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Polyvinyl Alcohol Modified Porous Graphitic Carbon Stationary Phase for Hydrophilic Interaction Liquid Chromatography Yanjie Hou, Feifang Zhang, Xinmiao Liang, Bingcheng Yang, Xiaodong Liu, and Purnendu K. Dasgupta Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04384 • Publication Date (Web): 07 Apr 2016 Downloaded from http://pubs.acs.org on April 8, 2016
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Polyvinyl Alcohol Modified Porous Graphitic Carbon Stationary Phase for Hydrophilic Interaction Liquid Chromatography Yanjie Hou,† Feifang Zhang,† Xinmiao Liang,† Bingcheng Yang,† * Xiaodong Liu,‡ and Purnendu K. Dasgupta§* †
School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China ‡
Thermo Fisher Scientific, 445 Lakeside Drive, Sunnyvale, CA 94087
§
Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX 76019-0065
*
Corresponding Authors:
Email:
[email protected];
[email protected] Fax: 86-21-64251830; 1-817-272-3812
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ABSTRACT. We report a polyvinyl alcohol (PVA)-coated porous graphitic carbon (PGC, HypercarbTM) packing as a novel stationary phase for hydrophilic interaction liquid chromatography (HILIC). The exterior and the pores of the PGC particles are coated with a thin layer of PVA by soaking the particles in a PVA solution, filtering, and thermally crosslinking the PVA. Such PVA coated PGC particles (5.7 m diameter), hereinafter called PVA-PGC are stable at least through pH 1.0-12.7, can be made in 25x in PVA-PGC. Scanning electron microscopy (SEM) shows no significant difference in the morphology or size. SEM imaging (n=5000 ea.) of PGC and PVA-PGC was performed and provided respective mean diameters of 5.690.75 and 5.720.74 m (Figure S3). The lack of a perceptible increase in size would indicate that the added PVA went largely to the pores of the particles. Based on the elemental analysis data, if the pores (pore volume 1.050.10 cm3/g, surface area 120 m2/g)11 were uniformly coated, the coating will be equivalent to a ~3 nm thick solid PVA layer (see Supporting Information for calculations) without any perceptible increase in the particle size. The operational thickness must be higher than this; PVA is well known for its propensity of PVA to imbibe very large amounts of water12 forming highly porous filamentous hydrogels.8
Chromatographic Behavior of PGC vs. PVA-PGC. The surface wetting characteristics of the PGC particles change dramatically upon PVA coating 5
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(Figure S4). This also indicates that the coating is not only in the pores but the exterior surface as well. Dinh et al. suggested kcytosine/kuracil as a function of [ACN] as a HILIC behavioral index.13 Figure 1a shows that PVA-PGC (but not PGC) clearly exhibits HILIC behavior. Figure 1b demonstrates a separation of seven nucleosides on both the bare PGC and the PVA-PGC phases at high [ACN]. The PVA-PGC phase exhibits excellent selectivity and better peak symmetry compared to the bare PGC; note also the significantly lower pressure drop (~50%). At the present time, the cause for this lower pressure drop (that has been observed for multiple columns packed with different batches of PGC coated with PVA) is under investigation. We only note here that microscopy shows no significant increase in particle size and poor packing is untenable as an explanation for the reduced pressure drop: poor efficiency that would have resulted is not observed. Given that the base particle size is 5.7 m, the efficiency of the present column is quite comparable to HILIC columns described in the literature. The PVA-PGC column produced ~70,000 plates/m for cytosine, compared to ~ 72000 plates/m on a 5 m silica based zwitterionic HILIC column, ~75,000 on PVA on 5 m silica,7 and 75200-114500 plates on different ionic liquid-based HILIC columns on 3 m silica substrates,14 for the same analyte.
Illustrative Separations.
The applicability of PVA-PGC as a selective
HILIC phase is demonstrated in Figure 2 for various phenols and aromatic carboxylic acids by UV detection and in Figure 3 for various sugars and amino acids. The present phase compares well with similar class separations on the 6
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carboxylated PGC phase.5 The eight carboxylic acids in Figure 2(a) range in pKa from 2.28 to 4.58. At pH 8.2, the PVA coating cannot possibly be positively charged; electrostatic contributions to retention should be negligible. Nicotinic acid elutes significantly after the other analytes and is the only one in this group based on a pyridine ring, rather than benzene and still shows the second best symmetry and efficiency among the group. The elution order is consistent overall with a HILIC retention mechanism; the same is true of the excellent separation of the phenols in Figure 2(b) where the retention follows the polarity order. The efficiency for resorcinol in Figure 2b is ~118,000 plates/m. Efficient separation of neutral sugars is challenging. In Fig. 3(a) the retention follows the polarity order as well with efficiencies of 30,000-40,000 plates/m. Retention of the amino acids (Figure 3b) follow their solubility order except for tyrosine which is retained more, likely due to additional H-bonding. An evaporative light scattering detector was used for the experiments in Figure 3; the low and highly stable baseline indicates that the PVA is not leached significantly from the column. Adherence to a Quantitative HILIC Model. One quantitative HILIC retention model15 posits: ln k = a + b lnφ + cφ
...(1)
where k is the retention factor, a, b, and c are constants, and φ is the volume fraction water in the eluent. Figure S5 demonstrates excellent fits (r2 0.9988-0.9996) for 4 nucleosides at 5 different eluent compositions (φ 0.10-0.30). 7
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Following Alpert’s original work,16 based on literature meta-analysis, Hemström and Irgum17 proposed partitioning as the primary retention mechanism in HILIC. Increased eluent electrolyte concentration apparently enhances the polarity of the partitioning water layer and/or increases the thickness of the same, causing increased retention; the same is presently observed (Figure S6). Selectivity vs. Other HILIC Phases. Lucy and coworkers5,18 have proposed simultaneous graphical depiction of the HILIC character and the negative electrostatic character of a phase. The HILIC character was represented by kcytosine/kuracil while the negative electrostatic character was determined by kBTMAC/kuracil, BTMAC being benzyltrimethylammonium chloride. We similarly depict the position of the present phase along with those of 7 other commercial phases as well as the PGC-COOH phase5 (Figure S7). PVA-PGC and PGC-COOH occupy the lowest and highest positions in their negative electrostatic character whereas the HILIC index for PVA-PGC is intermediate among the phases studied. However, the charge neutrality of the present phase is best examined by looking at its behavior towards a negatively charged analyte, e.g., p-toluenesulfonic acid (PTSA), a plot of kPTSA/kuracil vs. kBTMAC/kuracil uniquely locates the present phase closest to the origin, exhibiting the least interaction with either of these analytes. (Figure S8). Taking this teaching5 to the next step, we propose that selectivities can be depicted as a 3-D plot where in addition to kcytosine/kuracil and kBTMAC/kuracil, a third axis can represent kPTSA/kuracil as a measure of the positive electrostatic character of the phase (Figure 4). Note that PVA-PGC occupies a unique 8
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position in having the lowest charge based interaction but displaying very strong HILIC character.
Dinh et al.13 proposed kadenosine/kadenine as an index for
H-bonding affinity. While this should be high for a PVA-coated column, this index was not found to be high for the PVA-PGC phase (Figure S9). Stability of PVA-PGC. The PVA is not bonded to the PGC particles. Although it may be crosslinked, the longevity of such a coating would obviously be of concern. The retention of three model analytes (adenine, nicotinic acid, and BTMAC, respectively neutral, negatively, and positively charged) was studied over 40 h (2000 column volumes) of continuous operation with 75% acetonitrile-25% 20 mM ammonium formate (pH 7.8) as eluent. The retention times (tR) and plate counts (N) for all three model analytes were essentially constant (0.073%-0.473% rsd), see Figure 5. Intra-day and inter-day rsd’s of the retention time were in the range of 0.053-0.136% (n=9) and
