Alkanoic Alkanedioic Hydroxy Keto Unsaturated Arylalkanoic Acids and Other Related Acids of Biological Significance

n-Alkanoic acid The separation of these acids by the technique of TLC with respect to the lower homologous fatty acids has a historic precedent in that their separation in the vapour phase on a column coated with a stationary phase was the first published example of gas chromatography.

Although it might be generally considered that gas chromatography is more suitable than TLC for the separation of alkanoic acids, Table 1 shows some simple conditions that have been used in this series typical of a partition separation. Many of the values quoted in the ensuing tables have been adapted from extensive published information by Hanai (see Further Reading). For comparison, the hRF values of the dibasic acids malonic, succinic, glutaric and adipic in solvent a are 9, 14, 18 and 22 respectively, and that of glycolic acid, 38. The first four acids in the table have also been examined on crystalline cellulose impregnated with sodium bicarbonate in ethanol-water (100:20) and detection by dicyclohexyl carbodiimide to separate formic acid, acetic, propionic and butanoic acids having the hRF values 31, 37, 45 and 52 respectively.

n-Alkanedioic acids The saturated dibasic acids have been more widely studied on a variety of layers and solvents, as illustrated in Table 2 which again, as with the monobasic series, shows partition separations. In cases where a considerable number of solvents have been listed, the optimum conditions for the series of compounds have been given. For comparison, the hRF value of glycolic acid under the conditions of g was 38. In another separation on silica gel (sil G25, Macherey Nagel) with the solvent n-pentyl formate-chloroform-formic acid (70 : 15 : 15) and detection by bromocresol green, nonlinearity was found in that malonic, succinic, glutaric and adipic acids had hRF values of 40, 43, 54 and 48 respectively. Folic acid, which may be regarded as a 2-acylamino derivative of glutaric acid, had an hRF value of 0 compared with 78 for nicotinic acid on silica gel G in water as developing solvent.

Hydroxy acids It is convenient to classify this group of saturated acids as monohydroxy, monohydroxy-

Table 1 hRF values of homologous alkanoic acids on starch and on cellulose layers Alkanoic acid Conditions Detection b

Table 1 hRF values of homologous alkanoic acids on starch and on cellulose layers Alkanoic acid Conditions Detection b

Formic

52

8

Fluorescein UV 254 nm

Methyl red

Acetic

56

19

Fluorescein UV 254 nm

Methyl red

Propionic

66

28

Fluorescein UV 254 nm

Methyl red

Butanoic

71

37

Fluorescein UV 254 nm

Methyl red

Pentanoic (valeric)

78

48

Fluorescein UV 254 nm

Methyl red

Hexanoic (caproic)

85

59

Fluorescein UV 254 nm

Methyl red

a, Ethanol-water-concentrated ammonia (78:20:13), rice starch; b, light petroleum (40-60°C)-acetone (2:1) 95% saturated with ethane-1.2-diol, cellulose and Dowex® 50 W. (With acknowledgement to Hanai, 1982.)

a, Ethanol-water-concentrated ammonia (78:20:13), rice starch; b, light petroleum (40-60°C)-acetone (2:1) 95% saturated with ethane-1.2-diol, cellulose and Dowex® 50 W. (With acknowledgement to Hanai, 1982.)

Table 2 hRF values of n-alkane-a,«-dioic acids (dibasic acids) on various layers

Dibasic acid

Conditions

Table 2 hRF values of n-alkane-a,«-dioic acids (dibasic acids) on various layers

Dibasic acid

Conditions

a

b

c

d

e

f

g

Oxalic (C2)

16

0

6

Malonic (C3)

21

52

20

7.5

9

Succinic (C4)

37

63

38

27

25

59

14

Gultaric (C5)

46

71

47

32

31

74

18

Adipic (C6)

55

82

55

37

38

84

22

Pimelic (C7)

50

94

Suberic (C8)

58

100

Azelaic (C9)

67

Sebacic (C10)

72

Undecyl (C„)

82

a, Ethanol-concentrated ammonia-water (150:8:40), cellulose (Merck 5552); b, 2-ethyl-1-butanol-formic acid-water (40:12: 48); c, diethyl ether-light petroleum-CCl4-water-formic acid (50:20:20:8:1); polyamide 6; d, ethanol-concentrated ammonia-water (100:16:12), cellulose MN300; e, din-butyl ether-formic acid-water (65:25:2.2), cellulose (Merck 5716); f, toluene-propionic acid-water (47:47:4.9), silica gel (Merck 5721); g, ethanol-concentrated ammonia-water (78: 13: 20), rice starch. (With acknowledgement to Hanai, 1982.) The use of formic acid diminishes streaking sometimes found in the TLC of acids in neutral solvents. It is thought that in acidic solvents the formation of a dimeric intermolecularly hydrogen-bonded species is then favoured in the equilibrium with the monomericform, while in basic solvents the monomeric anion is largely present. Acidic adsorbents may likewise simulate acidic solvents.

hco2h _

(Modified with permission from Hanai, 1982.)

dibasic, monohydroxytribasic, dihydroxydibasic and polyhydroxy types. Table 3 lists the hRF values of a number of acids with this functionality. For comparison, the hRF value of malonic acid under condition f was 40 and in the aromatic series that of mandelic acid (a-hydroxyphenylacetic acid) was 57. In general, cellulose has been used as adsorbent in examples a to e and silica gel in f. In early work, silica gel G-kieselguhr (1:1), kieselguhr impregnated with polyethylene glycol and polyamide layers were also employed. It is possible that in acidic developing solvents certain of these acids are present as intra-molecularly hydrogen-bonded structures and that five-membered are likely to be more stable than six-membered rings. Thus glycolic and lactic acids would be expected to have high hRF values whereas acids having hydrogen-bonded rings and additional acidic groups would have lower values. Under basic conditions with ammonia the solutes are more polar and the polarity of the developing solvent has to be increased by the use of ethanol. The meso and dl forms of tartaric acid show a small difference of hRF which can be enhanced by the use of silica gel impregnated with boric acid. It is also possible to separate the enantiomers of racemic hydroxy acids by the incorporation of a chiral additive in the adsorbent layer. The role of impregnated layers has been reviewed by Hauck et al. (see Further Reading).

Keto acids The hRF values of a number of mono keto derivatives of monobasic and dibasic acids are given in Table 4. The compounds shown from top to bottom in the table are glyoxylic, pyruvic, 2-oxobutanoic, 2-oxovaleric, 2-oxoisocaproic, oxalo-acetic and 2-oxoglutaric acid. The need of formic acid in high concentration to effect a separation is illustrated in d compared with f. For comparison, the hRF values under conditions d of citric and malic acids were 44 and 56 respectively. Intramolecular hydrogen bonding may account for the higher hRF values of the monobasic compounds. The cis and trans 2,4-dinitrophenylhydrazones of a range of keto acids have been examined.

Unsaturated monobasic dibasic and polybasic acids

The unsaturated acids are a large group which have technical and medicinal uses. The majority are either di- or tribasic. Table 5 summarizes the hRF values of a selection of compounds. Extensive details of separations have been described by Hanai and also in early work a limited range of monobasic keto-, hydroxy acids and of dibasic acids was studied. The separation of cis and trans isomers, for example maleic and fumaric acids, appears to be generally straightforward and free of the requirement for argentation TLC, as in the case of unsaturated fatty acids. The stereochemistry of the glutaconic acid described in Table 5 was not stated. The formulae of (1) trans-aconitic acid, (2) itaconic acid, (3) trans-glutaconic acid, (4) mesaconic acid (trans) and (5) citraconic acid (cis) are depicted.

Table 3 hRF values of hydroxyacids on various layers

Acid Conditions a b c d e f

Glycolic, HOCH2CO2H 67 46 50 31

Lactic, HOCH(CH3)CO2H (dl) 76 72 73 89 36

Malic, HO2CCH2CH(OH)CO2H (dl) 29 30 32 35 50 26

Citramalic, HO2CCH2C(Me)(OH)CO2H 65

Citric, HO2CCH2C(CO2H)(OH)CH2CO2H 16 11 18 23 42 22

IsoCitric, HO2CCH(OH)CH(CO2H)CH2CO2H 40

Glyceric, HOCH2CH(OH)CO2H 60 32 24 36

Tartaric, HO2CCH(OH)CH(OH)CO2H (dl) 24 19 18 31 19

Quinic, 1 r,3r,4r,5r-Tetrahydroxycyclohexane carboxylic 18

Ascorbic 15

a, Diisopropyl ether-formic acid, (3:1), cellulose MN 300HR, detection by UV;

b, ethanol-concentrated ammonia-water, (150: 8: 40), cellulose, (Merck 5552), detection by bromocresol green or starch-iodine reagent; c, 2-ethyl-1-butanol-formic acid-water, (40:12:48), cellulose, (Merck 5552), detection as in b; d, diisopropyl ether-formic acid-water, (65:25:10), cellulose (Merck 5716), detection by aniline-xylose, furfural; e, propanol-methyl benzoate-90% formic acid-water, (7:3:2:1), cellulose, detection by Pelskova and Munk reagent; f, n-pentyl formate-chloroform-formic acid, (70:15:15), silG25, detection by bromocresol green. (With acknowledgement to Hanai, 1982.)

c02h

Arylalkanoic acids Prior to an account of the TLC properties of aromatic acids it is of interest to note those of the semi-aromatic group typified by phenylacetic acid and its homologues and analogues. The hRF values of a range of these compounds are shown in Table 6. The need to use the least polar combination of solvents is illustrated by the conditions with c and d where the latter is ineffective while the former affords a separation of homologous compounds. In the case of the unsaturated compound, the separation in conditions d would almost certainly be improved with argentated silica gel.

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