Investig&ons USA the mechanism chin iqws of polyamide CV. Chromatography HALINA
and
fietectiuiky of chromatography
of amino acids and complex
SZUMI-Z.0 u?d EDWARD
on
phenofic s&stances
.SO&EmSKI
Department of Inorganicwii Analyticai Ckemilrtry,institute of hbsic Chemicai Sciences, Medical ScAool, 2U-081 hbh (Pofaxd) (First
receivedDecemberlSth, 1975; m&d
rmmscript
receiv&
March 29t&, 1976)
In the preceding papers on polyamide thin-layer chromatography (TLC)*-‘, the mech-anism of chromatography in polyamide systems was investigated by the dilution method, using hydroxy derivatives of bee e and naphthalene as test solrrtes. In this paper, the results obtained for a number of phenolic acids and fiavones and also for strongly hydrophilic amino acids are reported_ The developing solvents were chosen based on experience with the chromatographic analysis of phenols and naphthols; as in preceding papers, binary solvent systems suitable for gradient elution were employed. All of the compounds investigated were strongly retained by the polyamide sorbent as a result of their molecular structure, especially the presence of proton donor groups (OH and COOH) capable of interaction with the peptide groups of the polymer. From the earlier investigations with phenols’- and literature data5m6, it wonid be expected that the compounds would require the use of polar solvents with considerable elution strength; the results co-ed these expectations. EXPERIMENTAL
The solutes were chromatographed on thin iayers of polyamide (Woelm, Eschwege, G.F.R.) sapported on glass plates’. Flavones and phenolic acids were detected with bis-diazotized benzidine and amino acids with mod&d ninhydrin reagent’ (a 0.3 % solution of ninhydrin in nbutanol containing 3 X of glacial acetic acid); the plates were sprayed with this reagent and then heated in an oven at 110” for 2-3 min. The & vaiues are given in Tables I and II . F=UL-IS
AND DISCUSSION
III the 6rst series of experimenti, binary solvents containing one-polar compozzent of class 3 or AB were used, with cyclohexane as diluent. owing to ‘Lhestrong sorption of phenolic acids .znd flavones, low & V&ES were obtained, except for high
-02
w
-08
02
XS &.
-06
04 I
-04
-02
-06
-04
-0.2
a
3i HCA 10
Fig. 1. RM versus log Xs rciationshipsfor phenolic acids for developing solvents composed of cyc!ohexane and ethanol (S). Fig. 2. RM versus log X, relationshipsfor phenolic acids for devetopingsoIventscomposed of cyclohexale and propalol (S).
of the
polar solvents (Figs. I and 2)_ The R,, wmm log X, relationships = 0.0. Therefore, in the present experiments binary mobile phases consisting of two polar solvents were employed : solutions of acetic acid in acetone CAB j B)‘, ethanol or propan (AI3 + AB). The RF values obtained for these systems (Figs. 3 and 4) were markedEy higher tkan for polar solvents diluted with cyclokexane. The R, versus log X, relationships (where S denotes the more polar component, acetic acid) were approximately linear for solutions of acetic acid in ethanol and propznol. On the other hand, for solutions of acetic acid in acetone the curves passed through concez&&ions
were linear and mostly in the rzmge above-&
maxima at higher concentrations of acetic acid (Fig. 4). The decrease in RF values at kigk X, v&es Is probably caused by increasing dimerization of acetic acid and swelling of the polyamide. The occurencc of maxima on the curves suggests that in gradient etution for systems of this type the concentration of acetic acid in the _-die& should not exceed ca. 40-50 vol.-%. An advantage of these systems is the
formation of compact, well defined spots of the solutes (good capacity and high efficiency of the chromatographic system). Three flavones (rutin, quercetin and morin) were chromatographed in the same solvent systems. For solve& systems of lower elution strength, suck as solutions of ethanol or propanol in cyclohexane, low RF values were obtained even at the highest concentrations of the polar soivent. For solvent systems such as cyclokexane + pyridine, prop=01 + acetic acid and acetone -I- acetic acid, the RF values were in tke range Q.L-0.5. It is worth noting the unexpected behaviour of rutin, which in all instances had kigk.er RF values than those of morin and quercetin. There are
rndOO0
Fig_ 3. ROW&tk.s log X, relationships for pheenoiic acids and flavones for devefoping solvents composed of propanol and acetic acid (S). Fig. 4_ Rew versus log X, relatio&xips for phenolic acids and flavones for developing solvents cornpa& of acetone and acetic acid (S).
-.‘0 t -08
c -04 -02
CO
02
04
06
,- 08
Fig_ 5. Ru- &~LS tog XS rdationships for phenolic acids and flavones for developing sokms ~&cd of cycfahkqne and pyridhe (s),
_-
com-
numemts hyd&yl grca~ps in the mofecufe of rutin and it w&d be ex$.xted th& it work% be more &ros@y sorb@ by polyamide thati t&e tw& other ffauones, w-i&h have fewer hydrtixyt&oups. However, the resultsindicated a relatively weak r$ention of r&in (Figs. 3-§}, wfiich snigbt be &used by a m&c&r S_;eveeE..t that cont%ols the petration of molecutes of &E&e+ moE*dar &nensions into the poEyamide ge19-f:. This effectplays a fkdamental role in the-sepqtion of molecties according to their size-in gel permeation chrom&ogra&y with pofyacrylamide, ~0Eystyreneand dextrangels, tid it might also occurfor pt3yamidegel. If the penetrationafthe larger mokcuks of rutin into the sorbei~t were limikd, this ~wouId explain its hi&er RF Values.
Although aliphatic amines (di-n-propylakine, diethylamine and triethylatie) are stronger bases than pyridine, it w&s found that the last solvent is a much better eluent of *be solutes in polyamide systems. For cyclohexane solutions of tines; the RF values of the phenolic acids Were equal OF close tu zero. On the other hand, for developing sok~~ts containing pyridine, the R,- values were mu& higher, the spots compact and well defined and the R,< versus log X, relationships linear. In the next series of experiments, strongly polar compotids (amino acids) interacted strongly with the a&de groupings of polyamide .arrdtheir small moiecilles were able easily to penetrate into the polyamidegel. Strong sorption of amino acids requiredthe application of s-p&al solvent systems,which, @thou& relatively sbp!e, could interact competitively with amino acids in the mobile phase. Most solvent sys-
p?hed ASP Fig.
6.
hexme
Ru
9ersxs
log
X.
1 reIation5~ps
for amine
acids for developing soivents.6omposc-d
Of Cycle-
for zmizo
acS.s for developing sokrZ~
Of CYdO-
and propionic acid(S).
Fig_ 7. &kus log Xs relatkmskips and propionic acid f.S)_
hexme
-P
_ _
k~mposed
_-
._--
m&i#e
Acetylglutamic
AbbreS&km
sid
Ac-GIu
iX--Ahdle
CcAla
/3-Akdne
g: Abu Arg Asp
p-Phenyfakmine a-Aminobotyrk acid Arginine As&c acid Aspvagine .Cysteic acid Cyzteine GIutamic acid Glutamine GSycine H&amine Histidine
Glu Gln
Creatinine Leucine Isoleucine Lysine
Hydroxylysine
Methioniue Omithine Proline Hydroxyproline Tbreocine ?-rypt0pbaII Tyrasice 3,$dZodotyrosiue Valtie Nomaline
Leu LYS
5Hyi
Fro
Cyciakeanre f
Benzetz -5
propionic
acetic acid (SJ
acid (S)
0.2
0.4
0.6
0.8
1.0
0.3
0.4
0.5
0.6
0.8
2 0 2 2 0 0 0 0 0 0 30 18 0 8 0 6
S ~6 10 11 3 2 2 825 0 5 60 51 6 17 4 25
22 21 42 27 23 7 8
54 43 62 52 53 20 17 50 0 34 75 80 33 m 46
67 64 82 64 71 44 32 70 0 48 86 82 48 67 67
11
25
9 27 9 15
20 37 22 27
43 35 50 32 45
63 47 62 52 58
79 65 74 65 72
0
Z
It
0 0 0 _
7 0 0
12 9 0 -
23 28 17 0 -
57 44 44 0 -
41 -
50 -
67 62 5.5 51 37 43 13 63 36 50 41 27 37 56 65
69 67 68 63 62 75 55 71 55 60 57 40 52 67 76
46 56 75 65 75 40 15 65 42 75 50 67 57
67 80 80 80 85 75 45 85 80 85 80 85 75
0 I5 70 74 I8 35 20
3 2 0 0 0 0 4 0 07 3 4
13 10 3 3 9 I 17 7 7 IL
56 43 30 12 :1 27 5 55 20 32 24 20
2 4
1; 15
22 3s 45
8
-
15 -
25 _ _--9 20 28 4 25 35 ci 60 17 34 47 20 35 55 2 7 15 0 2 7 15 22 40 2 s 16 34 50 64 2'..10 28 10 20 35 I4 27 6 ---7 20 27 10 22 35 ----
4.5 65 62 75
tems of the N -+- A and N -t_ B types gave very low RF values; good results were obtained with the binary solvent systems bemae + acetic acid and cyclohexane + propionic acid. Typical R, VIEWSleg X, retatiomhips for some of the ami~~o acids investigated are presented in Figs. 6-9. The relationships are linear over wide composition rakges (-I& = constant + nlog X,) and the slopes (n) are mostly high, usually in the range 3-5. Only for tyrosine, glutamic acid, glutamine, #Manine and proline were the Lines less steep (slope CQ.2.0); for amino acids with m aromatic ring the slope was ea. 3.8. The. use of non-aqueous solvents containing acetic acid for the chromatography of amino acids was found to be advantageous owing to satisfactory selectivity and compact,.well de&& spots. It seems that acetic and propionic acids give optimai swe!!L&gof pdtyamide and srrfiicient Ioosetig of its structure, w
and
fietectiuiky of chromatography
of amino acids and complex
SZUMI-Z.0 u?d EDWARD
on
phenofic s&stances
.SO&EmSKI
Department of Inorganicwii Analyticai Ckemilrtry,institute of hbsic Chemicai Sciences, Medical ScAool, 2U-081 hbh (Pofaxd) (First
receivedDecemberlSth, 1975; m&d
rmmscript
receiv&
March 29t&, 1976)
In the preceding papers on polyamide thin-layer chromatography (TLC)*-‘, the mech-anism of chromatography in polyamide systems was investigated by the dilution method, using hydroxy derivatives of bee e and naphthalene as test solrrtes. In this paper, the results obtained for a number of phenolic acids and fiavones and also for strongly hydrophilic amino acids are reported_ The developing solvents were chosen based on experience with the chromatographic analysis of phenols and naphthols; as in preceding papers, binary solvent systems suitable for gradient elution were employed. All of the compounds investigated were strongly retained by the polyamide sorbent as a result of their molecular structure, especially the presence of proton donor groups (OH and COOH) capable of interaction with the peptide groups of the polymer. From the earlier investigations with phenols’- and literature data5m6, it wonid be expected that the compounds would require the use of polar solvents with considerable elution strength; the results co-ed these expectations. EXPERIMENTAL
The solutes were chromatographed on thin iayers of polyamide (Woelm, Eschwege, G.F.R.) sapported on glass plates’. Flavones and phenolic acids were detected with bis-diazotized benzidine and amino acids with mod&d ninhydrin reagent’ (a 0.3 % solution of ninhydrin in nbutanol containing 3 X of glacial acetic acid); the plates were sprayed with this reagent and then heated in an oven at 110” for 2-3 min. The & vaiues are given in Tables I and II . F=UL-IS
AND DISCUSSION
III the 6rst series of experimenti, binary solvents containing one-polar compozzent of class 3 or AB were used, with cyclohexane as diluent. owing to ‘Lhestrong sorption of phenolic acids .znd flavones, low & V&ES were obtained, except for high
-02
w
-08
02
XS &.
-06
04 I
-04
-02
-06
-04
-0.2
a
3i HCA 10
Fig. 1. RM versus log Xs rciationshipsfor phenolic acids for developing solvents composed of cyc!ohexane and ethanol (S). Fig. 2. RM versus log X, relationshipsfor phenolic acids for devetopingsoIventscomposed of cyclohexale and propalol (S).
of the
polar solvents (Figs. I and 2)_ The R,, wmm log X, relationships = 0.0. Therefore, in the present experiments binary mobile phases consisting of two polar solvents were employed : solutions of acetic acid in acetone CAB j B)‘, ethanol or propan (AI3 + AB). The RF values obtained for these systems (Figs. 3 and 4) were markedEy higher tkan for polar solvents diluted with cyclokexane. The R, versus log X, relationships (where S denotes the more polar component, acetic acid) were approximately linear for solutions of acetic acid in ethanol and propznol. On the other hand, for solutions of acetic acid in acetone the curves passed through concez&&ions
were linear and mostly in the rzmge above-&
maxima at higher concentrations of acetic acid (Fig. 4). The decrease in RF values at kigk X, v&es Is probably caused by increasing dimerization of acetic acid and swelling of the polyamide. The occurencc of maxima on the curves suggests that in gradient etution for systems of this type the concentration of acetic acid in the _-die& should not exceed ca. 40-50 vol.-%. An advantage of these systems is the
formation of compact, well defined spots of the solutes (good capacity and high efficiency of the chromatographic system). Three flavones (rutin, quercetin and morin) were chromatographed in the same solvent systems. For solve& systems of lower elution strength, suck as solutions of ethanol or propanol in cyclohexane, low RF values were obtained even at the highest concentrations of the polar soivent. For solvent systems such as cyclokexane + pyridine, prop=01 + acetic acid and acetone -I- acetic acid, the RF values were in tke range Q.L-0.5. It is worth noting the unexpected behaviour of rutin, which in all instances had kigk.er RF values than those of morin and quercetin. There are
rndOO0
Fig_ 3. ROW&tk.s log X, relationships for pheenoiic acids and flavones for devefoping solvents composed of propanol and acetic acid (S). Fig. 4_ Rew versus log X, relatio&xips for phenolic acids and flavones for developing solvents cornpa& of acetone and acetic acid (S).
-.‘0 t -08
c -04 -02
CO
02
04
06
,- 08
Fig_ 5. Ru- &~LS tog XS rdationships for phenolic acids and flavones for developing sokms ~&cd of cycfahkqne and pyridhe (s),
_-
com-
numemts hyd&yl grca~ps in the mofecufe of rutin and it w&d be ex$.xted th& it work% be more &ros@y sorb@ by polyamide thati t&e tw& other ffauones, w-i&h have fewer hydrtixyt&oups. However, the resultsindicated a relatively weak r$ention of r&in (Figs. 3-§}, wfiich snigbt be &used by a m&c&r S_;eveeE..t that cont%ols the petration of molecutes of &E&e+ moE*dar &nensions into the poEyamide ge19-f:. This effectplays a fkdamental role in the-sepqtion of molecties according to their size-in gel permeation chrom&ogra&y with pofyacrylamide, ~0Eystyreneand dextrangels, tid it might also occurfor pt3yamidegel. If the penetrationafthe larger mokcuks of rutin into the sorbei~t were limikd, this ~wouId explain its hi&er RF Values.
Although aliphatic amines (di-n-propylakine, diethylamine and triethylatie) are stronger bases than pyridine, it w&s found that the last solvent is a much better eluent of *be solutes in polyamide systems. For cyclohexane solutions of tines; the RF values of the phenolic acids Were equal OF close tu zero. On the other hand, for developing sok~~ts containing pyridine, the R,- values were mu& higher, the spots compact and well defined and the R,< versus log X, relationships linear. In the next series of experiments, strongly polar compotids (amino acids) interacted strongly with the a&de groupings of polyamide .arrdtheir small moiecilles were able easily to penetrate into the polyamidegel. Strong sorption of amino acids requiredthe application of s-p&al solvent systems,which, @thou& relatively sbp!e, could interact competitively with amino acids in the mobile phase. Most solvent sys-
p?hed ASP Fig.
6.
hexme
Ru
9ersxs
log
X.
1 reIation5~ps
for amine
acids for developing soivents.6omposc-d
Of Cycle-
for zmizo
acS.s for developing sokrZ~
Of CYdO-
and propionic acid(S).
Fig_ 7. &kus log Xs relatkmskips and propionic acid f.S)_
hexme
-P
_ _
k~mposed
_-
._--
m&i#e
Acetylglutamic
AbbreS&km
sid
Ac-GIu
iX--Ahdle
CcAla
/3-Akdne
g: Abu Arg Asp
p-Phenyfakmine a-Aminobotyrk acid Arginine As&c acid Aspvagine .Cysteic acid Cyzteine GIutamic acid Glutamine GSycine H&amine Histidine
Glu Gln
Creatinine Leucine Isoleucine Lysine
Hydroxylysine
Methioniue Omithine Proline Hydroxyproline Tbreocine ?-rypt0pbaII Tyrasice 3,$dZodotyrosiue Valtie Nomaline
Leu LYS
5Hyi
Fro
Cyciakeanre f
Benzetz -5
propionic
acetic acid (SJ
acid (S)
0.2
0.4
0.6
0.8
1.0
0.3
0.4
0.5
0.6
0.8
2 0 2 2 0 0 0 0 0 0 30 18 0 8 0 6
S ~6 10 11 3 2 2 825 0 5 60 51 6 17 4 25
22 21 42 27 23 7 8
54 43 62 52 53 20 17 50 0 34 75 80 33 m 46
67 64 82 64 71 44 32 70 0 48 86 82 48 67 67
11
25
9 27 9 15
20 37 22 27
43 35 50 32 45
63 47 62 52 58
79 65 74 65 72
0
Z
It
0 0 0 _
7 0 0
12 9 0 -
23 28 17 0 -
57 44 44 0 -
41 -
50 -
67 62 5.5 51 37 43 13 63 36 50 41 27 37 56 65
69 67 68 63 62 75 55 71 55 60 57 40 52 67 76
46 56 75 65 75 40 15 65 42 75 50 67 57
67 80 80 80 85 75 45 85 80 85 80 85 75
0 I5 70 74 I8 35 20
3 2 0 0 0 0 4 0 07 3 4
13 10 3 3 9 I 17 7 7 IL
56 43 30 12 :1 27 5 55 20 32 24 20
2 4
1; 15
22 3s 45
8
-
15 -
25 _ _--9 20 28 4 25 35 ci 60 17 34 47 20 35 55 2 7 15 0 2 7 15 22 40 2 s 16 34 50 64 2'..10 28 10 20 35 I4 27 6 ---7 20 27 10 22 35 ----
4.5 65 62 75
tems of the N -+- A and N -t_ B types gave very low RF values; good results were obtained with the binary solvent systems bemae + acetic acid and cyclohexane + propionic acid. Typical R, VIEWSleg X, retatiomhips for some of the ami~~o acids investigated are presented in Figs. 6-9. The relationships are linear over wide composition rakges (-I& = constant + nlog X,) and the slopes (n) are mostly high, usually in the range 3-5. Only for tyrosine, glutamic acid, glutamine, #Manine and proline were the Lines less steep (slope CQ.2.0); for amino acids with m aromatic ring the slope was ea. 3.8. The. use of non-aqueous solvents containing acetic acid for the chromatography of amino acids was found to be advantageous owing to satisfactory selectivity and compact,.well de&& spots. It seems that acetic and propionic acids give optimai swe!!L&gof pdtyamide and srrfiicient Ioosetig of its structure, w
