separation of cephalosporins on thin silica gel layers impregnated with transition metal ions and by...

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Received 7 June 2001; accepted 2 July 2001

ABSTRACT: Cephalosporin antibiotics were separated on thin layer plates impregnated with transition metal ions, Mn2�, Fe2�,Co2�, Ni2� and Cu2�, using different concentrations. Various solvent systems were developed for the study and were used forseparation of these analytes. Impregnation was observed to have an effect on hRF values, removed tailing of analytes and improvedthe resolution. The results have been discussed for each metal ion and compared, and the best conditions of separation have beenidentified. Activation time of thin layer plates impregnated with 0.1% FeSO4 was found to affect both hRF values and resolution ofcephalosporins. New solvent systems are reported for both normal phase and reversed-phase TLC. Copyright � 2002 John Wiley &Sons, Ltd.

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Cephalosporins are a subgroup of �-lactam antibiotics.They contain a �-lactam ring fused to a six-membered-ring with a sulphur atom (Scheme 1) and are similar topenicillins in structure and action. Their structure can bemodified by addition of different side chains at position 7of the �-lactam ring and position 3 of the dihydrothiazinering. Reports show that modifications at position 7 areassociated with alteration in antibacterial activity, whilethose at position 3 are associated with changes inmetabolism and pharmacokinetic properties of the drug(Huber and Chauvette, 1972).

Cephalosporins are of low toxicity to man, have abroad spectrum of antimicrobial activity, and havepotential use in the treatment of individuals allergic topenicillins. They are relatively stable to dilute acids andare relatively resistant to penicillinase regardless of thenature of their side chain and their affinity for enzyme.Also, they dominate the antibiotics market worldwide.Their mode of action is by inhibition of synthesis ofbacteria cell wall and they do so at extremely lowconcentrations. This group of antibiotics have achievedgreat attention in medicinal science because of its wideapplication in checking the growth of both Gram-positive

and Gram-negative bacterial infections both in man anddomestic animals.

The origin and instability of many antibiotics led to asituation in which small amounts of structurally relatedcompounds and byproducts may be present together(Kreuzig, 1996). Further, separation of antibiotics suchas cephalosporins is difficult because individual cephalo-sporins have very small differences in polarity, areamphoteric and also lack chromophore or fluorophore,making it difficult to detect these compounds by directmethods (Dasenbrock and LaCourse, 1998). Cephalo-sporins have been analysed microbiologically, photo-chemically and iodometrically (Kreuzig, 1996), but thesetechniques lack specificity while microbiological tech-niques require a long time for sample analysis. Thus,there is great need for effective and rapid methods forseparation, detection and determination of individualcephalosporin antibiotics and their structurally relatedcompounds from fermentation broths, biological fluids aswell as in food products. There are many reports usingHPLC (Pehourcq and Jarry, 1998; Schuegerl and Seidel,1998), HPTLC (Agbaba et al., 1998) and capillary zoneelectrophoresis (Mrestani and Neubert, 1998) for separa-tion of cephalosporins.

The advantages of TLC such as simplicity, ability toutilize low volume of mobile phase as well as solventsunsuitable for HPLC, speed of separation and low costhave long been recognized. In analysis of cephalosporinsTLC is used in identification and purity control as well asin stability and metabolism studies. It is also the methodof choice for checking cross-contamination of cephalo-sporins with traces of penicillin. TLC is also an official

BIOMEDICAL CHROMATOGRAPHYBiomed. Chromatogr. 16: 165–174 (2002)DOI: 10.1002/bmc.122

*Correspondence to: R. Bhushan, Department of Chemistry, Uni-versity of Roorkee, Roorkee 247 667, India.†Present address: Department of Chemistry, Jomo Kenyatta Universityof Agriculture and Technology, P.O. Box 62000, Nairobi, Kenya.

Contract/grant sponsor: All India Council for Technical Education;contract/grant number: 8017/RDII/PHA/98.

Copyright 2002 John Wiley & Sons, Ltd.

ORIGINAL RESEARCH

method prescribed in the cephalosporium monograms ofthe European pharmacopoeia (1997). This prescriptioncontains mobile phases for separation of cephalosporins(Hoogmartens 1997).

The literature on qualitative and quantitative TLCanalysis of several antibiotics has been reviewed bySherma (Sherma, 1990, 1992, 1994, 1996, 1998, 2000;Sherma and Fried, 1982, 1984, 1986). Also a survey ofTLC separation of those antibiotics which are intherapeutic use has been presented by Kreuzig (1996),which covers papers published after 1980. Differentvisualizing agents such as H2SO4 or HNO3:H2SO4

(Hoogmartens et al., 1981), and fluorescamine (Fabre etal., 1985) have been used for detection of cephalosporinsby TLC on silica gel, while p-dimethylaminobenzalde-hyde, SnCl2 and I2 (Wang and Wang, 1975) have beenused for the same on both alumina and silica gel.

Literature reveals that cephalosporins were separatedby reversed-phase (RP-)TLC on silanized silica gel(Quintens et al., 1993), by normal phase on mixed layercomposed of a mixture of hydroxides of aluminium andmagnesium and silica gel (Qureshi et al., 1996) and onthin layer plates impregnated with ethylenediaminetetraacetic acid disodium salt hydrate (Bhushan andParshad, 1996). Retention behaviour of different cepha-losporins and penicillin derivatives on silica gel platesimpregnated with tri-caprylmethylammonium chloride(TCMA; Kovacs-Hadady and Szllagyi, 1991) has alsobeen reported.

There are few reports on separation of cephalosporinson untreated layers, impregnated layers and reversed-phase TLC. There have been no reports on separation ofcephalosporins on silica gel thin layer plates impregnatedwith transition metals. This paper describes separation of

Scheme 1. Chemical structures of cephalosporins investigated.

Copyright 2002 John Wiley & Sons, Ltd. Biomed. Chromatogr. 16: 165–174 (2002)

166 ORIGINAL RESEARCH R. Bhushan and G. T. Thiong’o

some cephalosporins using silica gel thin layer platesimpregnated with some transition metal sulphates. Newsolvent systems both for normal phase and reversed-phase TLC are reported. The effect of time for activationof thin layer plates on separation has also been studied.

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The antibiotics, cephalosporins, were purchased from variousmanufacturers. Both cefadroxil and cefazolin sodium were fromRanbaxy (India); both cephalexin and cefuroxime sodium werefrom Glaxo India Ltd (Bombay); cefotaxime sodium was fromRoussel India Ltd (Bombay); and cephradine was from SmithklineBeecham plc (London). Silica gel G with 13% calcium sulphate asbinder, having impurities of chloride, iron and lead up to 0.02%each and indicating pH 7.0 in 10% aqueous suspension, was fromE. Merck (Bombay, India). Sulphate salts of manganese (II), iron(II), cobalt (II), nickel (II) and copper (II) and solvents werepurchased from Sisco Research Laboratories (Bombay, India) andE. Merck (Bombay, India). The reversed-phase (Alugram, RP18 w/uv254) precoated TLC plates 20 � 10 cm � 0.15 mm were fromMacherey-Nagel (Duren, Germany).

Cephalosporin samples for analysis were prepared frominjection vials of individual components. The powdered content(100 mg) of each antibiotic was dissolved in the minimum amountof water, filtered and a few drops of absolute ethanol were addeduntil some turbidity appeared. The solution was allowed tocrystallize. The liquor was decanted and the crystals were washedwith a little ether. The standard solution (10�3

M) of each antibioticwas prepared in 70% ethanol and was applied to the plates using a25 �L Hamilton syringe.

TLC plates (10 � 20 cm � 0.5 mm) were prepared by spreadinga slurry of silica gel G in distilled water in a ratio of 1:2 usingStahl-type applicator. The plates were first dried at roomtemperature and then kept overnight in an oven at 60 � 2°C. Forimpregnated thin layer plates, the slurry was prepared in aqueoussolutions of different transition metal ions. The ions used wereMn2�, Fe2�, Co2�, Ni2� and Cu2�. Various amounts (0.1, 0.2, 0.3and 0.4%) of each ion were used.

All chromatograms were developed at 23 � 2°C in varioussolvent systems in a rectangular paper-lined glass chamber whichhad been pre-equilibrated for 15 min and were exposed to iodine

vapours for locating the spots. For normal-phase TLC, thechromatograms were developed for a 8.5 cm run in 40–60 min.

Reversed-phase (Alugram, RP-18 W/UV254) pre-coated TLCplates (20 � 10 cm � 0.15 mm) were generously donated byMacherey-Nagel (Duren, Germany). The chromatograms weredeveloped for a 9.5 cm run in 80–90 min.

%�� Various concentrations of cephalosporins werespotted on TLC plates using a 25 �L Hamilton syringe. The plateswere developed and spots were detected as above. The detectionlimit for both normal-phase and reversed-phase-TLC was 1.7 �g.

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The solvent systems S1–S6 were systematically workedout on untreated thin layer plates.

S1: propanol–H2O–butanol (15:3:1, v/v)S2: propanol–H2O–butanol (15:4:1; v/v)S3: propanol–H2O–butanol (16:3:1, v/v)S4: propanol–H2O–butanol (16:2:1, v/v)S5: butanol–methanol–H2O (10:4:2, v/v)S6: butanol–methanol–H2O (12:5:1, v/v)

The hRF (RF � 100) values of the cephalosporinsunder study using these solvents are shown on Table 1.Of these six solvent systems, S1 was found to be the best.It showed a resolution of all the six cephalosporins understudy with small and compact spots. The solvent systemsS2 and S3 showed slightly broad but compact spots,which nevertheless resolved five analytes. The solventsystems S4, S5 and S6 resolved only three analytes understudy and tailing was observed for cefadroxil, cephalexinand cephradine.

Impregnation of silica gel layers with transition metalions has been shown to improve separation of aminoacids (Bhushan and Parshad, 1994), antihistamines(Bhushan and Joshi, 1996), vitamin B complex and folicacid (Bhushan and Parshad, 1999). Some cephalosporinssuch as cefadroxil, are also known to form complexes

Table 1. hRF values of cephalosporins on untreated silica gel plates using various solvent systems

Sample no. Sample S1 S2 S3 S4 S5 S6

1 Cafadroxil 29 56 56 21 ST 44 ST 382 Cephalexin 32 60 63 21 ST 48 ST 41 T3 Cephradine 62 52 56 22 ST 45 ST 35 T4 Cefotaxime 57 79 72 56 72 795 Cefuroxime 47 81 69 79 71 866 Cefazolin 42 93 80 94 68 89

Solvent systems: S1, propanol–H2O–butanol (15:3:1, v/v); S4, propanol–H2O–butanol (16:2:1, v/v); S2, propanol–H2O–butanol (15:4:1, v/v); S5,butanol–methanol–H2O (10:4:2, v/v); S3, propanol–H2O–butanol (16:3:1, v/v); S6, butanol–methanol–H2O (12:5:1, v/v).T, tailing; ST, small tailing. Solvent front, 8.5 cm; temperature, 23 � 2°C; detection, iodine vapour; time, 40–60 min.


Separation of cephalosporins ORIGINAL RESEARCH 167

with metal ions such as Mn2�, Co2�, Ni2� and Cu2� andmany others (Shoukry et al., 1997).

The solvent systems S4–S6 showed poor separation ofthe cephalosporins under study on untreated plates.Consequently, these solvent systems S4–S6 were selectedfor further studies on separation of cephalosporins on thinsilica gel layers impregnated with transition metal ions.In the study, four different concentrations, viz. 0.1, 0.2,0.3 and 0.4% of each of the metal ions (Mn2�, Fe2�,Co2�, Ni2� and Cu2�) were tried. The results showing theinfluence of metal ions on chromatographic behavior ofcephalosporins are shown in Tables 2–6. The results ineach of the tables have been discussed and compared withthose on untreated thin layer plates.

The hRF values were affected by the concentration ofimpregnating reagents in all the solvent systems. Eachreported hRF value is average of at least three or moreidentical runs and had relative standard deviation valuesranging between �0.01 and �0.03. The spots were morecompact on impregnated layers than on plain silica gellayers. Resolution was considered to have occurred whenthe difference in hRF values of two spots was three units.The resolution possibility for each pair of cephalosporinswas calculated by dividing the distance between the twospot centres by the sum of two spot radii. A value of 1.5indicated a complete resolution whereas a value of 1.0 orbelow indicated incomplete resolution. The variation inhRF with different transition metal ion can be attributed

Table 2. hRF values of cephalosporins on plates impregnated with different concentrations of MnSO4

S4 (Concentration, %) S5 (Concentration, %) S6 (Concentration, %)

Sample Plain 0.1 0.2 0.3 0.4 Plain 0.1 0.2 0.3 0.4 Plain 0.1 0.2 0.3 0.4

Cafadroxil 21 ST 34 41 23 ST 21 T 44 ST 54 52 42 44 38 T 49 28 ST 18 22Cephalexin 21 ST 36 39 17 ST 20 T 48 ST 56 52 41 44 41 T 60 25 ST 15 23Cephradine 22 ST 32 34 20 ST 18 T 45 ST 55 50 39 45 35 T 50 23 ST 14 22Cefotaxime 56 65 55 40 38 T 72 74 69 66 62 79 77 44 33 38Cefuroxime 79 81 81 52 79 71 81 81 70 71 86 83 78 58 49Cefazolin 94 87 80 76 91 68 86 81 85 80 89 84 79 77 82

Solvent systems: S4, propanol–H2O–butanol (16:2:1, v/v); S5, butanol–methanol–H2O (10:4:2, v/v); S6, butanol–methanol–H2O (12:5:1, v/v).T, tailing; ST, small tailing. Solvent front, 8.5 cm; temperature, 23 � 2°C; detection, iodine vapour; time, 40–60 min.

Table 3. hRF values of cephalosporins on plates impregnated with different concentrations of FeSO4



Cafadroxil 21 ST 35 ST 09 10 T 0 44 ST 26 15 T 13 T 0 38 T 32 11 T 09 0Cephalexin 21 ST 35 07 11 T 0 48 ST 27 13 T 11 T 0 41 T 29 16 T 09 0Cephradine 22 ST 39 10 14 T 0 45 ST 28 16 T 13 T 0 35 T 29 10 T 08 0Cefotaxime 56 41 31 27 T 0 72 53 66 T 37 0 79 36 38 ST 42 0Cefuroxime 79 89 62 76 0 71 49 81 77 0 86 70 72 77 0Cefazolin 94 94 94 97 85 68 62 95 95 94 89 90 95 93 94


Table 4. hRF values of cephalosporins on plates impregnated with different concentrations of CoSO4



Cafadroxil 21 ST 22 8 T 13 T 25 44 ST 33 33 26 34 38 T 9 T 7 T 9 T 6Cephalexin 21 ST 22 7 T 12 T 31 48 ST 31 36 31 37 41 T 10 T 6 T 13 T 7Cephradine 22 ST 19 9 T 10 T 21 45 ST 30 30 29 40 35 T 9 T 7 T 15 T 6Cefotaxime 56 17 T 8 T 8 T 19 72 30 15 19 31 79 8 T 8 T 17 T 9Cefuroxime 79 64 64 78 89 71 70 72 74 74 86 44 67 64 61Cefazolin 94 69 80 ST 41 92 68 77 78 84 81 89 67 76 76 31 ST




to complex formation and variation in solubilities ofcomplexes in different solvent systems or their differentadsorption/partition coefficients during the developmentof the chromatogram. The effect of Ni2� in six solventsystems (S1–S6) at four different concentrations and thatfor other metal ions at four different concentrations inthree solvent systems (S4–S6) is discussed below.

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Table 2 shows hRF values for cephalosprins developedwith solvent systems S4, S5 and S6. In comparison tountreated thin layer plates, a general increase of hRF

values at concentration 0.1% and a general decrease ofhRF values from 0.2 to 0.4% for the solvent systems S4and S5 was observed. The solvent system S6 showed anincrease of hRF values for cefadroxil, cephalexin andcephradine and a decrease of the same for cefotaxime,cefuroxime and cefazolin at concentrations of 0.1%,while a general decrease of hRF values for all analyteswas observed from concentrations 0.2–0.4%. Unlike inthe case of untreated plates, none of the analytes showedtailing at concentrations 0.1 and 0.2% in S4, at allconcentrations in S5, and at concentrations 0.1, 0.3 and0.4% while using S6.

Table 2 shows that impregnation with MnSO4 resultedin separation of cephalosporins which had not been

resolved earlier on untreated plates. These cephalospor-ins are: cephalexin and cephradine at concentrations 0.1and 0.2% in solvent system S4; cefotaxime andcefuroxime at all concentrations in S5, and cefadroxiland cephradine at concentration 0.3% in S5; andcefadroxil, cephalexin and cephradine at 0.1% in S6.The best concentration was at 0.1% where none of theanalytes showed tailing in all solvent systems, while thebest solvent system was S5 at concentration 0.3% wherefive out of six analytes were resolved.

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In comparison to untreated thin layers, a decrease in hRF

values was observed on the thin layer impregnated withFeSO4 for the solvent systems S4, S5 and S6, except forsolvent system S4, which showed an increase in hRF

values at concentration 0.1% (Table 3). From concentra-tions 0.1–0.3% hRF values generally increased for allsolvents. It was interesting to observe that for all solventsystems studied all analytes remained at the origin atconcentration 0.4%, except cefazolin, which showed adecrease in hRF value in S4 and an increase in hRF in bothS5 and S6 at the same concentration of 0.4%. None of thecephalosporins under study showed tailing at concentra-tions 0.2% with solvent system S4, 0.1% with solventsystem S5, and 0.1 and 0.3% with solvent system S6.

Table 5. hRF values of cephalosporins on plates impregnated with different concentrations of NiSO4



Cafadroxil 21 ST 10 12 T 33 14 44 ST 36 39 47 22 ST 38 T 07 15 12 ST 27Cephalexin 21 ST 11 12 T 40 13 48 ST 39 41 54 28 ST 41 T 07 18 16 ST 34Cephradine 22 ST 12 13 T 30 10 45 ST 30 38 51 24 ST 35 T 05 16 12 ST 29Cefotaxime 56 47 89 T 56 37 72 44 59 71 45 79 21 63 41 66Cefuroxime 79 65 93 86 73 71 63 80 80 78 86 70 85 73 86Cefazolin 94 90 94 93 14 68 44 82 84 77 89 15 T 84 83 86


Table 6. hRF values of cephalosporins on untreated plates and plates impregnated with 0.3% NiSO4

Percentage concentrationS1 S2 S3

Sample no. Sample Plain 0.3 Plain 0.3 Plain 0.3

1 Cafadroxil 29 34 56 45 56 452 Cephalexin 32 41 60 52 63 483 Cephradine 62 46 52 38 56 454 Cefotaxime 57 57 79 64 72 565 Cefuroxime 47 85 81 88 69 836 Cefazolin 42 91 93 93 80 92

Solvent systems: S1, propanol–H2O–butanol (15:3:1, v/v); S2, propanol–H2O–butanol (15:4:1, v/v); S3, propanol–H2O–butanol (16:3:1, v/v).Solvent front, 8.5 cm; temperature, 23 � 2°C; time, 40–60 min; detection, iodine vapour.



It is evident from Table 3 that the cephalosporinscephalexin and cephradine in solvent system S4 atconcentrations 0.1 and 0.2%, cephradine in solventsystem S5 at concentration 0.1%, and cefadroxil andcephradine in solvent system S6 at concentrations 0.1%,which had earlier not resolved on untreated plates, werenow considered resolved on plates impregnated withFeSO4. The best concentration of FeSO4 was 0.1% for allsolvent systems. The solvent system S6 at concentration0.1% was the best where it resolved five of the analytesunder study with reasonably high hRF values.

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There was a general decrease of hRF values with CoSO4

impregnation in solvent systems S4, S5 and S6 incomparison to untreated plates (Table 4). However, allthe analytes except cefotaxime showed an increase in hRF

values in solvent systems S4 at concentration 0.4%, whilecefuroxime and cefazolin showed an increase in hRF

values in solvent system S5. No tailing of the analyteswas observed at concentrations 0.4% in solvent systemS4 and 0.1–0.4% in solvent system S5, while cefazolinonly showed slight tailing at 0.4% in solvent system S6.

It is clear from Table 4 that cephalosporins cefadroxil,cephalexin and cephradine in solvent systems S4 atconcentration 0.4%, and cefadroxil, cephalexin, cephra-dine, cefotaxime and cefuroxime at concentrations 0.2and 0.4% in solvent system S5, which had not beenresolved on untreated plates, were now consideredresolved on plates impregnated with CoSO4. The bestconcentration for all solvents was 0.4% and the bestsolvent system was S5 at concentration 0.4%.

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Table 5 shows the hRF values of cephalosporinsdeveloped on silica gel layers impregnated with NiSO4

using solvent systems S4, S5 and S6. In comparison tountreated plates the hRF values obtained using impreg-nated plates decreased at concentration 0.1% for solventsystems S4, S5 and S6, but increased from concentrations

0.1–0.3% and then decreased at concentrations 0.4% forsolvent systems S4 and S5. The hRF values increased ongoing from concentrations 0.1–0.4% for the solventsystem S6. None of the analytes showed tailing atconcentrations 0.1, 0.3 and 0.4% in solvent system S4,0.1, 0.2 and 0.3% in solvent system S5, and 0.2 and 0.4%in solvent system S6.

Table 5 shows that the cephalosporins cefadroxil,cephalexin and cephradine at concentration 0.3% andcefadroxil and cephradine at concentration 0.4% insolvent system S4, cefadroxil, cephalexin, cephradine,cefotaxime and cefuroxime at concentrations 0.1 and0.3% and also cephalexin, cephradine, cefotaxime andcefuroxime at concentration 0.2% in solvent system S5,and cefadroxil and cephalexin at concentration 0.2 and0.4% in solvent system S6, were considered resolvedalthough they had not earlier resolved on untreated plates.The best concentration was 0.3% for solvent system S4and S5 and 0.4% for solvent system S6. The best solventsystem was S5 at concentration 0.3% where it resolvedall analytes at relatively high hRF values.

In comparison to other impregnating reagents NiSO4

was found to be better under the experimental conditions.It showed the best resolutions at concentration 0.3% withS4 and S5. Thus, investigations were carried out atconcentration 0.3% NiSO4 also using the solvent systemsS1, S2 and S3. As mentioned earlier, these solventsystems had shown better resolutions on untreated plates.Table 6 shows hRF values of cephalosporins on platesimpregnated with 0.3% using solvent system S1, S2 andS3. The solvent system S1 resolved all the cephalosporinsunder study on impregnated plates with increase in hRF

values except cefuroxime, which did not show a changein hRF values, although resolved. In comparison tountreated plates, cefuroxime showed an increase in hRF

value while cefazolin showed no change, and the restshowed a decrease of hRF values in the solvent systemS2. Nevertheless, all cephalosporins under study wereconsidered resolved on the plates treated with 0.3%NiSO4. Therefore, the pair cefadroxil/cephalexin whichhad not resolved on untreated plates, was consideredresolved on plates impregnated with 0.3% NiSO4 using

Table 7. hRF values of cephalosporins on plates impregnated with different concentrations of CuSO4



Cafadroxil 21 ST 03 03 02 20 44 ST 04 02 05 16 38 T 02 02 02 21 STCephalexin 21 ST 03 01 01 17 48 ST 04 02 03 14 41 T 02 01 02 18 STCephradine 22 ST 03 03 01 13 45 ST 04 02 04 16 35 T 04 02 03 18 STCefotaxime 56 54 28 16 T 44 72 46 36 T 50 T 53 79 40 T 41 T 42 T 44 STCefuroxime 79 54 85 72 83 71 69 81 82 81 86 53 80 84 77Cefazolin 94 0 0 0 0 68 0 0 0 0 89 0 0 0 0

Solvent systems: S4, propanol–H2O–butanol (16:2:1, v/v); S5, butanol–methanol–H2O (10:4:2, v/v); S6, butanol–methanol–H2O (12:5:1, v/v).T, tailing; ST, small tailing. Solvent front, 8.5 cm; temperature, 23 � 2°C; time, 40–60 min; detection, iodine vapour.



solvent system S2. The solvent system S3 resolved fivecephalosporins; cefotaxime and cefuroxime showed anincrease of hRF values, but the cefadroxil/cephalexin pairthat had not resolved on untreated plates was also notresolved on impregnated layers.

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The hRF values generally decreased with CuSO4

impregnation in comparison to untreated plates in solventsystems S4, S5 and S6 (Table 7). The hRF valuesdecreased at 0.1% for solvent systems S4, S5 and S6. Itwas interesting to observe that cefazolin remained at theorigin at all concentrations of CuSO4, while cefuroximeshowed relatively high hRF values with all solventsystems at concentrations of 0.1, 0.2 and 0.3%. None ofthe analytes showed tailing at concentration 0.4% in bothsolvent systems S4 and S5.

Successfully resolved cephalosporins which were notresolved on untreated plates (Table 7) are cefadroxil,cephalexin and cephradine in solvent system S4 atconcentration 0.4%. No other solvent gave good resolu-tion of analytes at different concentrations of CuSO4. Thebest solvent system was S4 at concentration 0.4%, whereit resolved five of the analytes under study.

It can be assumed that, at the right concentrations,

metal ions influence the chromatographic behaviour bycomplex formation. The complex formed changed theadsorption/partition characteristics during the develop-ment of the chromatograms and better resolution ofcephalosporins was achieved. It can also be assumed thatweak electron donation from N, O or S atoms to the metalion affected chromatographic behaviour.

From the results it can be deduced that NiSO4 gave thebest results with solvent systems S4 and S5 at aconcentration of 0.3% (Table 5). CoSO4 also gave goodresults with solvent system S5 at concentrations 0.2 and0.3%. Both metal ions showed small and compact spots,but NiSO4 spots were more intense.

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The amount of water present in adsorbents such as silicais known to have a considerable effect on chromato-graphic properties (Lederer and Lederer, 1957). There-fore, studies were carried out to investigate the effect ofactivation time on resolution of cephalosporins on silicagel thin layers impregnated with 0.1% FeSO4. Thus,impregnated thin layer plates were prepared as mentionedabove, but activation was carried out as follows: (i) 0 hactivation—plates were kept at room temperature(23 � 2°C) for 48 h; (ii) plates were kept in an oven at

Table 8. hRF values of cephalosporins on plates impregnated with 0.1% FeSO4, activated for different times and developedwith S6

Activation time (h)Sample no. Sample Plain 0 2 4 6 Overnight

1 Cafadroxil 38 T 79 ST 53 T 28 T 12 T 322 Cephalexin 41 T 74 ST 61 T 28 T 12 T 293 Cephradine 35 T 74 ST 61 T 28 T 13 T 294 Cefotaxime 79 87 87 86 60 365 Cefuroxime 86 89 91 86 74 706 Cefazolin 89 94 91 86 89 90

Solvent system: S6, butanol–Methanol–H2O (12:5:1, v/v).T, tailing; ST, small tailing. Solvent front, 8.5 cm; temperature, 23 � 2°C; time, 40–60 min; detection, iodine vapour.




Solvent system: S1, propanol–H2O–butanol (15:3:1, v/v).Solvent front, 8.5 cm; temperature, 23 � 2°C; time, 40–60 min, detection, iodine vapour.



60 � 2°C for 2 h, 4 and 6 h and overnight. The plateswere then spotted with samples, developed with solventsystems S1, S2, S3 and S6 and detection was carried outin iodine vapour. The results are depicted in Tables 8–11and are discussed below.

The solvent system S6 gave the best resolution ofcephalosporins on layers impregnated with 0.1% FeSO4,while concentration 0.1% gave the best results for thesame. The solvent systems S1, S2 and S3 gave betterresults on untreated layers than S4, S5 and S6.

In comparison to plates activated overnight (14 h), thesolvent system S6 showed very high hRF values for allanalytes for plates activated for 0 h (Table 8). On goingfrom 0 to 6 h the hRF values decreased. The cephalo-sporins cefadroxil, cephalexin and cephradine showedtailed spots except on the plates activated overnight. Onlytwo pairs of analytes were resolved on plates activated for0 and 2 h, none for those activated for 4 h, and three pairsof analytes for those activated for 6 h. Five cephalospor-ins had been resolved on plates activated overnight. Thus,plates activated overnight gave better resolution of theanalytes than those activated for 0, 2, 4 and 6 h.

Table 9 shows hRF values of cephalosporins on silicagel plates impregnated with 0.1% FeSO4 activated fordifferent times and developed with the solvent system S1.Using this solvent system S1 cefuroxime and cefazolin

showed very little or no change in hRF values while therest showed an increase of hRF values for plates activatedfor 0, 2, 4 and 6 h in comparison to those activated forovernight. Plates activated for 0, 2 and 4 h showed aresolution of three cephalosporins while those activatedfor 6 h showed a resolution of four cephalosporins ascompared to six for similar plates activated for overnight.Thus, inactivated plates showed poor resolution with S1.

Similarly, the same study was carried out using solventsystem S2. In comparison to plates activated overnight,the analytes showed higher hRF values on plates activatedfor 0, 2 and 4 h, while those on plates activated for 6 hshowed low hRF values with the exception of cefuroxime,which showed no change in hRF values for platesactivated both for 6 h and overnight. The hRF valuesdecreased from plates activated for 0–6 h (Table 10). Ofthe six cephalosporins under study, five resolved onplates activated for 0, 4 and 6 h, while three cephalo-sporins were resolved on plates activated for 2 h. Platesactivated overnight resolved only four analytes and,therefore, in this case plates activated for 0, 4 and 6 hgave better resolutions. Similarly, when using S3 fordevelopment of plates activated for 0, 2, 4 and 6 h, theanalytes showed higher hRF values than those observedon plates activated overnight (Table 11). There was nodistinct trend of change of hRF values on increasing




Solvent system: S1, propanol–H2O–butanol (15:4:1, v/v).Solvent front, 8.5 cm; temperature, 23 � 2°C; time, 40–60 min; detection, iodine vapour.



1 Cafadroxil 56 43 58 59 58 23 T2 Cephalexin 63 41 56 54 55 23 T3 Cephradine 56 44 54 55 55 23 T4 Cefotaxime 72 72 76 54 79 915 Cefuroxime 69 92 84 85 85 856 Cefazolin 80 91 88 83 91 85

Solvent system: S3, propanol–H2O–butanol (16:3:1, v/v).Solvent front, 8.5 cm; temperature, 23 � 2°C; time, 40–60 min; detection, iodine vapour.



activation time of plates from 0 to 6 h. It was interestingto observe that cefadroxil, cephalexin and cephradine,which had not resolved on either untreated plates orplates activated overnight, were considered resolved onplates activated for 4 h. Plates activated for 0 and 4 hresolved four cephalosporins and plates activated for 2and 6 h resolved five, while plates activated overnightshowed tailing of spots for cefadroxil, cephalexin andcephradine. Consequently, 0 h activated plates gavebetter resolution than plates activated overnight.

Water used in preparation of TLC plates may fill thecavity system of silica gel or be held on the surface by theSiOH groups. Water held in the cavity may be consideredas capillary water. Drying of plates at temperatures below200°C removes only the capillary water and not waterheld on the surface by SiOH groups. Thus, in normal TLCusing silica gel, separation may be considered to occurdue to both partition and adsorption forces. Conse-quently, thin layer plates activated for lesser periods oftime will contain more cavity water and hence inclinemore to partition separation. Separation of analytes byadsorption will dominate on plates activated for periodslonger than overnight.

In the present study analytes showed high hRF valueson plates activated for 0, 2, 4 and 6 h than those activatedfor overnight. Plates activated for lesser periods thanovernight contain more cavity water and this water isavailable during development with the solvent system.Thus, the solvent system on such plates may beconsidered to have more water and hence be more polar,resulting in higher hRF values, as observed on platesactivated for shorter periods than overnight. However,cefadroxil, cephalexin and cephradine showed lower hRF

values on plates activated for 6 h than those activatedovernight using the solvent systems S2 and S6. Aplausible explanation could be that other factors comeinto play such as the solubility of the analytes in thedeveloping solvent system.

The solvent systems S1 and S6 gave better resolutionsof analytes on plates activated overnight than thoseactivated for 0, 2, 4 and 6 h. The solvent systems S2 andS3 showed better resolution of the analytes on platesactivated for less times than overnight. Thus S1 and S6may be considered better solvent systems where separa-tion of the cephalosporins is dominated by adsorption,whereas S2 and S3 may be considered better solventsystems where partition dominates separation.

��

The cephalosporins contain groups or atoms like S,�NH2 and �COOH. These groups make cephalosporinshighly polar, ionizing and also have high solubility inwater. All these properties favour the use of reversed-phase TLC separation of these compounds. Thus, it was

considered worthwhile to develop a method of separatingcephalosporins by this technique.

The solvent system propanol–H2O–butanol (15:3:1,v/v) resolved all cephalosporins under study by normal-phase TLC. This solvent system was considered to bereasonably polar and was therefore tried in RP mode inthe separation of cephalosporins. The solvent systemshowed small compact spots with the analytes. Of the sixcephalosporins under study, four were resolved. The pairsof cefadroxil/cephradine and cefuroxime/cefazolin whichhad resolved under normal-phase TLC did not resolve;the results are depicted in Table 1. Except for cephradine,all the analytes showed higher values of hRF in RP-TLCthan in normal-phase TLC (Tables 1 and 12). Develop-ment times of chromatograms were 40–60 min fornormal phase while for reversed phase it was 90 min.Solvent variation was tried so as to improve theseparations but did not yield good results.

Literature shows that mixtures of two solventsconsisting of water plus alcohol, acetonitrile, acetoneetc. (Sherma, 1996) have been used in separation ofvarious compounds using RP-TLC. Consequently, var-ious solvent compositions of water and methanol weretried and after extensive experimentation and withmodification using acetic acid, a new solvent systemR1 was worked out—methanol–H2O–acetic acid(4:16:0.2, v/v). The spots of the analytes were compactand small. All cephalosporins under study were resolved.

In normal phase mode the mobile phase is less polarthan the stationary phase. The sample–solvent interac-tions are relatively weak while sample–adsorbent inter-actions are strong. Thus, normal-phase TLC is favourablefor less polar samples (Snyder 1983). On the other handin reversed-phase TLC, the mobile phase is more polarthan the stationary phase. It is characterized by stronginteractions between the polar mobile phase and thevarious samples. The interactions between the sampleand stationary phase are weak and hence separation isdetermined by the strong interactions between the sampleand the solvent (Melander and Horvath, 1980). Thus, the

Table 12. hRF values of cephalosporins on reversed-phaseTLC plates

Solvent systemsSample no. Sample S1 R1a

1 Cafadroxil 51 672 Cephalexin 48 293 Cephradine 52 194 Cefotaxime 86 465 Cefuroxime 91 536 Cefazolin 91 37

Solvent systems: S1, propanol–H2O–butanol (15:3:1, v/v); R1,methanol–H2O–acetic acid (4:16:0.2, v/v).Solvent front, 9.5 cm; time, 80–90 min; detection, iodine vapour.a Successful solvent system.



solvent system applied on normal phase separations maynot be applied on reversed-phase conditions.

The method presented here is simple, rapid, reliable,economical and sensitive. The minimum detection limitwas 1.7 �g. It was successfully used in separation andidentification of six cephalosporins.

+� ��

The award of a fellowship by the Indian Council forCultural Relations, New Delhi, and award of leave ofabsence by Jomo Kenyatta University of Agriculture andTechnology, Kenya (to G.T.) are gratefully acknowl-edged. Financial assistance from the All India Council forTechnical Education, New Delhi (grant no. 8017/RDII/PHA/98) and a gift of RP-TLC pre-coated plates fromMacherey-Nagel, Duren, Germany (to R.B.) are alsogratefully acknowledged.

�'-'�'#�'�

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separation of cephalosporins on thin silica gel layers impregnated with transition metal ions and by...

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