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Flash column chromatograms estimated from thin-layer chromatography data
Journal of Chromatography A, 1211 (2008) 49–54
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Flash column chromatograms estimated from thin-layer chromatography data Justin D. Fair ∗ , Chad M. Kormos Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Unit 3060, Storrs, CT 06269-3060, USA
a r t i c l e
i n f o
Article history: Received 13 August 2008 Received in revised form 18 September 2008 Accepted 22 September 2008 Available online 1 October 2008 Keywords: TLC Thin-layer chromatography Open column chromatography Flash column chromatography Predictive chromatography Resolution
a b s t r a c t Given a sample mass and TLC data, a spreadsheet has been developed that provides information on the amount of silica gel needed, the optimal fraction size, and the degree of separation to be expected before flash chromatography is attempted. The spreadsheet is the first utility of its kind to accurately estimate the retention volume and band volume of analytes, as well as the fraction numbers expected to contain each analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted; ensuring a successful first separation. Published by Elsevier B.V.
1. Introduction Thin-layer chromatography (TLC), originally described by Kirchner et al. [1] in 1951, combined with liquid–solid column chromatography, discovered by Tswett [2] in 1906, provides a powerful method for the separation of organic compounds. The theory of transferring TLC data into retention times or, equivalently, retention volumes has been critically reviewed [3–5]. The theory of TLC transference has mainly been applied to reversedphase high-performance liquid chromatography (RP-HPLC) [6–8]. These methods suffer from the relatively large number of preliminary experiments that are required as well as a “case-specific” transference [8,9]. Since its description in 1978, flash column chromatography (FCC) has become a routinely used tool to separate fine chemicals in the organic laboratory [10]. This less efficient preparative system has only “rules of thumb” and uses ambiguous terms, such as difference in column volume (CV), for the transference between TLC and FCC [10,11]. Current automated FC systems on the market, starting at US$ 20 000, do not offer assistance in the selection of optimum separation conditions or provide any predictive information concerning an analyte’s band width or resolution. Even costly, commercially available predictive software designed for HPLC has yet to provide a means to predict the outcome of a flash column (FC) [12].
∗ Corresponding author. Tel.: +1 860 4866488; fax: +1 860 4862981. E-mail address: [email protected] (J.D. Fair). 0021-9673/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.chroma.2008.09.085
Therefore, it was our desire to develop and provide an innovative spreadsheet, based on this transference, to assist in the preparative separation of analytes via FCC that offers accurate information such as the resolution achieved from a particular solvent system. Herein, we describe the development of a spreadsheet that not only assists with solvent selection and flash column conditions, but also accurately determines the retention volume (VR ), band volume (Vb ), resolution (Rs ), and corresponding analyte fraction numbers. This information is useful in determining the predicted resolution before FCC is attempted; ensuring a greener, successful preparative separation generating less waste. The speed gained from such a predictive utility, from solvent system optimization to fraction analysis, proves useful for anyone carrying out preparative separations via FCC. A complete and functional spreadsheet, implementing the methods described below, is included as Supplementary Material.1 2. Experimental 2.1. Chemicals and equipment All samples prepared were made from analytically pure reagents supplied by Aldrich (Milwaukee, WI, USA) or Acros Organics (Mor-
1 Cells in the spreadsheet not needing user input have been protected by a password. If the user desires to alter the utility in any fashion, this feature can be unlocked. Password: chemistry.
50
J.D. Fair, C.M. Kormos / J. Chromatogr. A 1211 (2008) 49–54
Fig. 1. An example spreadsheet: cells B2–B6 and C2–C4 are required for data entry, cells B11 and B12 are for optional data entry, cells B7–B8, E3–E4, F3–F4, G7–G9, H7–H9, I7–I9, and J8–J9 are output relevant to separation optimization.
ris Plains, NJ, USA). Technical-grade solvents, supplied by J.T. Baker (Phillipsburg, NJ, USA), were used as received. Dry solvents, when used, were dried and distilled as recommended [13]. 2.2. Chromatography TLC was performed on 50 mm × 20 mm silica gel 60, F254 , aluminum backed TLC plates. TLC plates were supplied by EMD Chemicals (Gibbstown, NJ, USA). Plates were spotted 5–10 mm above the bottom of the plate and developed to no more than 5–10 mm from the top. Development was performed in a glass jar, with dimensions of 70 mm tall and 35 mm inner diameter, containing only a minimal volume of the solvent system and a TLC plate. Spotters were made from flamed and pulled capillary tubes; allowing for better spotting precision and elimination of spot broadening during sample application. Flash column chromatography was performed on silica gel, 40–63 m (230–400 mesh), 60 Å pore size, pH range of 6.5–7.5, in glass columns designed for FCC. Silica gel was supplied by Sorbent Technologies (Atlanta, GA, USA). Manually packed flash columns were packed using a plug of cotton and the slurry method. After the column was cool to the touch, a volume of the solvent system was pushed through the silica to remove any remaining air and to equilibrate the column. It was during the column equilibration when the bleed valve was adjusted to provide an average linear velocity of ∼2 in./min. Samples were loaded with a Pasteur pipette directly to the head of the column before elution unless otherwise noted. Fractions were collected in test tubes, graduated with the fraction volume, and visualized by spotting on TLC plates using a UV lamp (254 nm) and/or iodine staining. Experimental chromatograms were produced using the experimental analyte fraction numbers and fraction volumes. Commercially-packed flash columns were developed on a Horizon HPFC system supplied by Biotage (Charlottesville, VA, USA). Commercially-packed flash columns were supplied by Biotage, and Luknova (Mansfield, MA, USA). 2.3. Spreadsheet instructions Cell numbers refer to Fig. 1 as well as the spreadsheet file (Supplementary Material). Step 1: From the TLC, the distance traveled by each analyte is entered in the order of least retained to most retained in cells B2–B4; if there are only two analytes, “na” is entered in cell B4. The distance traveled by the solvent front is entered in cell B5. Distance units are arbitrary but they must be uniform.
Step 2: The total mass of sample, in grams, to be loaded is entered in cell B6. The approximate mass loading ratio, XA = analyte mass/total sample mass, is entered in cells C2–C4. The sum of these cells, C2–C4, should equal ∼1.0. The exact mass loading ratio does not need to be fully understood for the utility to function. As these values are entered, the spreadsheet populates the remaining cells and suggests a silica mass (cell B7) and fraction size (cell B8) for the column. Step 3: Alternative values of the silica mass and fraction size may be entered in cells B11 and B12, respectively. Step 4: The spreadsheet outputs the resolution between adjacent peaks (cells E3–E4 and F3–F4), information on the fractions expected to contain each analyte (columns G–I), the expected band volumes (J2–J4), and the number of fractions expected between adjacent bands (J7–J8). The spreadsheet also displays a visual representation of the developed TLC and the expected chromatogram.
3. Results and discussion 3.1. Transference of RF into k The current theory of transferring retention factor (RF ) data into solute capacity factor (k) assumes that both stationary phases consist of the same material and are developed under the same conditions. The RF found by TLC can be related to the corresponding value in column chromatography, viz. k in Eq. (1). The amount of solvent required to elute to the center of a peak, known as the retention volume (VR ), is proportional to 1 + k and the column void volume (Vv ). The void volume is the amount of solvent contained in a packed column bed prior to sample loading. A more direct relationship between RF and VR has been previously derived (Eq. (2)) [14]. It is important to note that RF and VR are inversely proportional. Since additives are contained in the TLC’s stationary phase and TLC relies on capillary action instead of gravity/pressure, this assumption has obvious errors. Reuke and Hauck analyzed the transference of TLC data to HPLC conditions for silica gel, diol, NH2 , CN, RP-18, and RP-8 stationary phases [8]. Good transference for NH2 , CN, RP-18, and RP-8 stationary phases was found, yet silica gel and diol phases seemed to deviate from a straightforward transference. Since Reuke and Hauk only investigated phosphoric esters on the silica gel stationary phase for transference to HPLC, it was decided to search for a common correction coefficient (C) for analytes containing a variety of functional groups for the transference to FCC. k = (1 − RF )/RF
(1)
VR = Vv · (1 + k) = Vv /RF
(2)
0.70 4.2
0.77 0.70 0.68 0.66 4.6 4.2 4.0 4.0
3.0 0.60
9.0 22.0 13.5 25.5 0.62 0.62 0.60 0.57
0.62
13.0 30.5 19.5 34.5 0.66 0.63 0.58 0.62
Middle fraction number
3.2 3.0 2.8 3.0
Middle fraction number
51
VR = Vv · (1 + k) · C = (Vv /RF ) · C
Correction coefficient (C)
Benzaldehyde
Vv equiv.
Correction coefficient (C)
Acetophenone
Vv equiv.
Correction coefficient (C)
J.D. Fair, C.M. Kormos / J. Chromatogr. A 1211 (2008) 49–54
(3)
A series of flash columns, with varying column sizes, were developed and summarized in Table 1. The experimental VR of each analyte was calculated by determining the volume of solvent used to reach the middle fraction in which the analyte eluted. The number of column void volume equivalents (Vv equiv.) taken to reach the experimental retention volume for each analyte is given in Table 1. This value remains approximately constant regardless of the column size. Although each analyte should have its own individual correction coefficient, it was of great interest, as seen in Table 1, to note that all three analytes shared the same approximate value within 6%. The average correction coefficient, calculated using Eq. (3) and taken over the three analytes, was found to be 0.64 for manually-packed columns. To further justify that our correction coefficient was not related to how well we packed our flash columns, the same compounds were developed in commercially-packed columns, where an average correction coefficient of 0.66 was found. This finding, coupled to the fact that the silica gel used in the construction of the manually- and commercially-packed columns were from three different vendors, indicated that no matter how a silica gel column was packed, either manually or commercially, the correction coefficient provided a general average for use in FCC. Many preparative separations involve technical grade solvents; thus, all separations summarized in Table 1 were conducted with a technical grade solvent system comprised of 5% ethyl acetate in hexanes. Additional experiments were also carried out using anhydrous 5% ethyl acetate in hexanes as well as with anhydrous 5% diethyl ether in hexanes. Although the RF values for each analyte varied with changes in the mobile phase composition providing corresponding k values, C remained 0.64. The presence or absence of trace water or the use of alternative solvent systems has no effect on the calculated correction coefficient so long as both the TLC and the column are developed using the same solvent system.
1.8
1.9 1.9 1.8 1.7 3 3 15 21 8.4 21.9 72.3 180.3
5.5 14.0 9.0 15.0
Vv equiv. Middle fraction number
Dibenzyl ether
Fraction size (mL) Column void volume, Vv (mL)
Still and co-workers [10] provided a table recommending an amount of silica gel to be used for a given mass of sample in terms of column diameter and height using a linear velocity of 2 in./min. More recent studies have shown there is little change in resolution when columns of varying height and diameter are used provided that the amounts of sorbent used and linear flow are maintained [15]. On this basis, linear functions (Eqs. (4) and (5)) were derived using the data provided by Still and co-workers. The mass of silica gel (SiO2g ) recommended to separate a given mass of sample (Sg ) depending on whether the separation is easy (RF ≥ 0.2; Eq. (4)) or difficult (0.1 ≤ RF < 0.2; Eq. (5)). Having determined the mass of silica to be used (Eq. (4) or (5)), the column void volume (Vv ) for a manually-packed column containing 4.5–150 g of silica gel may be estimated using Eq. (6), or the void volume may be measured experimentally. Once RF and Vv are known, the center of an
Table 2 Manually-packed column band widths. Mass silica (g)
Average
4.5 12.0 40.0 100.0 1 2 3 4
Mass silica (g)
Entry
Entry
Table 1 Manually-packed column retention volumes.
Retention volume (VR )
3.2. Column mass loading and column void volume
1 2 3 4
4.5 12.0 40.0 100.0
Band width (number of fractions) Dibenzyl ether
Benzaldehyde
Acetophenone
Exp.
Calc.
Exp.
Calc.
Exp.
Calc.
2 5 3 5
2 6 4 7
5 9 7 10
4 10 6 11
7 14 10 14
5 12 8 13
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J.D. Fair, C.M. Kormos / J. Chromatogr. A 1211 (2008) 49–54
Table 3 Assigning a relationship between variable mass loading and N. Entry
1 2 3 4 5
Mass of benzaldehyde loaded (mg) 22 33 63 82 166
a
Mass loading (XA ) Easy
Hard
0.13 0.20 0.37 0.49 1.00
0.33 0.53 1.00 1.31a 2.64a
Number of fractions containing analyte
Total band volume (Vb )
Efficiency (N)
8 9 10 11 12
24 27 30 33 36
90 64 52 43 36
Mass loading values (XA ) between 0.1 and 1.0 should be entered when utilizing the spreadsheet.
Rs = 2[(VR )B − (VR )A ]/[(Vb )B + (Vb )A ]
analyte’s elution band (VR ) may be calculated (Eq. (3)). SiO2g = 59.8 · Sg
(4)
SiO2g = 151.2 · Sg + 0.5
(5)
Vv = 1.8 · SiO2g + 0.3
(6)
3.3. Band broadening prediction in FCC systems The translation of spot broadening in TLC to band broadening in chromatographic columns is a complex process. Spot diffusion is a function of the original spot size, diffusion, and technique error [16]. Indeed, even the position of the origin in relation to the solvent entry level has been found to contribute to spot diffusion in thin layer systems [17]. Due to this complexity, the use of the van Deemter equation was attempted. Approximate values for a majority of terms contained in the van Deemter equation have been previously provided in the literature for high-performance liquid chromatography (HPLC) [18]; yet a more simplified approach was desirable. Band broadening is a function of how long an analyte remains on a column (VR ) and the column efficiency (N) [3,4,19]. Under ideal conditions, band shapes correspond to a Gaussian function. The band volume (Vb ) may be estimated using Eq. (7) from the calculated VR (Eq. (3)) and N. For the experiments summarized in Table 1, the estimated band volumes (Eq. (7)) are in good agreement with experimental values when N is set equal to 90; the estimated and experimentally observed band widths are summarized in Table 2. In short, a value of N = 90 correctly estimates the efficiency of all four column sizes and the assumption is made that this value for N may be used to estimate band volumes of any analyte having mass loading of 1/3 of the sample mass (Sg ) for a hard separation (Eq. (5)). The data shown in Table 2 all use the same mass loading ratio, 0.33, where XA = analyte mass/total sample mass. √ Vb = 4 · = 4 · VR / N (7) Changes in mass loading produce proportional changes in band volume [14]. In an effort to evaluate the effect of changes in mass loading on band volume, benzaldehyde was chromatographed using 5% ethyl acetate in hexanes at various mass loadings using 10 g of silica recommended by Eq. (4) for an easy separation and Eq. (5) for a hard separation, Table 3. The experimentally determined band volumes were converted into column efficiency for a given mass loading dictated by a hard separation (NHARD ) and for an easy separation (NEASY ). These data were fitted to functions of mass loading (XA ) to give Eqs. (8) and (9), respectively. Eqs. (8) and (9) provide a means to predict the band volumes as a function of mass loading relative to the separation difficulty. Entry 5 for an easy separation and entry 3 for a hard separation correctly demonstrate that a greater silica gel (SiO2g ) to sample mass (Sg ) ratio provides for a more efficient column. NHARD = 51.70 · (XA )−0.44 NEASY = 33.64 · (XA )
−0.44
3.4. Predicting resolution in FCC systems As previously stated, RF , and for that matter CV, provide an estimation of the degree of difficulty in a separation; however, these do not provide information regarding the resolution between adjacent peaks. Using the calculated retention volumes (Eq. (3)) and band widths (Eq. (7)), the Rs of adjacent bands may be evaluated using Eq. (10). A resolution of 1.0 affords a 98% separation; a value of ∼1.5 is required for a complete baseline separation between adjacent peaks [19]. The power gained through the prediction of band volumes of analytes allows a much more accurate prediction of the potential resolution a particular solvents system can provide regardless if the separation is an “easy” or “hard” separation. 3.5. Use of the predictive spreadsheet A spreadsheet, included as Supplementary Material, has been developed based on the discussion above to more accurately predict the success of a separation by providing information such as the mass of silica gel needed, the fraction size to take, the expected resolution between analytes, and the actual fraction number containing each analyte. To accomplish this, the spreadsheet utilizes user input such as mass loading, ratio of analyte mass, and distance data obtained from the TLC to produce an estimated chromatogram for a flash chromatographic separation. A representative, static snapshot of the interactive spreadsheet utility is shown in Fig. 1. From the TLC, the distance traveled by each analyte as well as the distance traveled by the solvent front, may be entered in any units as long as they are consistent. The total mass of sample is entered in grams and the analyte ratio should be in decimal form with the sum ∼1.0. When only two analytes are used, “na” (not applicable) can be entered into the distance for spot 3, eliminating all data for that spot. Due to the flexibility of applied spreadsheets, additional rows can be added if a separation contains more than three analytes. The spreadsheet automatically calculates the ideal mass of silica gel required for the separation as well as the ideal fraction size.2 From the calculated column void volume and TLC distances, the volumes at which each analyte will likely elute are calculated along with the estimated fraction numbers. For convenience and flexibility, the mass of silica and fraction size can be changed by the user in the “optional” section of the spreadsheet. The spreadsheet automatically recalculates new volumes and fractions based on this input. The recalculated band volumes assume the maximum loading described by Eqs. (4)–(9). The spreadsheet also calculates and displays the Rs between two adjacent peaks: “Bad” (Rs < 0.8), “Moderate” (0.8 ≤ Rs < 1.5), or “Good Separation” (Rs ≥ 1.5) is displayed depending on the numerical value calculated for Rs . A chromatogram showing the estimated
(8) (9)
(10)
2
The ideal fraction size is calculated to be 1/3 of the column void volume.
J.D. Fair, C.M. Kormos / J. Chromatogr. A 1211 (2008) 49–54
Fig. 2. (a) Estimated chromatogram and (b) experimentally observed chromatogram. Peaks 1 and 2 are methyl octanoate and octanol, respectively.
separation is produced by the spreadsheet assuming Gaussian-type peak shapes. In practice, the best solvent system for a given separation may be determined by simply entering new distance measurements obtained from TLC using various solvent systems. 3.6. Separation examples using the spreadsheet To provide evidence that the spreadsheet, as well as the correction coefficient and mass loading ratios, provide an elegant means for the prediction of FCC is perhaps best appreciated with reference to the following two examples. The separation of octanol and methyl octanoate represents an easy separation (RF > 0.2) of a two component, non-UV-active mixture. These analytes were chosen for their structural dissimilarity to the original benzyl-derived analytes on which the development of the spreadsheet was based. A TLC of the mixture was run in ethyl acetate/triethylamine/hexanes (15.0/0.1/84.9). The distances traveled by each compound as well as the solvent front were entered into the spreadsheet along with the mass loading ratio for each analyte and the total mass of the sample (Table 4). The spreadsheet returned a suggested silica gel mass of 9.6 g and recommended a fraction size of 6.1 mL. Values of 10.0 and 6.0 were entered into the cells labeled “optional grams of silica gel” and “optional fraction size”, respectively. The spreadsheet estimated the fractions that would contain each of the analytes (Table 4) and displayed an estimated chromatogram (Fig. 2a). As demonstrated by the results summarized in Table 4 and the experimental chromatogram (Fig. 2b), there was good agreement not only between the actual
53
Fig. 3. (a) Estimated chromatogram and (b) experimentally observed chromatogram. Peaks 1, 2 and 3 are 2-nitrophenol, phenol and 4-nitrophenol, respectively.
fraction numbers containing each analyte but also the actual resolution to those values estimated by the spreadsheet. It should be noted that the addition of a modifier such as triethylamine does not affect the predictive ability of the spreadsheet as long as both the TLC and FC are developed with the same solvent system. The resolution of a three-component mixture of phenol, 2nitrophenol and 4-nitrophenol was also explored as representative of a hard separation (RF < 0.2). A TLC of the mixture was run in diethyl ether/hexanes (8/92) and the data was entered into the spreadsheet as in the previous example (Table 5). The spreadsheet suggested the use of 39.8 g of silica gel and 24.1 mL fraction sizes. One gram of silica gel was used to dry-load the mixture. Thus, a sample containing 40% (w/w) 2-nitrophenol, 20% (w/w) phenol, and 40% (w/w) 4-nitrophenol with a total mass of 0.26 g was preabsorbed onto 1 g of silica gel. This silica gel was then added onto the head of a packed column before eluting. Therefore, values of 41 and 21 were entered into the cells labeled “optional grams of silica gel” and “optional fraction size”, respectively. The spreadsheet again estimated the fractions that would contain each of the analytes (Table 5) and displayed an estimated chromatogram (Fig. 3a). Good agreement was again observed between the experimental fraction numbers (Fig. 3b) and those predicted (Fig. 3a). 3.7. Errors in calculating VR and Vb As stated previously, errors may be attributable to minor differences in the stationary phases in the TLC and flash chromatography experiments. Indeed, the presence of binder in the TLC, minor differences in activity, pH, particle or pore size, and other factors,
Table 4 The separation of methyl octanoate and octanol. Analyte
Distance (mm)
RF
Mass loading (XA )
Estimated fractions numbers
Actual fractions numbers
Predicted resolution
Actual resolution
Methyl octanoate Octanol
21.5 7.0
0.61 0.20
0.3 0.7
2–4 7–13
4–5 11–15
1.7
1.6
Solvent front
35.0
Total mass: 0.16 g
Table 5 The separation of 2-nitrophenol, 4-nitrophenol and phenol. Analyte
Distance (mm)
RF
Mass loading (XA )
Estimated fractions numbers
Actual fractions numbers
Predicted resolution
Actual resolution
2-Nitrophenol Phenol 4-Nitrophenol
13.0 4.5 1.3
0.37 0.13 0.04
0.4 0.2 0.4
5–8 14–21 47–75
6–9 14–17 38–62
2.4 2.5
1.7 2.4
Solvent front
35.0
Total mass: 0.26 g
54
J.D. Fair, C.M. Kormos / J. Chromatogr. A 1211 (2008) 49–54
including the non-linear velocity profile of the solvent front during TLC development, may influence an analyte’s sorption isotherm. An analyte’s divergence from the average C will attribute to the error. Errors in the predicted VR of highly retained analytes may arise from systematic errors in TLC measurements and from an analyte’s divergence from the average correction coefficient. Greater error for more highly retained analytes stems from the inverse relationship between RF and VR and thus, errors are more likely to propagate. It is therefore recommended that the spreadsheet not be used for RF values less than ∼0.08; RF values less than ∼0.1 produce extremely broad peak shapes due to the broadening associated with a large VR . Due to the nature in which Vb and Rs are calculated, any error in the calculation of VR will be carried into both Vb and Rs . 4. Conclusion In summary, the spreadsheet included as Supplementary Material provides guidance in the development of an optimal system for preparative-scale FCC from easily obtained TLC data. The information gained through the use of the spreadsheet provides a level of detail superior to the “rule of thumb” currently used in the separation of fine chemicals. The implementation of this spreadsheet as a teaching tool in the undergraduate curriculum is currently being evaluated. Acknowledgements We are grateful to Dr. William F. Bailey, Dr. James D. Stuart, Dr. Nicholas E. Leadbeater and Dr. James M. Bobbitt of the University of Connecticut for helpful guidance and suggestions.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2008.09.085. References [1] J.G. Kirchner, J.M. Miller, G. Keller, Anal. Chem. 23 (1951) 420. [2] M. Tswett, Ber. Deutsch. Bot. Ges. 24 (1906) 384. [3] B. Karger, L.R. Snyder, C. Horvath, An Introduction to Separation Science, WileyInterscience, New York, 1973. [4] C.F. Poole, S.K. Poole, Chromatography Today, Elsevier, Amsterdam, New York, 1991. [5] H. Schlitt, F. Geiss, J. Chromatogr. 67 (1972) 261. [6] T. Baczek, M. Radkowska, B. Sparzak, J. Liq. Chromatogr. 30 (2007) 2963. [7] T. Cserhati, E. Forgacs, J. Chromatogr. A 668 (1994) 495. [8] S. Reuke, H.E. Hauck, Fresenius’ J. Anal. Chem. 351 (1995) 739. [9] T. Wennberg, I. Vovk, P. Vuorela, B. Simonovska, H. Vuorela, J. Planar Chromatogr. 19 (2006) 118. [10] W.C. Still, M. Kahn, A. Mitra, J. Org. Chem. 43 (1978) 2923. [11] Horizon HPFC System Operator’s Manual, Biotage, Charlottesville, VA, 2003. [12] L.R. Snyder, J.W. Dolan, D.C. Lommen, J. Chromatogr. 485 (2001) 65. [13] W.L.F. Armarego, C.L.L. Chai, Purification of Laboratory Chemicals, Butterworth–Heinemann, Boston, MA, 2003. [14] R.J. Gritter, J.M. Bobbitt, A.E. Schwarting, Introduction to Chromatography, Holden-Day, New York, 1985. [15] I.Z. Atamna, G.M. Muschick, H.J. Issaq, J. Liq. Chromatogr. 12 (1989) 285. [16] E. Cavalli, T.T. Truong, M. Thomassin, C. Guinchard, Chromatographia 35 (1993) 102. [17] C.F. Poole, W.P.N. Fernando, Planar Chromatogr. 5 (1992) 323. [18] R.W. Stout, J.J. DeStefano, J. Chromatogr. 282 (1983) 263. [19] J.M. Miller, Chromatography Concepts and Contrasts, Wiley–Interscience, New York, 2004.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
Flash column chromatograms estimated from thin-layer chromatography data Justin D. Fair ∗ , Chad M. Kormos Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Unit 3060, Storrs, CT 06269-3060, USA
a r t i c l e
i n f o
Article history: Received 13 August 2008 Received in revised form 18 September 2008 Accepted 22 September 2008 Available online 1 October 2008 Keywords: TLC Thin-layer chromatography Open column chromatography Flash column chromatography Predictive chromatography Resolution
a b s t r a c t Given a sample mass and TLC data, a spreadsheet has been developed that provides information on the amount of silica gel needed, the optimal fraction size, and the degree of separation to be expected before flash chromatography is attempted. The spreadsheet is the first utility of its kind to accurately estimate the retention volume and band volume of analytes, as well as the fraction numbers expected to contain each analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters for preparative-scale separations before the flash column itself is attempted; ensuring a successful first separation. Published by Elsevier B.V.
1. Introduction Thin-layer chromatography (TLC), originally described by Kirchner et al. [1] in 1951, combined with liquid–solid column chromatography, discovered by Tswett [2] in 1906, provides a powerful method for the separation of organic compounds. The theory of transferring TLC data into retention times or, equivalently, retention volumes has been critically reviewed [3–5]. The theory of TLC transference has mainly been applied to reversedphase high-performance liquid chromatography (RP-HPLC) [6–8]. These methods suffer from the relatively large number of preliminary experiments that are required as well as a “case-specific” transference [8,9]. Since its description in 1978, flash column chromatography (FCC) has become a routinely used tool to separate fine chemicals in the organic laboratory [10]. This less efficient preparative system has only “rules of thumb” and uses ambiguous terms, such as difference in column volume (CV), for the transference between TLC and FCC [10,11]. Current automated FC systems on the market, starting at US$ 20 000, do not offer assistance in the selection of optimum separation conditions or provide any predictive information concerning an analyte’s band width or resolution. Even costly, commercially available predictive software designed for HPLC has yet to provide a means to predict the outcome of a flash column (FC) [12].
∗ Corresponding author. Tel.: +1 860 4866488; fax: +1 860 4862981. E-mail address: [email protected] (J.D. Fair). 0021-9673/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.chroma.2008.09.085
Therefore, it was our desire to develop and provide an innovative spreadsheet, based on this transference, to assist in the preparative separation of analytes via FCC that offers accurate information such as the resolution achieved from a particular solvent system. Herein, we describe the development of a spreadsheet that not only assists with solvent selection and flash column conditions, but also accurately determines the retention volume (VR ), band volume (Vb ), resolution (Rs ), and corresponding analyte fraction numbers. This information is useful in determining the predicted resolution before FCC is attempted; ensuring a greener, successful preparative separation generating less waste. The speed gained from such a predictive utility, from solvent system optimization to fraction analysis, proves useful for anyone carrying out preparative separations via FCC. A complete and functional spreadsheet, implementing the methods described below, is included as Supplementary Material.1 2. Experimental 2.1. Chemicals and equipment All samples prepared were made from analytically pure reagents supplied by Aldrich (Milwaukee, WI, USA) or Acros Organics (Mor-
1 Cells in the spreadsheet not needing user input have been protected by a password. If the user desires to alter the utility in any fashion, this feature can be unlocked. Password: chemistry.
50
J.D. Fair, C.M. Kormos / J. Chromatogr. A 1211 (2008) 49–54
Fig. 1. An example spreadsheet: cells B2–B6 and C2–C4 are required for data entry, cells B11 and B12 are for optional data entry, cells B7–B8, E3–E4, F3–F4, G7–G9, H7–H9, I7–I9, and J8–J9 are output relevant to separation optimization.
ris Plains, NJ, USA). Technical-grade solvents, supplied by J.T. Baker (Phillipsburg, NJ, USA), were used as received. Dry solvents, when used, were dried and distilled as recommended [13]. 2.2. Chromatography TLC was performed on 50 mm × 20 mm silica gel 60, F254 , aluminum backed TLC plates. TLC plates were supplied by EMD Chemicals (Gibbstown, NJ, USA). Plates were spotted 5–10 mm above the bottom of the plate and developed to no more than 5–10 mm from the top. Development was performed in a glass jar, with dimensions of 70 mm tall and 35 mm inner diameter, containing only a minimal volume of the solvent system and a TLC plate. Spotters were made from flamed and pulled capillary tubes; allowing for better spotting precision and elimination of spot broadening during sample application. Flash column chromatography was performed on silica gel, 40–63 m (230–400 mesh), 60 Å pore size, pH range of 6.5–7.5, in glass columns designed for FCC. Silica gel was supplied by Sorbent Technologies (Atlanta, GA, USA). Manually packed flash columns were packed using a plug of cotton and the slurry method. After the column was cool to the touch, a volume of the solvent system was pushed through the silica to remove any remaining air and to equilibrate the column. It was during the column equilibration when the bleed valve was adjusted to provide an average linear velocity of ∼2 in./min. Samples were loaded with a Pasteur pipette directly to the head of the column before elution unless otherwise noted. Fractions were collected in test tubes, graduated with the fraction volume, and visualized by spotting on TLC plates using a UV lamp (254 nm) and/or iodine staining. Experimental chromatograms were produced using the experimental analyte fraction numbers and fraction volumes. Commercially-packed flash columns were developed on a Horizon HPFC system supplied by Biotage (Charlottesville, VA, USA). Commercially-packed flash columns were supplied by Biotage, and Luknova (Mansfield, MA, USA). 2.3. Spreadsheet instructions Cell numbers refer to Fig. 1 as well as the spreadsheet file (Supplementary Material). Step 1: From the TLC, the distance traveled by each analyte is entered in the order of least retained to most retained in cells B2–B4; if there are only two analytes, “na” is entered in cell B4. The distance traveled by the solvent front is entered in cell B5. Distance units are arbitrary but they must be uniform.
Step 2: The total mass of sample, in grams, to be loaded is entered in cell B6. The approximate mass loading ratio, XA = analyte mass/total sample mass, is entered in cells C2–C4. The sum of these cells, C2–C4, should equal ∼1.0. The exact mass loading ratio does not need to be fully understood for the utility to function. As these values are entered, the spreadsheet populates the remaining cells and suggests a silica mass (cell B7) and fraction size (cell B8) for the column. Step 3: Alternative values of the silica mass and fraction size may be entered in cells B11 and B12, respectively. Step 4: The spreadsheet outputs the resolution between adjacent peaks (cells E3–E4 and F3–F4), information on the fractions expected to contain each analyte (columns G–I), the expected band volumes (J2–J4), and the number of fractions expected between adjacent bands (J7–J8). The spreadsheet also displays a visual representation of the developed TLC and the expected chromatogram.
3. Results and discussion 3.1. Transference of RF into k The current theory of transferring retention factor (RF ) data into solute capacity factor (k) assumes that both stationary phases consist of the same material and are developed under the same conditions. The RF found by TLC can be related to the corresponding value in column chromatography, viz. k in Eq. (1). The amount of solvent required to elute to the center of a peak, known as the retention volume (VR ), is proportional to 1 + k and the column void volume (Vv ). The void volume is the amount of solvent contained in a packed column bed prior to sample loading. A more direct relationship between RF and VR has been previously derived (Eq. (2)) [14]. It is important to note that RF and VR are inversely proportional. Since additives are contained in the TLC’s stationary phase and TLC relies on capillary action instead of gravity/pressure, this assumption has obvious errors. Reuke and Hauck analyzed the transference of TLC data to HPLC conditions for silica gel, diol, NH2 , CN, RP-18, and RP-8 stationary phases [8]. Good transference for NH2 , CN, RP-18, and RP-8 stationary phases was found, yet silica gel and diol phases seemed to deviate from a straightforward transference. Since Reuke and Hauk only investigated phosphoric esters on the silica gel stationary phase for transference to HPLC, it was decided to search for a common correction coefficient (C) for analytes containing a variety of functional groups for the transference to FCC. k = (1 − RF )/RF
(1)
VR = Vv · (1 + k) = Vv /RF
(2)
0.70 4.2
0.77 0.70 0.68 0.66 4.6 4.2 4.0 4.0
3.0 0.60
9.0 22.0 13.5 25.5 0.62 0.62 0.60 0.57
0.62
13.0 30.5 19.5 34.5 0.66 0.63 0.58 0.62
Middle fraction number
3.2 3.0 2.8 3.0
Middle fraction number
51
VR = Vv · (1 + k) · C = (Vv /RF ) · C
Correction coefficient (C)
Benzaldehyde
Vv equiv.
Correction coefficient (C)
Acetophenone
Vv equiv.
Correction coefficient (C)
J.D. Fair, C.M. Kormos / J. Chromatogr. A 1211 (2008) 49–54
(3)
A series of flash columns, with varying column sizes, were developed and summarized in Table 1. The experimental VR of each analyte was calculated by determining the volume of solvent used to reach the middle fraction in which the analyte eluted. The number of column void volume equivalents (Vv equiv.) taken to reach the experimental retention volume for each analyte is given in Table 1. This value remains approximately constant regardless of the column size. Although each analyte should have its own individual correction coefficient, it was of great interest, as seen in Table 1, to note that all three analytes shared the same approximate value within 6%. The average correction coefficient, calculated using Eq. (3) and taken over the three analytes, was found to be 0.64 for manually-packed columns. To further justify that our correction coefficient was not related to how well we packed our flash columns, the same compounds were developed in commercially-packed columns, where an average correction coefficient of 0.66 was found. This finding, coupled to the fact that the silica gel used in the construction of the manually- and commercially-packed columns were from three different vendors, indicated that no matter how a silica gel column was packed, either manually or commercially, the correction coefficient provided a general average for use in FCC. Many preparative separations involve technical grade solvents; thus, all separations summarized in Table 1 were conducted with a technical grade solvent system comprised of 5% ethyl acetate in hexanes. Additional experiments were also carried out using anhydrous 5% ethyl acetate in hexanes as well as with anhydrous 5% diethyl ether in hexanes. Although the RF values for each analyte varied with changes in the mobile phase composition providing corresponding k values, C remained 0.64. The presence or absence of trace water or the use of alternative solvent systems has no effect on the calculated correction coefficient so long as both the TLC and the column are developed using the same solvent system.
1.8
1.9 1.9 1.8 1.7 3 3 15 21 8.4 21.9 72.3 180.3
5.5 14.0 9.0 15.0
Vv equiv. Middle fraction number
Dibenzyl ether
Fraction size (mL) Column void volume, Vv (mL)
Still and co-workers [10] provided a table recommending an amount of silica gel to be used for a given mass of sample in terms of column diameter and height using a linear velocity of 2 in./min. More recent studies have shown there is little change in resolution when columns of varying height and diameter are used provided that the amounts of sorbent used and linear flow are maintained [15]. On this basis, linear functions (Eqs. (4) and (5)) were derived using the data provided by Still and co-workers. The mass of silica gel (SiO2g ) recommended to separate a given mass of sample (Sg ) depending on whether the separation is easy (RF ≥ 0.2; Eq. (4)) or difficult (0.1 ≤ RF < 0.2; Eq. (5)). Having determined the mass of silica to be used (Eq. (4) or (5)), the column void volume (Vv ) for a manually-packed column containing 4.5–150 g of silica gel may be estimated using Eq. (6), or the void volume may be measured experimentally. Once RF and Vv are known, the center of an
Table 2 Manually-packed column band widths. Mass silica (g)
Average
4.5 12.0 40.0 100.0 1 2 3 4
Mass silica (g)
Entry
Entry
Table 1 Manually-packed column retention volumes.
Retention volume (VR )
3.2. Column mass loading and column void volume
1 2 3 4
4.5 12.0 40.0 100.0
Band width (number of fractions) Dibenzyl ether
Benzaldehyde
Acetophenone
Exp.
Calc.
Exp.
Calc.
Exp.
Calc.
2 5 3 5
2 6 4 7
5 9 7 10
4 10 6 11
7 14 10 14
5 12 8 13
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Table 3 Assigning a relationship between variable mass loading and N. Entry
1 2 3 4 5
Mass of benzaldehyde loaded (mg) 22 33 63 82 166
a
Mass loading (XA ) Easy
Hard
0.13 0.20 0.37 0.49 1.00
0.33 0.53 1.00 1.31a 2.64a
Number of fractions containing analyte
Total band volume (Vb )
Efficiency (N)
8 9 10 11 12
24 27 30 33 36
90 64 52 43 36
Mass loading values (XA ) between 0.1 and 1.0 should be entered when utilizing the spreadsheet.
Rs = 2[(VR )B − (VR )A ]/[(Vb )B + (Vb )A ]
analyte’s elution band (VR ) may be calculated (Eq. (3)). SiO2g = 59.8 · Sg
(4)
SiO2g = 151.2 · Sg + 0.5
(5)
Vv = 1.8 · SiO2g + 0.3
(6)
3.3. Band broadening prediction in FCC systems The translation of spot broadening in TLC to band broadening in chromatographic columns is a complex process. Spot diffusion is a function of the original spot size, diffusion, and technique error [16]. Indeed, even the position of the origin in relation to the solvent entry level has been found to contribute to spot diffusion in thin layer systems [17]. Due to this complexity, the use of the van Deemter equation was attempted. Approximate values for a majority of terms contained in the van Deemter equation have been previously provided in the literature for high-performance liquid chromatography (HPLC) [18]; yet a more simplified approach was desirable. Band broadening is a function of how long an analyte remains on a column (VR ) and the column efficiency (N) [3,4,19]. Under ideal conditions, band shapes correspond to a Gaussian function. The band volume (Vb ) may be estimated using Eq. (7) from the calculated VR (Eq. (3)) and N. For the experiments summarized in Table 1, the estimated band volumes (Eq. (7)) are in good agreement with experimental values when N is set equal to 90; the estimated and experimentally observed band widths are summarized in Table 2. In short, a value of N = 90 correctly estimates the efficiency of all four column sizes and the assumption is made that this value for N may be used to estimate band volumes of any analyte having mass loading of 1/3 of the sample mass (Sg ) for a hard separation (Eq. (5)). The data shown in Table 2 all use the same mass loading ratio, 0.33, where XA = analyte mass/total sample mass. √ Vb = 4 · = 4 · VR / N (7) Changes in mass loading produce proportional changes in band volume [14]. In an effort to evaluate the effect of changes in mass loading on band volume, benzaldehyde was chromatographed using 5% ethyl acetate in hexanes at various mass loadings using 10 g of silica recommended by Eq. (4) for an easy separation and Eq. (5) for a hard separation, Table 3. The experimentally determined band volumes were converted into column efficiency for a given mass loading dictated by a hard separation (NHARD ) and for an easy separation (NEASY ). These data were fitted to functions of mass loading (XA ) to give Eqs. (8) and (9), respectively. Eqs. (8) and (9) provide a means to predict the band volumes as a function of mass loading relative to the separation difficulty. Entry 5 for an easy separation and entry 3 for a hard separation correctly demonstrate that a greater silica gel (SiO2g ) to sample mass (Sg ) ratio provides for a more efficient column. NHARD = 51.70 · (XA )−0.44 NEASY = 33.64 · (XA )
−0.44
3.4. Predicting resolution in FCC systems As previously stated, RF , and for that matter CV, provide an estimation of the degree of difficulty in a separation; however, these do not provide information regarding the resolution between adjacent peaks. Using the calculated retention volumes (Eq. (3)) and band widths (Eq. (7)), the Rs of adjacent bands may be evaluated using Eq. (10). A resolution of 1.0 affords a 98% separation; a value of ∼1.5 is required for a complete baseline separation between adjacent peaks [19]. The power gained through the prediction of band volumes of analytes allows a much more accurate prediction of the potential resolution a particular solvents system can provide regardless if the separation is an “easy” or “hard” separation. 3.5. Use of the predictive spreadsheet A spreadsheet, included as Supplementary Material, has been developed based on the discussion above to more accurately predict the success of a separation by providing information such as the mass of silica gel needed, the fraction size to take, the expected resolution between analytes, and the actual fraction number containing each analyte. To accomplish this, the spreadsheet utilizes user input such as mass loading, ratio of analyte mass, and distance data obtained from the TLC to produce an estimated chromatogram for a flash chromatographic separation. A representative, static snapshot of the interactive spreadsheet utility is shown in Fig. 1. From the TLC, the distance traveled by each analyte as well as the distance traveled by the solvent front, may be entered in any units as long as they are consistent. The total mass of sample is entered in grams and the analyte ratio should be in decimal form with the sum ∼1.0. When only two analytes are used, “na” (not applicable) can be entered into the distance for spot 3, eliminating all data for that spot. Due to the flexibility of applied spreadsheets, additional rows can be added if a separation contains more than three analytes. The spreadsheet automatically calculates the ideal mass of silica gel required for the separation as well as the ideal fraction size.2 From the calculated column void volume and TLC distances, the volumes at which each analyte will likely elute are calculated along with the estimated fraction numbers. For convenience and flexibility, the mass of silica and fraction size can be changed by the user in the “optional” section of the spreadsheet. The spreadsheet automatically recalculates new volumes and fractions based on this input. The recalculated band volumes assume the maximum loading described by Eqs. (4)–(9). The spreadsheet also calculates and displays the Rs between two adjacent peaks: “Bad” (Rs < 0.8), “Moderate” (0.8 ≤ Rs < 1.5), or “Good Separation” (Rs ≥ 1.5) is displayed depending on the numerical value calculated for Rs . A chromatogram showing the estimated
(8) (9)
(10)
2
The ideal fraction size is calculated to be 1/3 of the column void volume.
J.D. Fair, C.M. Kormos / J. Chromatogr. A 1211 (2008) 49–54
Fig. 2. (a) Estimated chromatogram and (b) experimentally observed chromatogram. Peaks 1 and 2 are methyl octanoate and octanol, respectively.
separation is produced by the spreadsheet assuming Gaussian-type peak shapes. In practice, the best solvent system for a given separation may be determined by simply entering new distance measurements obtained from TLC using various solvent systems. 3.6. Separation examples using the spreadsheet To provide evidence that the spreadsheet, as well as the correction coefficient and mass loading ratios, provide an elegant means for the prediction of FCC is perhaps best appreciated with reference to the following two examples. The separation of octanol and methyl octanoate represents an easy separation (RF > 0.2) of a two component, non-UV-active mixture. These analytes were chosen for their structural dissimilarity to the original benzyl-derived analytes on which the development of the spreadsheet was based. A TLC of the mixture was run in ethyl acetate/triethylamine/hexanes (15.0/0.1/84.9). The distances traveled by each compound as well as the solvent front were entered into the spreadsheet along with the mass loading ratio for each analyte and the total mass of the sample (Table 4). The spreadsheet returned a suggested silica gel mass of 9.6 g and recommended a fraction size of 6.1 mL. Values of 10.0 and 6.0 were entered into the cells labeled “optional grams of silica gel” and “optional fraction size”, respectively. The spreadsheet estimated the fractions that would contain each of the analytes (Table 4) and displayed an estimated chromatogram (Fig. 2a). As demonstrated by the results summarized in Table 4 and the experimental chromatogram (Fig. 2b), there was good agreement not only between the actual
53
Fig. 3. (a) Estimated chromatogram and (b) experimentally observed chromatogram. Peaks 1, 2 and 3 are 2-nitrophenol, phenol and 4-nitrophenol, respectively.
fraction numbers containing each analyte but also the actual resolution to those values estimated by the spreadsheet. It should be noted that the addition of a modifier such as triethylamine does not affect the predictive ability of the spreadsheet as long as both the TLC and FC are developed with the same solvent system. The resolution of a three-component mixture of phenol, 2nitrophenol and 4-nitrophenol was also explored as representative of a hard separation (RF < 0.2). A TLC of the mixture was run in diethyl ether/hexanes (8/92) and the data was entered into the spreadsheet as in the previous example (Table 5). The spreadsheet suggested the use of 39.8 g of silica gel and 24.1 mL fraction sizes. One gram of silica gel was used to dry-load the mixture. Thus, a sample containing 40% (w/w) 2-nitrophenol, 20% (w/w) phenol, and 40% (w/w) 4-nitrophenol with a total mass of 0.26 g was preabsorbed onto 1 g of silica gel. This silica gel was then added onto the head of a packed column before eluting. Therefore, values of 41 and 21 were entered into the cells labeled “optional grams of silica gel” and “optional fraction size”, respectively. The spreadsheet again estimated the fractions that would contain each of the analytes (Table 5) and displayed an estimated chromatogram (Fig. 3a). Good agreement was again observed between the experimental fraction numbers (Fig. 3b) and those predicted (Fig. 3a). 3.7. Errors in calculating VR and Vb As stated previously, errors may be attributable to minor differences in the stationary phases in the TLC and flash chromatography experiments. Indeed, the presence of binder in the TLC, minor differences in activity, pH, particle or pore size, and other factors,
Table 4 The separation of methyl octanoate and octanol. Analyte
Distance (mm)
RF
Mass loading (XA )
Estimated fractions numbers
Actual fractions numbers
Predicted resolution
Actual resolution
Methyl octanoate Octanol
21.5 7.0
0.61 0.20
0.3 0.7
2–4 7–13
4–5 11–15
1.7
1.6
Solvent front
35.0
Total mass: 0.16 g
Table 5 The separation of 2-nitrophenol, 4-nitrophenol and phenol. Analyte
Distance (mm)
RF
Mass loading (XA )
Estimated fractions numbers
Actual fractions numbers
Predicted resolution
Actual resolution
2-Nitrophenol Phenol 4-Nitrophenol
13.0 4.5 1.3
0.37 0.13 0.04
0.4 0.2 0.4
5–8 14–21 47–75
6–9 14–17 38–62
2.4 2.5
1.7 2.4
Solvent front
35.0
Total mass: 0.26 g
54
J.D. Fair, C.M. Kormos / J. Chromatogr. A 1211 (2008) 49–54
including the non-linear velocity profile of the solvent front during TLC development, may influence an analyte’s sorption isotherm. An analyte’s divergence from the average C will attribute to the error. Errors in the predicted VR of highly retained analytes may arise from systematic errors in TLC measurements and from an analyte’s divergence from the average correction coefficient. Greater error for more highly retained analytes stems from the inverse relationship between RF and VR and thus, errors are more likely to propagate. It is therefore recommended that the spreadsheet not be used for RF values less than ∼0.08; RF values less than ∼0.1 produce extremely broad peak shapes due to the broadening associated with a large VR . Due to the nature in which Vb and Rs are calculated, any error in the calculation of VR will be carried into both Vb and Rs . 4. Conclusion In summary, the spreadsheet included as Supplementary Material provides guidance in the development of an optimal system for preparative-scale FCC from easily obtained TLC data. The information gained through the use of the spreadsheet provides a level of detail superior to the “rule of thumb” currently used in the separation of fine chemicals. The implementation of this spreadsheet as a teaching tool in the undergraduate curriculum is currently being evaluated. Acknowledgements We are grateful to Dr. William F. Bailey, Dr. James D. Stuart, Dr. Nicholas E. Leadbeater and Dr. James M. Bobbitt of the University of Connecticut for helpful guidance and suggestions.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.chroma.2008.09.085. References [1] J.G. Kirchner, J.M. Miller, G. Keller, Anal. Chem. 23 (1951) 420. [2] M. Tswett, Ber. Deutsch. Bot. Ges. 24 (1906) 384. [3] B. Karger, L.R. Snyder, C. Horvath, An Introduction to Separation Science, WileyInterscience, New York, 1973. [4] C.F. Poole, S.K. Poole, Chromatography Today, Elsevier, Amsterdam, New York, 1991. [5] H. Schlitt, F. Geiss, J. Chromatogr. 67 (1972) 261. [6] T. Baczek, M. Radkowska, B. Sparzak, J. Liq. Chromatogr. 30 (2007) 2963. [7] T. Cserhati, E. Forgacs, J. Chromatogr. A 668 (1994) 495. [8] S. Reuke, H.E. Hauck, Fresenius’ J. Anal. Chem. 351 (1995) 739. [9] T. Wennberg, I. Vovk, P. Vuorela, B. Simonovska, H. Vuorela, J. Planar Chromatogr. 19 (2006) 118. [10] W.C. Still, M. Kahn, A. Mitra, J. Org. Chem. 43 (1978) 2923. [11] Horizon HPFC System Operator’s Manual, Biotage, Charlottesville, VA, 2003. [12] L.R. Snyder, J.W. Dolan, D.C. Lommen, J. Chromatogr. 485 (2001) 65. [13] W.L.F. Armarego, C.L.L. Chai, Purification of Laboratory Chemicals, Butterworth–Heinemann, Boston, MA, 2003. [14] R.J. Gritter, J.M. Bobbitt, A.E. Schwarting, Introduction to Chromatography, Holden-Day, New York, 1985. [15] I.Z. Atamna, G.M. Muschick, H.J. Issaq, J. Liq. Chromatogr. 12 (1989) 285. [16] E. Cavalli, T.T. Truong, M. Thomassin, C. Guinchard, Chromatographia 35 (1993) 102. [17] C.F. Poole, W.P.N. Fernando, Planar Chromatogr. 5 (1992) 323. [18] R.W. Stout, J.J. DeStefano, J. Chromatogr. 282 (1983) 263. [19] J.M. Miller, Chromatography Concepts and Contrasts, Wiley–Interscience, New York, 2004.