Anal Bioanal Chem DOI 10.1007/s00216-015-8723-1
RESEARCH PAPER
Overcoming the equivalent-chain-length rule with pH-zone-refining countercurrent chromatography for the preparative separation of fatty acids Michael Englert 1 & Walter Vetter 1
Received: 30 January 2015 / Revised: 14 April 2015 / Accepted: 17 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Purification of individual fatty acids from vegetable oils by preparative liquid chromatography techniques such as countercurrent chromatography (CCC) is a challenging task due to the equivalent-chain-length (ECL) rule. It implies that one double bond equals two carbon atoms in the alkyl chain of a fatty acid and therefore causes co-elutions of saturated and unsaturated fatty acids. Accordingly, existing methods for the purification of individual fatty acids are cumbersome and time-consuming as two or more steps with different conditions are required. To avoid additional purification steps, we report a method utilizing pH-zone-refining CCC which enabled the purification of all major fatty acids from sunflower oil (purities >95 %) in one step by circumventing co-elutions caused by the ECL rule. This method is based on the involvement of acid strength and hydrophobicity of fatty acids during the separation process. By exploiting the preparative character of the pH-zone-refining mode, a tenfold sample amount of free fatty acids from sunflower oil could be separated in comparison to regular CCC.
Keywords Fatty acids . Equivalent-chain-length . Countercurrent chromatography . pH-zone-refining . Sunflower oil
Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8723-1) contains supplementary material, which is available to authorized users. * Walter Vetter [email protected] 1
Institute of Food Chemistry (170b), University of Hohenheim, Garbenstrasse 28, 70599 Stuttgart, Germany
Introduction Fatty acids (FAs) bound to the alcohol glycerol in the form of mono-, di-, and triacylglycerides represent the predominant constituents in terms of quantity in vegetable oils and fats [1]. Major FAs occurring therein include saturated species with an even number of carbon atoms in the typical range from 12 to 22 and linear unsaturated species with 18 carbon atoms and up to three double bonds [2]. FAs have a market in pharmaceuticals, cosmetics, and in health supplements. Furthermore, they give high use value to industrial products as renewable raw materials and improve the performance of paints, plastics, lubricants, or rubber products. Accordingly, there is a wide-ranging interest in isolating FAs in high purity from various vegetable oils and fats [3, 4]. Purification of selected FAs or FA esters on a preparative scale from various raw materials such as vegetable and fish oils has been accomplished by the solid support-free, liquid– liquid separation technique countercurrent chromatography (CCC) [5–7]. CCC involves the distribution of analytes between a liquid stationary and a liquid mobile phase and their transport by the latter. CCC can be considered favorable for purification purposes because of its unique advantages such as high recovery rates, no irreversible adsorption processes, and moderate solvent consumption in contrast to other liquid chromatography techniques [8]. Nevertheless, the purification of FAs and FA esters by liquid–liquid separation techniques proved to be laborious due to the equivalent-chain-length (ECL) rule [5] which implies that one double bond equals with two carbon atoms in the alkyl chain resulting in similar partitioning coefficients causing co-elutions of critical FA pairs [9–14]. FAs are representing weak carboxylic acids with relatively high hydrophobicities [15, 16]. Accordingly, they appeared to be well-suited target compounds for a variation of the regular
M. Englert, W. Vetter
CCC method known as pH-zone-refining CCC which has been introduced by Ito in the early 1990s [17]. Since then, pH-zone-refining CCC has been further developed into a powerful preparative technique for the separation of mixtures of ionizable target compounds such as organic acids and bases [17–21]. In pH-zone-refining CCC, the two-phase solvent system is modified prior the separation by rendering one of the solvent phases acidic and the other basic using a retainer and an eluter acid or base [17]. Ionizable analytes interact with the solvent system in terms of protonation and de-protonation which results in differing solubilities and partitioning behaviors of the neutral and ionized forms [17]. During isocratic elution, contiguous and rectangularly shaped peaks (pH-zones) are formed—each depending on the acid strength (pKa value) and hydrophobicity of the inherent analyte [22]. The involvement of both factors during the separation process appeared to offer a way to circumvent the co-elutions pursuant to the limitations in FA separations caused by the ECL rule. The goal of this study was the development of a method for the separation of free FAs obtained from sunflower oil in one step by pH-zone-refining CCC. Both possibilities, normal and the more widely used reverse displacement mode, were studied and compared with the results obtained for conventional CCC. The influence of eluter-to-retainer molar concentrations and the order of elution of the FAs are discussed.
Experimental Chemicals Acetonitrile, methanol, and n-hexane (all HPLC gradient grade) were from Th. Geyer (Renningen, Germany). Technical grade ethanol (BASF, Ludwigshafen, Germany) and ethyl acetate (Acros, Geel, Belgium) were distilled before use. Technical grade KOH (purity >85 %), anhydrous Na2SO4 (purity >99 %), NaCl (purity >99.8 %), and KCl (purity >99.8 %) were from Carl Roth (Karlsruhe, Germany). Concentrated H 2 SO 4 (purity >98 %) was from BASF (Ludwigshafen, Germany), and concentrated HCl (32 %), aqueous NH3 (25 %), and trifluoroacetic acid (TFA) (purity >99.8 %) were from Merck (Darmstadt, Germany). A 37component fatty acid methyl ester (FAME) mix for structural assignment, myristic acid (14:0), and pyridine (purity >99.8 %) were from Sigma-Aldrich (Steinheim, Germany). The recovery and syringe standards 10,11-dichloroundecanoic acid (10,11DC) and myristic acid ethyl ester (14:0-EE) were synthesized as shown elsewhere [23]. The trimethylsilylating reagent which consisted of 99 % N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane (TMCS) was from Supelco (Bellefonte, PA, USA). Helium and nitrogen (both
purity 99.999 %) were from WestfalenGas (Münster, Germany). Sample preparation Alkaline saponification of 102.0 g sunflower oil was performed in a 1-L flask by addition of 405 mL ethanol and 45 mL of 50 % aqueous KOH. The solution was heated under reflux for 6 h with stirring. After addition of 300 mL water, the unsaponifiable matter was extracted twice with 200 mL nhexane. Subsequent acidulation of the generated soap with 3 N HCl to pH 3 provided free FAs which were recovered by extraction with two portions of 400 mL n-hexane. Extracts were combined and washed with 400 mL deionized water. The organic phase was separated and dried over anhydrous Na2SO4, and the solvent was evaporated to dryness under vacuum, yielding 100.5 g of free FAs from sunflower oil. The dried residue was stored in an amber glass flask under nitrogen atmosphere in a refrigerator at 4 °C until needed. Portions thereof were used for shake flask tests and CCC separations (∼10 g). Gas chromatography coupled to electron ionization mass spectrometry (GC/MS) Prior to analysis of samples by means of GC/MS, the recovery standard 10,11-DC was added and FAs were derivatized to FAMEs by treatment of about 0.05–2 mg sample with 1 % H2SO4 in methanol for 2 h at 80 °C in a 10-mL tightly sealed derivatization tube with occasional shaking [23]. The solution was cooled down to room temperature, and 1 mL of deionized water and saturated NaCl solution was added. Subsequent addition of 2 mL n-hexane and thorough shaking transferred the FAMEs into the organic phase. An aliquot from the organic phase was mixed with the syringe standard 14:0-EE. Analysis of FAMEs was performed with a 5890 series II plus gas chromatograph interfaced to a 5971 MSD equipped with a 7673A autosampler (Hewlett-Packard/Agilent, Waldbronn, Germany). Chromatography was performed on two serially connected Rtx-2330 capillary columns (30 m length, 0.25 mm internal diameter, 0.1 μm film thickness, 10 % cyanopropylphenyl, 90 % biscyanopropyl polysiloxane; Restek, Bellefonte, PA, USA) with helium as carrier gas at constant flow rate of 1 mL/min. The initial oven temperature was 60 °C (held for 1 min) followed by a ramp of 6 °C/min to 150 °C, then the temperature was raised at 4 °C/min to 190 °C and at 7 °C/min to a final temperature of 250 °C, held for 7 min (total run time 41.6 min). Injector and transfer line temperatures were maintained at 250 and 170 °C, respectively. Samples (1 μL) were introduced using splitless injection. In full scan mode, a solvent delay of 8 min was applied and mass spectra were recorded from m/z 50 to m/z 500. In selected ion monitoring (SIM) mode, six fragment ions, m/z 74 and m/z 87
Overcoming the equivalent-chain-length rule
for saturated and monounsaturated methyl esters, m/z 79 and m/z 81 for di- and polyenoic methyl esters of FAs, and m/z 88 and m/z 101 for ethyl esters of the internal standard 14:0-EE, were recorded throughout the run [23]. The content of FAs was determined in the SIM mode using 14:0-EE and 10,11DC as internal standards [23]. CCC apparatus Separations were performed using a CCC-1000 instrument (Pharma-Tech Research, Baltimore, MD, USA) holding three self-constructed and serially connected semi-preparative multilayer coils (polytetrafluoroethylene (PTFE) tubing, 1.6 mm internal diameter, 60 mL coil volume) [24]. A ternary 655A12 pump (Merck, Darmstadt, Germany) was used for solvent delivery, and manual sample injection was done with a type 50 rotary valve (Rheodyne, Cotati, CA, USA) equipped with a 10-mL sample loop for pH-zone-refining or a 4-mL sample loop for regular CCC (PTFE tubing, 1.6 mm internal diameter). In pH-zone-refining, the effluent was monitored by a spectral photometer 87 UV/Vis detector (Knauer, Berlin, Germany) with the wavelength λ set at 220 nm. For regular CCC, UV/Vis was not sensitive enough to detect unsaturated FAs. Therefore, a micro-splitter valve was installed which divided 1/10 of the effluent stream to a PL-ELS 2100 (Polymerlabs, Amherst, MA, USA) evaporative light scattering detector (ELSD). The nebulizer temperature was set to 35 °C, and the nitrogen gas stream was 1.2 mL/min. Fractions were collected by an Isco Retriever 500 (Teledyne Isco, Lincoln, NE, USA), and pH values of each fraction collected were determined with a model 340i pH Meter (WTW, Weilheim, Germany). The electrode was filled with a mixture of methanol and water saturated with KCl for the determination of pH values in organic solvents. After each measurement, the electrode was rewetted to avoid drifting of the pH values. Selection of two-phase solvent systems for pH-zone-refining CCC The selection of an appropriate two-phase solvent system represents the most important step in pH-zone-refining CCC method development. Successful separations can be achieved if the solvent system provides partitioning coefficients K close to 1 for the target compound and an ideal range of the K values under both acidic (Ka ≪1) and basic (Kb ≫1) conditions for the target compounds [17]. Hence, FAs should show a very large difference between their neutral, protonated and ionized, deprotonated forms in terms of solubility and partitioning in the upper and lower phase of a two-phase solvent system. For testing the applicability of various solvent systems, portions of 0.1 mg FA extract were transferred into a 2-mL vial and 500 μL of upper and lower phases of the respective solvent systems was added. The vial was shaken for ∼30 s, and after
phase separation was reached, 100 μL of each phase was separated and the solvent was removed by a gentle stream of nitrogen at 40 °C. After derivatization into FAMEs, analysis by means of GC/MS-SIM was performed. For the determination of K values, peak areas of the main FAs in the upper phase were divided by that in the lower phase and evaluated from measurements in triplicate with accuracy estimated to be ±5 %. The procedure was repeated with addition of NH3 to the sample containing the equilibrated two-phase solvent system (pH ∼11) for the determination of Kb values. Likewise, TFA was added to the mixture (pH ∼1) and Ka values were subsequently determined. Preparation of solvent system and sample solutions The two-phase solvent system consisted of n-hexane/acetonitrile/methanol/water (4/7/1.4/0.5, v/v) and was prepared by combining the solvents in a 1-L separatory funnel. The mixture was vigorously shaken and equilibrated overnight. The two phases were separated and degassed for 20 min in an ultrasonic bath. For pH-zone-refining CCC, the lower phase was made basic by addition of NH3 (retainer), and the upper phase was made acidic with TFA (eluter). After preliminary tests, TFA was selected since it is most commonly used and rarely challenged in pH-zone-refining CCC with acidic analytes because it distributes almost completely in the organic phase, and it can be removed easily after the separation. As inorganic base, NH3 was selected since it is frequently used especially for carboxylic acids [17] and removal is also possible. Sample solutions for regular CCC were prepared by dissolving 50 mg of the FA extract in 2 mL of upper and 2 mL of lower phase of the twophase solvent system. In pH-zone-refining, 500 mg FA extract was either dissolved in 10 mL of lower phase containing 20 mM NH3 for normal displacement mode or in 10 mL upper phase containing 20 mM TFA for reverse displacement mode. Sample loadings were not increased since this measure results in broader pH-zones while the overall elution profile remains constant [17]. Separation procedure Separations in regular CCC were performed by filling the column with the stationary phase under rotation of the centrifuge at 1,000±10 rpm. Equilibration was done with mobile phase at 1 mL/min followed by sample injection after hydrodynamic equilibrium was reached. In pH-zone-refining, modes were initiated by entirely filling the column with the stationary phase which either represented the lower aqueous phase containing NH3 for normal displacement mode (tail-tohead elution) or the upper organic phase containing TFA as retainer for reverse displacement mode (head-to-tail elution). The centrifuge was rotated at 1,000±10 rpm and sandwich
M. Englert, W. Vetter
injection of 10-mL sample solution was done. Mobile phase containing the eluter was then pumped into the column at a flow rate of 2 mL/min. After the mobile front emerged at the column outlet, fractions were collected in intervals of 3 min (6 mL). Subsequently, pH values of each fraction were measured with a pH meter. Individual fractions were brought to dryness by an RVC 2-33 IR speed-vac concentrator (Christ, Osterode, Germany). Removal of residual TFA after normal displacement mode was accomplished by addition of 2 mL 5 % HCl to the dried fractions, followed by agitation and extraction of the FAs with 6 mL n-hexane. To recover the separated FAs after reverse displacement mode, 1 mL water and 2 mL 5 % HCl were added to each dried fraction to neutralize the soaps. Mixtures were agitated and extracted with 6 mL n-hexane. Dilutions of the each fraction in n-hexane were subjected to GC/MS-SIM analysis. After the separation was completed, the remaining column contents were collected into a graduated cylinder by applying pressurized nitrogen gas (0.5 MPa) to the inlet. In this way, the stationary phase retention Sf could be determined at the end of the separation.
Results and discussion Analysis of the fatty acid sample The free FAs obtained from sunflower oil were analyzed prior to the CCC fractionation by GC/MS to determine the FA profile (Fig. 1). The sample consisted mainly of the FAs 18:2n-6 (45.2 %), 18:1n-9 (24.2 %), and 16:0 (20.9 %) along with small amounts of 14:0 and 22:0 (Table S1, supplementary data). Full saponification of the sunflower oil sample was verified by high-temperature gas chromatography showing the absence of acylglycerols and other esterified compounds in comparison to the unsaponified oil (Fig. S1, supplementary data). This analysis was performed to ensure that the crude FA extract contained no neutral compounds which may interfere with the pH-zone-refining mode CCC through elution in the pH-zones of the target compounds and thereby contaminate 18:2n-6
Irel
the latter. The overlap of FAMEs with same ECL was avoided by using temperature programming of the GC oven [25].
Selection of solvent system for pH-zone-refining CCC Several non-polar aqueous solvent systems were first evaluated for their suitability in pH-zone-refining mode CCC. Nonaqueous solvent systems (e.g., n-hexane/acetonitrile) that are also suitable for the separation of FAs [13] were not selected because a minute amount of water was necessary in the lower phase to achieve solubility of the supplemented inorganic base NH3 and FAs being present in the ionized carboxylate state. The K values increased with increasing alkyl chain lengths and decreased with the numbers of double bonds present in the alkyl chain of the FAs (Table S2, supplementary data). In CCC, small K values (2.5) lead to peak band broadening [26]. The majority of the examined solvent systems provided suitable K values between 0.4≤K≤2.5 for the FAs, but nonideal Ka and Kb values were observed. The solvent system composed of n-hexane/acetonitrile/methanol/water (4/7/1.4/ 0.5, v/v/v/v) was selected because it provided K values in a suitable range for head-to-tail (0.40–1.35, Table 1) and tail-tohead (0.74–2.5) elution mode. This solvent system also fulfilled the criteria for pH-zone-refining CCC since the Ka and Kb values showed distinct partitioning behavior in dependence of the pH value of the solvent system. A strong partitioning of the protonated form of the FAs into the upper phase (Ka = 13.6–17.2) could be observed at pH ∼1, while the deprotonated form present at pH ∼11 showed high affinity for the lower phase (Kb =0.05–0.08) (Table 1). Determination of the normalized polarity parameters ETN according to the Reichardt polarity index [24] verified that the polarity of the
Table 1 Partitioning coefficients in the solvent system under neutral (K), acidic (Ka), and basic (Kb) conditions and separation factor α between consecutive major fatty acids in the solvent system used for CCC separation Fatty acid
K
Kaa
Kba
18:2n-6
0.40
16.3
0.05
αb
1.28 14:0
0.51
17.2
0.04
18:1n-9
0.75
14.4
0.06
16:0
0.78
16.8
0.07
18:0
1.35
13.6
0.08
18:1n-9
1.04
16:0
14:0
1.47
18:0
22:0
1.73 18.0
20.0
22.0
24.0
26.0
28.0
30.0
32.0
34.0[min]
Fig. 1 GC/MS-SIM chromatogram showing the fatty acid profile of the free FAs from sunflower oil after alkaline saponification (measured as FAMEs)
a
Mean values of triplicate determination
b
Calculated from neutral K values
Overcoming the equivalent-chain-length rule
14:0 with the difference however that a higher amount (∼80– 90 %) of pure 18:2n-6 could be isolated after (tail-to-head mode) or before (head-to-tail mode) elution of 14:0 due to the relatively low concentration of 14:0 compared to 18:2n6, respectively. Isolation of pure 14:0 was not possible with both modes as it was always accompanied by 18:2n-6. Nevertheless, isolation of pure 18:0 was possible in both modes because no critical FAs with similar ECL value (18) were present in the sample. The regular CCC separations also enabled the detection and isolation of the long-chain FAs 24:0 and 20:0 which were both initially not detected in the unfractionated sample (Fig. 1). Purification of all individual FAs in the sample by regular CCC was practically impossible in one step since FAs are subjected to the ECL rule.
upper (ETNUP =6.8) and lower phase (ETNLP =53.0) showed great difference (ΔETN=46.2) in the solvent system which confirms the distinct Ka and Kb values [27]. Regular CCC separation Fractions collected during regular CCC separations of 50 mg free FAs obtained from sunflower oil verified the co-elution of 18:2n-6 with 14:0 as well as 18:1n-9 with 16:0. This was expected from the low separation factors α between the consecutive FA pairs (Table 1) caused by same ECL values of 14 and 16, respectively. Insufficient chromatographic selectivity and strong overlapping were the case in both tail-to-head and head-to-tail mode. The FAs 18:1n-9 and 16:0 could not be separated from each other and showed nearly the same (co)elution behavior (Fig. 2, highlighted by rectangular zones). In tail-to-head mode, a small share (∼5 % of total content, collected in one fraction) of 18:1n-9 was collected with high purity (∼98 %) because the elution started slightly before 16:0 (Fig. 2a). Equally in head-to-tail mode, a fraction could be collected which contained 16:0 (purity ∼98 %) nearly without concomitant 18:1n-9 (Fig. 2b). Yet, this fraction also only represented ∼5 % of the total content of the FA. The same behavior was observed for the critical FA pairs 18:2n-6 and
The pH-zone-refining mode introduced the acid strength (pKa values) of the respective FAs as a second dimension in the separation process. The pKa values (measured in water) of 14:0, 16:0, 18:0, 18:1n-9, and 18:2n-6 show slight differences in dependence of the alkyl chain length and number of double bonds present in the FA molecule [15]. Plotting of pKa values against the K values of the FAs in the solvent system resulted
a)
16:0
ELSD signal [mV]
18:1n-9 16:0 18.0 20.0 22.0 24.0 26.0 28.0[min]
18:0 20:0 22:0 0
30
60
90
1
K=
24:0
120
150
2
180
[min]
3
18:2n-6
Irel
b)
14:0
18:1n-9 16:0 18.0 20.0 22.0 24.0 26.0 28.0[min]
18:2n-6 14:0
24:0 18:0 22:0 20:0
0 K=
18:1n-9
Irel
18:2n-6 14:0
ELSD signal [mV]
Fig. 2 CCC-ELSD chromatogram obtained during separation of free fatty acids from sunflower oil. a Head-to-tail mode and b tail-to-head mode with co-elutions of fatty acids highlighted as rectangular zones and exemplary GC/MS chromatograms of collected fractions containing the co-eluting pairs 14:0 and 18:2n-6 as well as 16:0 and 18:1n-9. Experimental conditions: CCC-1000 instrument with 60 mL coil volume and 1 mL/min mobile phase flow rate; rotation speed=1,000±10 rpm; solvent system: n-hexane/acetonitrile/methanol/water (4/7/1.4/ 0.5, v/v); sample size=50 mg. K points representing the column volume (Vc =60 mL, K=1) and the double (Vc =120 mL, K=2) and threefold column volume (Vc =180 mL, K=3) are additionally labeled on the x-axis
pH-zone-refining CCC
30
60 1
90
120 2
150
180 3
[min]
M. Englert, W. Vetter
CCC separations to find conditions where the ECL rule can be overcome and to investigate the effects on the separation (Table S-3). During the investigations, the retainer concentration was fixed at 20 mM because this parameter only affects the concentration of the analytes in the stationary phase, thus enhancing the retention of the FAs. Within both normal displacement and reverse displacement pH-zone-refining CCC, it could be observed that for a molar eluter-to-retainer ratio below 0.5, no pH-zones were formed and all of the FAs were immediately washed off the column. A value of 0.5 resulted in the formation of pH-zones with short retention times and incomplete resolution between all of the eluting zones. If the molar eluter-to-retainer ratio exceeded a value of 2, a long retention time was observed, and analysis by means of GC/ MS revealed that the elution order was exclusively determined by the acid strength (pKa values) of the FAs. In normal displacement pH-zone-refining CCC, best results could be obtained with 20 mM NH3 in the stationary phase (retainer) and 35 mM TFA in the mobile phase (eluter) corresponding to a molar eluter-to-retainer ratio of 1.75 (Fig. 4a). Online monitoring of the separation process by UV/Vis allowed detecting the TFA trace as well as FAs, whereas offline pH determination of the fractions allowed tracking the formation of individual pH-zones. After injection of 500 mg free FAs obtained from sunflower oil, the mobile front containing TFA appeared
18:0 18:1n-9 18:2n-6 16:0 14:0
1.4
Fig. 3 Acid strength (pKa value) of major fatty acids 14:0/16:0/18:0/ 18:1n-9 and 18:2n-6 plotted against partitioning coefficients K in the two-phase solvent system. Highlighted by rectangular zones are fatty acids 18:2n-6/14:0 and 18:1n-9/16:1 which showed co-elutions during regular CCC due to very similar K values
in a hypothetical, orthogonal separation mode as the selectivities of both separation processes are independent from each other (Fig. 3). Hence, 18:2n-6/14:0 and 18:1n-9/16:0 which co-eluted in regular CCC displayed sufficient distance to each other in this separation space which enabled to circumvent coelutions pursuant the ECL rule. For this purpose, we were running a series of studies in both normal and reverse displacement pH-zone-refining mode by interchanging the elution mode and hence the role of the mobile and stationary phase as well as that of the retainer and eluter. Seven different eluter-to-retainer molar concentration ratios were tested in
a)
12 Sf (0 min) = 77%
Absorbance (220 nm) [mV]
10
6
pH
8
14:0
4
0
30
60
90
120
150
180
2
0 [min] 210 240 270 300 12 Sf (0 min) = 75% 10 Sf (270 min) = 65%
6
20:0;22:0 24:0
4
0
30
60
90
120
150
2 0
180
210
240 [min]
pH
8
14:0;16:0
b)
Sf (330 min) = 68%
Absorbance (220 nm) [mV]
Fig. 4 Separation of 500 mg fatty acids from sunflower oil with pHzone-refining CCC in a normal displacement (20 mM NH3 (retainer) and 35 mM TFA (eluter)) and b reverse displacement mode (20 mM TFA (retainer) and 35 mM NH3 (eluter)) with UV/Vis signal and pH curve obtained by measuring each fraction. Experimental conditions: CCC-1000 instrument with 60 mL coil volume and 2 mL/min mobile phase flow rate; rotation speed=1,000±10 rpm; solvent system: n-hexane/acetonitrile/methanol/water (4/7/1.4/ 0.5, v/v) with TFA in the upper phase and NH3 in the lower phase
20:0 22:0 24:0
1.2
18:0
0.4 0.6 0.8 1.0 Partitioning coefficient K
18:2n-6
0.2
18:0
0
18:1n-9
8.0 7.5 7.0
18:2n-6
9.5 9.0 8.5
16:0
10.0
18:1n-9
Acid strength (pKa)
10.5
Overcoming the equivalent-chain-length rule Irel
Fig. 5 GC/MS analysis of selected fractions obtained during the separation of 500 mg fatty acids from sunflower oil with pHzone-refining CCC in normal displacement mode. Analyses were performed after conversion of fatty acids into fatty acid methyl esters
Irel
14:0
>98%
Irel
>95%
18:2n-6
18.0 20.0 22.0 24.0 26.0 28.0 [min] 18.0 20.0 22.0 24.0 26.0 28.0 [min] Irel
16:0
>98%
>96%
18.0 20.0 22.0 24.0 26.0 28.0 [min] Irel
>96% 18:0
Irel
18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 [min]
>98%
20:0
18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 [min]
18.0 20.0 22.0 24.0 26.0 28.0 Irel
22:0
Irel
>95%
18:1n-9
18.0 20.0 22.0 24.0 26.0 28.0 [min]
in the UV/Vis chromatogram at 15 min (fraction 1), and the initial pH value of the effluent (11.0, point A) slowly decreased to 10.2 (point B) due to neutralization of NH3 in the stationary phase. Slight phase bleeding occurred from 55 to 65 min (fractions 8–10) which caused a short irregularity of the pH level and a reduced overall yield, but this did not Table 2 Recovery, yield, and purity of the individual fatty acids isolated from 500 mg fatty acids from sunflower oil with pH-zonerefining CCC in normal displacement mode Fatty acid
Yield (mg)
Purity (%)
Recovery (%)
14:0 16:0 18:1n-9 18:2n-6 18:0 20:0 22:0 24:0
1.5 88.0 104.3 194.2 36.6 1.5 3.3 1.5
98 98 95 95 96 98 96 95
80.0 85.0 86.9 86.7 86.1 75.0 82.5 75.0
>95%
24:0
18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 [min]
impair the separation process and resolution. A pH-zone was observed at 70 min (fraction 10) when a pH plateau was formed and the pH decreased to ∼8.5 (point C). The separation was completed after 330 min with a stationary phase retention of Sf =68 % and a final pH value of ∼2.5 (point D). Analysis of the collected fractions by means of GC/MS revealed that the eight FAs present in the sample were eluted individually as consecutive rectangular pH-zones in nearly equal molar concentrations with minor overlap bands only (i.e., mixing zones) containing consecutive FAs. In general, the formation of a pH plateau during the separation represented the elution of a FA with high purity (>95 %). The elution order differed from the corresponding regular CCC separation in tail-to-head mode and the saturated FAs 14:0 and 16:0 eluted first, sharing the lowest pKa values of 8.1 and 8.6, respectively. This is consistent with the general behavior in pH-zone-refining of stronger acids eluting earlier [8]. Afterwards, the unsaturated FAs 18:1n-9 (pKa value 9.9, K=0.75) and 18:2n-6 (pKa value 9.2, K=0.40) formed two broad pH-zones as the concentration of them was highest in the sample. In this case, however, the weaker FA eluted first, suggesting that the hydrophobicity (K
M. Englert, W. Vetter
value) had a stronger impact on the elution sequence. In the end, the saturated FAs 18:0 < 20:0 < 22:0 < 24:0 were eluted with slight zone overlapping between them according to increasing alkyl chain length and hydrophobicity or decreasing pKa values. In this particular case, isolation of all FAs present in the sample could be achieved with high purities (Fig. 5) and with acceptable individual recovery rates of the FAs between 75.0 and 86.9 % and a total recovery rate of 86.2 % (Table 2). Figure 4b shows the separation in the more commonly applied reverse displacement pH-zone-refining CCC mode performed under the equal conditions with 500 mg free FAs obtained from sunflower oil but inversely 20 mM TFA in the stationary phase and 35 mM NH3 in the mobile phase. The initial pH value of the effluent was acidic (pH ∼2) and increasing during the slow neutralization by NH3 in the mobile phase. In this case, the separation was completed more quickly in 160 min but at the beginning of the separation (20 min) stationary phase bleeding was also observed and led to a reduced stationary phase retention of Sf =65 % at the end. The formation of pH plateaus could be observed, suggesting the elution of pure FAs. However, GC/MS analysis showed that the saturated FAs 20:0, 22:0, and 24:0 co-eluted in the starting zone since they represented the strongest bases in the sample. Under these conditions, the saturated FAs 14:0 and 16:0 eluted after a broad mixing zone where 20:0, 22:0, 24:0, 14:0, and 16:0 were present. A separation of them could be not achieved. Yet, the following zones contained 18:1n-9, 18:2n6, and 18:0 in this elution sequence with high purities. However, the mixing zones between them were broader than in the normal displacement mode leading to a reduced yield of the individual FAs.
Conflict of interest The authors declare no conflict of interest.
References 1. 2. 3.
4. 5. 6.
7.
8.
9.
10.
11.
12. 13.
Conclusion 14.
The ECL rule presents a challenge in the purification of FAs with liquid chromatographic techniques such as CCC. FAs with the same ECL value, e.g., 16:0 and 18:1n-9 or 14:0 and 18:2n-6, were difficult to separate from each other with regular CCC due to the similar partitioning coefficients. Timeconsuming separations in multiple steps would be a solution. We have introduced the acid strength of the FAs as a second dimension in the separation process by a variation known as pH-zone-refining CCC. All free FAs obtained from sunflower oil could be isolated in the normal displacement mode according to their respective acid strengths and partitioning coefficients in a tenfold amount compared to regular CCC. It should be noted that transfer of the method to another vegetable oil may require adjustment of the molar concentrations and ratio of retainer and eluter. Acknowledgments The authors would like to thank the state of BadenWürttemberg for providing a stipend grant to Michael Englert.
15.
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Zhang Q, Shu X, Jing F, Wang X, Lin C, Luo A (2014) Preparative separation of alkaloids from Picrasma quassioides (D. Don) Benn. by conventional and pH-zone-refining countercurrent chromatography. Molecules 19:8752 Ito Y, Ma Y (1996) pH-zone-refining countercurrent chromatography. J Chromatogr A 753:1 Thurnhofer S, Lehnert K, Vetter W (2008) Exclusive quantification of methyl-branched fatty acids and minor 18:1-isomers in foodstuff by GC/MS in the SIM mode using 10,11-dichloroundecanoic acid and fatty acid ethyl esters as internal standards. Eur Food Res Technol 226:975 Englert M, Vetter W (2014) Solvent systems with n-hexane and/or cyclohexane in countercurrent chromatography—physico-chemical
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RESEARCH PAPER
Overcoming the equivalent-chain-length rule with pH-zone-refining countercurrent chromatography for the preparative separation of fatty acids Michael Englert 1 & Walter Vetter 1
Received: 30 January 2015 / Revised: 14 April 2015 / Accepted: 17 April 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Purification of individual fatty acids from vegetable oils by preparative liquid chromatography techniques such as countercurrent chromatography (CCC) is a challenging task due to the equivalent-chain-length (ECL) rule. It implies that one double bond equals two carbon atoms in the alkyl chain of a fatty acid and therefore causes co-elutions of saturated and unsaturated fatty acids. Accordingly, existing methods for the purification of individual fatty acids are cumbersome and time-consuming as two or more steps with different conditions are required. To avoid additional purification steps, we report a method utilizing pH-zone-refining CCC which enabled the purification of all major fatty acids from sunflower oil (purities >95 %) in one step by circumventing co-elutions caused by the ECL rule. This method is based on the involvement of acid strength and hydrophobicity of fatty acids during the separation process. By exploiting the preparative character of the pH-zone-refining mode, a tenfold sample amount of free fatty acids from sunflower oil could be separated in comparison to regular CCC.
Keywords Fatty acids . Equivalent-chain-length . Countercurrent chromatography . pH-zone-refining . Sunflower oil
Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8723-1) contains supplementary material, which is available to authorized users. * Walter Vetter [email protected] 1
Institute of Food Chemistry (170b), University of Hohenheim, Garbenstrasse 28, 70599 Stuttgart, Germany
Introduction Fatty acids (FAs) bound to the alcohol glycerol in the form of mono-, di-, and triacylglycerides represent the predominant constituents in terms of quantity in vegetable oils and fats [1]. Major FAs occurring therein include saturated species with an even number of carbon atoms in the typical range from 12 to 22 and linear unsaturated species with 18 carbon atoms and up to three double bonds [2]. FAs have a market in pharmaceuticals, cosmetics, and in health supplements. Furthermore, they give high use value to industrial products as renewable raw materials and improve the performance of paints, plastics, lubricants, or rubber products. Accordingly, there is a wide-ranging interest in isolating FAs in high purity from various vegetable oils and fats [3, 4]. Purification of selected FAs or FA esters on a preparative scale from various raw materials such as vegetable and fish oils has been accomplished by the solid support-free, liquid– liquid separation technique countercurrent chromatography (CCC) [5–7]. CCC involves the distribution of analytes between a liquid stationary and a liquid mobile phase and their transport by the latter. CCC can be considered favorable for purification purposes because of its unique advantages such as high recovery rates, no irreversible adsorption processes, and moderate solvent consumption in contrast to other liquid chromatography techniques [8]. Nevertheless, the purification of FAs and FA esters by liquid–liquid separation techniques proved to be laborious due to the equivalent-chain-length (ECL) rule [5] which implies that one double bond equals with two carbon atoms in the alkyl chain resulting in similar partitioning coefficients causing co-elutions of critical FA pairs [9–14]. FAs are representing weak carboxylic acids with relatively high hydrophobicities [15, 16]. Accordingly, they appeared to be well-suited target compounds for a variation of the regular
M. Englert, W. Vetter
CCC method known as pH-zone-refining CCC which has been introduced by Ito in the early 1990s [17]. Since then, pH-zone-refining CCC has been further developed into a powerful preparative technique for the separation of mixtures of ionizable target compounds such as organic acids and bases [17–21]. In pH-zone-refining CCC, the two-phase solvent system is modified prior the separation by rendering one of the solvent phases acidic and the other basic using a retainer and an eluter acid or base [17]. Ionizable analytes interact with the solvent system in terms of protonation and de-protonation which results in differing solubilities and partitioning behaviors of the neutral and ionized forms [17]. During isocratic elution, contiguous and rectangularly shaped peaks (pH-zones) are formed—each depending on the acid strength (pKa value) and hydrophobicity of the inherent analyte [22]. The involvement of both factors during the separation process appeared to offer a way to circumvent the co-elutions pursuant to the limitations in FA separations caused by the ECL rule. The goal of this study was the development of a method for the separation of free FAs obtained from sunflower oil in one step by pH-zone-refining CCC. Both possibilities, normal and the more widely used reverse displacement mode, were studied and compared with the results obtained for conventional CCC. The influence of eluter-to-retainer molar concentrations and the order of elution of the FAs are discussed.
Experimental Chemicals Acetonitrile, methanol, and n-hexane (all HPLC gradient grade) were from Th. Geyer (Renningen, Germany). Technical grade ethanol (BASF, Ludwigshafen, Germany) and ethyl acetate (Acros, Geel, Belgium) were distilled before use. Technical grade KOH (purity >85 %), anhydrous Na2SO4 (purity >99 %), NaCl (purity >99.8 %), and KCl (purity >99.8 %) were from Carl Roth (Karlsruhe, Germany). Concentrated H 2 SO 4 (purity >98 %) was from BASF (Ludwigshafen, Germany), and concentrated HCl (32 %), aqueous NH3 (25 %), and trifluoroacetic acid (TFA) (purity >99.8 %) were from Merck (Darmstadt, Germany). A 37component fatty acid methyl ester (FAME) mix for structural assignment, myristic acid (14:0), and pyridine (purity >99.8 %) were from Sigma-Aldrich (Steinheim, Germany). The recovery and syringe standards 10,11-dichloroundecanoic acid (10,11DC) and myristic acid ethyl ester (14:0-EE) were synthesized as shown elsewhere [23]. The trimethylsilylating reagent which consisted of 99 % N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1 % trimethylchlorosilane (TMCS) was from Supelco (Bellefonte, PA, USA). Helium and nitrogen (both
purity 99.999 %) were from WestfalenGas (Münster, Germany). Sample preparation Alkaline saponification of 102.0 g sunflower oil was performed in a 1-L flask by addition of 405 mL ethanol and 45 mL of 50 % aqueous KOH. The solution was heated under reflux for 6 h with stirring. After addition of 300 mL water, the unsaponifiable matter was extracted twice with 200 mL nhexane. Subsequent acidulation of the generated soap with 3 N HCl to pH 3 provided free FAs which were recovered by extraction with two portions of 400 mL n-hexane. Extracts were combined and washed with 400 mL deionized water. The organic phase was separated and dried over anhydrous Na2SO4, and the solvent was evaporated to dryness under vacuum, yielding 100.5 g of free FAs from sunflower oil. The dried residue was stored in an amber glass flask under nitrogen atmosphere in a refrigerator at 4 °C until needed. Portions thereof were used for shake flask tests and CCC separations (∼10 g). Gas chromatography coupled to electron ionization mass spectrometry (GC/MS) Prior to analysis of samples by means of GC/MS, the recovery standard 10,11-DC was added and FAs were derivatized to FAMEs by treatment of about 0.05–2 mg sample with 1 % H2SO4 in methanol for 2 h at 80 °C in a 10-mL tightly sealed derivatization tube with occasional shaking [23]. The solution was cooled down to room temperature, and 1 mL of deionized water and saturated NaCl solution was added. Subsequent addition of 2 mL n-hexane and thorough shaking transferred the FAMEs into the organic phase. An aliquot from the organic phase was mixed with the syringe standard 14:0-EE. Analysis of FAMEs was performed with a 5890 series II plus gas chromatograph interfaced to a 5971 MSD equipped with a 7673A autosampler (Hewlett-Packard/Agilent, Waldbronn, Germany). Chromatography was performed on two serially connected Rtx-2330 capillary columns (30 m length, 0.25 mm internal diameter, 0.1 μm film thickness, 10 % cyanopropylphenyl, 90 % biscyanopropyl polysiloxane; Restek, Bellefonte, PA, USA) with helium as carrier gas at constant flow rate of 1 mL/min. The initial oven temperature was 60 °C (held for 1 min) followed by a ramp of 6 °C/min to 150 °C, then the temperature was raised at 4 °C/min to 190 °C and at 7 °C/min to a final temperature of 250 °C, held for 7 min (total run time 41.6 min). Injector and transfer line temperatures were maintained at 250 and 170 °C, respectively. Samples (1 μL) were introduced using splitless injection. In full scan mode, a solvent delay of 8 min was applied and mass spectra were recorded from m/z 50 to m/z 500. In selected ion monitoring (SIM) mode, six fragment ions, m/z 74 and m/z 87
Overcoming the equivalent-chain-length rule
for saturated and monounsaturated methyl esters, m/z 79 and m/z 81 for di- and polyenoic methyl esters of FAs, and m/z 88 and m/z 101 for ethyl esters of the internal standard 14:0-EE, were recorded throughout the run [23]. The content of FAs was determined in the SIM mode using 14:0-EE and 10,11DC as internal standards [23]. CCC apparatus Separations were performed using a CCC-1000 instrument (Pharma-Tech Research, Baltimore, MD, USA) holding three self-constructed and serially connected semi-preparative multilayer coils (polytetrafluoroethylene (PTFE) tubing, 1.6 mm internal diameter, 60 mL coil volume) [24]. A ternary 655A12 pump (Merck, Darmstadt, Germany) was used for solvent delivery, and manual sample injection was done with a type 50 rotary valve (Rheodyne, Cotati, CA, USA) equipped with a 10-mL sample loop for pH-zone-refining or a 4-mL sample loop for regular CCC (PTFE tubing, 1.6 mm internal diameter). In pH-zone-refining, the effluent was monitored by a spectral photometer 87 UV/Vis detector (Knauer, Berlin, Germany) with the wavelength λ set at 220 nm. For regular CCC, UV/Vis was not sensitive enough to detect unsaturated FAs. Therefore, a micro-splitter valve was installed which divided 1/10 of the effluent stream to a PL-ELS 2100 (Polymerlabs, Amherst, MA, USA) evaporative light scattering detector (ELSD). The nebulizer temperature was set to 35 °C, and the nitrogen gas stream was 1.2 mL/min. Fractions were collected by an Isco Retriever 500 (Teledyne Isco, Lincoln, NE, USA), and pH values of each fraction collected were determined with a model 340i pH Meter (WTW, Weilheim, Germany). The electrode was filled with a mixture of methanol and water saturated with KCl for the determination of pH values in organic solvents. After each measurement, the electrode was rewetted to avoid drifting of the pH values. Selection of two-phase solvent systems for pH-zone-refining CCC The selection of an appropriate two-phase solvent system represents the most important step in pH-zone-refining CCC method development. Successful separations can be achieved if the solvent system provides partitioning coefficients K close to 1 for the target compound and an ideal range of the K values under both acidic (Ka ≪1) and basic (Kb ≫1) conditions for the target compounds [17]. Hence, FAs should show a very large difference between their neutral, protonated and ionized, deprotonated forms in terms of solubility and partitioning in the upper and lower phase of a two-phase solvent system. For testing the applicability of various solvent systems, portions of 0.1 mg FA extract were transferred into a 2-mL vial and 500 μL of upper and lower phases of the respective solvent systems was added. The vial was shaken for ∼30 s, and after
phase separation was reached, 100 μL of each phase was separated and the solvent was removed by a gentle stream of nitrogen at 40 °C. After derivatization into FAMEs, analysis by means of GC/MS-SIM was performed. For the determination of K values, peak areas of the main FAs in the upper phase were divided by that in the lower phase and evaluated from measurements in triplicate with accuracy estimated to be ±5 %. The procedure was repeated with addition of NH3 to the sample containing the equilibrated two-phase solvent system (pH ∼11) for the determination of Kb values. Likewise, TFA was added to the mixture (pH ∼1) and Ka values were subsequently determined. Preparation of solvent system and sample solutions The two-phase solvent system consisted of n-hexane/acetonitrile/methanol/water (4/7/1.4/0.5, v/v) and was prepared by combining the solvents in a 1-L separatory funnel. The mixture was vigorously shaken and equilibrated overnight. The two phases were separated and degassed for 20 min in an ultrasonic bath. For pH-zone-refining CCC, the lower phase was made basic by addition of NH3 (retainer), and the upper phase was made acidic with TFA (eluter). After preliminary tests, TFA was selected since it is most commonly used and rarely challenged in pH-zone-refining CCC with acidic analytes because it distributes almost completely in the organic phase, and it can be removed easily after the separation. As inorganic base, NH3 was selected since it is frequently used especially for carboxylic acids [17] and removal is also possible. Sample solutions for regular CCC were prepared by dissolving 50 mg of the FA extract in 2 mL of upper and 2 mL of lower phase of the twophase solvent system. In pH-zone-refining, 500 mg FA extract was either dissolved in 10 mL of lower phase containing 20 mM NH3 for normal displacement mode or in 10 mL upper phase containing 20 mM TFA for reverse displacement mode. Sample loadings were not increased since this measure results in broader pH-zones while the overall elution profile remains constant [17]. Separation procedure Separations in regular CCC were performed by filling the column with the stationary phase under rotation of the centrifuge at 1,000±10 rpm. Equilibration was done with mobile phase at 1 mL/min followed by sample injection after hydrodynamic equilibrium was reached. In pH-zone-refining, modes were initiated by entirely filling the column with the stationary phase which either represented the lower aqueous phase containing NH3 for normal displacement mode (tail-tohead elution) or the upper organic phase containing TFA as retainer for reverse displacement mode (head-to-tail elution). The centrifuge was rotated at 1,000±10 rpm and sandwich
M. Englert, W. Vetter
injection of 10-mL sample solution was done. Mobile phase containing the eluter was then pumped into the column at a flow rate of 2 mL/min. After the mobile front emerged at the column outlet, fractions were collected in intervals of 3 min (6 mL). Subsequently, pH values of each fraction were measured with a pH meter. Individual fractions were brought to dryness by an RVC 2-33 IR speed-vac concentrator (Christ, Osterode, Germany). Removal of residual TFA after normal displacement mode was accomplished by addition of 2 mL 5 % HCl to the dried fractions, followed by agitation and extraction of the FAs with 6 mL n-hexane. To recover the separated FAs after reverse displacement mode, 1 mL water and 2 mL 5 % HCl were added to each dried fraction to neutralize the soaps. Mixtures were agitated and extracted with 6 mL n-hexane. Dilutions of the each fraction in n-hexane were subjected to GC/MS-SIM analysis. After the separation was completed, the remaining column contents were collected into a graduated cylinder by applying pressurized nitrogen gas (0.5 MPa) to the inlet. In this way, the stationary phase retention Sf could be determined at the end of the separation.
Results and discussion Analysis of the fatty acid sample The free FAs obtained from sunflower oil were analyzed prior to the CCC fractionation by GC/MS to determine the FA profile (Fig. 1). The sample consisted mainly of the FAs 18:2n-6 (45.2 %), 18:1n-9 (24.2 %), and 16:0 (20.9 %) along with small amounts of 14:0 and 22:0 (Table S1, supplementary data). Full saponification of the sunflower oil sample was verified by high-temperature gas chromatography showing the absence of acylglycerols and other esterified compounds in comparison to the unsaponified oil (Fig. S1, supplementary data). This analysis was performed to ensure that the crude FA extract contained no neutral compounds which may interfere with the pH-zone-refining mode CCC through elution in the pH-zones of the target compounds and thereby contaminate 18:2n-6
Irel
the latter. The overlap of FAMEs with same ECL was avoided by using temperature programming of the GC oven [25].
Selection of solvent system for pH-zone-refining CCC Several non-polar aqueous solvent systems were first evaluated for their suitability in pH-zone-refining mode CCC. Nonaqueous solvent systems (e.g., n-hexane/acetonitrile) that are also suitable for the separation of FAs [13] were not selected because a minute amount of water was necessary in the lower phase to achieve solubility of the supplemented inorganic base NH3 and FAs being present in the ionized carboxylate state. The K values increased with increasing alkyl chain lengths and decreased with the numbers of double bonds present in the alkyl chain of the FAs (Table S2, supplementary data). In CCC, small K values (2.5) lead to peak band broadening [26]. The majority of the examined solvent systems provided suitable K values between 0.4≤K≤2.5 for the FAs, but nonideal Ka and Kb values were observed. The solvent system composed of n-hexane/acetonitrile/methanol/water (4/7/1.4/ 0.5, v/v/v/v) was selected because it provided K values in a suitable range for head-to-tail (0.40–1.35, Table 1) and tail-tohead (0.74–2.5) elution mode. This solvent system also fulfilled the criteria for pH-zone-refining CCC since the Ka and Kb values showed distinct partitioning behavior in dependence of the pH value of the solvent system. A strong partitioning of the protonated form of the FAs into the upper phase (Ka = 13.6–17.2) could be observed at pH ∼1, while the deprotonated form present at pH ∼11 showed high affinity for the lower phase (Kb =0.05–0.08) (Table 1). Determination of the normalized polarity parameters ETN according to the Reichardt polarity index [24] verified that the polarity of the
Table 1 Partitioning coefficients in the solvent system under neutral (K), acidic (Ka), and basic (Kb) conditions and separation factor α between consecutive major fatty acids in the solvent system used for CCC separation Fatty acid
K
Kaa
Kba
18:2n-6
0.40
16.3
0.05
αb
1.28 14:0
0.51
17.2
0.04
18:1n-9
0.75
14.4
0.06
16:0
0.78
16.8
0.07
18:0
1.35
13.6
0.08
18:1n-9
1.04
16:0
14:0
1.47
18:0
22:0
1.73 18.0
20.0
22.0
24.0
26.0
28.0
30.0
32.0
34.0[min]
Fig. 1 GC/MS-SIM chromatogram showing the fatty acid profile of the free FAs from sunflower oil after alkaline saponification (measured as FAMEs)
a
Mean values of triplicate determination
b
Calculated from neutral K values
Overcoming the equivalent-chain-length rule
14:0 with the difference however that a higher amount (∼80– 90 %) of pure 18:2n-6 could be isolated after (tail-to-head mode) or before (head-to-tail mode) elution of 14:0 due to the relatively low concentration of 14:0 compared to 18:2n6, respectively. Isolation of pure 14:0 was not possible with both modes as it was always accompanied by 18:2n-6. Nevertheless, isolation of pure 18:0 was possible in both modes because no critical FAs with similar ECL value (18) were present in the sample. The regular CCC separations also enabled the detection and isolation of the long-chain FAs 24:0 and 20:0 which were both initially not detected in the unfractionated sample (Fig. 1). Purification of all individual FAs in the sample by regular CCC was practically impossible in one step since FAs are subjected to the ECL rule.
upper (ETNUP =6.8) and lower phase (ETNLP =53.0) showed great difference (ΔETN=46.2) in the solvent system which confirms the distinct Ka and Kb values [27]. Regular CCC separation Fractions collected during regular CCC separations of 50 mg free FAs obtained from sunflower oil verified the co-elution of 18:2n-6 with 14:0 as well as 18:1n-9 with 16:0. This was expected from the low separation factors α between the consecutive FA pairs (Table 1) caused by same ECL values of 14 and 16, respectively. Insufficient chromatographic selectivity and strong overlapping were the case in both tail-to-head and head-to-tail mode. The FAs 18:1n-9 and 16:0 could not be separated from each other and showed nearly the same (co)elution behavior (Fig. 2, highlighted by rectangular zones). In tail-to-head mode, a small share (∼5 % of total content, collected in one fraction) of 18:1n-9 was collected with high purity (∼98 %) because the elution started slightly before 16:0 (Fig. 2a). Equally in head-to-tail mode, a fraction could be collected which contained 16:0 (purity ∼98 %) nearly without concomitant 18:1n-9 (Fig. 2b). Yet, this fraction also only represented ∼5 % of the total content of the FA. The same behavior was observed for the critical FA pairs 18:2n-6 and
The pH-zone-refining mode introduced the acid strength (pKa values) of the respective FAs as a second dimension in the separation process. The pKa values (measured in water) of 14:0, 16:0, 18:0, 18:1n-9, and 18:2n-6 show slight differences in dependence of the alkyl chain length and number of double bonds present in the FA molecule [15]. Plotting of pKa values against the K values of the FAs in the solvent system resulted
a)
16:0
ELSD signal [mV]
18:1n-9 16:0 18.0 20.0 22.0 24.0 26.0 28.0[min]
18:0 20:0 22:0 0
30
60
90
1
K=
24:0
120
150
2
180
[min]
3
18:2n-6
Irel
b)
14:0
18:1n-9 16:0 18.0 20.0 22.0 24.0 26.0 28.0[min]
18:2n-6 14:0
24:0 18:0 22:0 20:0
0 K=
18:1n-9
Irel
18:2n-6 14:0
ELSD signal [mV]
Fig. 2 CCC-ELSD chromatogram obtained during separation of free fatty acids from sunflower oil. a Head-to-tail mode and b tail-to-head mode with co-elutions of fatty acids highlighted as rectangular zones and exemplary GC/MS chromatograms of collected fractions containing the co-eluting pairs 14:0 and 18:2n-6 as well as 16:0 and 18:1n-9. Experimental conditions: CCC-1000 instrument with 60 mL coil volume and 1 mL/min mobile phase flow rate; rotation speed=1,000±10 rpm; solvent system: n-hexane/acetonitrile/methanol/water (4/7/1.4/ 0.5, v/v); sample size=50 mg. K points representing the column volume (Vc =60 mL, K=1) and the double (Vc =120 mL, K=2) and threefold column volume (Vc =180 mL, K=3) are additionally labeled on the x-axis
pH-zone-refining CCC
30
60 1
90
120 2
150
180 3
[min]
M. Englert, W. Vetter
CCC separations to find conditions where the ECL rule can be overcome and to investigate the effects on the separation (Table S-3). During the investigations, the retainer concentration was fixed at 20 mM because this parameter only affects the concentration of the analytes in the stationary phase, thus enhancing the retention of the FAs. Within both normal displacement and reverse displacement pH-zone-refining CCC, it could be observed that for a molar eluter-to-retainer ratio below 0.5, no pH-zones were formed and all of the FAs were immediately washed off the column. A value of 0.5 resulted in the formation of pH-zones with short retention times and incomplete resolution between all of the eluting zones. If the molar eluter-to-retainer ratio exceeded a value of 2, a long retention time was observed, and analysis by means of GC/ MS revealed that the elution order was exclusively determined by the acid strength (pKa values) of the FAs. In normal displacement pH-zone-refining CCC, best results could be obtained with 20 mM NH3 in the stationary phase (retainer) and 35 mM TFA in the mobile phase (eluter) corresponding to a molar eluter-to-retainer ratio of 1.75 (Fig. 4a). Online monitoring of the separation process by UV/Vis allowed detecting the TFA trace as well as FAs, whereas offline pH determination of the fractions allowed tracking the formation of individual pH-zones. After injection of 500 mg free FAs obtained from sunflower oil, the mobile front containing TFA appeared
18:0 18:1n-9 18:2n-6 16:0 14:0
1.4
Fig. 3 Acid strength (pKa value) of major fatty acids 14:0/16:0/18:0/ 18:1n-9 and 18:2n-6 plotted against partitioning coefficients K in the two-phase solvent system. Highlighted by rectangular zones are fatty acids 18:2n-6/14:0 and 18:1n-9/16:1 which showed co-elutions during regular CCC due to very similar K values
in a hypothetical, orthogonal separation mode as the selectivities of both separation processes are independent from each other (Fig. 3). Hence, 18:2n-6/14:0 and 18:1n-9/16:0 which co-eluted in regular CCC displayed sufficient distance to each other in this separation space which enabled to circumvent coelutions pursuant the ECL rule. For this purpose, we were running a series of studies in both normal and reverse displacement pH-zone-refining mode by interchanging the elution mode and hence the role of the mobile and stationary phase as well as that of the retainer and eluter. Seven different eluter-to-retainer molar concentration ratios were tested in
a)
12 Sf (0 min) = 77%
Absorbance (220 nm) [mV]
10
6
pH
8
14:0
4
0
30
60
90
120
150
180
2
0 [min] 210 240 270 300 12 Sf (0 min) = 75% 10 Sf (270 min) = 65%
6
20:0;22:0 24:0
4
0
30
60
90
120
150
2 0
180
210
240 [min]
pH
8
14:0;16:0
b)
Sf (330 min) = 68%
Absorbance (220 nm) [mV]
Fig. 4 Separation of 500 mg fatty acids from sunflower oil with pHzone-refining CCC in a normal displacement (20 mM NH3 (retainer) and 35 mM TFA (eluter)) and b reverse displacement mode (20 mM TFA (retainer) and 35 mM NH3 (eluter)) with UV/Vis signal and pH curve obtained by measuring each fraction. Experimental conditions: CCC-1000 instrument with 60 mL coil volume and 2 mL/min mobile phase flow rate; rotation speed=1,000±10 rpm; solvent system: n-hexane/acetonitrile/methanol/water (4/7/1.4/ 0.5, v/v) with TFA in the upper phase and NH3 in the lower phase
20:0 22:0 24:0
1.2
18:0
0.4 0.6 0.8 1.0 Partitioning coefficient K
18:2n-6
0.2
18:0
0
18:1n-9
8.0 7.5 7.0
18:2n-6
9.5 9.0 8.5
16:0
10.0
18:1n-9
Acid strength (pKa)
10.5
Overcoming the equivalent-chain-length rule Irel
Fig. 5 GC/MS analysis of selected fractions obtained during the separation of 500 mg fatty acids from sunflower oil with pHzone-refining CCC in normal displacement mode. Analyses were performed after conversion of fatty acids into fatty acid methyl esters
Irel
14:0
>98%
Irel
>95%
18:2n-6
18.0 20.0 22.0 24.0 26.0 28.0 [min] 18.0 20.0 22.0 24.0 26.0 28.0 [min] Irel
16:0
>98%
>96%
18.0 20.0 22.0 24.0 26.0 28.0 [min] Irel
>96% 18:0
Irel
18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 [min]
>98%
20:0
18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 [min]
18.0 20.0 22.0 24.0 26.0 28.0 Irel
22:0
Irel
>95%
18:1n-9
18.0 20.0 22.0 24.0 26.0 28.0 [min]
in the UV/Vis chromatogram at 15 min (fraction 1), and the initial pH value of the effluent (11.0, point A) slowly decreased to 10.2 (point B) due to neutralization of NH3 in the stationary phase. Slight phase bleeding occurred from 55 to 65 min (fractions 8–10) which caused a short irregularity of the pH level and a reduced overall yield, but this did not Table 2 Recovery, yield, and purity of the individual fatty acids isolated from 500 mg fatty acids from sunflower oil with pH-zonerefining CCC in normal displacement mode Fatty acid
Yield (mg)
Purity (%)
Recovery (%)
14:0 16:0 18:1n-9 18:2n-6 18:0 20:0 22:0 24:0
1.5 88.0 104.3 194.2 36.6 1.5 3.3 1.5
98 98 95 95 96 98 96 95
80.0 85.0 86.9 86.7 86.1 75.0 82.5 75.0
>95%
24:0
18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 [min]
impair the separation process and resolution. A pH-zone was observed at 70 min (fraction 10) when a pH plateau was formed and the pH decreased to ∼8.5 (point C). The separation was completed after 330 min with a stationary phase retention of Sf =68 % and a final pH value of ∼2.5 (point D). Analysis of the collected fractions by means of GC/MS revealed that the eight FAs present in the sample were eluted individually as consecutive rectangular pH-zones in nearly equal molar concentrations with minor overlap bands only (i.e., mixing zones) containing consecutive FAs. In general, the formation of a pH plateau during the separation represented the elution of a FA with high purity (>95 %). The elution order differed from the corresponding regular CCC separation in tail-to-head mode and the saturated FAs 14:0 and 16:0 eluted first, sharing the lowest pKa values of 8.1 and 8.6, respectively. This is consistent with the general behavior in pH-zone-refining of stronger acids eluting earlier [8]. Afterwards, the unsaturated FAs 18:1n-9 (pKa value 9.9, K=0.75) and 18:2n-6 (pKa value 9.2, K=0.40) formed two broad pH-zones as the concentration of them was highest in the sample. In this case, however, the weaker FA eluted first, suggesting that the hydrophobicity (K
M. Englert, W. Vetter
value) had a stronger impact on the elution sequence. In the end, the saturated FAs 18:0 < 20:0 < 22:0 < 24:0 were eluted with slight zone overlapping between them according to increasing alkyl chain length and hydrophobicity or decreasing pKa values. In this particular case, isolation of all FAs present in the sample could be achieved with high purities (Fig. 5) and with acceptable individual recovery rates of the FAs between 75.0 and 86.9 % and a total recovery rate of 86.2 % (Table 2). Figure 4b shows the separation in the more commonly applied reverse displacement pH-zone-refining CCC mode performed under the equal conditions with 500 mg free FAs obtained from sunflower oil but inversely 20 mM TFA in the stationary phase and 35 mM NH3 in the mobile phase. The initial pH value of the effluent was acidic (pH ∼2) and increasing during the slow neutralization by NH3 in the mobile phase. In this case, the separation was completed more quickly in 160 min but at the beginning of the separation (20 min) stationary phase bleeding was also observed and led to a reduced stationary phase retention of Sf =65 % at the end. The formation of pH plateaus could be observed, suggesting the elution of pure FAs. However, GC/MS analysis showed that the saturated FAs 20:0, 22:0, and 24:0 co-eluted in the starting zone since they represented the strongest bases in the sample. Under these conditions, the saturated FAs 14:0 and 16:0 eluted after a broad mixing zone where 20:0, 22:0, 24:0, 14:0, and 16:0 were present. A separation of them could be not achieved. Yet, the following zones contained 18:1n-9, 18:2n6, and 18:0 in this elution sequence with high purities. However, the mixing zones between them were broader than in the normal displacement mode leading to a reduced yield of the individual FAs.
Conflict of interest The authors declare no conflict of interest.
References 1. 2. 3.
4. 5. 6.
7.
8.
9.
10.
11.
12. 13.
Conclusion 14.
The ECL rule presents a challenge in the purification of FAs with liquid chromatographic techniques such as CCC. FAs with the same ECL value, e.g., 16:0 and 18:1n-9 or 14:0 and 18:2n-6, were difficult to separate from each other with regular CCC due to the similar partitioning coefficients. Timeconsuming separations in multiple steps would be a solution. We have introduced the acid strength of the FAs as a second dimension in the separation process by a variation known as pH-zone-refining CCC. All free FAs obtained from sunflower oil could be isolated in the normal displacement mode according to their respective acid strengths and partitioning coefficients in a tenfold amount compared to regular CCC. It should be noted that transfer of the method to another vegetable oil may require adjustment of the molar concentrations and ratio of retainer and eluter. Acknowledgments The authors would like to thank the state of BadenWürttemberg for providing a stipend grant to Michael Englert.
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