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New solvents for paper chromatography of phenolic acids
619
NOTES
New
solvents for paper chromatography
of phenolic
acids
The two solvent systems: (A) isopropyl alcohol-ammonia-water (8 :I :I) and (13) the upper layer of the two phase mixture of benzene-propionic acid-water (2 : 2 :I) are reputed to give good separations in two-dimensional chromatography of phenolic acidsls2. In a study of urinary phenolic acids with this pair of solvents, crowding of spots was seen near the starting line for the second solvent. This was generally unavoidable if development was conducted at temperatures below IO'. Under these conditions there was hardly any movement of nz-hydroxyhippuric acid, g-hydroxyindoleacetic acid, $-hydroxymandelic acid and $-hydroxyhippuric acid, in the second solvent. In urine there are a number of diazo-reacting compounds with Rn values which are even less than those of the above-mentioned aromatic acids in solvent 13, therefore the solvent pair A-B is not of much use if one is especially interested in the study of these compounds. New solvents were therefore studied to overcome the difficulties mentioned above. Ex$erimental Urines were extracted according to SMITH2. Phenolic acids from 20 ml urine were extracted into 0.5 ml of 95 o/oalcohol. Extracts were pooled from three different urinary samples ancl the final solution used in this study was prepared by adding to the pooled mixture 5 mg each of o-hydroxybenzoic acid, ferulic acid and vanillic acid. Ten inch square sheets of filter paper, Whatman No. I, iYere used for chromatography. The following solvent systems were used in this study: I. Solvents A and E. 2. Ether-xylene-formic acid-water (50 : 30 : I o :3) ; solvent C. 3. Ether-ethyl acetate-ethyl alcohol-ammonia-water (5 : 5 : 5 : 2 :3) ; solvent D. This solvent was best prepared by first mixing all the constituents except ethyl alcohol in a stoppered cylinder and then adding etl;::ri alcohol gradually to obtain a clear solvent. The ether used in solvents C and D was kept over ferrous sulfate crystals. 4, Isopropyl alcohol-?z-butyl alcohol-tert.-butyl alcohol-ammonia-water (4 : 2 : 2:1:2); solvent E. Solvents D and E were used for the first dimension and solvent C for the second one. In order to test the utility of the solvents under extremes of temperature in winter and summer, chromatograms were run at 40” and at a temperature between 2O Xp values were calculated from the urinary chromatograms prepared by and -5”. the ascending technique of paper chromatography. Results and disczcssion RF values of various urinary phenolic acids in the above solvents at the two different temperatures are shown in Table I. Low temperature affected the usefulness of solvent B. the most. Once the temperature was less than IOO, there was hardly any migration of the spots I a, rb, 4, 5, 6 and 2 L (see Fig. L). In the case of solvent C, however, it was interesting to note that the RF values were higher at the lower temperature. This solvent gave similar resultsat all temperatures. Fig. I compares two urinary chromatograms, one obtained with J. Chromatog.,
xg (1965)
Gxg-62z
l
.
6r I5
8
i
55 3’ I7 I5 24 46 18 9 3 5
74 37
52 ;s 38 45 76 44 3’ 8 24 28 26 25
38 28
26 26
89 :;
-
7 7
0
38 86
0
69 :: 53 69 80
8
IO
ii 22
I3
2.5 Is
0
0
7I 6:
;
0
37 50 I9 44 55
42
2
29
15 II
327 7
50 4g
2
II
42 39
3h
27 29
I9
IS
6h
the
Temp. 20, tittte
Temp. 2' Temp. to --Jo, 400”: lime Y h
Temp. 4o", time
B
A
Rp ( x IUO)ia solvelat l l
-
5
87 :;
92 60
24 38 16
IO
29 52
0
26 57
0
56 49 46 56 49
25 ‘7
30 @ 34
30
20
I.25 h
tittze
Tewp. 4o”,
0
29 64
0
64 :: 58 63 59
“5
59 40 52
28 45
2O. little 2h
Temp.
C
* Solvents: A = Isopropyl alcohol-ammonia-water
’ Spot No. from the list of urinary phenolic acids published by SAINIet al!. (8: I :I). B = Upper layer from benzene-propionic acid-water (2:2:I). C = Ether-xylene-formic acid-water (50:30: 10:3). D = Ether-ethyl acetakethyl alcohol-ammonia-water (5 : 5 : j : 2 : 3). E = Isopropyi alcohol-n-butyl alcohol-tert.-butyl alcohol-ammonia-water (4: 2 :2 :I :2). ? Identity doubtful.
o-Hydroxybenzoic acid Ferulic acid
‘3 I4 I5 ‘7 I8 I9 20 21
II
26 25
p-Hydroxyhippuric acid
;
-m-Hydroxyphenylacetic acid o-Hydroxyhippuric acid p-Hydroxybenzoic acid ? Homovanillic acid Vanillic acid -! Feruoylglycine -. ? Vanilloylglycine
p-Hydroxyphenylacetic acid 5-Hydroxyindoleacetic acid
; 4
I 9 IO
wHydroxyhippuric acid p-Hydroxymandelic acid
Comfoued
I00)OI?URINARYPHENOLICACIDS IN DIFFERENTSOLVENTS
Ia Ib
spot No.’
Rp~.umS(x
TABLE I
61 22
33 59 29 16 9 I3 16 I5 I4
22
58 38 29
24 18
36 2 I9
26
I4 h
tittze
8r 50
2; 79 50 50 58 56 56 45 I3 35 45 39 35
38
2 62 44
55
4h
tittte
::
27 "3 :; 33 25 37 57 34 20 9 I5 20 I9 18
22
43
33 34
z
24 30 28 22
II
:; 43 32
:; 37 36
;:
48 47 3I
4’ 40
5h
tittte
I4 h
40'.
time
to-j',
Temp_ 2' Tewp_
40°,
Temp_2 O Tewp_ to-j',
E
D
621
NOTES
io!vents A and I3 and the other with the new solvents D and C. First development of ~3 hours in each case was carried out at a temperature varying from 2' to -5" and the distance covered by solvent D was 1.5 times the distance covered by solvent A. Account was taken of this fact in drawing Fig. I. The second development was conducted at 2'. The most remarkable feature of solvent C, however, was its usefulness for the study of urinary phenolic acids whose RF values are vexy low in solvent 53. Using this solvent, a number of spots in urinary chromatograms were encount.eredG which we Solvent
front
.
260
60
3% SO
30 140
180
I28
25c9’0 150
2:,” 6NO
‘i
e
Solvent
C
4
2 nd direction(Temp.
-E g
Solvent
C
v:
2”, time
2h 1
@OrIginI
;E .o G L ._ u t; P
front
a 80
7@2
110 250 150 Solvent
8
100
2 nd direction(Temp.
2: time 3h)
Fig. I. Two-dimensional chromatograms showing positions of various urinary phcnolic acids. Spot 6N is yellow (unstained chromatogram). reddish-brown, which fades, with sulfanilic reagent, deep purple with nitxanilinc reagent, reddish-brown to purple-brown with bcnzidine resgent3. Compounds corresponding t;o other numbers are mentioned in Table I. For solvents see the legend to Table I. J. Chvomatog.,
rg (1965) 619-622
‘.
622
NOTES
had not seen previously with solvents A and B*. Some of these spots were missed with solvent I3 because they were spread out diffusely instead of separating as discrete spots. This was at least true of a spot labelled as 6N in the figure, A combination of solvents I3 and C was found very ‘useful for rapid work. At a temperature of 25" a development of 20 cm could be accomplished with solvent D in 4-5 h; 2 h was sufficient for solvent C to run 20 cm. In fact these ether solvents were unsuitable for very long runs except at temperatures below 10~. These solvents evaporated off the papers in a matter of minutes and heating of the chromatograms in an oven was not required prior to staining with the diazo reagents. Solvent E was as good as solvent A at all temperatures although it migrated faster than solvent A. I am grateful to Dr. S. S. MAKHNI for some useful suggestions in this work, and wish to thank Mr. RAM NIWAS for his technical help. De$artment x M. 2
I.
oj Physiology,
Medical
D. ARMSTRONG,I<. N. j?. SMITH,
Cltromatogra+l~ic
SHAW
College, Rohtak
AND
I?.
(India)
A. S.
SAINI
E. WALL,J. Dial. Chem., 218 (1956) 293, Techniques, Vol. I, Heinemann, London, 1960,
awd Electvop7toretic
p.291.
3 A. S. SAINI, J. Amt. Sot. India, 12 (IgG3) 26. 4 A. S. SAINI, I. D. SINOH,V. R. BAJAJ AND A. SINGH, I&ia?z J. Med. RIM., 52 (1965) 1173, 3 A. S. SAINI, to be published.
Received July QIst, 1964 J.
Clwomato~.,
rg (1965)
Gig-G22
Evidence for the ninhydrin-positive
reaction of some ketoses*
The violet, red, or blue color produced when amino acids react under appropriate conditions with ninhydrin has been attributed to the presence of free a-amino groups, Recent reports indicate, however, that this test is not as specific as was once thought. SCHRAMet aZ.‘Jobtained a red color when ninhydrin was reacted with a non-nitrogencontaining carbohydrate fraction from a protein hydrolyzate. ZACHARLUSAND TALLEY~ have shown that levulinic acid reacts with ninhydrin to give a red color and also have reported that several other keto acids and related compounds give a color of relatively low intensity. While following by chromatographic examination the course of the oxidation of meso-erythritol to L-erythrulose by Acetobacter szcboxyda&, a faint reddish area was detected with ninhydrin and this did not correspond with amino acids. The area when located with an orcinol* or silver nitrateG reagent had the same I?pructose value as known r.,-erythrulose in two irrigant systems, ethyl acetate-pryidine-water (8 : 2 :I, * This work was partially supported by a of I-Eealth. J. Ckvoma’tog.,
xg (1963)
622-G23
Grant No. GM 077x4 from the National Institutes
NOTES
New
solvents for paper chromatography
of phenolic
acids
The two solvent systems: (A) isopropyl alcohol-ammonia-water (8 :I :I) and (13) the upper layer of the two phase mixture of benzene-propionic acid-water (2 : 2 :I) are reputed to give good separations in two-dimensional chromatography of phenolic acidsls2. In a study of urinary phenolic acids with this pair of solvents, crowding of spots was seen near the starting line for the second solvent. This was generally unavoidable if development was conducted at temperatures below IO'. Under these conditions there was hardly any movement of nz-hydroxyhippuric acid, g-hydroxyindoleacetic acid, $-hydroxymandelic acid and $-hydroxyhippuric acid, in the second solvent. In urine there are a number of diazo-reacting compounds with Rn values which are even less than those of the above-mentioned aromatic acids in solvent 13, therefore the solvent pair A-B is not of much use if one is especially interested in the study of these compounds. New solvents were therefore studied to overcome the difficulties mentioned above. Ex$erimental Urines were extracted according to SMITH2. Phenolic acids from 20 ml urine were extracted into 0.5 ml of 95 o/oalcohol. Extracts were pooled from three different urinary samples ancl the final solution used in this study was prepared by adding to the pooled mixture 5 mg each of o-hydroxybenzoic acid, ferulic acid and vanillic acid. Ten inch square sheets of filter paper, Whatman No. I, iYere used for chromatography. The following solvent systems were used in this study: I. Solvents A and E. 2. Ether-xylene-formic acid-water (50 : 30 : I o :3) ; solvent C. 3. Ether-ethyl acetate-ethyl alcohol-ammonia-water (5 : 5 : 5 : 2 :3) ; solvent D. This solvent was best prepared by first mixing all the constituents except ethyl alcohol in a stoppered cylinder and then adding etl;::ri alcohol gradually to obtain a clear solvent. The ether used in solvents C and D was kept over ferrous sulfate crystals. 4, Isopropyl alcohol-?z-butyl alcohol-tert.-butyl alcohol-ammonia-water (4 : 2 : 2:1:2); solvent E. Solvents D and E were used for the first dimension and solvent C for the second one. In order to test the utility of the solvents under extremes of temperature in winter and summer, chromatograms were run at 40” and at a temperature between 2O Xp values were calculated from the urinary chromatograms prepared by and -5”. the ascending technique of paper chromatography. Results and disczcssion RF values of various urinary phenolic acids in the above solvents at the two different temperatures are shown in Table I. Low temperature affected the usefulness of solvent B. the most. Once the temperature was less than IOO, there was hardly any migration of the spots I a, rb, 4, 5, 6 and 2 L (see Fig. L). In the case of solvent C, however, it was interesting to note that the RF values were higher at the lower temperature. This solvent gave similar resultsat all temperatures. Fig. I compares two urinary chromatograms, one obtained with J. Chromatog.,
xg (1965)
Gxg-62z
l
.
6r I5
8
i
55 3’ I7 I5 24 46 18 9 3 5
74 37
52 ;s 38 45 76 44 3’ 8 24 28 26 25
38 28
26 26
89 :;
-
7 7
0
38 86
0
69 :: 53 69 80
8
IO
ii 22
I3
2.5 Is
0
0
7I 6:
;
0
37 50 I9 44 55
42
2
29
15 II
327 7
50 4g
2
II
42 39
3h
27 29
I9
IS
6h
the
Temp. 20, tittte
Temp. 2' Temp. to --Jo, 400”: lime Y h
Temp. 4o", time
B
A
Rp ( x IUO)ia solvelat l l
-
5
87 :;
92 60
24 38 16
IO
29 52
0
26 57
0
56 49 46 56 49
25 ‘7
30 @ 34
30
20
I.25 h
tittze
Tewp. 4o”,
0
29 64
0
64 :: 58 63 59
“5
59 40 52
28 45
2O. little 2h
Temp.
C
* Solvents: A = Isopropyl alcohol-ammonia-water
’ Spot No. from the list of urinary phenolic acids published by SAINIet al!. (8: I :I). B = Upper layer from benzene-propionic acid-water (2:2:I). C = Ether-xylene-formic acid-water (50:30: 10:3). D = Ether-ethyl acetakethyl alcohol-ammonia-water (5 : 5 : j : 2 : 3). E = Isopropyi alcohol-n-butyl alcohol-tert.-butyl alcohol-ammonia-water (4: 2 :2 :I :2). ? Identity doubtful.
o-Hydroxybenzoic acid Ferulic acid
‘3 I4 I5 ‘7 I8 I9 20 21
II
26 25
p-Hydroxyhippuric acid
;
-m-Hydroxyphenylacetic acid o-Hydroxyhippuric acid p-Hydroxybenzoic acid ? Homovanillic acid Vanillic acid -! Feruoylglycine -. ? Vanilloylglycine
p-Hydroxyphenylacetic acid 5-Hydroxyindoleacetic acid
; 4
I 9 IO
wHydroxyhippuric acid p-Hydroxymandelic acid
Comfoued
I00)OI?URINARYPHENOLICACIDS IN DIFFERENTSOLVENTS
Ia Ib
spot No.’
Rp~.umS(x
TABLE I
61 22
33 59 29 16 9 I3 16 I5 I4
22
58 38 29
24 18
36 2 I9
26
I4 h
tittze
8r 50
2; 79 50 50 58 56 56 45 I3 35 45 39 35
38
2 62 44
55
4h
tittte
::
27 "3 :; 33 25 37 57 34 20 9 I5 20 I9 18
22
43
33 34
z
24 30 28 22
II
:; 43 32
:; 37 36
;:
48 47 3I
4’ 40
5h
tittte
I4 h
40'.
time
to-j',
Temp_ 2' Tewp_
40°,
Temp_2 O Tewp_ to-j',
E
D
621
NOTES
io!vents A and I3 and the other with the new solvents D and C. First development of ~3 hours in each case was carried out at a temperature varying from 2' to -5" and the distance covered by solvent D was 1.5 times the distance covered by solvent A. Account was taken of this fact in drawing Fig. I. The second development was conducted at 2'. The most remarkable feature of solvent C, however, was its usefulness for the study of urinary phenolic acids whose RF values are vexy low in solvent 53. Using this solvent, a number of spots in urinary chromatograms were encount.eredG which we Solvent
front
.
260
60
3% SO
30 140
180
I28
25c9’0 150
2:,” 6NO
‘i
e
Solvent
C
4
2 nd direction(Temp.
-E g
Solvent
C
v:
2”, time
2h 1
@OrIginI
;E .o G L ._ u t; P
front
a 80
7@2
110 250 150 Solvent
8
100
2 nd direction(Temp.
2: time 3h)
Fig. I. Two-dimensional chromatograms showing positions of various urinary phcnolic acids. Spot 6N is yellow (unstained chromatogram). reddish-brown, which fades, with sulfanilic reagent, deep purple with nitxanilinc reagent, reddish-brown to purple-brown with bcnzidine resgent3. Compounds corresponding t;o other numbers are mentioned in Table I. For solvents see the legend to Table I. J. Chvomatog.,
rg (1965) 619-622
‘.
622
NOTES
had not seen previously with solvents A and B*. Some of these spots were missed with solvent I3 because they were spread out diffusely instead of separating as discrete spots. This was at least true of a spot labelled as 6N in the figure, A combination of solvents I3 and C was found very ‘useful for rapid work. At a temperature of 25" a development of 20 cm could be accomplished with solvent D in 4-5 h; 2 h was sufficient for solvent C to run 20 cm. In fact these ether solvents were unsuitable for very long runs except at temperatures below 10~. These solvents evaporated off the papers in a matter of minutes and heating of the chromatograms in an oven was not required prior to staining with the diazo reagents. Solvent E was as good as solvent A at all temperatures although it migrated faster than solvent A. I am grateful to Dr. S. S. MAKHNI for some useful suggestions in this work, and wish to thank Mr. RAM NIWAS for his technical help. De$artment x M. 2
I.
oj Physiology,
Medical
D. ARMSTRONG,I<. N. j?. SMITH,
Cltromatogra+l~ic
SHAW
College, Rohtak
AND
I?.
(India)
A. S.
SAINI
E. WALL,J. Dial. Chem., 218 (1956) 293, Techniques, Vol. I, Heinemann, London, 1960,
awd Electvop7toretic
p.291.
3 A. S. SAINI, J. Amt. Sot. India, 12 (IgG3) 26. 4 A. S. SAINI, I. D. SINOH,V. R. BAJAJ AND A. SINGH, I&ia?z J. Med. RIM., 52 (1965) 1173, 3 A. S. SAINI, to be published.
Received July QIst, 1964 J.
Clwomato~.,
rg (1965)
Gig-G22
Evidence for the ninhydrin-positive
reaction of some ketoses*
The violet, red, or blue color produced when amino acids react under appropriate conditions with ninhydrin has been attributed to the presence of free a-amino groups, Recent reports indicate, however, that this test is not as specific as was once thought. SCHRAMet aZ.‘Jobtained a red color when ninhydrin was reacted with a non-nitrogencontaining carbohydrate fraction from a protein hydrolyzate. ZACHARLUSAND TALLEY~ have shown that levulinic acid reacts with ninhydrin to give a red color and also have reported that several other keto acids and related compounds give a color of relatively low intensity. While following by chromatographic examination the course of the oxidation of meso-erythritol to L-erythrulose by Acetobacter szcboxyda&, a faint reddish area was detected with ninhydrin and this did not correspond with amino acids. The area when located with an orcinol* or silver nitrateG reagent had the same I?pructose value as known r.,-erythrulose in two irrigant systems, ethyl acetate-pryidine-water (8 : 2 :I, * This work was partially supported by a of I-Eealth. J. Ckvoma’tog.,
xg (1963)
622-G23
Grant No. GM 077x4 from the National Institutes