Spectroscopic (FT-IR, FT-Raman, UV) and microbiological studies of di-substituted benzoates of alkali metals

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Spectrochimica Acta Part A 70 (2008) 126–135

Spectroscopic (FT-IR, FT-Raman, UV) and microbiological studies of di-substituted benzoates of alkali metals a , M. Borawska b , J. Piekut c , W. Lewandowski a,∗ ´ M. Kalinowska a , R. Swisłocka a

Department of Chemistry, Białystok Technical University, Zamenhofa 29, 15-435 Białystok, Poland b Department of Bromatology, Medical University, Kili´ nski 1, 15-089 Bialystok, Poland c Higher Vacational Scholl in Suwalki, 16-400 Suwałki, Teofila Noniewicza 10 street, Poland Received 3 April 2007; received in revised form 11 July 2007; accepted 15 July 2007

Abstract The FT-IR, FT-Raman and UV spectra of 3,5-dihydroxybenzoic and 3,5-dichlorobenzoic acids as well as lithium, sodium, potassium, rubidium, caesium 3,5-dihydroxy- and 3,5-dichlorobenzoates were recorded, assigned and compared. The theoretical geometries, Mulliken atomic charges, IR wavenumbers were obtained in B3LYP/6-311++G** level. On the basis of the gathered experimental and theoretical data the effect of metals and substituents on the electronic system of studied compounds were investigated. Moreover, the antimicrobiological activity of studied compounds against two species of bacteria: Bacillus subtilis, Staphylococus aureus and one species of yeast: Candida albicans were studied after 24 and 48 h of incubation. The attempt was made, to find out whether there is any correlation between the first principal component and the degree of growth inhibition exhibited by studied compounds in relation to selected microorganisms. © 2007 Elsevier B.V. All rights reserved. Keywords: 3,5-Dihydroxybenzoic acid; 3,5-Dichlorobenzoic acid; Alkali metal 3,5-dihydroxybenzoates; Alkali metal 3,5-dichlorobenzoates; Spectroscopy; Microbiology

1. Introduction Searching for new chemical compounds inhibiting the development of wide spectrum of microorganisms in low concentrations, do not possessing any allergic and carcinogenic properties and cheap and easy in production as well is justified by social and economic requirements. New disinfectants can replace presently applied ones which action is less and less effective (this may be due to the mutations of the organisms which growth used to be inhibited by these chemicals). Problem of transportation or storage, especially in case of products, for which physical methods (i.e. sterilisation, pasteurisation, draining) from different reasons cannot be applied, reduce to discovery of proper additives efficiently preventing the development of harmful microorganisms. Remarks concerning advantages and necessity of preservatives and disinfectants usage are presented in professional literature [1–3]. Many research centres performs researches on this field especially, that

∗

Corresponding author. Tel.: +48 85 469790; fax: +48 85 7469782. E-mail address: [email protected] (W. Lewandowski).

1386-1425/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.saa.2007.07.060

compounds showing properties inhibiting growth of microorganisms can find the use in the pharmaceutical, food, paper, cosmetic and textile industry. Benzoic acid is well known and applied for many years preservative, which activity is however limited, by the range of food pH and by sorts of microorganisms which growth it inhibits [4]. That is why we pay a large interest in benzoic acid derivatives. Some of them (e.g. esters) are applied in practice and posses even better preserving properties than acid [5]. Very large meaning has solubility of preservative in water or fats. Benzoic acid poorly dissolves in water (0, 29 g/100 g H2 O in 20 ◦ C), solubility of earlier mentioned esters decreases along with lengthening of aliphatic chain and therefore application of benzoic acid esters with aliphatic chain longer than seven to eight carbon atoms is difficult. The aim of this work is to study: (1) the molecular structure of differently di-substituted (hydroxy- and chloro-) benzoic acid derivatives and their alkali metal (Li, Na, K, Rb, Cs) salts by means of infrared (FT-IR), Raman (FT-Raman), UV methods and theoretical calculations; (2) the influence of these compounds on the growth of some harmful microorganisms; (3) dependencies between the molecular structure of investi-

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127

Fig. 1. The Mulliken atomic charges calculated for (a) 3,5-dihydro- (b) 3,5-dichlorobenzoic and (c) benzoic acids in the B3LYP/6-311++G**level.

gated molecules and their biological activity. In this way it may become possible to find compounds with specific activity not by a process of trial and error, but on the basis of strictly described dependencies between the structure and biological activity of compounds. Our previous investigations confirm the fact, that even little changes in spatial structure (e.g. change of position of the some substituents in the aromatic ring of benzoic acid) cause very large differences in the antimicrobial activity and that statistical dependencies occur between these values [6–8]. There are a few attempts of correlating biological activity of compounds (other than benzoic acid and its derivatives) with different physicochemical parameters [9–11] and the most

straight found dependence was correlation with frequency of bands observed in infrared spectrum [12]. In our study the statistical dependencies between antimicrobial properties of molecule and its electronic charge distribution (characterized by spectral parameters) will be studied. For microbial tests we chose the following bacteria Bacillus subtilis, Staphylococus aureus and one species of yeast’s Candida albicans. Seeking for new antimicrobial compounds one should pay attention to small molecules. Proposed by us molecules fulfil this condition—they have low molecular masses. For some of this compounds physicochemical properties have not been described yet in literature, and particularly

Fig. 2. The Mulliken atomic charges calculated for (a) 3,5-dihydro- and (b) 3,5-dichlorobenzoates in the B3LYP/6-311++G** level.

128

Table 1 The wavenumbers, intensities and assignments of bands from FT-IR spectra of 3,5-dihydroxybenoic acid and 3,5-dihydroxybenzoates 3,5-Dihydroxybenoic acid IR 3215 s

IR calc.

Raman

1106 w 1007 s 940 w 919 w 871 w 854 w 809 w – – 768 m 678 w 668 w 620 w 596 w – 564 w 532 vw 471 w

IR

IR

Raman

3220 w 3140 m

1789 vs 1662 m 1636 vs – 1514 m 1509 m – 1383 vs – 1372 w – – 1325 w 1305 s 1212 s 1194 w 1173 vs 1106 m 1010 w 940 m 890 w 785 m

1692 m 1631 m 1606 w 1516 vw – 1415 vw

1338 m 1302 w 1261 w 1219 w 1170 w 1108 w 1000 vs 946 w

674 m

602 w 588 m 586 s 554 w 523 w

3066 w 3044 w

Raman

IR

3061 w

2959 m

857 m

650 vw

3070 m

3276 s 3075 m

– – 772 vw – 670 w – 603 vw – 567 m 532 w 462 w

2574 m – 1611 m 1597 m 1545 s 1499 s – 1451 m – 1423 s 1360 vs 1302 vs – 1219 m 1159 s – 1104 w 1010 m – – 870 w 857 m – – 787 s – 684 vw 671 w 611 w – 581 m – – 481 m

s: strong; m: medium; w: weak; v: very; sh: shoulder

K 3,5-dihydroxybenzoate

3227 m 3072 w

Rb 3,5-dihydroxybenzoate

Raman

IR

3065 w

3229 m –

Raman 3058 w 3044 sh

Cs 3,5-dihydroxybenzoate IR 3163 m –

– 1421 m 1353 m 1329 w 1304 w – 1220 w – 1107 w 1001 vs 962 vw 874 vw

– 784 w – – – 613 vw 587 w 516 w 472 w

– 1623 m 1590 s 1565 vs 1509 m – 1449 w – 1413 s 1361 s 1334 m 1293 m – – 1205 w 1155 s – 1101 w 1004 m 953 w – 874 w 857 w – 839 m 792 m 774 m 686 w 668 w 614 w – 570 w – – 488 w

1634 m 1591 w 1565 w 1495 vw – – 1421 m 1354 w

–

1104 w 1004 vs 958 w 889 vw

839 vw 779 m –

609 vw 569 w 523 vw

– 1647 w 1620 s 1583 s 1510 m – 1455 m 1406 vs 1369 sh 1337 s 1301 m – – 1208 w 1164 m 1140 m 1105 w 1004 m 949 w – 8734 w 858 w – 843 w 790 m 763 m 696 vw 671 vw 616 vw – 562 vw – 522 vw 465 w

[18]

ν(OH) ν(CH)

20b

Raman 3059 w

ν(CH)ar ν(CH)ar

2928 w

1620 m 1600 sh 1551 vw – –

Assignments

1620 sh 1596 m – – –

1398 m

2562 w – 1649 m 1616 vs 1582 vs 1508 w – 1452 s 1398 s 1368 s

1348 m

–

– 1100 w 1002 vs 948 w

844 vw 783 vw 764 m 677 vw 615 w 573 w 530 w 471 w

1296 m – – 1204 m 1165 m 1139 s – 1003 m 947 w – 867 w 859 w – 845 m 787 m 764 s – 676 w 617 vw 602 vw 561 w – 522 w 462 w

1616 w 1594 w – – –

1395 m 1344 w 1304 vw –

1173 vw 1143 vw 1100 w 1000 vs 947 w 881 vw

846 vw 784 vw 763 m 679 vw 614 w 571 w 528 w 466 w

2638 m 2584 m – 1639 sh 1611 s 1579 s 1510 m – 1448 m

1625 sh 1607 m – 1500 vw –

1393 vs –

1394 m

1305 s – – 1213 m 1165 s 1140 s 1089 w 1011 m 944 w – 877 w 852 m – 835 w 790 m 762 s 697 w 685 w 616 w 604 W 560 W – 522 w 454 w

1303 vw – 1204 vw 1170 vw 1138 vw 1105 vw 1000 vs 946 w

– 793 vw 760 m 697 w 620 w 562 m 521 w 460 vw

ν(C O)ar ν(CC)ar ν(CC)ar νas (COO− ) ν(CC)ar ν(CC)ar , β(OH) ν(CC)ar β(OH) νs (COO− ) ν(CC)ar ν(OH) phenolic β(CH) β(OH) carboxylic ν(CC)ar β(CH) β(OH) phenolic β(CH) β(CH) β(CH) γ(CH) β(CH) γ(CH) γ(CH) ␴(OH) phenolic β(C O) γ(CH) βas (COO− ) φ(CC) α(CCC) φ(CC) α(CCC) βs (COO− ) γ(OH) α(CCC) α(CCC)

8a 8b 19b 19a

14 13

9b 18a 12 7b 17b

11 4 1

6a 6b

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1701 s 1686 vs 1609 vs – 1514 m 1481 m – 1420 s – – 1331 s 1302 s 1263 m 1221 m 1200 m 1163 s

Na 3,5-dihydroxybenzoate

3773 s 3068 vw

3007 m 2930 m

Li 3,5-dihydroxybenzoate

Table 2 The wavenumbers, intensities and assignments of bands from FT-IR spectra of 3,5-dichlorobenoic acid and 3,5-dichlorobenzoates 3,5-Dichlorobenoic acid

Li 3,5-dichlorobenoic acid

Na 3,5-dichlorobenoic acid

K 3,5-dichlorobenoic acid

Rb 3,5-dihydroxybenzoate

Cs 3,5-dihydroxybenzoate

IR

IR

IR

IR

IR

IR

3412 m

1030 w 1008 w 920 w 908 w 875 m 808 m 769 m 708 m 659 m 576 w 537 vw 474 vw 427 vw 414 vw

3769 s 3230 w 3119 w 3216 w 1793 vs

1623 w 1601 s 1459 m 1445 m 1358 vs 1331 w – 1269 w 1200 vs 1142 s 1121 w 1114 m 1013 w – – – 918 w – 890 m 802 s – 678 vw 656 m 592 s 539 vw 475 m 434 w 416 w

Raman

3424 w

3403 w

3080 w – 1688 w – – 1603 s 1563 vs 1441 s – – 1398 s 1381 sh 1297 w –

3084 w – 1631 m – – 1599 s 1558 vs 1435 m – – 1399 s 1379 vs 1290 w – 1230 vw – 1169 w 1099 w

1586 w –

– 1170 w 1103 w 1028 w 1005 vw 906 vw 898 vw 872 w 807 m 791 m 774 w 697 vw 664 w – 547 vw – 489 w 430 w 408 vw

999 m

–

– 999 vw 908 vw 896 vw 873 w 806 m 797 s – – 661 w – 557 w – 486 w – 419 w

Raman

3083 w

1584 w 1559 w 1438 w

1389 w 1375 w

1169 vw 1099 w 1055 w 1000 s

894 w

797 w

419 vw 372 m

3632 w 3320 w 3217 w 3083 w – 1642 w – 1614 m 1596 s 1558 vs 1431 w – – 1396 sh 1379 s 1289 w – 1229 vw – 1170 w 1098 w – 998 vw 910 vw 894 vw 868 w 804 m 789 m – – 662 w – 552 w – 483 w 430 vw 417 w

Raman

3084 vw

1585 w – 1431 w

1388 w –

– – – 1000 m

–

–

418 vw 366 m

Raman

3637 m 3302 m 3180 w 3083 w – 1652 m – – 1596 s 1558 vs 1431 w – – – 1379 s 1290 w – – – 1169 w 1097 w – – 909 vw 892 vw 867 w 803 m 790 m – – 663 w – 551 vw – 482 w – 415 vw

3083 w

1585 w 1562 w 1430 w

1382 w

1172 vw 1101 w 1064 w 1001 s

893 vw

791 w

3635 m 3294 m 3146 m 3080 m – 1642 m – – 1594 s 1558 vs 1429 m – – – 1377 s 1290 w – – – 1167 w 1096 w – 1000 w 909 w 889 vw 866 w 803 m 789 m – – 663 w – 553 vw

Raman

3082 vw

1584 w 1559 w 1430 w

1379 w 1379 w

1170 vw 1103 w 1064 w 999 s

–

–

475 w 416 m 364 m

413 w

415 w 360 m

ν(OH) ν(CH) ν(CH) ν(CH) ν(C O) ν(CC)ar ν(CC)ar νas (COO− ) ν(CC)ar ν(CC)ar β(OH) ν(CC)ar νs (COO− ) β(CH) ν(OH) β(CH) β(CH) β(CH) γ(CH) γ(CH) γ(CH) γ(OH) γ(CH) βs (COO− ) γ(CH) γ(CH) γ s (COO− ) γ(CH) β(C O) γ(C O) βas (COO− ) α(CCC) α(CCC) α(CCC) α(CCC)

8a

8b 19a 19b 4

9b 12

6b

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3084 m 1706 vs – 1654 m 1636 m 1609 w 1570 s 1448 s 1426 m 1403 m – – – 1287 s 1236 m 1199 m 1163 m 1099 w

IR calc.

Assignments [18]

4

16a 16b

s: strong; m: medium; w: weak; v: very; sh: shoulder 129

471 w

520 w –

469 m

617 vw 466 m

M. Kalinowska et al. / Spectrochimica Acta Part A 70 (2008) 126–135

spectral data are missing. Moreover, the antimicrobial studies are fragmentary and taken in different conditions what make difficult to compare the results from various scientific centres.

4 φ(CC)

684 m 699 s

701 s

678 s –

678 w

942 sh 965 w

974 vw

886 s –

871 w

2. Experimental

17b γ(CH)

6b α(CCC)

130

2.1. Sample preparation The metal 3,5-dihydroxy- and 3,5-dichlorobenzoates were prepared by dissolving appropriate weighed amount of 3,5dihydroxy- and 3,5-dichlorobenzoic acid in aqueous solution of alkali metal hydroxides in a stoichiometric ratio 1:1. Then, water was evaporated at 120 ◦ C in a dryer.

– 1106 w 1163 s 1514 m 1686 vs 3,5-Dihydroxybenzoic acid

1701 s

1609 vs

– – 1077 m – 1296 m – 1509 m – 1601 vs – 1700 vs –

– –

1275 m 1063 m 1323 w 1483 s 1574 s 1669 vs

1634 vs

1101 w 1252 vs 1027 m 1144 vw 1290 vs 1296 vs 1455 s 1485 vs 1586 m 1615 s 1604 m 1581 w 1693 vs 1665 vs

Benzoic acid [16] 2-Hydroxybenzoic acid [16] 2,6-Dihydroxybenzoic acid 3-Hydroxybenzoic acid

19b ν(CC)ar 8b ν(CC)ar 8a ν(CC)ar ν(C O)

Table 3 The wavenumbers and intensities of selected wavenumbers from the FT-IR spectra of studied acids [4]

ν(C O)

18a β(CH)

7a β(CH)

2.2. Measurement The IR spectra were recorded with an Equinox 55 spectrometer within the range of 400–4000 cm−1 . Samples in the solid state were measured in KBr matrix pellets obtained with hydraulic press under 739 MPa pressure. The sample:KBr mass ratio was 1:300. Raman spectra of solid samples in capillary tubes were recorded in the range of 100–4000 cm−1 with a FTRaman accessory of a Perkin-Elmer system 2000. The resolution of spectrometer was 1 cm−1 . The UV spectra were recorded on a DR 4000U HACH spectrophotometer between 190 and 300 nm. The compounds were studied in aqueous solutions with concentrations 5 × 10−5 mol/l. To calculate optimized geometrical structures of studied compounds, a Density Functional Theory in B3LYP/6-311++G(d,p) level was used. Theoretical calculations were performed using the GAUSSIAN 98 W package of programs running on a PC computer. No imaginary frequencies for calculated structures were reported. Two species of bacteria: B. subtilis, S. aureus and one species of yeast’s: C. albicans were used for antimicrobial tests. (B. subtilis, ATCC 6633; S. aureus, ATCC 29213; C. albicans, ATCC; 10231.) The studied microorganisms were inoculated on broth medium and stored in 35 ◦ C for 24 h. The growth of tested cells were standardized using turbidimetry method by measuring optical density at 600 nm for B. subtilis, S. aureus and C. albicans with a V-2001 HITACHI spectrophotometer. The microbiological tests for studied compounds (the final concentration was 0.01% expressed as concentration of appropriate acid) were carried out in water medium. The ultra pure water was used (deionised water, resistance 18.2 M cm−1 ). The samples were incubated in 35 ◦ C for bacteria and 25 ◦ C for yeast. The increase in the number of colonies was estimated similarly 24 and 48 h after incubation. 3. Results and discussion 3.1. Assignments The wavenumbers, intensities and assignment of the bands occurring in the IR and Raman spectra of di-substituted acid and alkali metal 3,5-dihydroxy- and 3,5-dichlorobenzoates are presented in Tables 1 and 2, respectively. For ligands the theoretical IR are also included. The spectral assignments were

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Table 4 The wavenumbers and intensities of selected wavenumbers from the FT-IR spectra of studied sodium salts of benzoic acids

Na benzoate [16] Na 2-hydroxybenzoate [16] Na 2,6-dihydroxybenzoate Na 3-hydroxybenzoate Na 3,5-dihydroxybenzoate

8a ν(CC)ar

8b ν(CC)ar

νas (COO− )

νs (COO− )

18b

18a

17b

6b

ν(COO− )

– 1581 vs 1587 vs 1621 m 1623 m

1597 vs – 1621 vs – 1600 sh

1554 vs 1597 vs 1604 vs 1561 vs 1565 vs

1413 vs 1377 vs 1348 s 1408 vs 1413 s

1068 m 1030 w 1024 s 1108 w –

1029 m 1142 w 1068 w 1079 vw 1101 w

919 w 944 w – 883 w 874 w

617 m 467 vw 440 w 513 vw 488 w

141 220 256 153 152

done on the basis of the literature data [13–17] and theoretically calculated IR frequencies for ligands. The symbol “ν” denotes stretching deformation, “β” denotes in-plane deformation, “γ” denotes out-of-plane deformation; “φ(CC)” denotes the aromatic ring out-of-plane deformation, and “α(CCC)” denotes the aromatic ring in-plane deformation. Normal vibrations of the aromatic ring were given by Varsanyi [18]. Replacement of the carboxylic group hydrogen with a metal ion, brought about characteristic changes in the IR and Raman spectra of the metal 3,5-disubsituted benzoates in comparison with the spectra of ligands. The disappearance of bands of the symmetric and asymmetric valence vibrations: stretching v(C O) as well as γ(C O) of the carbonyl group; disappearance of stretching vibrations ν(O H) and deformation vibrations β(O H) at 1409 (IR) cm−1 ; appearance of bands of the asymmetric and symmetric vibrations of the carboxylate anion νas (COO− ), νs (COO− ) as well as βas (COO− ), βs (COO− ), and disappearance or changes in positions and intensities of some aromatic bands was observed. 3.2. The effect of alkali metal ions on the electronic structure of 3,5-dihydroxy- and 3,5-dichlorobenzoic acids 3.2.1. 3,5-Dihydroxybenzoic acid 3.2.1.1. IR and Raman spectra. The wavenumbers of the asymmetric and symmetric stretching vibrations of the carboxylic

anion in the spectra of 3,5-dihydroxybenzoates occurred in the range of 1583–1545 (IR) cm−1 , 1551–1565 (Raman) cm−1 and 1423–1393 (IR) cm−1 , 1421–1394 (Raman) cm−1 , respectively. The wavenumbers of the νs (COO− ) band from the IR and Raman spectra of dihydroxybenzoates decrease in along the series: Li → Na → K → Rb → Cs. Moreover an explicit increase in the difference between wavenumbers of asymmetric and symmetric stretches of the carboxylic anion vibrations ν(COO− ) from the IR spectra along the same series is noticeable. The magnitudes of the separation ν(COO− ) amount to: 122, 152, 177, 184 and 186 cm−1 for Li, Na, K, Rb and Cs 3,5-dihydroxybenzoates, respectively. Increasingly higher value of ν(COO− ) indicate an increase in the asymmetrization of the carboxylate anion structure. The wavenumbers of the asymmetric and symmetric in-plane deformations of the carboxylic anion in the spectra of 3,5-dihydroxybenzoates occurred in the range of 792–787 (IR) cm−1 , 793–779 (Raman) cm−1 and 581–560 (IR) cm−1 , 587–562 (Raman) cm−1 , respectively. The band βs (COO− ) from the IR spectra of dihydroxybenzoates was shifted towards lower wavenumbers in the series Li → Na → K → Rb → Cs. The difference between wavenumbers of βas (COO− ) and βs (COO− ) [i.e. β(COO− )] from the IR and Raman spectra of studied compounds changed in a regular fashion as well. Namely, along the series: Li → Na → K → Rb → Cs 3,5-dihydroxybenzoates the value of β(COO− ) changed in the following way: 206, 222, 228, 226,

Fig. 3. The structures of (a) 2,6-dihydroxybenzoic acid, (b) 2-hydroxybenzoic acid and (c) benzoic acid (the dashed line denotes the hydrogen bonds).

132

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illustrates the tendency of alkali metal cations for destabilizing the electronic system of benzoic and hydroxybenzoic acids.

Fig. 4. The degree of growth inhibition exhibited by studied compounds in relation to B. subtilis evaluated by means of turbidimetry method by measuring optical density of water solutions at 600 nm after 24 h incubation.

230 cm−1 (IR spectra) and 197, 210, 210, 213, 231 cm−1 (Raman spectra). Therefore, the influence of alkali metals on the carboxylic anion structure of 3,5-dihydroxybenzoic acid is clearly visible. The bands of the stretching vibrations of aromatic ring were located at 1686–1360 cm−1 (IR) and 1620–1353 cm−1 (Raman). The wavenumbers of bands no. 8a increase in the IR spectra of 3,5-dihydroxybenzoates in the series: Li → Na → K → Rb, and no. 14 along with Li → Na → K. The bands of in-plane deformations β(CH) occurred in the range 1305–949 cm−1 (IR) and 1304–962 cm−1 (Raman). One of these bands, namely 7b was shifted towards lower wavenumbers in the IR and Raman spectra of 3,5-dihydroxybenzoates along the series: Li → Na → K → Rb → Cs. The bands derived from the out-of-plane deformations γ(CH) were located in the range 919–835 cm−1 (IR) and 889–839 cm−1 (Raman). No regular shifts of these bands were observed in the spectra of studied compounds. The bands of the aromatic ring out-ofplane deformations φ(CC) occurred at 697–611 cm−1 (IR) and 697–609 cm−1 (Raman). The wavenumbers of bands no. 4 and φ(CC) located at 611–617 cm−1 increase in the IR spectra of 3,5dihydroxybenzoates in the series: Li → Na → K → Rb → Cs. The bands derived from the in-plane deformations of the aromatic ring α(CCC) were located in the range 685–454 (IR) cm−1 and 679–460 cm−1 (Raman). The band no. 6b was shifted in the direction of lower wavenumbers in the IR and Raman spectra of 3,5-dihydroxybenzoates along the same as above series. 3.2.1.2. UV spectra. The hipsochromic shifts of ␲ → ␲* bands in the UV spectra of sodium salts compared to UV spectra of ligands were observed. Benzoic acid absorbed at 227 nm, while sodium benzoate at 224 nm. For 2-hydroxybenzoic acid the maximum of ␲ → ␲* band was at 234 nm, and for sodium 2-hydroxybenzoate at 230 nm. 3,5-Dihydroxybenzoic acid absorbed at 243 nm, whereas sodium 3,5-dihydroxybenzoate at 241 nm. The hipsochromic shifts is caused by a increase in the gap between bonding and antibonding orbitals, what

3.2.2. 3,5-Dichlorobenzoic acid 3.2.2.1. IR and Raman spectra. The wavenumbers of the asymmetric and symmetric stretching vibrations of the carboxylic anion in the spectra of 3,5-dichlorobenzoates occurred in the range of 1563–1558 cm−1 (IR), 1562–1559 cm−1 (Raman) and 1381–1379 cm−1 (IR), 1377–1375 cm−1 (Raman), respectively. The wavenumbers of the symmetric and asymmetric in-plane deformations of the carboxylic anion in the spectra of 3,5dihydroxybenzoates occurred in the range 896–889 cm−1 (IR), 894–893 cm−1 (Raman) and 557–574 cm−1 (IR), respectively. Only the band βs (COO− ) was shifted towards lower wavenumbers. The values of ν(COO− ) and β(COO− ) did not change in a regular fashion in the series: Li → Na → K → Rb → Cs, and amounted respectively 182, 179, 179, 179, 181 cm−1 [ν(COO− )] and 351, 339, 342, 341, 336 cm−1 [β(COO− )]. In case of the others bands occurred in the IR and Raman spectra, a regular decrease in the wavenumbers of the following bands were observed: ν(CC) = 3424–3294 cm−1 (IR), 8a (IR), 19a (IR), 4 (Raman), 12 (IR), 6b (IR), 16b (IR), γ(CH) 873–866 cm−1 (IR), α(CCC) 419–413 cm−1 (IR, Raman), whereas band no. 12 (Raman) was shifted towards higher wavenumbers in the series Li → Na → K → Rb → Cs 3,5dichlorobenzoates. 3.2.2.2. Calculations. The structures and Mulliken atomic charges for benzoic acid, 3,5-dihydroxy- and 3,5-dichlorobenzoic acids as well as lithium, sodium and potassium 3,5-dihydroxyand 3,5-dichlorobenzoates were calculated in B3LYP/6311++G** level (Figs. 1 and 2). In the molecules of di-substituted acids the electronic charge distribution around carbon atoms in meta position the most changed in comparison with benzoic acid molecule. Moreover, the electron density around C2, C6, C4 and C7 atoms is clearly higher in dichlorobenzoic

Fig. 5. The degree of growth inhibition exhibited by studied compounds in relation to B. subtilis evaluated by means of turbidimetry method by measuring optical density of water solutions at 600 nm after 48 h incubation.

M. Kalinowska et al. / Spectrochimica Acta Part A 70 (2008) 126–135

Fig. 6. The degree of growth inhibition exhibited by studied compounds in relation to C. albicans evaluated by means of turbidimetry method by measuring optical density of water solutions at 600 nm after 24 h incubation.

acid than in benzoic acid molecule, whereas in dihydroxybenzoic acid the electron density around the same carbon atoms is lower in comparison with benzoic acid molecule. Alkali metal cations affect not only the electronic charge distribution in carboxylic anion, aromatic ring, but also in the substituents. Moreover, alkali metal ions stronger affect the electronic charge distribution in dichlorobenzoates than in dihydroxybenzoates. 3.3. Effect of the substituents on the electronic structures of 3,5-dihydroxy- and 3,5-dichlorobenzoic acids and its alkali metal salts 3.3.1. IR spectra The wavenumbers of selected bands which changed in a regular fashion in certain ligand series and sodium salts were gathered in Tables 3 and 4, respectively. In the series: benzoic acid → 2-hydroxybenzoic acid → 2,6dihydroxybenzoic acid bands no. 8b, 7a, 17b, 4 and ν(C O) were shifted towards higher wavenumbers, whereas bands no. 8a underwent displacement in the direction of lower wavenumbers. In the next series: benzoic acid → 3-hydroxybenzoic acid → 3,5-dihydroxybenzoic acid, the wavenumbers of the bands no. 19b, 18a and ν(C O) increased and the wavenumbers of the bands no. 17b, 6b decreased. In the series sodium benzoate → sodium 2-hydroxybenzoate → sodium 2,6dihydroxybenzoate the bands νasym (COO− ) and νsym (COO− ) were shifted towards higher wavenumbers, whereas bands no. 18b and 6b towards lower wavenumbers. Along the next series sodium benzoate → sodium 3-hydroxybenzoate → sodium 3,5-dihydroxybenzoate the wavenumbers of the bands no. 18a and νasym (COO− ) increased, and the wavenumbers of the bands no. 17b, 6b decreased. Moreover the value of ν(COO− ) increased in a distinct manner in the series: sodium benzoate (141 cm−1 ) → sodium 2-hydroxybenzoate (220 cm−1 ) → sodium 2,6-dihydroxybenzoate (256 cm−1 ). In the following series: sodium benzoate (141 cm−1 ) → sodium 3-hydroxybenzoate (153 cm−1 ) → sodium 3,5-

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dihydroxybenzoate (152 cm−1 ) there is a slight increase in the value of ν(COO− ). The presented results suggested that the hydroxyl substituent in the ortho position affects the electronic structure of carboxylic group stronger than in the meta position. This is probably due to the presence of intra-molecular hydrogen linkages in molecule, the same which exists in the 2-hydroxy and 2,6-dihydroxybenzoic acid (Fig. 3). Moreover the structure of carboxylic group in alkali metal benzoates depends on the number of hydroxyl substituents in ortho position. In the series: sodium benzoate → sodium 2-hydroxybenzoate → sodium 2,6dihydroxybenzoate the value of ν(COO− ) increases. This is probably caused by the formation of intra-molecular hydrogen bonds by two-hydroxyl group in dihydroxy-substituted benzoate on the contrary to the single intra-molecular linkage in monohydroxy-substituted benzoate. 3.3.2. UV spectra The bathochromic shifts in the UV spectra of studied compounds were observed in the following series: (1) benzoic acid (227 nm) → 2-hydroxybenzoic acid (234 nm) → 2,6-dihydroxybenzoic acid (246 nm); (2) benzoic acid (227 nm) → 3-hydroxybenzoic acid (230 nm) → 3,5-dihydroxybenzoic acid (243 nm); (3) sodium benzoate (224 nm) → sodium 2-hydroxybenzoate (230 nm) → sodium 2,6-dihydroxybenzoate (246 nm); (4) sodium benzoate (224 nm) → sodium 3-hydroxybenzoate (230 nm) → sodium 3,5-dihydroxybenzoate (241 nm). The bathochromic shifts in the above series are due to a decrease in the gap between bonding and antibonding orbitals, and an increase in the delocalization energy. This suggest that an increase in the number of hydroxyl substituents in the benzene ring causes an increase in the stabilization of the aromatic system of molecule. The main reason of the stabilization is probably the formation of intra-molecular hydrogen linkages in hydroxybenzoic acids and its alkali salts.

Fig. 7. The degree of growth inhibition exhibited by studied compounds in relation to C. albicans evaluated by means of turbidimetry method by measuring optical density of water solutions at 600 nm after 48 h incubation.

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M. Kalinowska et al. / Spectrochimica Acta Part A 70 (2008) 126–135 Table 5 The correlation coefficients between first principal component (performed on the set of five and eight bands, respectively) and antimicrobial activities of alkali metal 3,5-dihydroxybenzoates and 3,5-dichlorobenzoates exhibited against B. subtilis, S. aureus and C. albicans after 24 h and 48 h of incubation

Fig. 8. The degree of growth inhibition exhibited by studied compounds in relation to S. aureus evaluated by means of turbidimetry method by measuring optical density of water solutions at 600 nm after 24 h incubation.

3.4. Microbiological study of di-substituted benzoic acids and their alkali metal salts The results of microbiological tests for: 3,5-dihydroxybenzoic acid and its alkali salts; 3,5-dichlorobenzoates of alkali metals; 2,6-dihydroxybenzoic acid and sodium 2,6dihydroxybenzoate; 2,6-dichlorobenzoic acid and sodium 2,6-dichlorobenzoate; sodium 2-hydroxybenzoate and sodium benzoate, after 24 h and 48 h incubations are shown in Figs. 4–9. The presented results suggest that none of the selected compounds exhibited growth inhibition against C. albicans. Among studied compounds the highest antimicrobial activity against B. subtilis and S. aureus possessed alkali metal 3,5dichlorobenzoates, whereas 3,5-dihydroxybenzates were less microbiological active. The studied compounds caused higher growth inhibition after 24 h than after 48 h of inhibition. The extensive research data showed that the replacement of substituent in the benzene ring caused higher changes in the microbial activity of compound than the change of metal bounded with the carboxylic group. Namely alkali metal

B. subtilis

S. aureus

C. albicans

24 h

48 h

24 h

48 h

24 h

48 h

Five bands 3,5-Dichlorobenzoates 3,5-Dichlorobenzoates

0.719 0.359

0.991 0.71

0.791 0.176

0.997 0.244

0.215 0.126

0.558 0.751

Eight bands 3,5-Dihydroxybenzoates 3,5-Dichlorobenzoates

0.418 0.456

0.969 0.489

0.982 0.365

0.998 0.16

0.524 0.303

0.458 0.981

dichlorobenzoates exhibited higher antimicrobial activity than alkali metal dihydroxybenzoates, sodium 2-hydroxybenzoate and sodium benzoate. Moreover the position of substituent in the benzene ring also affected the microbial activity of compound. Sodium 3,5-dichlorobenzoate caused higher growth inhibition than sodium 2,6-dichlorobenzote. In case of Na 3,5-dihydroxyand 2,6-dihydroxybenzoates this tendency was not clearly visible. The antimicrobial activity slightly changed along metal series, mainly in the series Li → Na → K 3,5-dichlorobenzoates against B. subtilis and S. ureus after 24 h of incubation. In order to describe the dependency between electronic structure of compound and its antimicrobial activity, the correlations between particular band wavenumbers and antimicrobial activity along metal series of 3,5-dichloro- and 3,5-dihydroxybenzoates were done. The obtained correlation coefficients differed in case of various bands, and it was difficult to decide which of the band was the most important. Therefore the principal component analysis on the wavenumbers of characteristic bands which occurred in the spectrum of every studied compounds and often changed in a regular way along metal series, i.e. five bands no. 8a, 19a and νas (COO− ), νs (COO− ), βas (COO− ) and eight bands no. 8b, 8a, 19a and νas (COO− ), νs (COO− ), βas (COO− ), βs (COO− ) and γ s (COO− ). In Table 5 the correlation coefficients calculated between first principal components and microbial activity described by the optical density measured at 600 nm. The correlation coefficients were relatively high for 3,5dihydroxybenzoates and B. subtilis and S. aureus. For 3,5-dichlorobenzoates the obtained correlation coefficients were low (except C. albicans after 48 h of incubation). 3.5. Conclusions

Fig. 9. The degree of growth inhibition exhibited by studied compounds in relation to S. aureus evaluated by means of turbidimetry method by measuring optical density of water solutions at 600 nm after 24 h incubation.

1. Among studied compounds the highest antimicrobial activity towards B. subtilis and S. aureus show alkali metal 3,5-dichlorobenzoates. 3,5-Dihydroksybenzoates are less antimicrobial active. 2. None of studied compounds inhibit growth of C. albicans. 3. The change of substituent in the benzene ring caused more explicit changes in the antimicrobial activity of compounds than the substitution of alkali metal ion in the carboxylic anion.

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4. Sodium 3,5-dichlorobenzoate possesses higher antimicrobial properties than sodium 2,6-dichlorobenzoate. This suggests that the position of subtituent in the benzene ring influence the biological activity of compound. 5. In the FT-IR and FT-Raman spectra of studied alkali metal compounds some bands undergo regular displacement in the series: Li → Na → K → Rb → Cs. 6. The spectroscopic data suggest that an increase in the number of hydroxyl substituents in the benzene ring causes an increase in the stabilization of the aromatic system of molecule. The hydroxyl substituent affects the structure of carboxylic group to higher degree than the halogens. 7. The calculations, which were done for studied acids and its selected alkali metal salts, showed the perturbation effect of these metal cations on the electronic structure of ligands. The Mulliken’s population analysis gave us an idea about the influence of alkali metal cations on the electronic charge distribution. Namely, alkali metal cations affect not only the electronic charge distribution in carboxylic anion, aromatic ring, but also in the hydroxyl’s substituent. Alkali metal ions stronger affect the electronic charge distribution in dichlorobenzoates compared with dihydroxybenzoates. Acknowledgements This work was supported by the Polish Scientific Committee (KBN’s grant no. 3 T09D 025 27, 2 PO5F 024 28) and ACK CYFRONET AGH (grant no. MNiI/SGI2800/PBia łystok/058/2005).

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References [1] M. Jager, Food Market. Technol. 8 (1994) 4. [2] J. Coast, R.D. Smith, DDT 1 (2003) 1. [3] L. Gr¨onholm, G. Wirtanen, K. Ahlgren, K. Nordstr¨om, A.M. Sj¨oberg, Z Lebensm Unters Forsch A 208 (1999) 289. [4] A.J. Dunn, Food Chem. 1 (1997) 187. [5] D.E. Wilcox, A.G. Porras, Y.T. Hwang, K. Lerch, M.E. Winkler, E.I. Solomon, J. Am. Chem. Soc. 107 (1985) 4015. [6] P. Koczo´n, J. Piekut, M. Borawska, W. Lewandowski, J. Mol. Struct. 651–653 (2003) 651. ´ [7] P. Koczo´n, J. Piekut, M. Borawska, R. Swisłocka, W. Lewandowski, Spectrochim. Acta A 61 (2005) 1917. ´ [8] P. Koczo´n, J. Piekut, M. Borawska, W. Swisłocka, W. Lewandowski, Anal. Bioanal. Chem. 384 (2005) 302. [9] V. Frecer, Bioorg. Med. Chem. 14 (2006) 6065. [10] S. Mud-Ronda, M.T. Salabert-Salvador, M.J. Duart, G.M. Ant´on-Fos, Bioorg. Med. Chem. Lett. 13 (2003) 2699. [11] N. Ostberg, Y. Kaznessis, Peptides 26 (2005) 197. [12] X. Cui, C. Joannou, M.N. Hughes, R. Cammack, FEMS Microbiol. Lett. 98 (1992) 67. [13] B. Humbert, M. Alnot, F. Quil`es, Spectrochim. Acta A 54 (1998) 465. [14] V. Aletras, A. Karaliota, M. Kamariotaki, D. Hatzipanayioti, N. Hadjiliadis, Inorg. Chim. Acta 312 (2001) 151. [15] A.S.-L. Lee, Y.-S. Li, Spectrochim. Acta A 52 (1996) 173. [16] W. Lewandowski, B. Dasiewicz, P. Koczo´n, J. Skierski, K. Dobrosz´ Teperek, R. Swisłocka, L. Fuks, W. Priebe, A.P. Mazurek, J. Mol. Struct. 604 (2002) 189. [17] P. Koczo´n, H. Bara´nska, W. Lewandowski, J. Inorg. Chem. 12 (1996) 41. [18] G. Varsanyi, Assignments for Vibrational Spectra of 700 Benzene Derivatives, Akad´emiai Kiad´o, Budapest, 1973.