A comparison of the parameters of the chromatographic separation of the mixture of dyes obtained by caLC on the adsorbents type RP-8 and RP-18

Abstract

The aim of the presented research was to compare the chromatographic separation process, a mixture of food dyes obtained in capillary action liquid chromatography (caLC) columns packed with adsorbent type RP-8 and RP-18 and using as the eluent: methanol-water-acetic acid (4:5:1, v/v). The obtained retention data were compared with separation in caLC columns with different internal diameters, different distance of migration and the different mode of development the chromatograms.

Introduction

In recent years, emphasis has been placed on chemical analyses that reduce the amount of used solvents that are harmful for the environment (Gałuszka et al., 2012). Capillary action liquid chromatography (caLC) is one method that uses such a restriction the total effluent. The principle of this technique has been described in articles (Gierak et al., 2015; Zhang et al., 2009). Our previous work presents the results of comparative tests proceeded in caLC column and chromatographic plates (TLC). In this paper, we present a comparison of retention data received in columns on two different types of adsorbents, i.e. RP-8 and RP-18, (the dimensions of the Merck adsorbents d₅₀ = 10 µm,).

Experimental

In our investigation, the caLC column was made of quartz tubes supplied by the Department of Fiber Optics Technology (UMCS Lublin, Poland). The method of preparing the column was presented in a previous work (Gierak et al., 2015). The mixture of food dyes: allura red, patent blue, brilliant black in methanol (0.05%) have been separated. The volume of the sample applied on caLC column was 0.1 µL, and the volume of the used mobile phase was 1.0 mL. The bandwidth of dyes was measured using optical loupes with an accuracy of 0.1 mm.

Results and discussion

In this work, the retention data obtained in the caLC columns were compared with the different distance of development the chromatogram (L = 40; 50, 60; 70 mm). We used caLC columns with an internal diameter of 1.4 mm and we developed chromatogram in the ascending method. From the chromatograms, retention parameters were calculated, which are summarized in the tables. The retention data obtained from compared packings type RP-8 or RP-18 with the application as mobile phase mixture: methanol–water–acetic acid (4:5:1, v/v) were compared. The mixture chromatograms of the analyzed dyes obtained on columns caLC (with RP-8 and RP-18 packings) are shown in Figure 1(a) and (b).

Figure 1.

Chromatograms obtained in caLC columns on adsorbent RP-8 (a) and RP-18 (b). The mobile phase: methanol-water-acetic acid (4:5:1, v/v). Internal diameter of capillary 1.4 mm, distance of development: a – 40 mm, b – 50 mm, c – 60 mm, d – 70 mm. Analyzed dyes: 1 – Patent blue, 2 – Allura red, 3 – Brilliant black.

From the analysis of the chromatograms obtained on adsorbent RP-8, it can be observed that the way development does not have a major impact on the retardation factors, hR_f (Table 1). It does not affect significantly on the chromatographic bands blur. Analysis of the height equivalent of a theoretical plates (H), which is a measure of the efficiency of the chromatographic columns shows that, for example for the allura red it is lower on sorbent RP-8 than on the RP-18 and even reaches 0.3–0.5 µm (Figures 1 and 2, Tables 1 and 2). The reason for this might be the phenomenon of demixion, that is, the formation of two fronts of migration of the constituents of the mobile phase. This can affect the narrow bands of these substances that migrate after the forehead. These retention data are summarized in Table 1. According to Geiss (1987), almost the same performance of the separation processes (H in the range of 5 up to 10 µm) can be obtained in chromatography on columns with an open tube capillary (OTC). This is where the chromatography adsorbent is embedded in the form of a thin layer on the inner surface of the column.

Table 1.

Retention parameters obtained in caLC on RP-8 and RP-18 on different distance of development.

	RP-8				RP-18
	hRf	R S	H (µm)	t, min	hRf	R S	H (µm)	t, min
Distance of migration mobile phase 40 mm
Patent blue	30 ± 2	5.69 ± 0.19 3.50 ± 0.00	643 ± 80	28 ± 5	33 ± 3	3.77 ± 0.14 1.11 ± 0.48	950 ± 182	152 ± 5
Allura red	83 ± 2		5.1 ± 0.2		80 ± 2		9.9 ± 0.4
Brilliant black	91 ± 2		0.5 ± 0.0		84 ± 3		2.2 ± 0.2
Distance of migration mobile phase 50 mm
Patent blue	35 ± 1	6.12 ± 0.19 4.00 ± 0.00	491 ± 39	46 ± 7	29 ± 1	5.37 ± 0.32 1.22 ± 0.48	712 ± 68	216 ± 10
Allura red	87 ± 1		3.7 ± 0.1		78 ± 1		8.3 ± 0.3
Brilliant black	95 ± 1		0.3 ± 0.0		81 ± 1		1.9 ± 0.1
Distance of migration mobile phase 60 mm
Patent blue	33 ± 2	6.32 ± 0.00 5.17 ± 1.90	601 ± 75	68 ± 5	33 ± 2	6.00 ± 0.00 1.24 ± 0.41	601 ± 75	298 ± 15
Allura red	83 ± 2		3.4 ± 0.2		83 ± 2		6.0 ± 0.3
Brilliant black	92 ± 1		0.3 ± 0.0		87 ± 1		3.1 ± 0.1
Distance of migration mobile phase 70 mm
Patent blue	31 ± 1	6.70 ± 0.14 5.50 ± 2.48	744 ± 49	86 ± 5	31 ± 2	6.06 ± 0.14 1.58 ± 0.36	580 ± 65	395 ± 17
Allura red	81 ± 2		3.0 ± 0.1		77 ± 1		9.4 ± 0.3
Brilliant black	89 ± 2		0.3 ± 0.0		81 ± 2		3.0 ± 0.1

Figure 2.

Chromatograms obtained in caLC columns on adsorbent RP-8 (a) and RP-18 (b). The mobile phase: methanol-water-acetic acid (4:5:1, v/v). Distance of development 60 mm, column internal diameter: a – 1.4 mm, b – 1.7 mm, c – 2.5 mm. Analyzed dyes: 1 – Patent blue, 2 – Allura red, 3 – Brilliant black.

Table 2.

Retention parameters obtained in caLC on RP-8 and RP-18 on different column internal diameter.

	RP-8				RP-18
	hRf	R S	H (µm)	t, min	hRf	R S	H (µm)	t, min
Internal diameter of column 1.4 mm
Patent blue	33 ± 2	6.32 ± 0.00 5.17 ± 1.90	601 ± 75	68 ± 5	33 ± 2	6.00 ± 0.00 1.24 ± 0.41	601 ± 75	298 ± 15
Allura red	83 ± 2		3.4 ± 0.2		83 ± 2		6.0 ± 0.3
Brilliant black	92 ± 2		0.3 ± 0.0		87 ± 1		3.1 ± 0.1
Internal diameter of column 1.7 mm
Patent blue	33 ± 1	5.78 ± 0.36 2.86 ± 0.00	621 ± 45	70 ± 8	28 ± 2	6.41 ± 0.14 1.05 ± 0.41	832 ± 122	172 ± 8
Allura red	83 ± 2		9.4 ± 0.5		84 ± 1		9.1 ± 0.3
Brilliant black	92 ± 1		1.2 ± 0.1		88 ± 2		1.4 ± 0.1
Internal diameter of column 2.5 mm
Patent blue	29 ± 1	6.49 ± 0.40 3.24 ± 0.41	589 ± 47	61 ± 4	28 ± 1	8.81 ± 0.15 0.95 ± 0.42	649 ± 56	171 ± 10
Allura red	81 ± 2		10.0 ± 0.5		82 ± 1		9.7 ± 0.3
Brilliant black	90 ± 1		1.3 ± 0.0		85 ± 1		1.5 ± 0.0

The data analysis shows that for the brilliant black on column with adsorbent type RP-8, we obtain very high performance (H = 0.3–0.5 µm only). We observe a somewhat lower efficiency on the column with adsorbent type RP-18 (H = 2.2 to 3.1 µm). This is probably the result of the difference in polarity of the adsorbent types RP-8 and RP-18. Adsorbent RP-18 is less polar than adsorbent RP-8. We have received quite different results for band of the patent blue on adsorbents RP-8 and RP-18, for which the hR_f was in the range 29–35 (depending on the distance migration of the mobile phase, which was used to develop of the chromatogram – Figure 1 and Table 1). For this substance (patent blue), we get much lower efficiency (H = 490–950 µm, Tables 1 and 2). It is three orders of magnitude lower (1000×) than for brilliant black.

For an explanation of this effect, we must consider the fact that patent blue apparently changes the form of the presence depending on the pH of the eluent. In the eluent methanol–water–acetic acid, dye occurs mainly in the polar form. In this phase (methanol–water–acetic acid 4:5:1 (v/v)), patent blue is in the form that has the protonated nitrogen atoms of the amide group. Due to the presence of positive charge on the amide group and the negative charge on the sulphonyl groups, this molecule has clearly the largest dipole moment. This results in stronger interaction with the stationary phase than with the eluent and leads to the small value of the hR_f. The high retention rate of chromatographic band (small hR_f, for patent blue) gives a very fuzzy chromatographic band and in consequence a large value of H (i.e. low efficiency).

The retention data received on RP-18-caLC (hR_f, Figure 1(b)) are repeated on all the analyzed distances of development (Table 1). Some deviation occurs for brilliant black on the distance to develop 60 mm (hR_f = 87). In addition, on caLC column with RP-18 for allura red does not have a clear relationship between the efficiency of the column (expressed as H) and the way of development, this value reaches its minimum on the distance L = 60 mm (H = 6.0 µm).

Significant limitation analysis of caLC on RP-18 is a very long time to develop the chromatograms (from 150 to 400 min). When compared to the chromatography in the TLC on RP-8, it is a longer four to five times higher (30–90 min).

The effect of column diameter

We examined the effect of diameter of caLC column on the efficiency and time development of the chromatograms. Zhang et al. (2009) used the columns with internal diameters of 0.2–0.5 mm. So, small column diameter creates problems with their packaging. Zhang et al. have used the columns of the ratio of the internal diameter to the size of the grains: 50:1 up to 380:1. During our realization studies, we used the columns with internal diameters (ϕ) 1.4, 1.7 and 2.5 mm, with the ratio of their internal diameter to the size of the grains: 140:1 (1.4 mm) and increased to 250:1 for column 2.5 mm, and we have used adsorbents RP-8 and RP-18 (with a diameter of 10 µm grains). Chromatograms were developed on the distance 60 mm, in ascending mode.

Analyzing the retardation factors (hR_f) obtained for the dyes on the RP-8, one can notice that their values do not change with an increase in the diameter of the column (Figure 2). We examined the effect of inner diameter of the column on the time development of the chromatograms. It was found that there not relationship between these parameters, and the time development of chromatograms is almost constant (about 65 min).

We compared the resolution (R_s) and the height equivalent of a theoretical plate (H), for the separation of mixtures of food dyes in the caLC column of various diameters. For columns with diameters of ϕ = 1.4 mm, resolution of these bands is the total (R_s > 2) and greater than those obtained in the larger diameter columns (i.e. 1.7 and 2.5 mm) (Table 2).

For columns with the larger diameter (1.7 and 2.5 mm) more blur of bands is observed, and hence lower efficiency (higher H) than in the column with a diameter of 1.4 mm. The greater diameter of the columns can be associated with a greater transverse diffusion. The obtained values of the efficiency and resolution for the analytes do not differ significantly from those which have been obtained by Zhang et al. (2009) during tests on the columns of lower diameters (0.3–0.5 mm).

For the dyes migrated in caLC column as the second band, the best performance was obtained for allura red, where H ≈ 3.4 µm and presents the results of the analysis in a column with a diameter of 1.4 mm (Table 2). Probably this substance could have migrated in demixion zone, like the first separated dyes. This suggestion results from the fact that H is lower than 2 × d_p (where d_p means particle diameter).

Also, we carried out the test for caLC columns with diameters 1.4, 1.7 and 2.5 mm, packed with RP-18, using the same mobile phase as were used for columns with RP-8 packing. The value of hR_f coefficients for each of the tested dyes is repeatable and independent of column diameter. By comparing the resolution obtained on RP-18, we do not observe influence this parameter from the diameter of the column. For the eluent, methanol–water–acetic acid resolution increases with an increase in diameter of the column, for the pair: patent blue-allura from 6.00 (for ϕ = 1.4 mm) to 8.81 (for ϕ = 2.5 mm) (Table 2). The resolution of a pair of allura and brilliant black is not full and decreases with increasing diameter of the columns: from the 1.24 (for column 1.4 mm) to 0.95 (for column 2.5 mm).

Migration time of the mobile phase in the column caLC on adsorbent RP-18 is very long. This may be due to the low wettability of RP-18 adsorbent by eluent. Time of development of chromatograms is the longest in the columns with a diameter of 1.4 mm (approx. 300 min). In the columns with diameters between 1.7 and 2.5 mm analysis, time is almost constant (about 170 min).

By analyzing the separation process parameters in columns with different diameters, one can notice that the most optimal and repeatable results data retention was obtained in columns with a diameter of 1.4 mm. Therefore, in all comparative studies, we used columns of this diameter.

Mode development caLC columns

In the TLC chromatographic chambers, it is possible to develop the chromatograms in the ways: horizontal, ascending and descending. In caLC, you can also develop the chromatogram in different directions and, therefore it was examined as a way of developing in caLC which affects the efficiency of separation. The columns were placed in the vial filled with eluent and sealed with silicone septa, and then were set in the right position. All chromatograms were developed in the caLC columns with a diameter of 1.4 mm, 60 mm.

Analyzing the parameters of the retention obtained for allura red, we can observe that the retardation factor (hR_f) obtained after ascending and descending way is similar, while the analysis in horizontal position gives lower values hR_f (Figure 3; Table 3). The highest efficiency on the RP-8 for allura red was obtained after the analysis of the ascending way (H approx. 3.4 µm). No clear relationship was observed between the time of analysis and the direction of the development of chromatograms; the shortest time was receiving in horizontal mode (approx. 28 min for RP-8).

Figure 3.

Chromatograms obtained in caLC columns on RP-8 (a) and RP-18 (b). The mobile phase: methanol-water-acetic acid (4:5:1, v/v). Distance of development 60 mm, internal diameter 1.4 mm, mode of development: a – ascending, b – descending, c – horizontal. Analyzed dyes: 1 – Patent blue, 2 – Allura red, 3 – Brilliant black.

Table 3.

Retention parameters obtained in caLC on RP-8 and RP-18 on different mode of development.

	RP-8				RP-18
	hRf	R S	H [µm]	t, min	hRf	R S	H [µm]	t, min
Ascending mode development
Patent blue	33 ± 2	6.32 ± 0.00 5.17 ± 1.90	601 ± 75	68 ± 5	33 ± 2	6.00 ± 0.00 1.24 ± 0.41	601 ± 75	298 ± 15
Allura red	83 ± 2		3.4 ± 0.2		83 ± 2		6.0 ± 0.3
Brilliant black	92 ± 1		0.3 ± 0.0		87 ± 1		3.1 ± 0.1
Descending mode development
Patent blue	31 ± 2	4.72 ± 0.11 2.06 ± 0.26	889 ± 120	79 ± 2	33 ± 1	6.35 ± 0.15 1.50 ± 0.00	467 ± 34	346 ± 12
Allura red	82 ± 1		24.8 ± 0.7		83 ± 2		9.4 ± 0.5
Brilliant black	91 ± 1		2.8 ± 0.1		88 ± 2		3.0 ± 0.1
Horizontal mode development
Patent blue	25 ± 1	4.09 ± 0.25 1.49 ± 0.25	1 706 ± 168	28 ± 2	33 ± 2	5.40 ± 0.00 1.13 ± 0.29	460 ± 57	368 ± 17
Allura red	76 ± 2		45.3 ± 2.5		78 ± 2		15.3 ± 0.8
Brilliant black	84 ± 1		3.3 ± 0.1		83 ± 1		6.0 ± 0.2

While analyzing during ascending and descending mode on the RP-18, we received reproducible hR_f value (Figure 3(b); Table 3). On the chromatograms developed in horizontal position only blue patent has repeatable value (hR_f = 33) while allura red and brilliant black show stronger retention (hR_f 78 and 83, respectively) than on the chromatograms obtained in the ascending and descending mode.

The resolution for the last separated pair of dyes (red allura and brilliant black) for the chromatograms develops in horizontal and ascending way on column with RP-18 are low (R_s ≈ 1.2) (Figure 3(b) and Table 3). Better resolution (R_s) is obtained after analysis on this adsorbent in descending mode (R_s = 1.5) (Figure 3(b) and Table 3).

The big drawback analysis caLC on RP-18, compared to fill the RP-8, is the time to develop, which is very long and equal 150 to 370 min. The reason for this may be less wettability mobile phase of the more hydrophobic RP-18 packing.

Conclusions

The advantage of the technique of caLC is the ability to use a small amount of mobile phase, and hence the formation of small quantities of waste to be disposed of. This is perfectly connecting within the scope of “Green Chemistry”. caLC technique can be an alternative for TLC, giving similar efficiency of separation of mixtures of dyes.