absorption and development are carried on in a special glass enclosed box under controlled moisture and temperature conditions. The best temperature is 200 to 25o C. The moisture 30% to 40% at the beginning.

Results of Chromatography: (The second repetitive (2 5 49 days) sample were used.)

A. 1. Turmeric Powder, Untreated: 1 gr. extracted in 20cc. aqua dest.
Outer circular edge: Chocolate brown, radius 55 to 57 mm.
Lobular edge zone (33 lobes, average), in waves, with rounded forms, color light gray-brown.
Second ring: Lighter yellowish brown, rays lighter, radius 30 to 33 mm.
Inner zone: Bluish - violet pinkish color, turning lighter and pinkish towards center, radius 22 mm.
Center halo: Yellow-pinkish, yellow irregular, radius 3 to 5 mm.

2. Turmeric Powder, Treated: 1 gr. extracted in 20 cc. aqua dest.

Outer circular edge: Light chocolate brown, radius 60mm.
Lobular edge zone with rounded forms: Less gray than in same zone of "untreated", 40 lobes average, little more organized than (1), radius 51 to 53 mm.
Second ring: Yellowish-brown, separation from lobular ring less sharp,
against inner ring more pronounced with a sharper reddish edge, radius 40 mm.
Inner zone: More intense pink, radius 24 to 28 mm.
Center halo: More intensive yellow color, radius about 4 mm.

B. 1. Figs. Untreated: 3 gr. extracted in 30 cc. aqua dest.

Outer ring: Dark brown, radius 54 to 58 mm.
Lobular ring: Inverted medium brown, irregular, formed like "villi", 18 to 20 l lobes; the inner zone extends in small colorless lobes into it, radius
30 to 55 mm.
Lobular ring: Inverted medium brown, irregular, formed like "villi", 18 to 20
lobes; a light brown halo around the villi; the inner zone extends in small colorless lobes into it, radius 30 to 55 mm.
Second ring: Violet, thin sharp ring, radius 45 mm.
Inner zone: Pale brown, radius 30 to 40 mm.
Center halo: Very slightly darkened.

2. Figs. Treated: 3 gr. extracted in 30cc. aqua dest.
Outer ring: Less dark brown than the untreated, radius 54 to 55 mm.
Lobular ring: Inverted with regular rounded lobes, radius 37 to 53 mm.,
light brown, 23 lobes.
Inner ring: Vermillion color, bright sharp demarcation lines, radius 31 to 38
mm., lost color on exposure to light, not present in "untreated".
Inner zone: Reddish, faint hue, radius 30 to 31 mm.
Halo: Faint pink.
All reddish, pink and yellowish colors faded or darkened upon exposure to
light and aging. It does not now show its original brightness. The untreated
sample was "dull" from the beginning.

C. 1. Raisins, Untreated: 3gr. extracted from 30 cc. aqua dest.
Lobular edge zone: Irregular, dark brown with bluish tinge and yellow brown small halo, radius 54 to 60 mm., 30 to 32 indentations ("twins" counted as one).
Outer zone: Irregular, radial, brown color, radius 40 to 54 mm., some extensive, lobular, but small into inner zone.
Inner zone: Yellow brown, light, radius 40 mm., a few spots here and there.
Due to exposure to light, there was considerable darkening and discoloration, especially of the inner zone.

2. Raisins, Treated: 3gr. extracted with 30 cc. aqua dest.
Lobular edge zone: Entirely different from "untreated".
Very much serrated, deep indentations with a brown (now darkened) borderline on the inside of the indentations, radius 42 to 60 mm. 32 indentations on the average (: twins: counted as one).
Second outer zone: None but a violet ring, sharp, disappearing through
exposure to light.
Inner zone: Yellow-brown, radial, very bright. All areas have darkened
considerable through aging.

The difference in color and pattern as compared with the untreated is quite
remarkable and speaks for the strongest effect of the treatment as against the other three samples.

D. 1. Almonds, Untreated: 2 gr. extracted in 40 cc. aqua dest.
Edge zone: Medium dark brown with a small lighter brown halo, no differentiation, and radius 54mm. on the average.
Second zone: Weavy, undulated, small; light brown with bluish tinge,
inside lighter halo, radius 44 to 50 mm.
Third zone: Strongly undulated with faint radial spots, radius 39 to 44 mm.
Inner zone: Very light chocolate brown; radial lobes, small on the outside, radius 18 to 40mm.
Large white halo, radius 15 to 20 mm.
Spotty vermillion halo, irregular, radius 10 to 15 mm.

Most colors have either darkened or faded with aging.

2. Almonds, Treated: 2 gr. extracted in 40 cc. aqua dest.
Edge zone: Reddish chocolate brown with lighter outer halo and lighter
inner edge, radius 40 to 45 mm. Much more contracted as against (1).
Second zone: Light yellow brown, undulated sharp edge zone with
inverted faint dark brown lobulation; undulated area about 10 to 12 mm. in thickness.
Third zone: Inside of undulated area; light reddish brown, radius 23 to 30
mm.
Halo: Bright mild pink, a little irregular, radius 13 mm. Faded through
exposure to light and aging.

Summary:

In each of the four cases a significant reaction of the treatment could be observed in formation as well as in color. The bright colors of the treated samples have suffered upon exposure to light colors of the treated samples have suffered upon exposure to light and by aging, and the present colors do not resemble th e original ones. Raisins and figs showed spectacular differences. Almonds showed the least differences.

Under ultraviolet light the zones show different fluorescence.
From a chemical point of view it may be said that the differences are due to different proteins and amino acids, which could be identified.

The zones can be cut out, dissolved again and the individual characteristics determined in a spectrophotometer, with the addition of specific reagents. We did not follow up this separation since the macroscopic effect was spectacular enough to show the differences.

E.E. Pfeiffer

June 29, 1954.

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