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Column Chromatography of Dyes: Microanalysis with a Thin Layer Chromatography Scanner Works to Beat the Bands
MICROCHEMICAL JOURNAL ARTICLE NO.
55, 145–150 (1997)
MJ961407
Column Chromatography of Dyes: Microanalysis with a Thin Layer Chromatography Scanner Works to Beat the Bands JUDITH M. BONICAMP1
AND
ELIZABETH B. MOLL
Department of Chemistry, Middle Tennessee State University, Murfreesboro, Tennessee 37132 Quantitative analysis students have performed the following experiment in our laboratories for 15 yr with generally disappointing results: column chromatographic separation of a dye mixture on alumina followed by spectrophotometric analysis of the eluates and quantification of the dyes using Beer’s law. Previous workers claimed that these dyes, Victoria Blue R, methylene blue, and fluorescein, are separable under the chromatography conditions, but we have always had loss of dye on the column or poor separation or both. We have formulated a replacement dye mixture (Oil Red O, Victoria Blue R, fluorescein) that separates cleanly with inexpensive solvents. For preliminary studies we used TLC plates with novel sample application discs and a TLC scanner to measure lmax values for microgram quantities of the dyes. The dyes mentioned can be recovered nearly quantitatively from alumina columns. The mud-colored dye mixture becomes aesthetically pleasing as the dyes move down the column and separate into brightly colored bands, making this experiment useful for classroom demonstrations of the column chromatographic technique. q 1997 Academic Press
INTRODUCTION
For years we have used a column chromatography experiment in quantitative analysis to separate a dye mixture prior to spectrophotometric analysis (1, 2). Students inoculate a basic alumina column with a mixture of Victoria Blue R, methylene blue, and fluorescein (about 0.1 mg/mL of each dye), and then separate the dyes with 95% ethanol to elute the Victoria Blue R and methylene blue and then with dilute ammonia to elute the fluorescein. The experiment is safe and cheap, but it has flaws, the main problem being poor separation of Victoria Blue R and methylene blue. Due to the similar colors of the blue dyes, it is difficult for the student to detect when the methylene blue begins to elute. Since the dyes have similar lmax’s, poorly separated fractions do not lend themselves well to the technique of simultaneous quantification of two components by Beer’s law (3). Another flaw is that close to 50% of the methylene blue is retained on the seasand that holds the alumina in the column. We wanted to continue using an introductory chromatography experiment in quantitative analysis, so we redesigned the experiment to make it work better. We sought a replacement for methylene blue that would separate cleanly from Victoria Blue R, avoiding the need for solving simultaneous equations. We had available among others the two dyes new methylene blue and Oil Red O, and will describe their use here. Oil Red O is less polar than Victoria Blue R, and new methylene blue, with a structure different from that of methylene blue, is reported to separate better from a variety of other dyes (4). 1
To whom correspondence should be addressed. 145 0026-265X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Absorption spectrum of Oil Red O acquired with a TLC scanner in the absorbance–reflectance mode from a TLC plate inoculated with 10 mL of 0.3 mg/L solution (--- background, r – r spot spectrum, — difference spectrum).
MATERIALS AND METHODS
Stock solutions of Victoria Blue R, new methylene blue, methylene blue, and fluorescein (Aldrich Chemical Co.) contained 1.0 mg/mL. Oil Red O stock solutions (Sigma Chemical Co.) contained 3.0 mg/mL. The solvent was 95% ethanol except that fluorescein was dissolved in dilute ammonia. Working solutions, either of pure compounds or mixtures, were 10-mL aliquots from the stock solutions diluted to 100 mL with 95% ethanol. The concentrations of the standard solutions were 1.0 mg/L except that Oil Red O was 3.0 mg/L. We used a scanning densitometer (CS930 TLC Scanner, Shimadzu Instruments, Columbia, MD) to determine the lmax’s for Oil Red O and new methylene blue. Glass microfiber TLC plates impregnated with silica gel (Ansys, Inc., Irvine, CA) were inoculated by depositing 10 mL of working solution on small discs of chromatography medium, the solvent was removed by heat and evaporation, and the discs were inserted into small holes at the lower end of the TLC plates. The plates were migrated in ethyl acetate or in 95% ethanol and the solvent was removed by heating gently. Plots of the absorbance for the background as the wavelength changed from 370 nm to 700 nm produced the dashed line shown in Fig. 1. A scan of the absorbance of the Oil Red O spot over the same wavelength range produced the dash–dot–dashed line in the figure. The instrument then subtracted the background absorbance from the dye absorbance to produce the corrected absorbance spectrum shown by the solid line in Fig. 1. The wavelength corresponding to the maximum absorbance, lmax , was read directly from the chart. Figures 2 and 3 present the corrected absorbance spectra of new methylene blue and Victoria blue R. Scheme 1 gives a summary of the chromatography procedure. The column is packed with basic alumina (Brockman activity 1, 60-325 mesh, Fisher Co.). Very small
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FIG. 2. Difference absorption spectrum of new methylene blue acquired with a TLC scanner from a plate inoculated with 10 mL of 0.1 mg/L dye solution.
amounts of the solvent are used to rinse the interior walls of the column after inoculation. If large volumes are used, the bands will be broad and separation will be much less complete. Instead of the suggested buret (1), we use a three-part chromatography column (Fig. 4) to run the separations. This column has two advantages: first, it is much easier to clean than a buret; and second, the cost of a sturdy small chromatography column is $21.10 (Fisher Co.), one-fourth that of a fragile class-A buret. Working solutions for testing were mixtures of either new methylene blue, Victoria
FIG. 3. Difference absorption spectrum of Victoria Blue R acquired with a TLC scanner from a plate inoculated with 10 mL of 0.1 mg/L dye solution.
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Column Preparation Insert glass wool plug and 3-5 mm sea sand. Add 20 cm alumina continuously with tapping. Place filter paper circle on top of alumina. Pre wash column with elution solvent. Inoculation Pipette 1.00 mL of dye mixture working solution onto column. Rinse interior walls with 1-mL aliquots of solvent. Elution Add elution solvent in 5-mL portions. Collect dye fractions in 100-mL volumetric flasks. Change flasks after each dye emerges and dilute to volume with solvent. Change solvents as needed to elute constituents. Spectral Analysis and Quantification Set Spectronic 20 to wavelength of maximum absorbance. Measure absorbance of dye solution from column and compare with absorbance of standard solution. Calculate mg/mL dye in unknown using Beer’s Law. SCHEME 1. Procedure for separation of dyes by chromatography.
Blue R, and fluorescein, or Oil Red O, Victoria Blue R, and fluorescein. Based on the knowledge from thin layer chromatography that Oil Red O migrates with the solvent front in ethyl acetate, we attempted using a small amount of ethyl acetate to separate the Oil Red O and Victoria Blue R. A variety of other elution solvents were investigated, including 95% ethanol, ethanol–ethyl acetate mixtures, absolute ethanol, and 2-propanol, but in all the experiments fluorescein was eluted with dilute ammonia. Recovery from the column was assessed by spectrophotometry. The percent recoveries of the dyes were calculated from the ratio of the absorbance of the diluted solutions recovered from the column divided by the absorbance of the standard solutions. RESULTS
New methylene blue elutes before the Victoria Blue R with 95% ethanol, which is different from the behavior of methylene blue. The two dyes did not separate, as evidenced by the low recovery of new methylene blue (87%) and the extremely high recovery of Victoria Blue R (124%). Very likely the spectrophotometer was reading both new methylene blue and Victoria Blue R in the Victoria Blue R fraction due to mixing and to the closeness of the lmax values for the dyes (625 nm for NMB and 600 nm for VBR). In some experiments 30% of the new methylene blue was retained on the seasand. Oil Red O has a lmax of 518 nm as determined with the TLC scanner. In the experiments in which Oil Red O, Victoria Blue R, and fluorescein were eluted, the colored bands enabled us to see when to change collection flasks. There sometimes was mixing between Oil Red O and Victoria Blue R using 95% ethanol. A 10-mL aliquot of ethyl acetate to start the elution improved the separation of Oil Red O and Victoria Blue R, but, unfortunately, the ethyl acetate interfered with the subsequent
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DYES BY CHROMATOGRAPHY/TLC SCANNER
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FIG. 4. Proper setup for separation using alumina in a three-part chromatography column with Teflon stopcock.
elution of fluorescein. A 1:9 mixture of ethyl acetate and 95% ethanol gave a better separation than 95% ethanol, and fluorescein was unaffected by the mixture. Absolute ethanol yielded a better separation than 95% ethanol, but poorer than the ethyl acetate–
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ethanol mixture. The best separation was with 2-propanol, but its flow rate is slower than ethanol’s, and the separation required about 5 h on a 20-cm column. DISCUSSION
The spectrophotometric data show that of the five dyes, new methylene blue and methylene blue are retained to the highest degree on the seasand. Their recoveries were only 67% and 48%, respectively, using 95% ethanol, while Oil Red O, Victoria Blue R, and fluorescein had recoveries of 90% or greater. This observation alone did not impel us to reject the new methylene blue. The Victoria Blue R and new methylene blue separate poorly and the closeness of the lmax’s of the two dyes prevents a straightforward mathematical analysis of the spectrophotometric data from the mixtures. The best combination of dyes was Oil Red O, Victoria Blue R, and fluorescein. Oil Red O and Victoria Blue R separate well with 95% ethanol. Two solvent systems that separate the two dyes better and are feasible for use in a teaching laboratory situation are a 1:9 mixture of ethyl acetate with 95% ethanol and absolute ethanol. The ethyl acetate mixture needs to be used in a hood, and absolute ethanol costs more than 95% ethanol, offsetting its usefulness. Since 95% ethanol is cheap and safe to use, we recommend it for separating this dye mixture. Determination of the lmax values for dyes using a TLC scanner is rapid and convenient. The TLC plates with the novel sample application discs allow us to analyze microgram samples of the dyes. The plates give lmax values for Oil Red O and new methylene blue that agree with those reported in the supplier’s catalogs. When students compare the spectra obtained using the TLC scanner (Figs. 1 and 3), they can see the separation of the maxima for Oil Red O and Victoria Blue R in addition to the extent by which their absorption spectra overlap. The lmax values differ by about 80 nm. If the chromatographic separation is less than ideal, the fractions can now be analyzed using the method of simultaneous quantification of two components by Beer’s law. ACKNOWLEDGMENT ANSYS, Inc., Irvine, California, 92718-2002 donated chromatography supplies for the project. The University Noninstructional Assignment Committee funded released time for preparation of the manuscript. Elizabeth B. Moll is a chemistry major who plans to teach high school chemistry.
REFERENCES 1. Dilts, R. V. Analytical Chemistry: Methods of Separation, pp. 358–363. Van Nostrand, New York, 1974. 2. Shott, J.; Heine, W. H. J. Chem. Ed., 1951, 28, 39–40. 3. Huber, W.; Ewing, G. W.; Kriger, J. J. Am. Chem. Soc., 1945, 67, 609–617. 4. Reynolds, R. C.; O’Dell, C. A. J. Chem. Ed., 1992, 69, 989–991.
ah06$$1407
01-29-97 22:16:04
mica
AP: MICROCHEM
55, 145–150 (1997)
MJ961407
Column Chromatography of Dyes: Microanalysis with a Thin Layer Chromatography Scanner Works to Beat the Bands JUDITH M. BONICAMP1
AND
ELIZABETH B. MOLL
Department of Chemistry, Middle Tennessee State University, Murfreesboro, Tennessee 37132 Quantitative analysis students have performed the following experiment in our laboratories for 15 yr with generally disappointing results: column chromatographic separation of a dye mixture on alumina followed by spectrophotometric analysis of the eluates and quantification of the dyes using Beer’s law. Previous workers claimed that these dyes, Victoria Blue R, methylene blue, and fluorescein, are separable under the chromatography conditions, but we have always had loss of dye on the column or poor separation or both. We have formulated a replacement dye mixture (Oil Red O, Victoria Blue R, fluorescein) that separates cleanly with inexpensive solvents. For preliminary studies we used TLC plates with novel sample application discs and a TLC scanner to measure lmax values for microgram quantities of the dyes. The dyes mentioned can be recovered nearly quantitatively from alumina columns. The mud-colored dye mixture becomes aesthetically pleasing as the dyes move down the column and separate into brightly colored bands, making this experiment useful for classroom demonstrations of the column chromatographic technique. q 1997 Academic Press
INTRODUCTION
For years we have used a column chromatography experiment in quantitative analysis to separate a dye mixture prior to spectrophotometric analysis (1, 2). Students inoculate a basic alumina column with a mixture of Victoria Blue R, methylene blue, and fluorescein (about 0.1 mg/mL of each dye), and then separate the dyes with 95% ethanol to elute the Victoria Blue R and methylene blue and then with dilute ammonia to elute the fluorescein. The experiment is safe and cheap, but it has flaws, the main problem being poor separation of Victoria Blue R and methylene blue. Due to the similar colors of the blue dyes, it is difficult for the student to detect when the methylene blue begins to elute. Since the dyes have similar lmax’s, poorly separated fractions do not lend themselves well to the technique of simultaneous quantification of two components by Beer’s law (3). Another flaw is that close to 50% of the methylene blue is retained on the seasand that holds the alumina in the column. We wanted to continue using an introductory chromatography experiment in quantitative analysis, so we redesigned the experiment to make it work better. We sought a replacement for methylene blue that would separate cleanly from Victoria Blue R, avoiding the need for solving simultaneous equations. We had available among others the two dyes new methylene blue and Oil Red O, and will describe their use here. Oil Red O is less polar than Victoria Blue R, and new methylene blue, with a structure different from that of methylene blue, is reported to separate better from a variety of other dyes (4). 1
To whom correspondence should be addressed. 145 0026-265X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
ah06$$1407
01-29-97 22:16:04
mica
AP: MICROCHEM
146
BONICAMP AND MOLL
FIG. 1. Absorption spectrum of Oil Red O acquired with a TLC scanner in the absorbance–reflectance mode from a TLC plate inoculated with 10 mL of 0.3 mg/L solution (--- background, r – r spot spectrum, — difference spectrum).
MATERIALS AND METHODS
Stock solutions of Victoria Blue R, new methylene blue, methylene blue, and fluorescein (Aldrich Chemical Co.) contained 1.0 mg/mL. Oil Red O stock solutions (Sigma Chemical Co.) contained 3.0 mg/mL. The solvent was 95% ethanol except that fluorescein was dissolved in dilute ammonia. Working solutions, either of pure compounds or mixtures, were 10-mL aliquots from the stock solutions diluted to 100 mL with 95% ethanol. The concentrations of the standard solutions were 1.0 mg/L except that Oil Red O was 3.0 mg/L. We used a scanning densitometer (CS930 TLC Scanner, Shimadzu Instruments, Columbia, MD) to determine the lmax’s for Oil Red O and new methylene blue. Glass microfiber TLC plates impregnated with silica gel (Ansys, Inc., Irvine, CA) were inoculated by depositing 10 mL of working solution on small discs of chromatography medium, the solvent was removed by heat and evaporation, and the discs were inserted into small holes at the lower end of the TLC plates. The plates were migrated in ethyl acetate or in 95% ethanol and the solvent was removed by heating gently. Plots of the absorbance for the background as the wavelength changed from 370 nm to 700 nm produced the dashed line shown in Fig. 1. A scan of the absorbance of the Oil Red O spot over the same wavelength range produced the dash–dot–dashed line in the figure. The instrument then subtracted the background absorbance from the dye absorbance to produce the corrected absorbance spectrum shown by the solid line in Fig. 1. The wavelength corresponding to the maximum absorbance, lmax , was read directly from the chart. Figures 2 and 3 present the corrected absorbance spectra of new methylene blue and Victoria blue R. Scheme 1 gives a summary of the chromatography procedure. The column is packed with basic alumina (Brockman activity 1, 60-325 mesh, Fisher Co.). Very small
ah06$$1407
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DYES BY CHROMATOGRAPHY/TLC SCANNER
147
FIG. 2. Difference absorption spectrum of new methylene blue acquired with a TLC scanner from a plate inoculated with 10 mL of 0.1 mg/L dye solution.
amounts of the solvent are used to rinse the interior walls of the column after inoculation. If large volumes are used, the bands will be broad and separation will be much less complete. Instead of the suggested buret (1), we use a three-part chromatography column (Fig. 4) to run the separations. This column has two advantages: first, it is much easier to clean than a buret; and second, the cost of a sturdy small chromatography column is $21.10 (Fisher Co.), one-fourth that of a fragile class-A buret. Working solutions for testing were mixtures of either new methylene blue, Victoria
FIG. 3. Difference absorption spectrum of Victoria Blue R acquired with a TLC scanner from a plate inoculated with 10 mL of 0.1 mg/L dye solution.
ah06$$1407
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Column Preparation Insert glass wool plug and 3-5 mm sea sand. Add 20 cm alumina continuously with tapping. Place filter paper circle on top of alumina. Pre wash column with elution solvent. Inoculation Pipette 1.00 mL of dye mixture working solution onto column. Rinse interior walls with 1-mL aliquots of solvent. Elution Add elution solvent in 5-mL portions. Collect dye fractions in 100-mL volumetric flasks. Change flasks after each dye emerges and dilute to volume with solvent. Change solvents as needed to elute constituents. Spectral Analysis and Quantification Set Spectronic 20 to wavelength of maximum absorbance. Measure absorbance of dye solution from column and compare with absorbance of standard solution. Calculate mg/mL dye in unknown using Beer’s Law. SCHEME 1. Procedure for separation of dyes by chromatography.
Blue R, and fluorescein, or Oil Red O, Victoria Blue R, and fluorescein. Based on the knowledge from thin layer chromatography that Oil Red O migrates with the solvent front in ethyl acetate, we attempted using a small amount of ethyl acetate to separate the Oil Red O and Victoria Blue R. A variety of other elution solvents were investigated, including 95% ethanol, ethanol–ethyl acetate mixtures, absolute ethanol, and 2-propanol, but in all the experiments fluorescein was eluted with dilute ammonia. Recovery from the column was assessed by spectrophotometry. The percent recoveries of the dyes were calculated from the ratio of the absorbance of the diluted solutions recovered from the column divided by the absorbance of the standard solutions. RESULTS
New methylene blue elutes before the Victoria Blue R with 95% ethanol, which is different from the behavior of methylene blue. The two dyes did not separate, as evidenced by the low recovery of new methylene blue (87%) and the extremely high recovery of Victoria Blue R (124%). Very likely the spectrophotometer was reading both new methylene blue and Victoria Blue R in the Victoria Blue R fraction due to mixing and to the closeness of the lmax values for the dyes (625 nm for NMB and 600 nm for VBR). In some experiments 30% of the new methylene blue was retained on the seasand. Oil Red O has a lmax of 518 nm as determined with the TLC scanner. In the experiments in which Oil Red O, Victoria Blue R, and fluorescein were eluted, the colored bands enabled us to see when to change collection flasks. There sometimes was mixing between Oil Red O and Victoria Blue R using 95% ethanol. A 10-mL aliquot of ethyl acetate to start the elution improved the separation of Oil Red O and Victoria Blue R, but, unfortunately, the ethyl acetate interfered with the subsequent
ah06$$1407
01-29-97 22:16:04
mica
AP: MICROCHEM
DYES BY CHROMATOGRAPHY/TLC SCANNER
149
FIG. 4. Proper setup for separation using alumina in a three-part chromatography column with Teflon stopcock.
elution of fluorescein. A 1:9 mixture of ethyl acetate and 95% ethanol gave a better separation than 95% ethanol, and fluorescein was unaffected by the mixture. Absolute ethanol yielded a better separation than 95% ethanol, but poorer than the ethyl acetate–
ah06$$1407
01-29-97 22:16:04
mica
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BONICAMP AND MOLL
ethanol mixture. The best separation was with 2-propanol, but its flow rate is slower than ethanol’s, and the separation required about 5 h on a 20-cm column. DISCUSSION
The spectrophotometric data show that of the five dyes, new methylene blue and methylene blue are retained to the highest degree on the seasand. Their recoveries were only 67% and 48%, respectively, using 95% ethanol, while Oil Red O, Victoria Blue R, and fluorescein had recoveries of 90% or greater. This observation alone did not impel us to reject the new methylene blue. The Victoria Blue R and new methylene blue separate poorly and the closeness of the lmax’s of the two dyes prevents a straightforward mathematical analysis of the spectrophotometric data from the mixtures. The best combination of dyes was Oil Red O, Victoria Blue R, and fluorescein. Oil Red O and Victoria Blue R separate well with 95% ethanol. Two solvent systems that separate the two dyes better and are feasible for use in a teaching laboratory situation are a 1:9 mixture of ethyl acetate with 95% ethanol and absolute ethanol. The ethyl acetate mixture needs to be used in a hood, and absolute ethanol costs more than 95% ethanol, offsetting its usefulness. Since 95% ethanol is cheap and safe to use, we recommend it for separating this dye mixture. Determination of the lmax values for dyes using a TLC scanner is rapid and convenient. The TLC plates with the novel sample application discs allow us to analyze microgram samples of the dyes. The plates give lmax values for Oil Red O and new methylene blue that agree with those reported in the supplier’s catalogs. When students compare the spectra obtained using the TLC scanner (Figs. 1 and 3), they can see the separation of the maxima for Oil Red O and Victoria Blue R in addition to the extent by which their absorption spectra overlap. The lmax values differ by about 80 nm. If the chromatographic separation is less than ideal, the fractions can now be analyzed using the method of simultaneous quantification of two components by Beer’s law. ACKNOWLEDGMENT ANSYS, Inc., Irvine, California, 92718-2002 donated chromatography supplies for the project. The University Noninstructional Assignment Committee funded released time for preparation of the manuscript. Elizabeth B. Moll is a chemistry major who plans to teach high school chemistry.
REFERENCES 1. Dilts, R. V. Analytical Chemistry: Methods of Separation, pp. 358–363. Van Nostrand, New York, 1974. 2. Shott, J.; Heine, W. H. J. Chem. Ed., 1951, 28, 39–40. 3. Huber, W.; Ewing, G. W.; Kriger, J. J. Am. Chem. Soc., 1945, 67, 609–617. 4. Reynolds, R. C.; O’Dell, C. A. J. Chem. Ed., 1992, 69, 989–991.
ah06$$1407
01-29-97 22:16:04
mica
AP: MICROCHEM