2.1. Chemicals and materials
HPLC grade chloroform (CHCl3), methanol (MeOH), acetonitrile (ACN), n-hexane, acetone, and anhydrous ethanol (EtOH) were purchased from Tedia (Fairfield, CT, USA). Analytical grade petroleum ether (PE) and ethyl acetate (EA) were purchased from Sinopharm (Beijing, China). All reference compounds were obtained from Innochem Scientific Co. Ltd (Beijing, China). The dried toad venom of Bufo bufo gargarizans, dammar resin and propolis were purchased from Luyan Pharmaceuticals (Xiamen, China) and identified by Professor Ying-Kun Qiu (Xiamen University, Xiamen, China).
2.2. Instrumentation
The first dimensional NPLC was performed on an Agilent 1200 analytical HPLC system, containing a quaternary pump and a DAD detector. The second NPLC system consists of a binary pump, a DAD detector and an integrated SE interface (Fig. 1a). The SE interface includes two Agilent 1290 Infinity Thermostatted column compartments, whose highest column temperature could reach 99°C. A 2-position 10-port switching valve (Valve A) and a 2-position 6-port switching valve (Valve B) were furnished in one of the Thermostatted column compartments for switching the flow path. Agilent Chemstation Rev. B.04.03 was used for data acquisition and processing. 2D-LC spectra were visualized by using ACD/Spectrus 2016 (Advanced Chemistry Development Inc., Toronto, Canada).
Insert Fig. 1 here
2.3. Sample preparation
The reference compounds were mixed in EtOH to afford the reference compounds solution with concentrations as shown Fig. 2a.
Ground toad venom, dammar resin, and propolis (1.0 g) were extracted with EA (20 mL) for 1.5 h using Soxhlet extractor, respectively. Each solution was concentrated under vacuum at 40˚C to obtain the corresponding extract. Then the three extracts were dissolved in CHCl3/MeOH (10:1) to obtain the test solution at concentration of 20 mg/mL, respectively.
2.4. SE interface setup
As shown in Fig. 1a, the two NPLC systems were coupled through the novel designed SE interface, which was applied to eliminate the NP solvents and adsorb the solute. In the SE interface, two equivalent Cosmosil silica gel enrichment columns (10 mm × 4.6 mm i.d., 5 µm) were furnished in the 2-position 10-port switching valve (Valve A) in one of the Agilent 1290 Thermostatted column compartment. In the NP enrichment column, the elevated column temperature was up to 99°C, and vacuum condition was applied to remove the NP solvent. The 2-port 6-position valve (Valve B) was equipped in another Thermostatted column compartment, which was designed not only to introduce the second dimensional NP mobile phase to the NP enrichment column (position A), but also to regenerate the NP enrichment column.
2.5. Optimization of the SE interface
To optimize the SE interface, a batch of 20 µL reference compounds mixture solutions were directly transferred to the NP enrichment column in different flow rates, by different mobile phases systems: CHCl3-MeOH (5:1), CHCl3-acetone (5:1), n-hexane-acetone (3: 1), n-hexane-anhydrous EtOH (3:1), and n-hexane-EA (2: 1). In the NP enrichment column, the solvent was evaporated at different temperature (30°C, 60°C, 90°C), with (SE interface) or without vacuum. The retained solutes were then eluted and analyzed in the second dimensional NPLC. Analysis on the second dimension was performed on an Inertsil SIL-100A column (150 mm × 4.6 mm i.d., 5 µm, Shimadzu Corporation, Kyoto Japan) with mobile phase of n-hexane (A) and anhydrous EtOH (B) in a gradient as follows: 24–52% B (0-3.5 min), 52–100% B (3.5-4 min), 100–100% (4.01-5.0 min), before returning to initial conditions at 5.1min. The flow rate was 3.0 mL/min. The column was maintained at 50°C and the detection wavelength was in 300 nm.
2.6. On-line 2D NPLC × NPLC separation
After optimization of SE interface, 20 µL of toad venom, dammar resin, and propolis test solutions were injected and analyzed on the 2D NPLC × NPLC system, respectively.
A HPLCONE silica gel column (250 mm × 4.6 mm i.d., 5 µm, Microwants Co. Ltd., Suzhou, China) was used for the first dimension. In the second dimension, a Cosmosil silica gel column (50 mm × 4.6 mm i.d., 5 µm, Nakalai Tesque Co. Ltd., Kyoto Japan) or a Luna silica gel column (150 mm × 4.6 mm i.d., 3 µm, Phenomenex Co. Ltd.) was chosen. In the SE interface, the NP enrichment column was kept at 99°C, and column regeneration time was 10 sec. As shown in Fig. 1b, the switching procedure (separation case of toad venom as example) of valve was as follows: Valve A was switched every 220 sec; Valve B was remained in position A for 210 sec and in position B for 10 sec, respectively. At elevated temperature and vacuum condition, the flow of the first NPLC effluent into the NP enrichment experienced rapid solvent evaporation, which rendered fast adsorption of solute in the NP enrichment column. With the switching of Valve A, the effluent from the NPLC continued to flow into the other silica gel enrichment column. Simultaneously, Valve B was kept at position A and the second dimensional NP mobile phase was delivered to the previous column to transfer the solute for the further NPLC separation. Once the second dimensional NPLC separation completed, Valve B was switched to another position, to establish an association between the NP enrichment column and vacuum pump for column regeneration (10 sec). The separations were carried out according to the conditions shown in Table 1.
Table 1
The analysis conditions of 2D NPLC × NPLC
No. | 1st Dimension a | 2nd Dimension b | DAD (nm) |
1a | (B/A)% c: 0–200 min: from 0–100%; v: 0.2 mL/min; | (C/D)% d: 0–3.5 min: from 5–20%; 3.5–3.51 min: run back to 5%; 3.51-4.0 min: 5% v: 3 mL/min | 300 |
1b | (E/F)% e: 0–300 min: from 2–12%; v: 0.2 mL/min | 300 |
2a | (E/F)% e: 0–200 min: from 0–19%; v: 0.2 mL/min | (C/D)% d: 0–3.5 min: from 2–38%; 3.5–3.51 min: run back to 2%; 3.51-4.0 min: 2%; v: 3 mL/min | 254 |
3a | (E/F)%e: 0–200 min: from 0–20%; v: 0.05 mL/min | (G/H)% f: 0–3 min: from 20–65%; 3-3.5 min: keep 65%; 3.5–3.51 min: run back to 20%; 3.51-4.0 min: 20%; v: 2.1 mL/min | 280 |
3b | (E/F)%e: 0–200 min: from 0–20%; v: 0.05 mL/min | (G/H)% f: 0–2.5 min: from 20–65%; 2.5-4.0 min: keep 65%; 4.0–4.21 min: run back to 20%; 4.1–4.5 min: 20%; v: 2.1 mL/min | 280 |
Test solution of ethyl acetate extract was injected in separation of No. 1–3, with of |
sample loading 20 µL. The sample solution of No. 1, No. 2 and was No. 3 was toad |
venom, dammar resin and propolis, respectively. |
aAll separation of the second dimension was performed on a Cosmosil 3SL column (50 mm × 4.6 mm i.d., 5 µm, Nakalai Tesque Co. Ltd., Kyoto Japan), except for the |
separation case of 3b, in which a Luna silica gel column (150 mm × 4.6 mm i.d., 3 µm, Phenomenex Co. Ltd.) was chosen. |
*Solvent system: CHCl3 (A) / acetone (B); n-hexane (C) / anhydrous ethanol (D); CHCl3 (E) / MeOH (F); PE (G) / EA (H). |
Insert Table 1 here
2.7. On-line 2D RPLC × RPLC separation
Test solution of the sample EA extract was injected in separation with of sample loading 20 µL. First dimension was performed by a Cosmosil C18 column (250 mm × 4.6 mm i.d., 5 µm, Nakalai Tesque Co. Ltd., Kyoto Japan). The mobile phase was constituted of MeOH as solvent C, and water (H2O) as solvent A. The gradient was: 0 min (60% C), 200 min (85% C). The flow rate was 0.1 ml/min. The separation of the second dimension was performed on a Kinetex XB-C18 column (Phenomenex, 50 × 4.6 mm i.d., 5 µm). The mobile phase was constituted of ACN as solvent B, and H2O as solvent A. The gradient elution was as follows: 0–6 min, from 15 to 80% B; 6–6.1 min: from 80 to 15% B; 4.51-5 min, 15% B. Flow rate and temperature were set to 0.1 ml/min and 50°C, respectively. UV chromatograms were recorded at 254 nm. The interface modulation time is 5 min. Enrichment columns were used in the 2D-LC interface, H2O was pumped according to the dilution ratio (100% A) at 0.1 ml/min, to dilute the eluents from the 1st dimension and to trap them onto the enrichment columns.