Multiple Development

This mode of development is seldom applied in practice, in comparison to conventional development, but its significance is increasing owing to its greatly increased separation efficiency. A characteristic feature of this mode is repetitive development of the chromatogram on the same plate. Each development is followed by evaporation of eluent from the plate to prepare it for the next chromatographic process. The resolution provided is greater than conventional or continuous development, and mixtures of wide polarity can easily be separated. The spots to be separated are compact, which leads to better detectability.There are three types of multiple development:

Figure 8 Comparison of (A) conventional and (B) multiple development (4x5)+ (3 x 7) min for the separation of a mixture of PAH standards. The mobile phase was methanol-water (4:1) and the stationary phase octadecylsilanized silica gel. 1, Cor-onene; 2, benzo[g,h,i]perylene; 3, benzo[a]pyrene; 4, benzo[a] anthracene; 5, fluoranthene. (Reproduced with permission from Butler HT, Coddens ME, Khatib S and Poole CF (1985) Determination of polycyclic aromatic hydrocarbons in environmental samples by high performance thin-layer chromatography and fluorescence scanning densitometry. Journal of Chromatographic Science 23: 200.)

Figure 8 Comparison of (A) conventional and (B) multiple development (4x5)+ (3 x 7) min for the separation of a mixture of PAH standards. The mobile phase was methanol-water (4:1) and the stationary phase octadecylsilanized silica gel. 1, Cor-onene; 2, benzo[g,h,i]perylene; 3, benzo[a]pyrene; 4, benzo[a] anthracene; 5, fluoranthene. (Reproduced with permission from Butler HT, Coddens ME, Khatib S and Poole CF (1985) Determination of polycyclic aromatic hydrocarbons in environmental samples by high performance thin-layer chromatography and fluorescence scanning densitometry. Journal of Chromatographic Science 23: 200.)

Figure 9 Chromatograms of PTH-amino acids after multiple development on silica gel plate. (A) First development with methylene chloride; (B) Second development with methylene chloride-isopropanol (99 : 1); (C) third development with methylene chloride-iso-propanol (99:1); (D) fourth development with methylene chloride-isopropanol (97:3); (E) fifth development with ethyl acetate-acetonitrile-glacial acetic acid (74.3:25:0.7). (Reproduced with permission from Schuette SA and Poole CF (1982) Unidimensional, sequential separation of PTH-amino acids by high-performance thin-layer chromatography. Journal of Chromatography 239: 251.)

Figure 9 Chromatograms of PTH-amino acids after multiple development on silica gel plate. (A) First development with methylene chloride; (B) Second development with methylene chloride-isopropanol (99 : 1); (C) third development with methylene chloride-iso-propanol (99:1); (D) fourth development with methylene chloride-isopropanol (97:3); (E) fifth development with ethyl acetate-acetonitrile-glacial acetic acid (74.3:25:0.7). (Reproduced with permission from Schuette SA and Poole CF (1982) Unidimensional, sequential separation of PTH-amino acids by high-performance thin-layer chromatography. Journal of Chromatography 239: 251.)

1. repetitive development with the same solvent in the same direction

2. repetitive development with the different solvents in the same direction

3. single or repetitive development in one direction with a given solvent, followed by single or repetitive development in the second direction perpendicular to it with another solvent (two-dimensional development)

The first mode is especially applied to the separation of poorly resolved spots, the second to mixtures of a wide range of polarity, and the third mode to separation of complex mixtures with components of similar polarity and/or different polarity.

The example of separation of polyaromatic hydrocarbons (PAH) by repetitive development with the same eluent is demonstrated in Figure 8B. The chromatogram was obtained with octadecyl silica layer and methanol-water (4:1) as eluent. Chrom-atogram developments were performed in an SB/CD chamber, position 4 (Regis Chemical Co.). The first four developments were performed for 5 min each and the next three for 7 min each. Between developments the plate was dried using a stream of purified nitrogen. The same PAH mixture was also separated applying conventional single development with the same plate and eluent. Figure 8B clearly shows the advantage of multiple development, in comparison to conventional development (Figure 8A).

Multiple development using change in eluent strength (stepwise gradient development) of each development stage is suitable for the separation of samples with a wide range of polarities. An example of this approach is shown in Figure 9 for the separation of PTH-amino acid derivatives. Chromatography was performed on a 10 x 10 cm HPTLC plate coated with silica gel. The spots were applied 0.5 cm from the lower edge of the plate. The plate was developed in a short-bed continuous development chamber. The first development was made with methylene chloride for 5 min with a 3.5 cm development distance (Figure 9A). At this stage, only PTH-proline is well separated from the other derivatives. After evaporation of the methylene chloride, the second development was performed with methylene chloride-isop-ropanol (99: 1 for 10 min with a 7.5 cm development distance). Figure 9B illustrates that five amino acid derivatives can be identified. The third consecutive development was made in the same way as the second (Figure 9C). The fourth step was obtained by development with methylene chloride-isopropanol (97:3) for 10 min (Figure 9D). The most polar PTH-amino acid derivatives are not resolved. Their resolution was achieved in the fifth step with ethyl acetate-

Figure 10 Separation of a mixture of oestrogens by multiple chromatography with fixed solvent entry position (A) and by multiple development with fixed (B) and variable (C) solvent entry position. Conditions are given in the text. The oestrogens, in order of migration, are 17^-dihydroequilenin, 17a-dihydroequilenin, 17^-oestradiol, 17a-oestradiol, equilenin and oestrone. (Reproduced with permission from Poole SK and Poole CF (1992) Insights into mechanism and applications of unidimensional multiple development in thin layer chromatography. Journal of Planar Chromatography 5: 221.)

Figure 10 Separation of a mixture of oestrogens by multiple chromatography with fixed solvent entry position (A) and by multiple development with fixed (B) and variable (C) solvent entry position. Conditions are given in the text. The oestrogens, in order of migration, are 17^-dihydroequilenin, 17a-dihydroequilenin, 17^-oestradiol, 17a-oestradiol, equilenin and oestrone. (Reproduced with permission from Poole SK and Poole CF (1992) Insights into mechanism and applications of unidimensional multiple development in thin layer chromatography. Journal of Planar Chromatography 5: 221.)

acetonitrile-glacial acetic acid (74.3 : 25 : 0.7); only two derivatives (GLU, GLN) are not separated (Figure 9E).

The separation efficiency of conventional multiple development can be further improved by moving the solvent entry to a higher position on the chromato-graphic plate for each successive development. Figure 10 shows the separation of a mixture of six

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Figure 11 Two-dimensional chromatogram on RP-18 plate. Eluents: in the first direction, hexane-ethyl acetate-acetic acid (80:18:2); in the second direction, 1 mol L~1 ammonia #3% potassium chloride in 60% methanol. SP = Starting point. 1, DNP-Gly; 2, DNP-Ala; 3, DNP-Ser; 4, DNP-Thr; 5, DNP-Val; 6, DNP-Leu; 7, DNP-Ile; 8, DNP-Pro; 9, DNP-Met-O2; 10, DNP-Trp; 11, DNP-Phe; 12, Di-DNP-Tyr; 13, DNP-Asp; 14, DNP-Glu; 15, DNP-CySO3Na; 16, Di-DNP-Lys; 17, a-N-DNP-Arg; 18, Di-DNP-His; 19, DNP-OH; 20, DNP-NH2. (Reproduced with permission from Lepri L, Desideri PG and Heimler D (1982) High-performance thin-layer chromatography of 2,4-dinitrophenyl-amino acids on layers of RP-8, RP-18 and ammonium tungstophos-phate. Journal of Chromatography 235: 411.)

Figure 11 Two-dimensional chromatogram on RP-18 plate. Eluents: in the first direction, hexane-ethyl acetate-acetic acid (80:18:2); in the second direction, 1 mol L~1 ammonia #3% potassium chloride in 60% methanol. SP = Starting point. 1, DNP-Gly; 2, DNP-Ala; 3, DNP-Ser; 4, DNP-Thr; 5, DNP-Val; 6, DNP-Leu; 7, DNP-Ile; 8, DNP-Pro; 9, DNP-Met-O2; 10, DNP-Trp; 11, DNP-Phe; 12, Di-DNP-Tyr; 13, DNP-Asp; 14, DNP-Glu; 15, DNP-CySO3Na; 16, Di-DNP-Lys; 17, a-N-DNP-Arg; 18, Di-DNP-His; 19, DNP-OH; 20, DNP-NH2. (Reproduced with permission from Lepri L, Desideri PG and Heimler D (1982) High-performance thin-layer chromatography of 2,4-dinitrophenyl-amino acids on layers of RP-8, RP-18 and ammonium tungstophos-phate. Journal of Chromatography 235: 411.)

oestrogens on silica gel plates, 5x10 cm with a mobile phase of cyclohexane-ethyl acetate (3 : 1, v/v). The chromatograms were scanned at 280 nm. The poorest separation was obtained with simple multiple chromatography (Figure 10A); seven 7 cm developments with fixed solvent entry position at the origin of the plate were used. Separation was improved using multiple development with incrementing times (or distances) of development.

Figure 12 (A) Chromatographic plate prepared for separation of four samples by two-dimensional mode of development. (B) Four two-dimensional chromatograms of hormones on silica gel HPTLC plate, 10 x 10 cm. Eluents: the first direction (two developments simultaneously), heptane-diethyl ether-dichloro-methane (4 : 3 : 2); the second direction (two developments simultaneously), chloroform-ethanol-benzene (36 : 1 : 4). 1, Zeranol; 2, trans-diethylstilbestrol and c/s-diethylstilboestrol; 3 dienoestrol. (Part A reproduced from De Brabander HF, Smets F and Pottie G (1998) Faster and cheaper two-dimensional HPTLC using the '4 x 4' mode. Journal of Planar Chromatography 1: 369.)

Figure 13 (A) Circular and (B) anticircular development. (Reproduced with permission from Bauer K, Gros L and Saur W (1991) Thin Layer Chromatography - An introduction, p. 36. Heidelberg: Huthig Buch).

Figure 12 (A) Chromatographic plate prepared for separation of four samples by two-dimensional mode of development. (B) Four two-dimensional chromatograms of hormones on silica gel HPTLC plate, 10 x 10 cm. Eluents: the first direction (two developments simultaneously), heptane-diethyl ether-dichloro-methane (4 : 3 : 2); the second direction (two developments simultaneously), chloroform-ethanol-benzene (36 : 1 : 4). 1, Zeranol; 2, trans-diethylstilbestrol and c/s-diethylstilboestrol; 3 dienoestrol. (Part A reproduced from De Brabander HF, Smets F and Pottie G (1998) Faster and cheaper two-dimensional HPTLC using the '4 x 4' mode. Journal of Planar Chromatography 1: 369.)

Figure 13 (A) Circular and (B) anticircular development. (Reproduced with permission from Bauer K, Gros L and Saur W (1991) Thin Layer Chromatography - An introduction, p. 36. Heidelberg: Huthig Buch).

Figure 10B shows the chromatogram using nine developments with an incremental increase of the time of each successive development according to the sequence 5, 6, 7, 8, 9, 10, 12, 13, 14 min. However, the best separation was achieved with an incremental increase in the development time, as above, and a variable solvent entry position (0.5 cm below the slowest zone in each development; Figure 10C).

In two-dimensional development the sample is spotted at the corner of the chromatographic plate and developed with the first eluent (in the first direc tion). After this development, the eluent is evaporated from the plate; the spots are positioned along the edge of the chromatographic plate. The plate is then rotated through 90° and the next development is performed with the second eluent from the edge with the separated spots of the first development towards the opposite edge. The mixture can be redistributed on the entire plate surface if both eluents (or chromato-graphic systems) show a dramatic change in selectivity. An example of this mode of separation is shown in Figure 11. Twenty DNP-amino acids were separated using a reversed-phase layer. The sample volume was 0.2-0.3 |L. The spots were visualized in UV light (360 nm with a dried plate or 254 nm when wet). The migration distance was 6 cm. The separations were carried out at 25°C using a Desaga thermostating chamber. The elution in the first direction was performed with hexane-ethyl acetate-acetic acid (80 : 18 : 2) and in the second direction with 1 mol L"1 ammonia +30% potassium chloride in 60% methanol.

Another variant of two-dimensional development is the separation of four samples on one plate instead of one sample on one plate. Figure 12 shows the application of this method to the separation of hormones. The silica gel plate, 10x10 cm, is divided into four sample zones and four reference zones, as shown in Figure 12A. The four samples (S1, S2, S3, S4) are spotted at each corner of the plate and the reference solutes on the four reference zones (R1, R2, R3, R4). The plate is introduced into a horizontal DS-chamber (Chromdes) or a linear developing chamber (Camag), which allows the development of the plate from two opposite directions simultaneously with eluent

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